Clinical evaluation of the Roche distributed SD Biosensor SARS-CoV-2 & Flu A/B Rapid Antigen Test amongst mild symptomatic people during the 2022/2023 winter season. (preprint)

Both influenza and SARS-CoV-2 are seasonal respiratory illnesses with similar symptoms, however distinguishing one from the other can have benefits for the patient and have different implications in various settings. In this study we have evaluated the clinical performance of the Roche distributed SD Biosensor SARS-CoV-2 & Flu A/B Rapid Antigen Test during the 2022/2023 winter season, in a non-hospitalized, mild symptomatic population, comparing results with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Participants also filled in a short questionnaire about their symptom onset, symptoms, vaccination status for both influenza and SARS-CoV-2. We could include 290 people with complete records with female majority (72%, 209/290). Age ranged from 18 years old (minimum age for inclusion) to 71 years (mean age was 40.4 years). From the 290 inclusions 93 tested positive with SARS-CoV-2 PCR, 12 by influenza A and 6 by influenza B PCR. For SARS-CoV-2 overall sensitivity was 72.0% (confidence interval, CI 61.8-80.9%) and specificity 99.5% (CI 97.2-99.9%). SARS-CoV-2 RDT performed best up to and including PCR ct value of 25 (sensitivity 96% CI 85.8-99.5%), but could also detect samples less or equal to PCR ct 33, however with lower sensitivity (sensitivity 80.0% CI 69.6-88.1%). For influenza limited amount of samples were available; the RDT detected influenza A with 58.3% sensitivity (CI 27.7-84.8) and 100% specificity (CI 98.6-100.0%). In case of influenza B the inclusions were too low to calculate sensitivity reliably (2/6, 33.3% CI 4.3-77.7%); specificity was 98.2% (5/274, CI 95.8-99.4%). No cross reaction between SARS-CoV-2 and Flu A/B was experienced. As was shown before, SARS-CoV-2 could be determined with high sensitivity in recent onset and lower than ct 25 samples. In spite of performing the study throughout the influenza season, we had sub optimal inclusions for determining RDT clinical performance; further studies are needed.

Diagnostic accuracy of SARS-CoV-2 rapid antigen self-tests in asymptomatic individuals in the Omicron period: cross sectional study (preprint)

Objectives To assess the performances of three commonly used rapid antigen diagnostic tests (Ag-RDTs) used as self-tests in asymptomatic individuals in the Omicron period. Design Cross-sectional diagnostic test accuracy study. Setting Three public health service COVID-19 test sites in the Netherlands. Participants 3,600 asymptomatic individuals aged ≥16 years presenting for SARS-CoV-2 testing for any reason except confirmatory testing after a positive self-test. Interventions Participants were sampled for RT-PCR (reference test) and received one self-test (either Acon Flowflex (Flowflex), MP Biomedicals (MPBio), or Siemens-Healthineers Clinitest (Clinitest)) to perform unsupervised at home within three hours and blinded to the RT-PCR result. Main Outcome(s) and Measures(s) Diagnostic accuracies (sensitivity, specificity, positive and negative predictive values) of each self-test compared to RT-PCR. Results Overall sensitivities of the three self-tests were 27.5% (95% CI: 21.3-34.3%) for Flowflex, 20.9% (13.9-29.4%) for MPBio, and 25.6% (19.1-33.1%) for Clinitest. After applying a viral load cut-off (≥5.2 log10 SARS-CoV-2 E-gene copies/mL), sensitivities increased to 48.3% (95% CI: 37.6-59.2%), 37.8% (22.5-55.2%), and 40.0% (29.5-51.2%), respectively. No consistent differences were found in sensitivities by COVID-19 vaccination status, having had a prior SARS-CoV-2 infection, gender or age across the three self-tests. Specificities were >99% for all tests in most analyses. Conclusions The sensitivities of three commonly used SARS-CoV-2 Ag-RDTs when used as self-tests in asymptomatic individuals in the Omicron period, were very low. Our findings indicate that Ag-RDT self-testing in asymptomatic individuals may only detect the minority of infections at that point in time and may not be sufficient to prevent the spreading of the virus to other (vulnerable) persons. Repeated self-testing in case of a negative self-test is advocated to improve the diagnostic yield of the self-tests, and individuals should certainly be advised to re-test when symptoms develop. Summary box What is already known on this topic If sufficiently reliable, SARS-CoV-2 self-testing by asymptomatic persons prior to admission in places where groups gather could have a huge public health impact by lowering the reproduction number or keep it below one for longer periods. Current evidence suggests that SARS-CoV-2 rapid antigen diagnostic tests (Ag-RDTs) when used as self-tests by asymptomatic individuals perform suboptimal, but sample sizes of the previous studies were too small to draw robust conclusions, and also empirical data on the accuracy of Ag-RDT self-tests in asymptomatic individuals during the Omicron period are scarce. What this study adds Compared to RT-PCR testing, overall sensitivities of three commercially available SARS-CoV-2 Ag-RDTs when used as self-tests by asymptomatic individuals (primary analysis population of non-confirmatory testers; n= 3600, 87% of full analysis population) in the Omicron period, were very low: 27.5% (95% CI: 21.3-34.3%) for the Acon Flowflex test, 20.9% (13.9-29.4%) for the MP Biomedicals test, and 25.6% (19.1-33.1%) for the Siemens Healthineers Clinitest Ag-RDT, which increased to 48.3% (95% CI: 37.6-59.2%), 37.8% (22.5-55.2%), and 40.0% (29.5-51.2%), respectively, when applying a viral load cut-off (≥5.2 log10 SARS-CoV-2 E-gene copies/mL). Our findings indicate that Ag-RDT self-testing in asymptomatic individuals may only detect the minority of infections at that point in time and may not be sufficient to prevent the spreading of the virus to other (vulnerable) persons. Repeated self-testing in case of a negative self-test is advocated to improve the diagnostic yield of the self-tests, and individuals should certainly be advised to re-test when symptoms develop.

SARS-CoV-2 outbreaks in secondary school settings in the Netherlands during fall 2020; silent circulation (preprint)

Background: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces. Methods: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 hrs and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible. Results: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive. Conclusion: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks.

Clinical characteristics and outcome of immunocompromised patients with COVID-19 caused by the Omicron variant: a prospective observational study (preprint)

Summary Background In the general population, illness after infection with the SARS-CoV-2 Omicron variant is less severe compared with previous variants. Data on the disease burden of Omicron in immunocompromised patients are lacking. We investigated the clinical characteristics and outcome of a cohort of immunocompromised patients with COVID-19 caused by Omicron. Methods Solid organ transplant recipients, patients on anti-CD20 therapy, and allogenic hematopoietic stem cell transplantation recipients on immunosuppressive therapy infected with the Omicron variant, were included. Patients were contacted regularly until symptom resolution. Clinical characteristics of consenting patients were collected through their electronic patient files. To identify possible risk factors for hospitalization, a univariate logistic analysis was performed. Findings A total of 114 consecutive immunocompromised patients(88% after a solid organ transplant) were enrolled. Eighty-nine percent had previously received three mRNA vaccinations. While only one patient died, 23(20%) required hospital admission for a median of 11 days. A low SARS-CoV-2 IgG antibody response(<300 BAU/mL) at diagnosis, higher age, being a lung transplant recipient, more comorbidities and a higher frailty were associated with hospital admission(all p<0.01). At the end of follow-up, 25% had still not fully recovered. Of the 23 hospitalized patients, 70% had a negative and 92% a low IgG (<300 BAU/mL) antibody response at admission. Sotrovimab was administered to 17 of them, of which one died. Interpretation While the mortality in immunocompromised patients infected with Omicron was low, hospital admission was frequent and the duration of symptoms often prolonged. Besides vaccination, other interventions are needed to limit the morbidity from COVID-19 in immunocompromised patients. Funding None.

Admissions to a large tertiary care hospital and Omicron BA.1 and BA.2 SARS-CoV-2 PCR positivity: primary, contributing, or incidental COVID-19 (preprint)

SARS-CoV-2 Omicron variants BA.1 and BA.2 seem to show reduced clinical severity. We classified 172 COVID-19 Omicron patient admissions. 66.2% of patients were admitted with primary or admission-contributing COVID-19. We therefore must be careful to base healthcare and public health decisions on the total number of hospitalized COVID-19 patients alone.

Accuracy of COVID-19 self-tests with unsupervised nasal or nasal plus oropharyngeal self-sampling in symptomatic individuals in the Omicron period (preprint)

BackgroundPerformances of rapid antigen diagnostic tests (Ag-RDTs) with nasal self-sampling, and oropharyngeal plus nasal (OP-N) self-sampling, in the Omicron period are unknown. MethodsProspective diagnostic accuracy study among 6,497 symptomatic individuals aged >16 years presenting for SARS-CoV-2 testing at three test-sites. Participants were sampled for RT-PCR (reference test) and received one Ag-RDT to perform unsupervised with either nasal self-sampling (during the emergence of Omicron, and after Omicron share was >90%, phase-1) or with OP-N self-sampling (in a subsequent phase-2; Omicron share >99%). The evaluated tests were Acon Flowflex (Flowflex; phase-1 only), MP Biomedicals (MPBio), and Siemens-Healthineers Clinitest (Clinitest). FindingsDuring phase-1, 45% of Flowflex, 29% of MPBio, and 35% of Clinitest participants were confirmatory testers (previously tested positive by a self-test at own initiative). Overall sensitivities with nasal self-sampling were 79.0% (95% CI: 74.7-82.8%) for Flowflex, 69.9% (65.1-74.4%) for MPBio, and 70.2% (65.6-74.5%) for Clinitest. Sensitivities were substantially higher in confirmatory testers (93.6%, 83.6%, and 85.7%, respectively) than in those who tested for other reasons (52.4%, 51.5%, and 49.5%, respectively). Sensitivities decreased by 6.1 (p=0.16 by Chi-square test), 7.0 (p=0.60), and 12.8 (p=0.025) percentage points, respectively, when transitioning from 29% to >95% Omicron. During phase-2, 53% of MPBio, and 44% of Clinitest participants were confirmatory testers. Overall sensitivities with OP-N self-sampling were 83.0% (78.8%-86.7%) for MPBio and 77.3% (72.9%-81.2%) for Clinitest. Comparing OP-N to nasal sampling, sensitivities were slightly higher in confirmatory testers (87.4% and 86.1%, respectively), and substantially higher in those testing for other reasons (69.3% and 59.9%, respectively). InterpretatioSensitivities of three Ag-RDTs with nasal self-sampling decreased during Omicron emergence but was only statistically significant for Clinitest. Sensitivities were substantially influenced by the proportion of confirmatory testers. Addition of oropharyngeal to nasal self-sampling improved sensitivities of MPBio and Clinitest. FundingDutch Ministry of Health, Welfare, and Sport. Research into contextO_ST_ABSEvidence before this studyC_ST_ABSSARS-CoV-2 rapid antigen diagnostic tests (Ag-RDTs) require no or minimal equipment, provide a result within 15-30 minutes, and can be used in a range of settings including for self-testing at home. Self-testing may potentially lower the threshold to testing and allows individuals to obtain a test result quickly and at their own convenience, which could support the early detection of infectious cases and reduce community transmission. Real world evidence on the performance of unsupervised nasal and oropharyngeal plus nasal (OP-N) self-sampling in the Omicron variant period is needed to accurately inform end-users and policymakers. Therefore, we conducted a large prospective diagnostic accuracy study of three commercially available Ag-RDTs with self-sampling (the Acon Flowflex test, the MP Biomedicals test, and the Siemens-Healthineers Clinitest) during and after the emergence of Omicron using RT-PCR as the reference standard. Our aims were to evaluate whether the accuracies of Ag-RDTs with nasal self-sampling changed over time with the emergence of Omicron; and to determine whether addition of oropharyngeal to nasal self-sampling with the same swab yielded higher diagnostic accuracies. What this study addsThe large comprehensive study was conducted in almost 6,500 participants with symptoms when presenting for routine SARS-CoV-2 testing at three public health service COVID-19 test-sites in the Netherlands. During the study, conducted between 21 December 2021 and 10 February 2022, the percentage of the Omicron variant in samples from the national SARS-CoV-2 pathogen surveillance increased from 29% in the first week to 99% in the last week of the study. The period during which the Omicron variant was dominant was divided into a nasal sampling phase (phase-1; Omicron present in >90% of surveillance samples) and an OP-N sampling phase (phase-2; Omicron share was >99%). In phase-1, 45% of Flowflex, 29% of MPBio, and 35% of Clinitest participants visited the test-site because of a positive self-test (confirmatory testers). Overall sensitivities with nasal self-sampling were 79.0% (95% CI: 74.7-82.8%) for the Flowflex, 69.9% (65.1-74.4%) for the MPBio, and 70.2% (65.6-74.5%) for the Clinitest Ag-RDT. Sensitivities were 94%, 84%, and 86%, respectively, for confirmatory testers, and 52%, 52%, and 50%, respectively, for those who had other reasons for getting tested. Sensitivities were 87.0% (79.7-92.4%), 83.1% (72.9-90.7%), and 80.0% (51.9-95.7%), respectively, in the first week, and decreased by 6.1 (p=0.16 by Chi-square test), 7.0 (p=0.60), and 12.8 (p=0.025) percentage points in the final week of the study. In Phase-2, 53% of MPBio and 44% of Clinitest participants were confirmatory testers. Overall sensitivities with OP-N self-sampling were 83.0% (78.8%-86.7%) for MPBio and 77.3% (72.9%-81.2%) for Clinitest. When comparing OP-N to nasal sampling, sensitivities were slightly higher in confirmatory testers (87.4% and 86.1%, respectively), and substantially higher in those testing for other reasons (69.3% and 59.9%). Implications of all the available evidenceThe sensitivities of three commercially available Ag-RDTs performed with nasal self-sampling decreased during the emergence of Omicron, but this trend was only statistically significant for Clinitest. Addition of oropharyngeal to nasal self-sampling improved the sensitivity of the MPBio and Clinitest, most notably in individuals who visited the test-site for other reasons than to confirm a positive self-test. Based on these findings, the manufacturers of MPBio and Clinitest may consider extending their instructions for use to include combined oropharyngeal and nasal sampling, and other manufacturers may consider evaluating this as well.

Omicron BA.1 and BA.2 are antigenically distinct SARS-CoV-2 variants (preprint)

The emergence and rapid spread of SARS-CoV-2 variants may impact vaccine efficacy significantly. The Omicron variant termed BA.2, which differs genetically substantially from BA.1, is currently replacing BA.1 in several countries, but its antigenic characteristics have not yet been assessed. Here, we used antigenic cartography to quantify and visualize antigenic differences between SARS-CoV-2 variants using hamster sera obtained after primary infection. Whereas early variants are antigenically similar, clustering relatively close to each other in antigenic space, Omicron BA.1 and BA.2 have evolved as two distinct antigenic outliers. Our data show that BA.1 and BA.2 both escape (vaccine-induced) antibody responses as a result of different antigenic characteristics. Close monitoring of the antigenic changes of SARS-CoV-2 using antigenic cartography can be helpful in the selection of future vaccine strains.

Accuracy and usability of saliva and nasal rapid antigen self-testing for detection of SARS-CoV-2 infection in the general population: a head-to-head comparison (preprint)

BackgroundSARS-CoV-2 self-tests may lower the threshold of testing and produce a result quickly. This could support the early detection of infectious cases and reduce further community transmission. However, the diagnostic accuracy of (unsupervised) self-testing with rapid antigen diagnostic tests (Ag-RDTs) is mostly unknown. We therefore conducted a large-scale head-to-head comparison of the diagnostic accuracy of a self-performed SARS-CoV-2 saliva and nasal Ag-RDT, each compared to a molecular reference test, in the general population in the Netherlands. MethodsIn this cross-sectional study we consecutively included individuals aged 16 years and older presenting for SARS-CoV-2 testing at three Dutch public health service test sites irrespective of their indication for testing, vaccination status, and symptomatology. Participants were sampled for molecular testing at the test site and received two self-tests (the Hangzhou AllTest saliva self-test and the SD Biosensor nasal self-test by Roche Diagnostics) to perform at home within a few hours without knowledge of their molecular test result. Information on presence and type of symptoms, user experiences, and results of both self-tests were collected via an online questionnaire. For each self-test, sensitivity, specificity, positive and negative predictive values were determined with molecular testing as reference standard. FindingsThe SARS-CoV-2 molecular reference test positivity rate was 6.5% in the 2,819 participants. Overall sensitivities with 95% confidence intervals were 46.7% (85/182; 39.3%-54.2%) for the saliva Ag-RDT, and 68.9% (124/180; 61.6%-75.6%) for the nasal Ag-RDT. With a viral load cut-off ([≥]5.2 log10 SARS-CoV-2 E-gene copies/mL) as a proxy of infectiousness, sensitivities increased to 54.9% (78/142; 46.4%-63.3%) for the saliva Ag-RDT and 83.9% (120/143; 76.9%-89.5%) for the nasal Ag-RDT. For the nasal Ag-RDT, sensitivities were 78.5% [71.1%-84.8%] and 22.6% [9.6%-41.1%] in those with and without symptoms at the time of sampling, which increased to 90.4% (113/125; 83.8%-94.9%) and 38.9% (7/18; 17.3%-64.3%) after applying the viral load cut-off. In those with and without prior confirmed SARS-CoV-2, sensitivities were 36.8% [19/372; 16.3%-61.6%] and 72.7% [161/2437; 65.1%-79.4%] for the nasal Ag-RDT, which increased to 100% (7/7; 59.0%-100%) and 83.1% (113/126; 75.7%-89.0%) after applying the viral load cut-off. The diagnostic accuracy of the nasal Ag-RDT did not differ by COVID-19 vaccination status, sex, and age. Specificities were >99%, positive predictive values >70% and negative predictive values >95%, for the saliva Ag-RDT, and >99%, >90%, and >95% for the nasal Ag-RDT, respectively, in most analyses. Interpreting the results was considered (very) easy for both self-tests. InterpretationThe Hangzhou AllTest self-performed saliva Ag-RDT is not reliable for SARS-CoV-2 infection detection overall nor in the studied subgroups. The SD Biosensor self-performed nasal Ag-RDT had high sensitivity in individuals with symptoms and in those without a prior SARS-CoV-2 infection. The overall accuracy in individuals with symptoms was comparable to that found in previous studies with professional sampling for this Ag-RDT. The extremely low sensitivity of the nasal Ag-RDT in asymptomatic individuals and in individuals who had had a prior SARS-CoV-2 infection is an important finding and warrants further investigation. FundingDutch Ministry of Health, Welfare, and Sport.

Diagnostic accuracy of three prevailing rapid antigen tests for detection of SARS-CoV-2 infection in the general population: cross sectional study (preprint)

Objective To assess the diagnostic accuracy of three rapid antigen tests (Ag-RDTs) for detecting SARS-CoV-2 infection in the general population. Design Cross-sectional study with follow-up using pseudonymised record linkage. Setting Three Dutch public health service COVID-19 test sites. Participants Consecutively included individuals aged 16 years and older presenting for SARS-CoV-2 testing. Main outcome measures Sensitivity, specificity, positive and negative predictive values of BD-Veritortm System (Becton Dickinson), PanBio (Abbott), and SD-Biosensor (Roche Diagnostics), applying routinely used sampling methods (combined oropharyngeal and nasal [OP-N] or nasopharyngeal [NP] swab), with molecular testing as reference standard. For SDBiosensor, the diagnostic accuracy with OP-N sampling was also assessed. A viral load cutoff ([≥]5.2 log10 SARS-CoV-2 E-gene copies/mL) served as a proxy of infectiousness. Results SARS-CoV-2 prevalence and overall sensitivities with 95% confidence intervals were 188/1441 (13.0%) and 129/188 (68.6% [61.5%-75.2%]) for BD-Veritor, 173/2056 (8.4%) and 119/173 (68.8% [61.3%-75.6%]) for PanBio, and 215/1769 (12.2%) and 160/215 (74.4% [68.0%-80.1%]) for SD-Biosensor with routine sampling, and 164/1689 (9.7%) and 123/164 (75.0% [67.7%-81.4%]) for SD-Biosensor with OP-N sampling. In those symptomatic or asymptomatic at sampling, sensitivities were 72.2%-83.4% and 54.0%-55.9%, respectively. With a viral load cut-off, sensitivities were 125/146 (85.6% [78.9%-90.9%]) for BD-Veritor, 108/121 (89.3% [82.3%-94.2%]) for PanBio, 160/182 (87.9% [82.3%-92.3%]) for SD-Biosensor with routine sampling, and 118/141 (83.7% [76.5%-89.4%]) with OP-N sampling. Specificities were >99%, and positive and negative predictive values >95%, for all tests in most analyses. 61.3% of false negative Ag-RDT participants returned for testing within 14 days (median of 3 days, interquartile range 3) of whom 90.3% tested positive. Conclusions The overall sensitivities of the three Ag-RDTs were 68.6%-75.0%, increasing to at least 85.6% after the viral load cut-off was applied. For SD-Biosensor, the diagnostic accuracy with OP-N and NP sampling was comparable. Over 55% of false negative Ag-RDT participants tested positive during follow-up.

Virological characteristics of SARS-CoV-2 vaccine breakthrough infections in health care workers (preprint)

BackgroundSARS-CoV-2 vaccines are highly effective at preventing COVID-19-related morbidity and mortality. As no vaccine is 100% effective, breakthrough infections are expected to occur. MethodsWe analyzed the virological characteristics of 161 vaccine breakthrough infections in a population of 24,706 vaccinated healthcare workers (HCWs), using RT-PCR and virus culture. ResultsThe delta variant (B.1.617.2) was identified in the majority of cases. Despite similar Ct-values, we demonstrate lower probability of infectious virus detection in respiratory samples of vaccinated HCWs with breakthrough infections compared to unvaccinated HCWs with primary SARS-CoV-2 infections. Nevertheless, infectious virus was found in 68.6% of breakthrough infections and Ct-values decreased throughout the first 3 days of illness. ConclusionsWe conclude that rare vaccine breakthrough infections occur, but infectious virus shedding is reduced in these cases.

Droplet Digital RT-PCR to detect SARS-CoV-2 variants of concern in wastewater (preprint)

Wastewater surveillance has shown to be a valuable and efficient tool to obtain information about the trends of COVID-19 in the community. Since the recent emergence of new variants, associated with increased transmissibility and/or antibody escape (variants of concern), there is an urgent need for methods that enable specific and timely detection and quantification of the occurrence of these variants in the community. In this study we demonstrate the use of RT-ddPCR on wastewater samples for specific detection of mutation N501Y. This assay enabled simultaneous enumeration of the concentration of variants with the 501Y mutation and Wild Type (WT, containing 501N) SARS-CoV-2 RNA. Detection of N501Y was possible in samples with mixtures of WT with low proportions of lineage B.1.351 (0.5%). The method could accurately determine the proportion of N501Y and WT in mixtures of SARS-CoV-2 RNA. The application to raw sewage samples from the cities of Amsterdam and Utrecht demonstrated that this method can be applied to determine the concentrations and the proportions of WT and N501Y containing SARS-CoV-2 RNA in wastewater samples. The emergence of N501Y in Amsterdam and Utrecht wastewater aligned with the emergence of B.1.1.7 as causative agent of COVID-19 in the Netherlands, indicating that RT-ddPCR of wastewater samples can be used to monitor the emergence of the N501Y mutation in the community. It also indicates that RT-ddPCR could be used for sensitive and accurate monitoring of current (like K417N, E484K) or future mutations present in SARS-CoV-2 variants of concern. Monitoring emergence of these mutations in the community via wastewater is rapid, efficient and valuable in supporting public health decision-making.

Diagnostic accuracy of rapid antigen tests in pre-/asymptomatic close contacts of individuals with a confirmed SARS-CoV-2 infection (preprint)

Background Pre-/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. Methods We 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 VeritorTM 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. Results Of 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. Conclusions The 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.

Temporal kinetics of RNAemia and associated systemic cytokines in hospitalized COVID-19 patients (preprint)

COVID-19 is associated to a wide range of extra-respiratory complications, of which the pathogenesis is currently not fully understood. In this study we report the temporal kinetics of viral RNA and inflammatory cytokines and chemokines in serum during the course of COVID-19. We show that a RNAemia occurs more frequently and lasts longer in patients that develop critical disease compared to patients that develop moderate or severe disease. Furthermore we show that concentrations of IL-10 and MCP-1--but not IL-6--are associated with viral load in serum. However, higher levels of IL-6 were associated with the development of critical disease. The direct association of inflammatory cytokines with viral load or disease severity highlights the complexity of systemic inflammatory response and the role of systemic viral spread.

SNPnexus COVID: Facilitating the analysis of COVID-19 host genetics (preprint)

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has presented a crisis for global healthcare systems. Human SARS-CoV-2 infection can result in coronavirus disease 2019 (COVID-19), which has been characterised as an acute respiratory illness, with most patients displaying flu-like symptoms, such as a fever, cough and dyspnoea. However, the range and severity of individual symptoms experienced by patients can vary significantly, indicating a role for host genetics in impacting the susceptibility and severity of COVID-19 disease. Whilst most symptomatic infections are known to manifest in mild to moderate respiratory symptoms, severe pneumonia and complications including cytokine release syndrome, which can lead to multi-organ dysfunction, have also been observed in cases worldwide. Global initiatives to sequence the genomes of patients with COVID-19 have driven an expanding new field of host genomics research, to characterise the genetic determinants of COVID-19 disease. The functional annotation and analysis of incoming genomic data, within a clinically relevant turnaround time, is therefore imperative given the importance and urgency of research efforts to understand the biology of SARS-CoV-2 infection and disease. To address these requirements, we developed SNPnexus COVID. This is a web-based variant annotation tool, powered by the SNPnexus software.

assayM: a web application to monitor mutations in COVID-19 diagnostic assays. (preprint)

Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) is the gold standard as diagnostic assays for the detection of COVID-19 and the specificity and sensitivity of these assays depend on the complementarity of the RT-PCR primers to the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the virus mutates over time during replication cycles, there is an urgent need to continuously monitor the virus genome for appearances of mutations and mismatches in the PCR primers used in these assays. Here we present assayM, a web application to explore and monitor mutations introduced in the primer and probe sequences published by the World Health Organisation (WHO) or in any custom-designed assay primers for SARS-CoV-2 detection assays in globally available SARS-CoV-2 genome datasets.

Clinical evaluation of the Roche/SD Biosensor rapid antigen test with symptomatic, non-hospitalized patients in a municipal health service drive-through testing site. (preprint)

BackgroundRapid detection of infectious individuals is essential in stopping the further spread of SARS-CoV-2. Although rapid antigen test is not as sensitive as the gold standard RT-PCR, the time to result is decreased by day(s), strengthening the effectiveness of contact tracing. MethodsThe Roche/SD Biosensor lateral flow antigen rapid test was evaluated in a mild symptomatic population at a large drive through testing site. A second nasopharyngeal swab was directly tested with the rapid test on site and results were compared to RT-PCR and virus culture. Date of onset and symptoms were analysed using data from a clinical questionnaire. ResultsWe included 970 persons with complete data. Overall sensitivity and specificity were 84.9% (CI95% 79.1-89.4) and 99.5% (CI95% 98.7-99.8) which translated into a positive predictive value of 97.5% (CI95% 94.0-99.5) under the current regional PCR positivity of 19.2%. Sensitivity for people with high loads of viral RNA (ct <30, 2.17E+05 E gene copy/ml) and who presented within 7 days since symptom onset increased to 95.8% (CI95% 90.5-98.2). Band intensity and time to result correlated strongly with viral load thus strong positive bands could be read before the recommended time. Around 98% of all viable specimen with ct <30 were detected successfully indicating that the large majority of infectious people can be captured with this test. ConclusionAntigen rapid tests can detect mildly symptomatic cases in the early phase of disease thereby identifying the most infectious individuals. Using this assay can have a significant value in the speed and effectiveness of SARS-CoV-2 outbreak management. SummaryO_LIPeople with early onset and high viral load were detected with 98.2% sensitivity. C_LIO_LI97% of individuals in which virus could be cultured were detected by the rapid test. C_LIO_LIThis test is suitable to detect mild symptomatic cases. C_LI

From more testing to smart testing: data-guided SARS-CoV-2 testing choices (preprint)

We present an in-depth analysis of data from drive through testing stations using rapid antigen detection tests (RDTs), RT-PCR and virus culture, to assess the ability of RDTs to detect infectious cases. We show that the detection limits of five commercially available RDTs differ considerably, impacting the translation into the detection of infectious cases. We recommend careful fit-for-purpose testing before implementation of antigen RDTs in routine testing algorithms as part of the COVID-19 response.

Jumping back and forth: anthropozoonotic and zoonotic transmission of SARS-CoV-2 on mink farms (preprint)

The zoonotic origin of the SARS-CoV-2 pandemic is still unknown. Animal experiments have shown that non-human primates, cats, ferrets, hamsters, rabbits and bats can be infected by SARS-CoV-2. In addition, SARS-CoV-2 RNA has been detected in felids, mink and dogs in the field. Here, we describe an in-depth investigation of outbreaks on 16 mink farms and humans living or working on these farms, using whole genome sequencing. We conclude that the virus was initially introduced from humans and has evolved, most likely reflecting widespread circulation among mink in the beginning of the infection period several weeks prior to detection. At the moment, despite enhanced biosecurity, early warning surveillance and immediate culling of infected farms, there is ongoing transmission between mink farms with three big transmission clusters with unknown modes of transmission. We also describe the first animal to human transmissions of SARS-CoV-2 in mink farms. One sentence summarySARS-CoV-2 transmission on mink farms.

Shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (COVID-19): duration and key determinants (preprint)

BackgroundLong-term shedding of viral RNA in COVID-19 prevents timely discharge from the hospital or de-escalation of infection prevention and control practices. Key questions are the duration and determinants of infectious virus shedding. We assessed these questions using virus cultures of respiratory tract samples from hospitalized COVID-19 patients as a proxy for infectious virus shedding. MethodsClinical and virological data were obtained from 129 hospitalized COVID-19 patients (89 intensive care, 40 medium care). Generalized estimating equations were used to identify if viral RNA load, detection of viral subgenomic RNA, serum neutralizing antibody response, duration of symptoms, or immunocompromised status were predictive for a positive virus culture. FindingsInfectious virus shedding was detected in 23 of the 129 patients (17,8%). The median duration of shedding was 8 days post onset of symptoms (IQR 5 - 11) and the probability of detecting infectious virus dropped below 5% after 15,2 days post onset of symptoms (95% confidence interval (CI) 13,4 - 17,2). Multivariate analyses identified viral loads above 7 log10 RNA copies/mL (odds ratio [OR]; CI 14,7 (3,57-58,1; p<0,001) as independently associated with isolation of infectious SARS-CoV-2 from the respiratory tract. A serum neutralizing antibody titre of at least 1:20 (OR of 0,01 (CI 0,003-0,08; p<0,001) was independently associated with non-infectious SARS-CoV-2. InterpretationInfection prevention and control guidelines should take into account that patients with severe or critical COVID-19 may shed infectious virus for longer periods of time compared to what has been reported for in patients with mild COVID-19. Infectious virus shedding drops to undetectable levels below a viral RNA load threshold and once serum neutralizing antibodies are present, which warrants the use of quantitative viral RNA load assays and serological assays in test-based strategies to discontinue or de-escalate infection prevention and control precautions. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed, bioRxiv, and medRxiv for articles that reported on shedding of infectious virus in COVID-19 patients using the search terms ("coronavirus" OR "SARS" OR "SARS-CoV-2" OR "COVID-19") AND ("shedding" OR "infectivity" OR "infectious" OR "virus culture") with no language or time restrictions. A detailed study on nine patients with mild COVID-19 reported that infectious virus could not be isolated after more than eight days of symptoms. The probability of isolating infectious virus was less than 5% when viral loads dropped below 6,51 Log10 RNA copies/mL. Similar results were obtained with a larger diagnostic sample set, but that study did not report on clinical parameters such as disease severity. Finally there is a report of a single patient shedding infectious virus up to 18 days after onset of symptoms. No published works were found on the shedding of infectious virus in patients with severe or critical COVID-19, and no published works were found on factors independently associated with shedding of infectious virus. Added value of this studyWe assessed the duration and determinants of infectious virus shedding in 129 patients with severe or critical COVID-19. The duration of infectious virus shedding ranged from 0 to 20 days post onset of symptoms (median 8 days, IQR 5 - 11). The probability of detecting infectious virus dropped below 5% after 15,2 days post onset of symptoms (95% confidence interval (CI) 13,4 - 17,2). Viral loads above 7 log10 RNA copies/mL were independently associated with detection of infectious SARS-CoV-2 from the respiratory tract (odds ratio [OR]; CI 14,7 (3,57-58,1; p<0,001). A serum neutralizing antibody titre of at least 1:20 (OR of 0,01 (CI 0,003-0,08; p<0,001) was independently associated with non-infectious SARS-CoV-2. Implications of all the available evidenceInfection prevention and control guidelines should take into account that patients with severe or critical COVID-19 may shed infectious virus for longer periods of time compared to what has been reported for in patients with mild COVID-19. Quantitative viral RNA load assays and serological assays should be used for test-based strategies to discontinue or de-escalate infection prevention and control precautions.

COVID-19 in healthcare workers in three hospitals in the South of the Netherlands, March 2020 (preprint)

Background: Ten days after the first reported case of SARS-CoV-2 infection in the Netherlands, 3.9% of healthcare workers (HCWs) in nine hospitals located in the South of the Netherlands tested positive for SARS-CoV-2 RNA. The extent of nosocomial transmission that contributed to the HCW infections was unknown. Methods: We combined epidemiological data, collected by means of structured interviews of HCWs, with whole genome sequencing (WGS) of SARS-CoV-2 in clinical samples from HCWs and patients in three of nine hospitals that participated in the HCW screening, to perform an in-depth analysis of sources and modes of transmission of SARS -CoV-2 in HCWs and patients. Results: A total of 1,796 out of 12,022 HCWs (15%) of the three participating hospitals were screened, based on clinical symptoms, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete genome sequences of 50 HCWs and 18 patients. Most sequences grouped in 3 clusters, with 2 clusters displaying local circulation within the region. The observed patterns are most consistent with multiple introductions into the hospitals through community acquired infections, and local amplification in the community. Conclusions: Although direct transmission in the hospitals cannot be ruled out, the data does not support widespread nosocomial transmission as source of infection in patients or healthcare workers.