The unified proposal for classification of human respiratory syncytial virus below the subgroup level (preprint)

A globally implemented unified classification for human respiratory syncytial virus (HRSV) below the subgroup level remains elusive. Here, we formulate the global consensus of HRSV classification based on the challenges and limitations of our previous proposals and the future of genomic surveillance. From a high-quality dataset of 1,480 HRSV-A and 1,385 HRSV-B genomes submitted to NCBI and GISAID up to March 2023, we categorized HRSV-A/B sequences into lineages based on phylogenetic clades and amino acid markers. We defined 24 lineages within HRSV-A and 16 within HRSV-B, providing guidelines for prospective lineages definition. Our classification demonstrated robustness in its applicability to both complete and partial genomes. In addition, it allowed the observation of notable lineage replacements and the identification of lineages exclusively detected since the COVID-19 pandemic. We envision that this unified HRSV classification proposal will strengthen and facilitate HRSV molecular epidemiology on a global scale.

Seropositivity to Nucleoprotein to detect SARS-CoV-2 infections: a tool to detect breakthrough infections after COVID-19 vaccination (preprint)

BackgroundWith COVID-19 vaccine roll-out ongoing in many countries globally, monitoring of breakthrough infections is of great importance. Antibodies persist in the blood after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since COVID-19 vaccines induce immune response to the Spike protein of the virus, which is the main serosurveillance target to date, alternative targets should be explored to distinguish infection from vaccination. MethodsMultiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) with a primary infection and no vaccination history were used to determine the accuracy of Nucleoprotein-specific immunoglobulin G (IgG) in detecting past SARS-CoV-2 infection. We also described Spike S1 and Nucleoprotein-specific IgG responses in 230 COVID-19 vaccinated individuals (Pfizer/BioNTech). ResultsThe sensitivity of Nucleoprotein seropositivity was 85% (95% confidence interval: 80-90%) for mild COVID-19 in the first two months following symptom onset. Sensitivity was lower in asymptomatic individuals (67%, 50-81%). Participants who had experienced a SARS-CoV-2 infection up to 11 months preceding vaccination, as assessed by Spike S1 seropositivity or RT-qPCR, produced 2.7-fold higher median levels of IgG to Spike S1 [≥]14 days after the first dose as compared to those unexposed to SARS-CoV-2 at [≥]7 days after the second dose (p=0.011). Nucleoprotein-specific IgG concentrations were not affected by vaccination in naive participants. ConclusionsSerological responses to Nucleoprotein may prove helpful in identifying SARS-CoV-2 infections after vaccination. Furthermore, it can help interpret IgG to Spike S1 after COVID-19 vaccination as particularly high responses shortly after vaccination could be explained by prior exposure history.

SARS-CoV-2 RNA and antibody dynamics in a Dutch household study with dense sampling frame (preprint)

This study investigated the dynamics of SARS-CoV-2 infection and diagnostics in household members of different ages and with different symptom severity after SARS-CoV-2 exposure during the early phase of the pandemic. Households with a SARS-CoV-2 confirmed positive case and at least one child in the Netherlands were followed for 6 weeks. Naso (NP)- and oropharyngeal (OP) swabs, oral fluid and feces specimens were analyzed for SARS-CoV-2 RNA and serum for SARS-CoV-2-specific antibodies. The dynamics of the presence of viral RNA and the serological response was modeled to determine the sampling time-frame and sample type with the highest sensitivity to confirm or reject a SARS-CoV-2 diagnosis. Transmission of SARS-CoV-2 between adults and children within a household was correlated with symptom severity of index cases. In children higher viral loads compared to adults were detected at symptom onset. Early in infection, higher viral loads were detected in NP and OP specimens, while RNA in especially feces were longer detectable. SARS-CoV-2-specific antibodies have a 90% probability of detection from 7 days (total Ig) and 18 days (IgG) since symptom onset. In conclusion this study has shown that on average, children carry higher loads of virus as compared to adults early after infection. For highest probability of detection in SARS-CoV-2 diagnostics early in infection, RT-PCR on NP and OP specimens are more sensitive than on oral fluid and feces. For SARS-CoV-2 diagnostics late after infection, RT-PCR on feces specimens and serology are more valuable.

Children and adults with mild COVID-19 symptoms develop memory T cell immunity to SARS-CoV-2 (preprint)

BackgroundSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to considerable morbidity/mortality worldwide, but most infections, especially among children, have a mild course. However, it remains largely unknown whether infected children develop cellular immune memory. MethodsTo determine whether a memory T cell response is being developed as an indicator for long-term immune protection, we performed a longitudinal assessment of the SARS-CoV-2-specific T cell response by IFN-{gamma} ELISPOT and activation marker expression analyses of peripheral blood samples from children and adults with mild-to-moderate COVID-19. ResultsUpon stimulation of PBMCs with heat-inactivated SARS-CoV-2 or overlapping peptides of spike (S-SARS-CoV-2) and nucleocapsid proteins, we found S-SARS-CoV-2-specific IFN-{gamma} T cell responses in most infected children (83%) and all adults (100%) that were absent in unexposed controls. Frequencies of SARS-CoV-2-specific T cells were higher in infected adults, especially in those with moderate symptoms, compared to infected children. The S-SARS-CoV-2 IFN-{gamma} T cell response correlated with S1-SARS-CoV-2-specific serum IgM, IgG, and IgA antibody concentrations. Predominantly, effector memory CD4+ T cells of a Th1 phenotype were activated upon exposure to SARS-CoV-2 antigens, which persisted for 4-8 weeks after symptom onset. We detected very low frequencies of SARS-CoV-2-reactive CD8+ T cells in these individuals. ConclusionsOur data indicate that an antigen-specific memory CD4+ T cell response is induced in children and adults with mild SARS-CoV-2 infection. T cell immunity induced after mild COVID-19 could contribute to protection against re-infection.

Pathology and immunity after SARS-CoV-2 infection in male ferrets is affected by age and inoculation route (preprint)

Improving COVID-19 intervention strategies partly relies on animal models to study SARS-CoV-2 disease and immunity. In our pursuit to establish a model for severe COVID-19, we inoculated young and adult male ferrets intranasally or intratracheally with SARS-CoV-2. Intranasal inoculation established an infection in all ferrets, with viral dissemination into the brain and gut. Upon intratracheal inoculation only adult ferrets became infected. However, neither inoculation route induced observable COVID-19 symptoms. Despite this, a persistent inflammation in the nose was prominent in especially young ferrets and follicular hyperplasia in the bronchi developed 21 days post infection. These effects -if sustained- might resemble long-COVID. Respiratory and systemic cellular responses and antibody responses were induced only in animals with an established infection. We conclude that intranasally-infected ferrets resemble asymptomatic COVID-19 and possibly aspects of long-COVID. Combined with the increasing portfolio to measure adaptive immunity, ferrets are a relevant model for SARS-CoV-2 vaccine research.

Potential environmental transmission routes of SARS-CoV-2 inside a large meat processing plant experiencing COVID-19 clusters (preprint)

Worldwide exceptionally many COVID-19 clusters were observed in meat processing plants. Many contributing factors, promoting transmission, were suggested, including climate conditions in cooled production rooms favorable for environmental transmission but actual sampling studies are lacking. We aimed to assess SARS-CoV-2 contamination of air and surfaces to gain insight in potential environmental transmission in a large Dutch meat processing plant experiencing COVID-19 clusters. We performed SARS-CoV-2 screening of workers operating in cooled production rooms and intensive environmental sampling during a two-week study period in June 2020. Sampling of air (both stationary and personal), settling dust, ventilation systems, and sewage was performed. Swabs were collected from high-touch surfaces and workers hands. Screening of workers was done using oronasopharyngeal swabs. Samples were tested for presence of SARS-CoV-2 RNA by RT-qPCR. Of the 76 (predominantly asymptomatic) workers tested, 27 (35.5%) were SARS-CoV-2 RNA positive with modest to low viral loads (Ct[≥]29.7). In total, 6 out of 203 surface swabs were positive (Ct [≥]38), being swabs taken from communal touchscreens/handles. One of the 12 personal air samples and one of the 4 sewage samples were positive, RNA levels were low (Ct[≥]38). All other environmental samples tested negative. Although one-third of workers tested SARS-CoV-2 RT-PCR positive, environmental contamination was limited. Hence widespread transmission of SARS-CoV-2 via air and surfaces was considered unlikely within this plant at the time of investigation in the context of strict COVID-19 control measures in place.

Self-testing for the detection of SARS-CoV-2 infection with rapid antigen tests. (preprint)

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 RDTs 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.

High infection attack rates of SARS-CoV-2 in Dutch households revealed by dense sampling (preprint)

Background Indoor environments are considered a main setting for transmission of SARS-CoV-2. Households in particular present a close-contact environment with high probability of transmission between persons of different ages and with different roles in society. Methods Complete households with a laboratory-confirmed SARS-CoV-2 positive case in the Netherlands (March-May 2020) were included. At least three home visits were performed during 4-6 week of follow-up, collecting naso- and oropharyngeal swabs, oral fluid, faeces and blood samples for molecular and serological analyses of all household members. Symptoms were recorded from two weeks before the first visit up to the last visit. Secondary attack rates (SAR) were estimated with logistic regression. A transmission model was used to assess transmission routes in the household. Results A total of 55 households with 187 household contacts were included. In 17 households no transmission took place, and in 11 households all persons were infected. Estimated SARs were high, ranging from 35% (95%CI: 24%-46%) in children to 51% (95%CI: 39%-63%) in adults. Estimated transmission rates in the household were high, with reduced susceptibility of children compared to adolescents and adults (0.67; 95%CI: 0.40-1.1). Conclusion Estimated SARs were higher than reported in earlier household studies, presumably owing to a dense sampling protocol. Children were shown to be less susceptible than adults, but the estimated SAR in children was still high. Our results reinforce the role of households as main multiplier of SARS-CoV-2 infection in the population. Key points We analyze data from a SARS-CoV-2 household study and find higher secondary attack rates than reported earlier. We argue that this is due to a dense sampling strategy that includes sampling at multiple time points and of multiple anatomical sites.

Absence of association between 2019–20 influenza vaccination and COVID-19: results of the European I-MOVE-COVID-19 primary care project, March–August 2020 (preprint)

Background: Claims of influenza vaccination increasing COVID-19 risk are circulating. Within the I-MOVE-COVID-19 primary care multicentre study, we measured the association between 2019–20 influenza vaccination and COVID-19. Methods We conducted a multicentre test-negative case-control study at primary care level, in study sites in five European countries, from March–August 2020. Patients presenting with acute respiratory infection were swabbed, with demographic, 2019–20 influenza vaccination and clinical information documented. Using logistic regression we measured the adjusted odds ratio (aOR), adjusting for study site and age, sex, calendar time, presence of chronic conditions. The main analysis included patients swabbed ≤7 days after onset from the three countries with <15% of missing influenza vaccination. In secondary analyses, we included five countries, using multiple imputation with chained equations to account for missing data. Results We included 257 COVID-19 cases and 1631 controls in the main analysis (three countries). The overall aOR between influenza vaccination and COVID-19 was 0.93 (95% CI: 0.66–1.32). The aOR was 0.92 (95% CI: 0.58–1.46) and 0.92 (95%CI: 0.51–1.67) among those aged 20–59 and ≥60 years, respectively. In secondary analyses, we included 6457 cases and 69272 controls. The imputed aOR was 0.87 (95% CI: 0.79–0.95) among all ages and any delay between swab and symptom onset. Conclusions There was no evidence that COVID-19 cases were more likely to be vaccinated against influenza than controls. Influenza vaccination should be encouraged among target groups for vaccination. I-MOVE-COVID-19 will continue documenting influenza vaccination status in 2020-21, in order to learn about effects of recent influenza vaccination.

Exposure assessment for airborne transmission of SARS-CoV-2 via breathing, speaking, coughing and sneezing (preprint)

Background Evidence for indoor airborne transmission of SARS-CoV-2 is accumulating. If SARS-CoV-2 also spreads via aerosols, this has implications for measures taken to limit transmission. Objectives The aim of this study is to assess exposure to airborne SARS-CoV-2 particles from breathing, speaking, coughing and sneezing in an indoor environment. Methods An exposure assessment model was developed to estimate numbers of SARS-CoV-2 particles in aerosol droplets, expelled during breathing, speaking, coughing and sneezing by an infected person in an unventilated indoor environment, and subsequent inhalation by one or more persons. Scenarios encompass a range of virus concentrations, room sizes and exposure times. Results The calculated total volume of expelled aerosol droplets was highest for a sneeze, followed by a cough and speaking for 20 minutes, and lastly breathing for 20 minutes. A few to as much as tens of millions of virus particles were expelled. Exposure probability strongly depends on the viral concentration in mucus, as well as on the scenario. Exposure probabilities were generally below 1% at a virus concentration in mucus below 10^5 per mL for all scenarios, increasing steeply at different higher concentrations. According to nose / throat swab data collected from patients, 75%, 50% and 5% of infected individuals carry an estimated number of SARS-CoV-2 per mL mucus of at least 10^5, 10^6 and 10^8, respectively. Discussion Exposure to SARS-CoV-2 via aerosols generated during breathing, speaking, coughing and sneezing in an unventilated indoor environment is possible. This study forms a basis to estimate probabilities of exposure to SARS-Cov-2 by airborne transmission in indoor spaces. As long as it is uncertain what fraction of the airborne virus particles is infectious and as long as a dose response relation is lacking, it is recommended to be precautious.

Rapid SARS-CoV-2 whole genome sequencing for informed public health decision making in the Netherlands (preprint)

SARS-CoV-2 is a novel coronavirus that has rapidly spread across the globe. In the Netherlands, the first case of SARS-CoV-2 has been notified on the 27th of February. Here, we describe the first three weeks of the SARS-CoV-2 outbreak in the Netherlands, which started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also, outside the South of the Netherlands. The timely generation of whole genome sequences combined with epidemiological investigations facilitated early decision making in an attempt to control local transmission of SARS-CoV-2 in the Netherlands.

Comparison of commercial RT-PCR diagnostic kits for COVID-19 (preprint)

The final months of 2019 witnessed the emergence of a novel coronavirus in the human population. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has since spread across the globe and is posing a major burden on society. Measures taken to reduce its spread critically depend on timely and accurate identification of virus-infected individuals by the most sensitive and specific method available, i.e. real-time reverse transcriptase PCR (RT-PCR). Many commercial kits have recently become available, but their performance has not yet been independently assessed. The aim of this study was to compare basic analytical and clinical performance of selected RT-PCR kits from seven different manufacturers (Altona Diagnostics, BGI, CerTest Biotec, KH Medical, PrimerDesign, R-Biopharm AG, and Seegene). We used serial dilutions of viral RNA to establish PCR efficiency and estimate the 95% limit of detection (LOD95%). Furthermore, we ran a panel of SARS-CoV-2-positive clinical samples (n=16) for a preliminary evaluation of clinical sensitivity. Finally, we used clinical samples positive for non-coronavirus respiratory viral infections (n=6) and a panel of RNA from related human coronaviruses to evaluate assay specificity. PCR efficiency was [≥]96% for all assays and the estimated LOD95% varied within a 6-fold range. Using clinical samples, we observed some variations in detection rate between kits. Importantly, none of the assays showed cross-reactivity with other respiratory (corona)viruses, except as expected for the SARS-CoV-1 E-gene. We conclude that all RT-PCR kits assessed in this study may be used for routine diagnostics of COVID-19 in patients by experienced molecular diagnostic laboratories.