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The persistence of SARS-CoV-2 in pharyngeal swabs belonging to patients affected by COVID-19 is not a rare occurrence. Indeed, the viral load determined through real-time reverse-transcriptase PCR (rRT-PCR) test peaks at the onset of symptoms and decreases to undetectable level within 1–3 weeks.1 Nevertheless, few studies investigated the potential infectivity of the clinical specimens, and to date viral culture isolation failed after some weeks from the first symptoms,1 being the 18th day the last timing in which virus replication in vitro was achieved in immunocompetent host.2 Here we report the case of SARS-CoV-2 infection positivity at the molecular test after 42 days, describing successful viral replication in vitro obtained after 27 days from the first positive swab. A 58-year-old man resulted positive to SARS-CoV-2 during routine screening for health workers; he was initially asymptomatic. He underwent isolation and was treated with lactoferrin, vitamin C and D. The subsequent day after testing positive, swab specimens from his wife and daughter yielded positive for SARS-CoV-2 infection; however, they returned negative after 15 and 10 days, respectively. After 4 days he developed fever (max 38.7°C) and cough and was treated with paracetamol and levocloperastine. At the seventh day after the swab positivity he aggravated and presented dyspnoea exertional, although with 94%–97% saturation and no pulmonary abnormalities evidenced by direct lung auscultation; treatment with azithromycin (500 mg/day) for 6 days was started. After 3 days of treatment, he did not show fever. After 23 days he continued to have a cough that was successfully treated with prednisone (50 mg/day for 4 days, 25 mg/day for 2 days, 12.5 mg/day for 2 days). At the haematologic analysis the unique alteration regarded an increment of D-dimer (D-dimer=996 ng/mL), a characteristic associated with a hypercoagulative status often found in severe cases of COVID-19.3 Treatment with fodaparinux (2.5 mg/day for 10 days) was initiated. The leucocytes …

BackgroundPerformance characteristics of SARS-CoV-2 antigen tests among children are limited despite the need for point-of-care testing in school and childcare settings. We describe children seeking SARS-CoV-2 testing at a community site and compare antigen test performance to real-time reverse transcription-polymerase chain reaction (RT-PCR) and viral culture.MethodsTwo anterior nasal specimens were self-collected for BinaxNOW antigen and RT-PCR testing, along with demographics, symptoms, and exposure information from individuals ≥5 years at a community testing site. Viral culture was attempted on residual antigen or RT-PCR positive specimens. Demographic and clinical characteristics, and the performance of SARS-CoV-2 antigen tests, were compared among children (ResultsAbout one in ten included specimens were from children (225/2110); 16.4% (37/225) were RT-PCR positive. Cycle threshold values were similar among RT-PCR positive specimens from children and adults (22.5 vs 21.3, p=0.46) and among specimens from symptomatic and asymptomatic children (22.5 vs 23.2, p=0.39). Sensitivity of antigen test compared to RT-PCR was 73.0% (27/37) among specimens from children and 80.8% (240/297) among specimens from adults; among specimens from children, specificity was 100% (188/188), positive and negative predictive value were 100% (27/27) and 94.9% (188/198) respectively. Virus was isolated from 51.4% (19/37) of RT-PCR positive pediatric specimens; all 19 had positive antigen test results.ConclusionsWith lower sensitivity relative to RT-PCR, antigen tests may not diagnose all positive COVID-19 cases; however, antigen testing identified children with live SARS-CoV-2 virus.

Summary Background Lateral flow devices (LFDs) for rapid antigen testing are set to become a cornerstone of SARS-CoV-2 mass community testing, although their reduced sensitivity compared with PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is, therefore, essential for successful implementation. We evaluated six commercial LFDs and assessed their correlation with infectious virus culture and PCR cycle threshold (Ct) values. Methods In a single-centre, laboratory evaluation study, we did a head-to-head comparison of six LFDs commercially available in the UK: Innova Rapid SARS-CoV-2 Antigen Test, Spring Healthcare SARS-CoV-2 Antigen Rapid Test Cassette, E25Bio Rapid Diagnostic Test, Encode SARS-CoV-2 Antigen Rapid Test Device, SureScreen COVID-19 Rapid Antigen Test Cassette, and SureScreen COVID-19 Rapid Fluorescence Antigen Test. We estimated the specificities and sensitivities of the LFDs using stored naso-oropharyngeal swabs collected at St Thomas' Hospital (London, UK) for routine diagnostic SARS-CoV-2 testing by real-time RT-PCR (RT-rtPCR). Swabs were from inpatients and outpatients from all departments of St Thomas' Hospital, and from health-care staff (all departments) and their household contacts. SARS-CoV-2-negative swabs from the same population (confirmed by RT-rtPCR) were used for comparative specificity determinations. All samples were collected between March 23 and Oct 27, 2020. We determined the limit of detection (LOD) for each test using viral plaque-forming units (PFUs) and viral RNA copy numbers of laboratory-grown SARS-CoV-2. Additionally, LFDs were selected to assess the correlation of antigen test result with RT-rtPCR Ct values and positive viral culture in Vero E6 cells. This analysis included longitudinal swabs from five infected inpatients with varying disease severities. Furthermore, the sensitivities of available LFDs were assessed in swabs (n=23; collected from Dec 4, 2020, to Jan 12, 2021) confirmed to be positive (RT-rtPCR and whole-genome sequencing) for the B.1.1.7 variant, which was the dominant genotype in the UK at the time of study completion. Findings All LFDs showed high specificity (≥98·0%), except for the E25Bio test (86·0% [95% CI 77·9–99·9]), and most tests reliably detected 50 PFU/test (equivalent SARS-CoV-2 N gene Ct value of 23·7, or RNA copy number of 3 × 106/mL). Sensitivities of the LFDs on clinical samples ranged from 65·0% (55·2–73·6) to 89·0% (81·4–93·8). These sensitivities increased to greater than 90% for samples with Ct values of lower than 25 for all tests except the SureScreen fluorescence (SureScreen-F) test. Positive virus culture was identified in 57 (40·4%) of 141 samples; 54 (94·7%) of the positive cultures were from swabs with Ct values lower than 25. Among the three LFDs selected for detailed comparisons (the tests with highest sensitivity [Innova], highest specificity [Encode], and alternative technology [SureScreen-F]), sensitivity of the LFDs increased to at least 94·7% when only including samples with detected viral growth. Longitudinal studies of RT-rtPCR-positive samples (tested with Innova, Encode, and both SureScreen-F and the SureScreen visual [SureScreen-V] test) showed that most of the tests identified all infectious samples as positive. Test performance (assessed for Innova and SureScreen-V) was not affected when reassessed on swabs positive for the UK variant B.1.1.7. Interpretation In this comprehensive comparison of antigen LFDs and virus infectivity, we found a clear relationship between Ct values, quantitative culture of infectious virus, and antigen LFD positivity in clinical samples. Our data support regular testing of target groups with LFDs to supplement the current PCR testing capacity, which would help to rapidly identify infected individuals in situations in which they would otherwise go undetected. Funding King's Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation, UK Department of Health, National Institute for Health Research Comprehensive Biomedical Research Centre.

Background Vertical transmission of SARS-CoV-2 has been reported but does not appear to be common. This study aims to systematically review the evidence for vertical transmission of SARS-CoV-2. Methods This review is part of an Open Evidence Review on the transmission dynamics of SARS-CoV-2 and the role of intrauterine mother to fetus transmission. Literature searches were performed in the WHO Covid-19 Database, LitCovid, medRxiv, and Google Scholar for SARS-CoV-2 using keywords and associated synonyms, search date up to 20 December 2020, no language restrictions. Results We included 106 studies assessing vertical transmission of SARS-CoV-2 from pregnant women to their neonates: these studies comprised 40 reviews (21 fulfilled systematic review methodology, including risk of bias assessment of included studies) and 66 primary studies including 32 case reports (of up to two cases) and 34 prospective and retrospective cohort studies, prospective and retrospective case series, observational studies (including asymptomatic screening), database studies and a quality improvement project. Almost all were conducted in a hospital setting. The 32 case reports were considered to be at high risk of bias, due to the study design; across the 34 remaining primary studies, risk of bias was low to moderate. Sixteen case reports examined vertical transmission, which was not related to maternal symptomatology. For the cohort and case series studies, the percentage of positive neonates ranged from 0% to 22% across the studies. Twenty studies reported no positive vertical transmission. Three studies that reported the highest positivity rates of 11%, 15% and 22% had specifically selected neonates with a positive test (within up to 35 days) within the study population and were therefore more selective populations. Across the cohort and case series studies there were 65/2391 (2.7%) neonates born to mothers with a diagnosis of COVID-19 tested positive for SARS-CoV-2 within 24 hours of birth. No evidence correlated maternal symptomatology to vertical transmission. Mode of delivery did not correlate with rates of vertical transmission. Of 25 studies, 7 identified SARS-CoV-2 in placental tissue; some of these did not demonstrate vertical transmission to the neonate. No study reported the results of viral culture to detect SARS-CoV-2. Conclusions The results of these studies indicate that vertical transmission is possible, but is not frequent, and factors that influence when vertical transmission occurs are unknown. Further studies using standardised methods to establish viral infection are needed to establish vertical transmission rates and to assess clinical and other conditions affecting transmission.

The identification and isolation of highly infectious SARS-CoV-2-infected individuals is an important public health strategy. Rapid antigen detection tests (RADT) are promising tools for large-scale screenings due to timely results and feasibility for on-site testing. Nonetheless, the diagnostic performance of RADT in detecting infectious individuals is not yet fully determined. In this study, RT-qPCR and virus culture of RT-qPCR-positive samples were used to evaluate and compare the performance of the Standard Q COVID-19 Ag test in detecting SARS-CoV-2-infected and possibly infectious individuals. To this end, two combined oro- and nasopharyngeal swabs were collected at a routine SARS-CoV-2 diagnostic center. A total of 2,028 samples were tested, and 118 virus cultures were inoculated. SARS-CoV-2 infection was detected in 210 samples by RT-qPCR, representing a positive rate of 10.36%. The Standard Q COVID-19 Ag test yielded a positive result in 92 (4.54%) samples resulting in an overall sensitivity and specificity of 42.86 and 99.89%, respectively. For adjusted CT values of

Objectives This study compares the infectivity of SARS-CoV-2 in respiratory samples from patients with mild COVID-19 with those from hospitalised patients with severe bilateral pneumonia. In severe COVID-19, we also analysed the presence of neutralising activity in paired sera. Methods We performed cell cultures on 193 real-time reverse transcription polymerase chain reaction respiratory samples, positive for SARS-CoV-2, obtained from 189 patients at various times, from clinical diagnosis to follow-up. Eleven samples were obtained from asymptomatic individuals, 91 samples from 91 outpatients with mild forms of COVID-19, and 91 samples from 87 inpatients with severe pneumonia. In these patients, neutralising activity was analysed in 30 paired sera collected after symptom onset >10 days. Results We detected a cytopathic effect (CPE) in 91 (91/193, 47%) samples. Viral viability was maintained for up to 10 days in the patients with mild COVID-19. In the patients with severe COVID-19, the virus remained viable for up to 32 days after the onset of symptoms. Patients with severe COVID-19 presented infectious virus at a significantly higher rate in the samples with moderate to low viral load (cycle threshold value >26): 32/75 (43%) versus 14/63 (22%) for mild cases (P < 0.01). We observed a positive CPE despite the presence of clear neutralising activity (NT50 >1:1024 in 10% (3/30) of samples. Conclusions Patients with severe COVID-19 might shed viable virus during prolonged periods of up to 4 weeks after symptom onset, even when presenting high cycle threshold values in their respiratory samples and despite having developed high neutralising antibody titres.

South Korea has experienced FMD outbreaks almost every year since 2014. Therefore, a novel local vaccine that can cover various topotypes of viruses is required. Two virus strains, O/Boeun/SKR/2017 and A/Yeoncheon/SKR/2017, were cultured up to the pilot scale based on the optimized conditions set up on the flask scale. FMDV particles (146S) of 2 µg/mL or more were obtained from the virus culture supernatant using a 100 L bioreactor. The viruses were fully inactivated using binary ethylenimine within 16 h through two inactivation cycles and mixed with an adjuvant into a bivalent vaccine (types O and A) consisting of 15 µg viruses per strain. The experimental bivalent vaccine showed a broad spectrum of high neutralizing antibody titers against heterologous viruses, including type O Cathay strain and type A Asia topotypes, except for GVII. The 50% protective dose was determined as 12.5 for O/Boeun/SKR/2017 and 15.6 for A/Yeoncheon/SKR/2017. Collectively, we expect that the bivalent vaccine could protect against FMDV types O and A circulating in South Korea and neighboring countries. To our knowledge, this is the first report demonstrating that the vaccine strains could be successfully scaled-up to a 100 L bioreactor, with the determination of its protective efficacy in pigs.

BackgroundData on viral factors causing pediatric disease and guidance for pediatric-specific considerations have lagged behind adults throughout the COVID-19 pandemic. As COVID-19 infections and deaths increase in the pediatric population, characterization of SARS-CoV-2 viral dynamics in children would enable data-driven public health guidance. MethodsNasal swabs collected from children with COVID-19 were analyzed. Viral load was quantified by RT-PCR; viral culture was assessed by direct observation of cytopathic effects and semiquantitative viral titers. Correlations with age or symptom duration were analyzed. SARS-CoV-2 whole genome amplification was compared with contemporaneous Massachusetts sequences to assess for clustering patterns. ResultsNinety-seven children with COVID-19 (median age 10 years, range 2 weeks-22 years) were included in this study. Age did not impact SARS-CoV-2 viral load in nasal secretions: children of all ages were equally likely to carry live, replicating virus. Children within the first five days of illness had higher viral loads and rates of culture positivity, and viral load in hospitalized children (n=30) did not differ from hospitalized adults (n=21) with similar duration of symptoms. While pediatric SARS-CoV-2 sequences were representative of those in the community, novel variants were identified. ConclusionsChildren can carry high quantities of live, replicating virus, creating a potential reservoir for transmission and evolution of genetic variants. As guidance around social distancing and masking evolves following vaccine uptake in older populations, a clear understanding of SARS-CoV-2 infection dynamics in children is critical for rational development of public health policies and vaccination strategies to mitigate the impact of COVID-19. Key points- Infants, children and adolescents are equally capable of carrying live, replicating SARS-CoV-2 in their nasal secretions. - Respiratory secretions of infants, children and adolescents with COVID-19 are most infectious before symptoms develop and during the first five days of illness. - Children can harbor SARS-CoV-2 variants that could have implications for disease severity and vaccine efficacy.

BackgroundIvermectin, an anti-parasitic agent, also has anti-viral properties. Our aim was to assess whether ivermectin can shorten the viral shedding in patients at an early-stage of COVID-19 infection.MethodsThe double-blinded trial compared patients receiving ivermectin 0·2 mg/kg for 3 days vs. placebo in non-hospitalized COVID-19 patients. RT-PCR from a nasopharyngeal swab was obtained at recruitment and then every two days. Primary endpoint was reduction of viral-load on the 6th day (third day after termination of treatment) as reflected by Ct level>30 (non-infectious level). The primary outcome was supported by determination of viral culture viability.ResultsEighty-nine patients were eligible (47 in ivermectin and 42 in placebo arm). Their median age was 35 years. Females accounted for 21·6%, and 16·8% were asymptomatic at recruitment. Median time from symptom onset was 4 days. There were no statistical differences in these parameters between the two groups.On day 6, 34 out of 47 (72%) patients in the ivermectin arm reached the endpoint, compared to 21/ 42 (50%) in the placebo arm (OR 2·62; 95% CI: 1·09-6·31). In a multivariable logistic-regression model, the odds of a negative test at day 6 was 2.62 time higher in the ivermectin group (95% CI: 1·06–6·45). Cultures at days 2 to 6 were positive in 3/23 (13·0%) of ivermectin samples vs. 14/29 (48·2%) in the placebo group (p=0·008).ConclusionsThere were significantly lower viral loads and viable cultures in the ivermectin group, which could lead to shortening isolation time in these patients.The study is registered at ClinicalTrials.gov NCT 044297411.

Tests that detect the presence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigen in clinical specimens from the upper respiratory tract can provide a rapid means of coronavirus disease 2019 (COVID-19) diagnosis and help identify individuals who may be infectious and should isolate to prevent SARS-CoV-2 transmission. This systematic review assesses the diagnostic accuracy of SARS-CoV-2 antigen detection in COVID-19 symptomatic and asymptomatic individuals compared to quantitative reverse transcription polymerase chain reaction (RT-qPCR) and summarizes antigen test sensitivity using meta-regression. In total, 83 studies were included that compared SARS-CoV-2 rapid antigen-based lateral flow testing (RALFT) to RT-qPCR for SARS-CoV-2. Generally, the quality of the evaluated studies was inconsistent; nevertheless, the overall sensitivity for RALFT was determined to be 75.0% (95% confidence interval: 71.0–78.0). Additionally, RALFT sensitivity was found to be higher for symptomatic vs. asymptomatic individuals and was higher for a symptomatic population within 7 days from symptom onset compared to a population with extended days of symptoms. Viral load was found to be the most important factor for determining SARS-CoV-2 antigen test sensitivity. Other design factors, such as specimen storage and anatomical collection type, also affect the performance of RALFT. RALFT and RT-qPCR testing both achieve high sensitivity when compared to SARS-CoV-2 viral culture.

Rapid detection of infection is essential for stopping the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Roche SD Biosensor rapid antigen test for SARS-CoV-2 was evaluated in a nonhospitalized symptomatic population. We rapid-tested a sample onsite and compared results with those from reverse transcription PCR and virus culture. We analyzed date of onset and symptoms using data from a clinical questionnaire. Overall test sensitivity was 84.9% (95% CI 79.1-89.4) and specifi city was 99.5% (95% CI 98.7-99.8). Sensitivity increased to 95.8% (95% CI 90.5-98.2) for persons who sought care within 7 days of symptom onset. Test band intensity and time to result correlated strongly with viral load; thus, strong positive results could be read before the recommended time. Approximately 98% of all viable specimens with cycle threshold

Tests that detect the presence of SARS-CoV-2 antigen in clinical specimens from the upper respiratory tract can provide a rapid means of COVID-19 disease diagnosis and help identify individuals that may be infectious and should isolate to prevent SARS-CoV-2 transmission. This systematic review assesses the diagnostic accuracy of SARS-CoV-2 antigen detection in COVID-19 symptomatic and asymptomatic individuals compared to RT-qPCR, and summarizes antigen test sensitivity using meta-regression. In total, 83 studies were included that compared SARS-CoV-2 rapid antigen lateral flow testing (RALFT) to RT-qPCR for SARS-CoV-2. Generally, the quality of the evaluated studies was inconsistent, nevertheless, the overall sensitivity for RALFT was determined to be 75.0% (95% confidence interval [CI]: 71.0-78.0). Additionally, RALFT sensitivity was found to be higher for symptomatic versus asymptomatic individuals and was higher for a symptomatic population within 7 days from symptom onset (DSO) compared to a population with extended days of symptoms. Viral load was found to be the most important factor for determining SARS-CoV-2 antigen test sensitivity. Other design factors, such as specimen storage and anatomical collection type, also affect the performance of RAFLT. RALFT and RT-qPCR testing both achieve high sensitivity when compared to SARS-CoV-2 viral culture.

Rapid antigen tests (RATs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have advantages over viral culture in terms of cost and rapidity of testing, but they have low sensitivity. In addition, RATs tend to be negative from approximately 11 days after symptom onset. To determine whether the antigen-negative state indicates a lack of infectiousness, we assessed the association between viral culture and RAT results. Viral culture, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and rapid antigen testing were performed on stored nasopharyngeal samples with threshold cycle values

Replication-competent virus has not been detected in individuals with mild to moderate coronavirus disease 2019 (COVID-19) more than 10 days after symptom onset. It is unknown whether these findings apply to nursing home residents. Of 273 specimens collected from nursing home residents10 days from the initial positive test, none were culture positive.

Background Serial screening is critical for restricting spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by facilitating timely identification of infected individuals to interrupt transmission. Variation in sensitivity of different diagnostic tests at different stages of infection has not been well documented. Methods In a longitudinal study of 43 adults newly infected with SARS-CoV-2, all provided daily saliva and nasal swabs for quantitative reverse transcription polymerase chain reaction (RT-qPCR), Quidel SARS Sofia antigen fluorescent immunoassay (FIA), and live virus culture. Results Both RT-qPCR and Quidel SARS Sofia antigen FIA peaked in sensitivity during the period in which live virus was detected in nasal swabs, but sensitivity of RT-qPCR tests rose more rapidly prior to this period. We also found that serial testing multiple times per week increases the sensitivity of antigen tests. Conclusions RT-qPCR tests are more effective than antigen tests at identifying infected individuals prior to or early during the infectious period and thus for minimizing forward transmission (given timely results reporting). All tests showed >98% sensitivity for identifying infected individuals if used at least every 3 days. Daily screening using antigen tests can achieve approximately 90% sensitivity for identifying infected individuals while they are viral culture positive.

Background: Around 5% of patients with coronavirus disease ( COVID-19) caused by severe acute respiratory syndrome coronavirus 2 develop severe COVID-19. Severe COVID-19 requires respiratory management with mechanical ventilation and an extended period of treatment. Prolonged infectious virus shedding is a concern in severe COVID-19 cases, but few reports have examined the duration of infectious virus shedding. We investigated the duration of infectious virus shedding in patients transferred to Hiroshima University Hospital with severe COVID-19 requiring mechanical ventilation. Methods: Nasopharyngeal swab specimens were collected and analyzed using both viral culture and reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) tests between December 2020 and February 2021. Findings: Of the 23 patients tested, the percentage of those with positive test results at first specimen collection (the median number of days to first specimen collection after symptom onset was ten) on RT-qPCR and viral culture was 95 ·7% (n = 22) and 30·4% (n = 7), respectively. All six patients with positive viral culture test results who were followed-up tested negative by day 24 after symptom onset but remained positive on RT-qPCR. Specimen viral loads based on PCR testing did not decrease over time, but viral loads determined via culture tests loads decreased over time. The longest negative conversion time was observed in a dialysis patient on immunosuppressive drugs. Interpretation: This study indicated that patients with severe COVID-19 remain culture positive ≥ ten days after symptom onset. The work also suggests that immunosuppressed patients with severe COVID-19 could consider isolation for ≥ 20 days. Funding: None Declaration of Interests: We declare no competing interests. Ethics Approval Statement: This research was approved by the Ethical Committee for Epidemiology of Hiroshima University (approval number: E-2157).

BackgroundSARS-CoV-2 infections of infants and toddlers are usually mild but can result in life-threatening disease. SARS-CoV-2 RNA been detected in the breast milk of lactating women, but the potential role of breastfeeding in transmission to infants has remained uncertain.MethodsBreast milk specimens were examined for the presence of the virus by RT-PCR and/or culture. Specimens that contained viral RNA (vRNA) were examined for the presence of subgenomic coronavirus RNA (sgRNA), a putative marker of infectivity. Culture methods were used to determine the thermal stability of SARS-CoV-2 in human milk.ResultsBreast milk samples from 110 women (65 confirmed with a SARS-CoV-2 diagnostic test, 36 with symptoms but without tests, and 9 with symptoms but a negative SARS-CoV-2 diagnostic test) were tested by RT-PCR (285 samples) and/or viral culture (160 samples). Although vRNA of SARS-CoV-2 was detected in the milk of 7 of 110 (6%) women with either a confirmed infection or symptomatic illness, and in 6 of 65 (9%) of women with a positive SARS-CoV-2 diagnostic test, virus was not detected in any culture. None of the 7 milk specimens with detectable vRNA contained sgRNA. Notably, when artificially added to human milk in control experiments, infectious SARS-CoV-2 could be cultured despite several freeze-thaw cycles, as occurs in the storage and usage of human milk.ConclusionsSARS-CoV-2 RNA can be found infrequently in the breastmilk of women with recent infection, but we found no evidence that breastmilk contains infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants.Key PointsQuestionSARS-CoV-2 RNA has been detected in a small number of human milk samples collected from recently infected women. The role of breastfeeding in transmission of the virus to infants has remained uncertain due to the small number of specimens analyzed in any study published thus far.FindingsIn a total study group of 110 women, SARS-CoV-2 RNA was detected in milk from 6 of 65 women (9.2%) with recent confirmed infection. Neither infectious virus nor subgenomic RNA (a marker of virus infectivity) were detected in any of the samples.MeaningWe found no evidence that infectious SARS-CoV-2 is present milk from recently infected women, even if SARS-CoV-2 PCR tests are positive, providing reassurance of the safety of breastfeeding.

BackgroundSustained molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the upper respiratory tract (URT) in mild to moderate coronavirus disease 2019 (COVID-19) is common. We sought to identify host and immune determinants of prolonged SARS-CoV-2 RNA detection.MethodsNinety-five symptomatic outpatients self-collected midturbinate nasal, oropharyngeal (OP), and gingival crevicular fluid (oral fluid) samples at home and in a research clinic a median of 6 times over 1–3 months. Samples were tested for viral RNA, virus culture, and SARS-CoV-2 and other human coronavirus antibodies, and associations were estimated using Cox proportional hazards models.ResultsViral RNA clearance, as measured by SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR), in 507 URT samples occurred a median (interquartile range) 33.5 (17–63.5) days post–symptom onset. Sixteen nasal-OP samples collected 2–11 days post–symptom onset were virus culture positive out of 183 RT-PCR-positive samples tested. All participants but 1 with positive virus culture were negative for concomitant oral fluid anti-SARS-CoV-2 antibodies. The mean time to first antibody detection in oral fluid was 8–13 days post–symptom onset. A longer time to first detection of oral fluid anti-SARS-CoV-2 S antibodies (adjusted hazard ratio [aHR], 0.96; 95% CI, 0.92–0.99; P = .020) and body mass index (BMI) ≥25 kg/m2 (aHR, 0.37; 95% CI, 0.18–0.78; P = .009) were independently associated with a longer time to SARS-CoV-2 viral RNA clearance. Fever as 1 of first 3 COVID-19 symptoms correlated with shorter time to viral RNA clearance (aHR, 2.06; 95% CI, 1.02–4.18; P = .044).ConclusionsWe demonstrate that delayed rise of oral fluid SARS-CoV-2-specific antibodies, elevated BMI, and absence of early fever are independently associated with delayed URT viral RNA clearance.

It has been hoped that rapid SARS-CoV-2 antigen testing could reduce the tragic toll of COVID-19 in nursing homes. The performance of the BinaxNOW antigen test in a nursing home during an ongoing SARS-CoV-2 outbreak was evaluated., Visual Abstract. Serial SARS-CoV-2 Rapid Antigen Testing During a Nursing Home Outbreak. It has been hoped that rapid SARS-CoV-2 antigen testing could reduce the tragic toll of COVID-19 in nursing homes. The performance of the BinaxNOW antigen test in a nursing home during an ongoing SARS-CoV-2 outbreak was evaluated., Background: To address high COVID-19 burden in U.S. nursing homes, rapid SARS-CoV-2 antigen tests have been widely distributed in those facilities. However, performance data are lacking, especially in asymptomatic people. Objective: To evaluate the performance of SARS-CoV-2 antigen testing when used for facility-wide testing during a nursing home outbreak. Design: A prospective evaluation involving 3 facility-wide rounds of testing where paired respiratory specimens were collected to evaluate the performance of the BinaxNOW antigen test compared with virus culture and real-time reverse transcription polymerase chain reaction (RT-PCR). Early and late infection were defined using changes in RT-PCR cycle threshold values and prior test results. Setting: A nursing home with an ongoing SARS-CoV-2 outbreak. Participants: 532 paired specimens collected from 234 available residents and staff. Measurements: Percentage of positive agreement (PPA) and percentage of negative agreement (PNA) for BinaxNOW compared with RT-PCR and virus culture. Results: BinaxNOW PPA with virus culture, used for detection of replication-competent virus, was 95%. However, the overall PPA of antigen testing with RT-PCR was 69%, and PNA was 98%. When only the first positive test result was analyzed for each participant, PPA of antigen testing with RT-PCR was 82% among 45 symptomatic people and 52% among 343 asymptomatic people. Compared with RT-PCR and virus culture, the BinaxNOW test performed well in early infection (86% and 95%, respectively) and poorly in late infection (51% and no recovered virus, respectively). Limitation: Accurate symptom ascertainment was challenging in nursing home residents; test performance may not be representative of testing done by nonlaboratory staff. Conclusion: Despite lower positive agreement compared with RT-PCR, antigen test positivity had higher agreement with shedding of replication-competent virus. These results suggest that antigen testing could be a useful tool to rapidly identify contagious people at risk for transmitting SARS-CoV-2 during nascent outbreaks and help reduce COVID-19 burden in nursing homes. Primary Funding Source: None.

Introduction Rapid antigen detection (RAD) tests are convenient tools for detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinics, and testing using saliva samples could decrease the risk of infection during sample collection. This study aimed to assess the accuracy of the SARS-CoV-2 RAD for testing of nasopharyngeal swab specimens and saliva samples in comparison with the RT-PCR tests and viral culture for detecting viable virus. Methods One hundred seventeen nasopharyngeal swab specimens and 73 saliva samples with positive results on RT-PCR were used. Residual samples were assayed using a commercially available RAD test immediately, and its positivity was determined at various time points during the clinical course. The concordance between 54 nasopharyngeal swab samples and saliva samples that were collected simultaneously was determined. Viral culture was performed on 117 samples and compared with the results of the RAD test. Results The positive rate of RAD test using saliva samples was low throughout the clinical course. Poor concordance was observed between nasopharyngeal swab specimens and saliva samples (75.9%, kappa coefficient 0.310). However, a substantially high concordance between the RAD test and viral culture was observed in both nasopharyngeal swab specimens (86.8%, kappa coefficient 0.680) and saliva samples (95.1%, kappa coefficient 0.643). Conclusions The sensitivity of the SARS-CoV-2 RAD test was insufficient, particularly for saliva samples. However, a substantially high concordance with viral culture suggests its potential utility as an auxiliary test for estimating SARS-CoV-2 viability.

Highlights • Evaluation the quality of disinfection in hospital during COVID-19 pandemic • Revealed the places easy to be ignored when performed disinfection procedures • Routine disinfection could be helpful in preventing the HCWs from getting infected, Background Coronavirus disease 2019 (COVID-19) has spread globally and been a public health emergency worldwide. It is important to reduce the risk of healthcare associated infections among the healthcare workers and patients. This study aimed to investigate the contamination of environment in isolation wards and sewage, and assess the quality of routine disinfection procedures in our hospital. Methods Routine disinfection procedures were performed three-times a day in general isolation wards and six-times a day in isolated ICU wards in our hospital. Environmental surface samples and sewage samples were collected for viral RNA detection. SARS-CoV-2 RNA detection were performed with quantitative reverse transcription PCR (qRT-PCR). Results A total of 163 samples were collected from February 6th to April 4th. Among 122 surface samples, two were positive for SARS-CoV-2 RNA detection. One was collected from the flush button of the toilet bowl, and the other was collected from a hand-basin. Although 10 of the sewage samples were positive for viral RNA detection, all positive samples were negative for viral culture. Conclusion These results revealed the routine disinfection procedures in our hospital were effective in reducing the potential risk of healthcare associated infection. Two surface samples were positive for viral detection, suggesting that more attention should be paid when disinfecting places easy to be ignored.

Highlights • SARS-CoV-2 is not fully inactivated by heating to 56 °C or 60 °C for up to 60 min. • Complete inactivation occurred at 80 °C for 90 min or 95 °C for 1 or 5 min. • Heating SARS-CoV-2 to 80 °C for more than 30 min results in increased Ct values. • Significant variation in inactivation efficacy occurred between replicate samples. • Due to variability heat-inactivation protocols require local validation., The development of safe diagnostic protocols for working with SARS-CoV-2 clinical samples at Biosafety Level 2 (BSL2) requires understanding of the effect of heat-treatment on SARS-CoV-2 viability and downstream RT-PCR sensitivity. In this study heating SARS-CoV-2/England/2/2020 to 56 °C and 60 °C for 15, 30 and 60 min reduced the virus titre by between 2.1 and 4.9 log10 pfu/mL (as determined by plaque assay). Complete inactivation did not occur and there was significant variability between replicates. Viable virus was detected by plaque assay after heat-treatment at 80 °C for 15 or 30 min but not 60 or 90 min. After heat-treatment at 80 °C for 60 min infectious virus was only detected by more sensitive virus culture. No viable virus was detected after heating to 80 °C for 90 min or 95 °C for 1 or 5 min. RT-PCR sensitivity was not compromised by heating to 56 °C and 60 °C. However, RT-PCR sensitivity was reduced (≥3 Ct value increase) after heating the virus to 80 °C for 30 min or longer, or 95 °C for 1 or 5 min. In summary we found that the efficacy of heat-inactivation varies greatly depending on temperature and duration. Local validation of heat-inactivation and its effects downstream is therefore essential for molecular testing.

Objectives The development of a rapid diagnostic test for viable SARS-CoV-2 is important for infection control. Real-time RT-PCR assays detect non-viable virus and cell culture differentiates viable virus but it takes several weeks and is labor-intensive. Subgenomic RNAs may reflect replication-competent virus. We therefore evaluated the usefulness of subgenomic RNAs for diagnosing viable SARS-CoV-2 in patients with COVID-19. Methods Patients with various severities of confirmed COVID-19 were enrolled at a tertiary hospital between February and December 2020. RT-PCR assay results for genomic and subgenomic RNA of SARS-CoV-2 from nasopharyngeal swab, sputum, and saliva specimens were compared with cell culture results. Results A total 189 specimens from 20 COVID-19 patients were tested in genomic and subgenomic PCR assays, and cultured on Vero cells. Of these 189 samples, 62 (33%) gave positive culture results, 93 (49%) negative results, and the remaining 34 (18%) indeterminate results. Compared with cell culture results, the sensitivities of genomic RNA and subgenomic RNA of the N and S genes were comparable as 100%, but the specificity of subgenomic RNA (N, 65% and S, 68%) was higher than that of genomic RNA (N, 23% and S, 17%, p value < 0.001). The mean durations of positive culture and subgenomic RNA were 11.39 ± 10.34 and 13.75 ± 11.22 days after symptom onset (p value = 0.437), respectively, while that of genomic RNA was 22.85 ± 11.83 days after symptom onset (p value < 0.001). Conclusion Our comparison of subgenomic RNA detection with symptom duration and SARS-CoV-2 culture positivity provides a significant advancement on the transmissibility-based approach beyond the detection of SARS-CoV-2 genomic RNA, and warrants further studies on the development of better diagnostic strategy.

BackgroundPre-/asymptomatic close contacts of SARS-CoV-2 infected individuals were tested at day 5 after contact by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic accuracy of antigen-detecting rapid diagnostic tests (Ag-RDT) in pre-/asymptomatic close contacts was up till now unknown.MethodsWe performed a prospective cross-sectional diagnostic test accuracy study. Close contacts (e.g. selected via the test-and-trace program or contact tracing app) aged ≥16 years and asymptomatic when requesting a test, were included consecutively and tested at day 5 at four Dutch public health service test sites. We evaluated two Ag-RDTs (BD Veritor™ System Ag-RDT (BD), and Roche/SD Biosensor Ag-RDT (SD-B)) with RT-PCR as the reference standard. Virus culture was performed in RT-PCR positive individuals to determine the viral load cut-off above which 95% was culture positive, as a proxy of infectiousness.ResultsOf 2,678 BD-tested individuals, 233 (8.7%) were RT-PCR positive and BD detected 149 (sensitivity 63.9%; 95% confidence interval 57.4%-70.1%). Out of 1,596 SD-B-tested individuals, 132 (8.3%) were RT-PCR positive and SD-B detected 83 (sensitivity 62.9%; 54.0%-71.1%). When applying an infectiousness viral load cut-off ≥ 5.2 log10 gene copies/mL, the sensitivity was 90.1% (84.2%-94.4%) for BD, 86.8% (78.1% to 93.0%) for SD-B overall, and 88.1% (80.5%-93.5%) for BD, 85.1% (74.3%-92.6%) for SD-B for those still asymptomatic at the actual time of sampling. Specificity was >99% for both Ag-RDTs in all analyses.ConclusionsThe sensitivity for detecting SARS-CoV-2 of both Ag-RDTs in pre-/asymptomatic close contacts is over 60%, increasing to over 85% after applying an infectiousness viral load cut-off.Trial registration numberNot applicable. A study protocol is available upon request.

Con el avance de la pandemia por COVID-19, la aparición de pacientes con un segundo episodio confirmado por reacción en cadena de la polimerasa, con transcripción inversa (rt-PCR) compatible con reinfección, puso de manifiesto la falta de recomendaciones para su abordaje.Presentamos un estudio descriptivo multicéntrico retro-prospectivo de una serie de doce casos atendidos entre el 01/06/2020 y el 20/10/2020. En la misma, diez casos presentaron el segundo episodio en un período de tiempo menor a 90 días. Por su complejidad, la confirmación de una reinfección no está al alcance en la práctica diaria. Esto requiere de estudios que incluyan comparaciones genómicas de cepas virales involucradas en ambos episodios, determinación de la infectividad del ARN por cultivo viral y análisis molecular. Es necesario establecer definiciones operativas y algoritmos clínicos para la atención de los segundos episodios.

SummaryBackgroundThere is increasing concern that persistent infection of SARS-CoV-2 within immunocompromised hosts could serve as a reservoir for mutation accumulation and subsequent emergence of novel strains with the potential to evade immune responses.MethodsWe describe three patients with acute lymphoblastic leukemia who were persistently positive for SARS-CoV-2 by real-time polymerase chain reaction. Viral viability from longitudinally-collected specimens was assessed. Whole-genome sequencing and serological studies were performed to measure viral evolution and evidence of immune escape.FindingsWe found compelling evidence of ongoing replication and infectivity for up to 162 days from initial positive by subgenomic RNA, single-stranded RNA, and viral culture analysis. Our results reveal a broad spectrum of infectivity, host immune responses, and accumulation of mutations, some with the potential for immune escape.InterpretationOur results highlight the need to reassess infection control precautions in the management and care of immunocompromised patients. Routine surveillance of mutations and evaluation of their potential impact on viral transmission and immune escape should be considered.FundingThe work was partially funded by The Saban Research Institute at Children’s Hospital Los Angeles intramural support for COVID-19 Directed Research (X.G. and J.D.B.), the Johns Hopkins Center of Excellence in Influenza Research and Surveillance HHSN272201400007C (A.P.), NIH/NIAID R01AI127877 (S.D.B.), NIH/NIAID R01AI130398 (S.D.B.), NIH 1U54CA260517 (S.D.B.), an endowment to S.D.B. from the Crown Family Foundation, an Early Postdoc.Mobility Fellowship Stipend to O.F.W. from the Swiss National Science Foundation (SNSF), and a Coulter COVID-19 Rapid Response Award to S.D.B. L.G. is a SHARE Research Fellow in Pediatric Hematology-Oncology.

BackgroundRapid antigen lateral flow devices (LFDs) are set to become a cornerstone of SARS-CoV-2 mass community testing. However, their reduced sensitivity compared to PCR has raised questions of how well they identify infectious cases. Understanding their capabilities and limitations is therefore essential for successful implementation. To address this, we evaluated six commercial LFDs on the same collection of clinical samples and assessed their correlation with infectious virus culture and cycle threshold (Ct) values.MethodsA head-to-head comparison of specificities and sensitivities was performed on six commercial rapid antigen tests using combined nasal/oropharyngeal swabs, and their limits of detection determined using viral plaque forming units (PFU). Three of the LFDs were selected for a further study, correlating antigen test result with RT-PCR Ct values and positive viral culture in Vero-E6 cells. This included sequential swabs and matched serum samples obtained from four infected individuals with varying disease severities. Detection of antibodies was performed using an IgG/IgM Rapid Test Cassette, and neutralising antibodies by infectious virus assay. Finally, the sensitivities of selected rapid antigen LFTs were assessed in swabs with confirmed B.1.1.7 variant, currently the dominant genotype in the UK.FindingsMost of the rapid antigen LFDs showed a high specificity (>98%), and accurately detected 50 PFU/test (equivalent N1 Ct of 23.7 or RNA copy number of 3×106/ml). Sensitivities of the LFDs performed on clinical samples ranged from 65 to 89%. These sensitivities increased in most tests to over 90% for samples with Cts lower than 25. Positive virus culture was achieved for 57 out of 141 samples, with 80% of the positive cultures from swabs with Cts lower than 23. Importantly, sensitivity of the LFDs increased to over 95% when compared with the detection of infectious virus alone, irrespective of Ct. Longitudinal studies of PCR-positive samples showed that most of the tests identified all infectious samples as positive, but differences in test sensitivities can lead to missed cases in the absence of repeated testing. Finally, test performance was not impacted when re-assessed against swabs positive for the dominant UK variant B.1.1.7.InterpretationIn this comprehensive comparison of antigen LFD and virus infectivity, we demonstrate a clear relationship between Ct values, quantitative culture of infectious virus and antigen LFD positivity in clinical samples. Our data support regular testing of target groups using LFDs to supplement the current PCR testing capacity, to rapidly identify infected individuals in situations where they would otherwise go undetected.FundingKing’s Together Rapid COVID-19, Medical Research Council, Wellcome Trust, Huo Family Foundation.

Background Patients with end-stage-renal-disease (ESRD) undergoing hemodialysis (HD) represent a vulnerable population for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, due to their intrinsic fragility and increased exposure to the virus. Therefore, applying effective screening strategies and infection control measures is essential to control the spread of the epidemic within hemodialysis centers. Objective Description and evaluation of the efficacy of systematic screening by rt-PCR and viral cultures, in addition to triage to limit the spread of the epidemic. Evaluation of the performance of these tests using “post-hoc” SARS-CoV-2 serology as a surrogate marker of infection. Methods One hundred and forty-four patients undergoing hemodialysis in the Nephrology-Hemodialysis center of CHU Brugmann, Brussels, benefited from systematic virological screening using viral cultures in asymptomatic patients, or molecular tests (rt-PCR) for symptomatic ones, in addition to general prevention measures. Post-hoc serology was performed in all patients. Results Thirty-eight (26.3%) individuals were infected with SARS-CoV-2. Seventeen infected patients (44.7%) were asymptomatic and thus detected by viral culture. Our strategy allowed us to detect and isolate 97.4% of the infected patients, as proven by post-hoc serology. Only one patient, missed by clinical screening and sequential viral cultures, had a positive serology. Conclusion The implementation of a control and prevention strategy based on a systematic clinical and virological screening showed its effectiveness in limiting (and shortening) the spread of the SARS-CoV-2 epidemic within our hemodialysis unit. Graphic abstract

IntroductionSelf-testing for COVID-19 infection with lateral flow assay SARS-CoV-2 rapid antigen detection tests (RDT), provides rapid results and could enable frequent and extensive testing in the community, thereby improving the control of SARS-CoV-2. The objective of this study is to evaluate the performance of self-testing using RDT without assistance.MethodsParticipants visiting a municipal SARS-CoV-2 testing centre, received self-testing kits containing either the BD Veritor System (BD RDT) or Roche SARS-CoV-2 antigen detection test (Roche RDT). Oro-nasopharyngeal swabs were collected from the participants for qRT-PCR testing. As a proxy for contagiousness, viral culture was performed on a selection of qRT-PCR positive samples to determine the Ct-value at which the chance of a positive culture was dropping below 0.5 (Ct-value cut-off). Sensitivity and specificity of self-testing were compared to qRT-PCR with a Ct-value below the Ct value cut-off. Determinants independently associated with a false-negative self-test result were determined.ResultsA total of 3,215 participants were included (BD RDT n=1604; Roche RDT n=1611). Sensitivity and specificity of self-testing compared to the qRT-PCR results with Ct-value below the Ct-value cut-off was 78.0% (95% CI:72.5-82.8) and 99.4% (95%CI: 99.0-99.6) respectively. Determinants independently associated with a false-negative self-testing results were: higher age, low viral load and finding self-testing difficult.DiscussionSelf-testing using currently available RDT’s has a high specificity and relatively high sensitivity to identify individuals with a high probability of contagiousness. The performance of two tests were comparable. This application has the potential for frequent and extensive testing which may be an aid to lift restrictions to society while controlling the spread of SARS-CoV-2.

Duration of Positive SARS-CoV-2 on RT-PCR and Culture In 21 consecutive patients with confirmed Covid-19, the median times from symptom onset to negative viral culture and negative real-time RT-PCR...

We report the performance of a variety of commercially available SARS-CoV-2 PCR kits, used in several different sites across Ireland to determine if Ct values across platforms are comparable. We also investigate whether a Ct value, a surrogate for calculated viral loads in the absence of viral culture of > 34 can be used to exclude SARS-CoV-2 infection and its complications. We found a variation in Ct values from different assays for the same calculated viral load; this should be taken into consideration for result interpretation.

Background Guidelines for stopping coronavirus disease 2019 patient isolation are mainly symptom-based, with isolation for 10 to 20 days depending on their condition. Methods In this study, we describe 3 deeply immunocompromised patients, each with different clinical evolutions. We observed (1) the patients’ epidemiological, clinical, and serological data, (2) infectiousness using viral culture, and (3) viral mutations accumulated over time. Results Asymptomatic carriage, symptom resolution, or superinfection with a second severe acute respiratory syndrome coronavirus 2 strain were observed, all leading to prolonged infectious viral shedding for several months. Conclusions Understanding underlying mechanisms and frequency of prolonged infectiousness is crucial to adapt current guidelines and strengthen the use of systematic polymerase chain reaction testing before stopping isolation in immunocompromised populations.

Green tea extracts effectively inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro in a dose-dependent manner. Ten-fold serially diluted solutions of catechin mixture reagent from green tea were mixed with the viral culture fluid at a volume ratio of 9:1, respectively, and incubated at room temperature for 5 min. The solution of 10 mg/mL catechin reagent reduced the viral titer by 4.2 log and 1.0 mg/mL solution by one log. Pre-infection treatment of cells with the reagent alone did not affect viral growth. In addition, cells treated with only the reagent were assayed for host cell viability using the WST-8 system, and almost no host cell damage by the treatment was observed. These findings suggested that the direct treatment of virus with the reagent before inoculation decreased the viral activity and that catechins might have the potential to suppress SARSCoV-2 infection.

Background The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID‐19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS‐CoV‐2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory. Methods An enzyme‐linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings. Results Preliminary data from 10 sera (5 patients with COVID‐19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS‐CoV‐2‐positive individuals. Conclusion This ELISA appears to be a specific and reliable method for detecting COVID‐19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus., The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has generated a pandemic with alarming rates of fatality worldwide. This ELISA appears to be a specific and reliable method for detecting COVID‐19 antibodies (IgG, IgM and IgA), and provides a useful tool for identifying individuals which have developed immunity to the virus.

Introduction Acute respiratory diseases account for a substantial number of outpatient visits and hospitalizations among U.S. military personnel, significantly affecting mission readiness and military operations. We conducted a retrospective analysis of respiratory viral pathogen (RVP) samples collected from U.S. military personnel stationed in Hawaii and tested at Tripler Army Medical Center from January 2014 to May 2019 in order to describe the etiology, distribution, and seasonality of RVP exposure in a military population. Materials and Methods Samples were analyzed by viral culture or multiplex PCR. Distribution of respiratory viruses over time was analyzed as well as subject demographic and encounter data. Presenting signs and symptoms were evaluated with each RVP. Results A total of 2,576 military personnel were tested, of which 726 (28.2%) were positive for one or more RVP. Among positive tests, the three most common viral pathogens detected were influenza A (43.0%), rhinovirus (24.5%), and parainfluenza (7.6%). Symptoms were generally mild and most frequently included cough, fever, and body aches. Conclusion Our study evaluated respiratory virus prevalence, seasonality, and association with clinical symptoms for military personnel in an urban tropical setting in Oahu, HI, over a 5-year period. We show that viral prevalence and seasonality in Hawaii are distinct from those of the CONUS. Results contribute to the broader understanding of seasonality, clinical manifestation, and demographics of RVP among active duty military personnel stationed in Hawaii.

BackgroundPatients have been shown to shed SARS-CoV-2 viral RNA in nasopharyngeal (NP) specimens for over 100 days after resolution of clinical disease (1, 2). How this relates to anterior nares and oral fluid specimens has not previously been investigated.MethodsWe prospectively collected oral fluid, anterior nares, NP swab and serum specimens from 1,326 individuals at 2 “drive-through” testing locations. The Curative SARS-CoV-2 Assay (Curative Assay)(3) on oral fluid and anterior nares specimens was compared to the EURORealTime SARS-CoV-2 Assay (EuroRT Assay)(4) on anterior nares and NP specimens. Viral culture and IgG serology were used to assess infectious potential and stage of disease.Additionally we investigated differences in viral RNA detection between specimen types, both early (< 21 days) and late (> 21 days) in SARS-CoV-2 infection, by using an employee surveillance program with daily SARS-CoV-2 testing to precisely determine infection date, even without symptoms. We prospectively collected oral fluid, anterior nares and NP swab specimens from 165 subjects with early infections and 22 subjects with late infections. Specimens were tested using the Curative Assay with the “high-sensitivity” Hologic Aptima SARS-CoV-2 Assay (Hologic Assay)(5) on an NP swab used as the comparator. Late infection specimens were also tested with EuroRT and Zymo Quick SARS-CoV-2 rRT-PCR Kit (Zymo) (6) Assays.ResultsThe “drive-through” study showed similar sensitivities of oral fluid and anterior nares specimens on the Curative Assay to anterior nares specimens tested with the EuroRT Assay. However NP specimens tested with the same EuroRT assay produced 20-30% more positives. Incorporating viral culture and serology data to exclude NP RT-PCR positives that are not infectious or late in the course of disease showed a Positive Percent Agreement (PPA) for of 98.2% and 96.2% and Negative Percent Agreement (NPA) of 97.6% and 98.1% for anterior nares and oral fluid specimens, respectively.Within 21 days of infection, the Curative Assay showed a PPA and NPA of 100% and 100%, respectively for oral fluid; of 100% and 99% respectively for anterior nares; and of 98.2% and 99.0%, respectively in nasopharyngeal specimens compared to an NP specimen on the Hologic Assay. 29 positives were asymptomatic and showed 100% PPA and 100% NPA for all specimen types. After 21 days from infection onset, significant divergence between NP and other specimen types occurred on all 4 assays. Out of 22 paired sample sets, 18, 13, 8 and 4 NP specimens were positive on the Curative, Zymo, Hologic and EuroRT assays, respectively, compared to only 3, 2, 0 and 1 positive anterior nares specimens. Only one oral fluid sample was positive in both the Curative and Zymo assays.ConclusionsWe used a unique population to show significant divergence between NP specimens and anterior nares or oral fluid specimens >21 days from SARS-CoV-2 infection, which appears to be biological variation and is independent of assay used. This has significant public health implications for the use of NP specimens in community testing programs and policy implications for evaluation of novel specimen types and tests where the use of NP swabs as a comparator may say more about the study population than the assay or specimen type to be evaluated and may unnecessarily limit access to testing.

BackgroundSARS-CoV-2 surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (Genscript) is the first such commercially available assay, detecting antibodies that block RBD/ACE-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared to anti-RBD ELISA assays.MethodsSerum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD IgG, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection.ResultsCompared to PRNT-50, cPass sensitivity ranged from 77% - 100% and specificity was 95% - 100%. Sensitivity was also high compared to the pseudotyped lentiviral neutralization assay (93% [95%CI 85-97]), but specificity was lower (58% [95%CI 48-67]). Highest agreement between cPass and ELISA was for anti-RBD IgG (r=0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99% [95%CI 94-100]) was very similar to that of cPass, but overall specificity was lower (37% [95%CI 28-47]). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical.ConclusionsThe added value of cPass compared to an IgG anti-RBD ELISA was modest.

Background:Understanding the extent of aerosol-based transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important for tailoring interventions for control of the coronavirus disease 2019 (COVID-19) pandemic. Multiple studies have reported the detection of SARS-CoV-2 nucleic acid in air samples, but only one study has successfully recovered viable virus, although it is limited by its small sample size.Objective:We aimed to determine the extent of shedding of viable SARS-CoV-2 in respiratory aerosols from COVID-19 patients.Methods:In this observational air sampling study, air samples from airborne-infection isolation rooms (AIIRs) and a community isolation facility (CIF) housing COVID-19 patients were collected using a water vapor condensation method into liquid collection media. Samples were tested for presence of SARS-CoV-2 nucleic acid using quantitative real-time polymerase chain reaction (qRT-PCR), and qRT-PCR-positive samples were tested for viability using viral culture.Results:Samples from 6 (50%) of the 12 sampling cycles in hospital rooms were positive for SARS-CoV-2 RNA, including aerosols ranging from 4 µm in diameter. Of 9 samples from the CIF, 1 was positive via qRT-PCR. Viral RNA concentrations ranged from 179 to 2,738 ORF1ab gene copies per cubic meter of air. Virus cultures were negative after 4 blind passages.Conclusion:Although SARS-CoV-2 is readily captured in aerosols, virus culture remains challenging despite optimized sampling methodologies to preserve virus viability. Further studies on aerosol-based transmission and control of SARS-CoV-2 are needed.

We performed viral culture of respiratory specimens in 118 severe acute respiratory coronavirus virus 2 (SARS-CoV-2)–infected healthcare workers (HCWs), ∼2 weeks after symptom onset. Only 1 HCW (0.8%) had a positive culture. No factors for prolonged viral shedding were identified. Infectivity is resolved in nearly all HCWs ∼2 weeks after symptom onset.

An Italian male with no link to China SARS-CoV-2 epidemic presented at Emergency Room with severe respiratory impairment. The RT-PCR on 20th February, 2020, nasopharyngeal swab revealed SARS-CoV-2 infection, confirmed with viral culture and sequencing. This was the first identified autochthonous SARS-CoV-2 transmission in Italy, that unveiled global pathogen diffusion.

IntroductionThe COVID-19 pandemic has led to high demand of diagnostic tools. Rapid antigen detection tests have been developed and many have received regulatory acceptance such as CE IVD or FDA markings. Their performance needs to be carefully assessed.Materials and Methods158 positive and 40 negative retrospective samples collected in saline and analyzed by a laboratory-developed RT-PCR test were used to evaluate Sofia (Quidel), Standard Q (SD Biosensor), and Panbio™ (Abbott) rapid antigen detection tests (RADTs). A subset of the specimens was subjected to virus culture.ResultsThe specificity of all RADTs was 100% and the sensitivity and percent agreement was 80% and 85% for Sofia, 81% and 85% for Standard Q, and 83% and 86% for Panbio™, respectively. All three RADTs evaluated in this study reached a more than 90% sensitivity for samples with a high viral load as estimated from the low Ct values in the reference RT-PCR. Virus culture was successful in 80% of specimens with a Ct value ConclusionsAs expected, the RADTs were less sensitive than RT-PCR. However, they benefit from the speed and ease of testing, and lower price as compared to RT-PCR. Repeated testing in appropriate settings may improve the overall performance.

Background. The highly variable manifestation of the COVID-19 disease, from completely asymptomatic to fatal, is both a clinical and a public health issue. The criteria for discharge of hospitalized patients have been based so far on the negative result of Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) tests, but the persistence of viral fragments may exceed that of the integral virus by weeks. The aim of our study was to verify the clearance of the virus at viral culture in patients hospitalized for COVID-19 that have clinically recovered but are still positive on nasopharyngeal swab. Methods. The study was conducted in hospitalized patients with positive RT-PCR on nasopharyngeal swab. Patients included were from asymptomatic to severe cases and performed nasopharyngeal control swabbing on day 14 for asymptomatic patient or at least three days after remission of symptoms. RT-PCR positive specimens were sent to a biosafety level 3 laboratory for viral culture. Results. We performed a combined analysis of RT-PCR and a highly sensitive in vitro culture from 84 samples of hospitalized patients. The average age was 46 &plusmn, 20.29, and 40.5% of the subjects had radiologically confirmed pneumonia, with average PaO2 of 72.35 &plusmn, 12.12and P/F ratio of 315 &plusmn, 83.15. Ct values for the N gene were lower in the first swab than in the control one (p &lt, 0.001). The samples from 83 patients were negative at viral culture, and RT-PCR on the respective supernatants always confirmed the absence of viral growth. Conclusions. Our preliminary results demonstrate that patients clinically recovered for at least three days show the viral clearance at viral culture, and presumably they continued to not be contagious.

Introduction: The B.1.617.2 SARS-CoV-2 or Delta variant, first detected in India, has shown a rapid global spread due to its high transmissibility and now represents more than 99% of the currently circulating variants in Europe. Methods and result: In May 2021, two ships that had recently arrived in the Port of Antwerp reported crew members with COVID-like symptoms. SARS-CoV-2 RNA was detected in nasopharyngeal swabs in 30 out of 45 skippers and the B.1.617.2 variant was identified via whole genome sequencing. Crew members were isolated or quarantined and repeatedly tested to assess the evolution of their SARS-CoV-2 viral load based on the cycle threshold (CT) values of the PCR reaction. Viral cultures were also taken at day 7 to detect viable virus and were compared with the subjects CT value at that moment. The shipper's clinical condition was closely observed using a digital home monitoring tool. Eleven crew members (37%) required hospitalization, with CT values of SARS-CoV-2 RNA being a good predictive factor for the hospitalization need. Furthermore, a clear correlation between CT values and positive viral culture was observed, hinting infectiousness even longer than 10 days after the intitial positive PCR test. Conclusion: Our study of 2 Delta variant clusters shows that the initial CT value is a good predictor for hospitalization need and suggests that patients infected with this variant may remain infectious for a longer time period.

Background. In SARS-CoV-2 infection, viral RNA may persist in respiratory samples for several weeks after the resolution of symptoms. Criteria to assess the end of infectivity are not unequivocally defined. In some countries, time from diagnosis is the unique criterion used, in addition to symptom cessation. This study evaluates the role of the Lumipulse® Antigen Assay (LAA) for the safe end of isolation of patients ≥21 days after the diagnosis of infection. Methods. A total of 671 nasopharyngeal swabs from patients diagnosed with infection at least 21 days before were assessed by RT-PCR and LAA, and the role of LAA in predicting the absence of infectivity was evaluated by virus cell culture. Results. Viable virus was present in 10/138 cultured samples. Eight out of ten infective patients suffered from a concomitant disease, predisposing them to long-term shedding of infective virus. In particular, infectious virus was isolated from 10/20 RT-PCR+/LAA+ cultured samples, whereas no viable virus was found in all 118 RT-PCR+/LAA– cultured swabs. LLA and RT-PCR agreed in 484/671 (72.1%) samples, with 100% and 26.7% concordance in RT-PCR negative and positive samples, respectively. Conclusions. Viable virus can be found ≥21 days after diagnosis in immunocompromised or severely ill patients. LAA better than RT-PCR predicts non-infectivity of patients and can be safely used to end isolation in cases with long persistence of viral RNA in the respiratory tract.

Conjunctivitis is a frequent ocular disorder caused by human adenoviruses (HAdVs). Only a few of the 45 HAdV-D species are associated with epidemic keratoconjunctivitis, including HAdV-D8. Nosocomial outbreaks due to HAdV-D8 have been rarely described, because keratoconjunctivitis cases are clinically diagnosed and treated without having to characterize the causative agent. Moreover, molecular typing is tedious when using classical techniques. In this study, a hospital outbreak of conjunctivitis caused by HAdV-D8 was characterized using the recently developed whole-genome sequencing (WGS) method. Of the 363 patients attending the Ophthalmology Department between July 13 and August 13, 2018, 36 may have acquired intrahospital conjunctivitis. Also, 11 of 22 samples sent to the Virology section were selected for WGS analysis. The WGS results revealed that 10 out of 11 HAdV-D8 strains were closely related. The remaining strain (Case 28) was more similar to a strain from an outbreak in Germany obtained from a public sequence database. WGS results showed that outbreak HAdV-D8 strains had a minimum percentage of identity of 94.3%. WGS is useful in a clinical setting, because it avoids carrying out viral culture or specific polymerase chain reaction sequencing. The public availability of sequence reads makes it easier to compare clusters in circulation. In conclusion, WGS can play an important role in standard routines to describe viral outbreaks.

Background Prolonged SARS-CoV-2 shedding has been described in immunocompromised COVID-19 patients, resulting in protracted disease and poor outcome. Specific therapy to improve viral clearance and outcome for this group of patients is currently unavailable. Methods Five critically ill COVID-19 patients with severe defects in cellular immune responses, high SARS-CoV-2 viral RNA loads, and no respiratory improvement were treated with interferon gamma, 100 μg subcutaneously, thrice weekly. Bronchial secretion was collected every 48 hours for routine diagnostic SARS-CoV-2 RT-PCR and viral culture. Findings Interferon gamma administration was followed by a rapid decline in SARS-CoV-2 load and a positive to negative viral culture conversion. Four patients recovered and no signs of hyperinflammation were observed. Conclusions Interferon gamma may be considered as adjuvant immunotherapy in a subset of immunocompromised COVID-19 patients. Funding AvL and RvC are supported by National Institute of Health [R01AI145781]. GJO and RPvR are supported by a VICI grant [016.VICI.170.090] from the Dutch Research Council (NWO). WFA is supported by Clinical Fellowship grant [#9071561]) of Netherlands Organization for Health Research and Development. MGN is supported by an ERC Advanced Grant [#833247] and a Spinoza Grant of the Netherlands Organization for Scientific Research., Graphical Abstract, Van Laarhoven and Kurver et al. report five COVID-19 patients with impaired cellular immunity and persistently high SARS-CoV-2 loads, treated with last-resort interferon gamma immunotherapy, which was followed by viral clearance in five and clinical recovery in four patients. No signs of hyperinflammation were observed.