In vitro fitness of SARS-CoV-2 variants as assessed by competition experiments followed by ddRT-PCR and whole genome sequencing

Objective To develop a new method for reliable and rapid determination of the fitness of SARS-CoV-2 variants of concern. Methods Competition experiments between two SARS-CoV-2 variants were performed in cells of the upper (nasal human airway epithelium) and lower (Calu-3 cells) respiratory tracts followed by quantification of variant ratios by droplet digital reverse transcription (ddRT)-PCR. Results In competition experiments, the delta variant outcompeted the alpha variant in both cells of the upper and lower respiratory tracts. A 50/50% mixture of delta and omicron variants indicated a predominance of omicron in the upper respiratory tract whereas delta predominated in the lower respiratory tract. There was no evidence of recombination events between variants in competition as assessed by whole gene sequencing. Conclusion Differential replication kinetics were shown between variants of concern which may explain, at least partly, the emergence and disease severity associated with new SARS-CoV-2 variants.

Duration of isolation and contagiousness in coronavirus disease 2019 (COVID-19) patients receiving tocilizumab and dexamethasone: A case series.

We describe 10 patients with severe coronavirus disease 2019 (COVID-19) who received tocilizumab and dexamethasone. We correlated isolation duration with cycle thresholds (Ct) values of nucleic acid amplification tests, clinical state and viral cultures. Isolation duration exceeded 21 days for 7 patients due to positive viral cultures or Ct values <30.

Pre-Omicron seroprevalence, seroconversion, and seroreversion of infection-induced SARS-CoV-2 antibodies among a cohort of children and teenagers in Montréal, Canada.

OBJECTIVES: To use serological testing to assess the pre-Omicron seroprevalence, seroconversion, and seroreversion of infection-induced SARS-CoV-2 antibodies in children and adolescents in Montréal, Canada. DESIGN: This analysis is from a prospective cohort study of children aged 2-17 years (at baseline) that included blood spots for antibody detection. The serostatus of participants was determined by enzyme-linked immunosorbent assays using the receptor-binding domain from the spike protein and the nucleocapsid protein as antigens. We estimated seroprevalence, seroconversion rates, and the likelihood of seroreversion at 6 months and 1 year. RESULTS: The baseline (October 2020 to April 2021) seroprevalence was 5.8% (95% confidence interval [CI] 4.8-7.1), which increased to 10.5% (May to September 2021) and 11.0% (November 2021 to March 2022) for the respective follow-ups (95% CI 8.6-12.7; 95% CI 8.8-13.5). The crude rate of seroconversion over the study period was 12.8 per 100 person-years (95% CI 11.0-14.7). The adjusted hazard rates of seroconversion by child characteristics showed higher rates in children who were female, whose parent identified as a racial or ethnic minority, and in households with incomes in the lowest tercile of our study population. The likelihood of remaining seropositive at 6 months was 68% (95% CI 60-77%) and dropped to 42% (95% CI 32-56%) at 1 year. CONCLUSION: Serological studies continue to provide valuable contributions for infection prevalence estimates and help us better understand the dynamics of antibody levels after infection.

Symptomatology during previous SARS-CoV-2 infection and serostatus before vaccination influence the immunogenicity of BNT162b2 COVID-19 mRNA vaccine.

Public health vaccination recommendations for COVID-19 primary series and boosters in previously infected individuals differ worldwide. As infection with SARS-CoV-2 is often asymptomatic, it remains to be determined if vaccine immunogenicity is comparable in all previously infected subjects. This study presents detailed immunological evidence to clarify the requirements for one- or two-dose primary vaccination series for naturally primed individuals. The main objective was to evaluate the immune response to COVID-19 mRNA vaccination to establish the most appropriate vaccination regimen to induce robust immune responses in individuals with prior SARS-CoV-2 infection. The main outcome measure was a functional immunity score (zero to three) before and after vaccination, based on anti-RBD IgG levels, serum capacity to neutralize live virus and IFN-γ secretion capacity in response to SARS-CoV-2 peptide pools. One point was attributed for each of these three functional assays with response above the positivity threshold. The immunity score was compared based on subjects' symptoms at diagnosis and/or serostatus prior to vaccination. None of the naïve participants (n=14) showed a maximal immunity score of three following one dose of vaccine compared to 84% of the previously infected participants (n=55). All recovered individuals who did not have an immunity score of three were seronegative prior to vaccination, and 67% had not reported symptoms resulting from their initial infection. Following one dose of vaccine, their immune responses were comparable to naïve individuals, with significantly weaker responses than individuals who were symptomatic during infection. These results indicate that the absence of symptoms during initial infection and negative serostatus prior to vaccination predict the strength of immune responses to COVID-19 mRNA vaccine. Altogether, these findings highlight the importance of administering the complete two-dose primary regimen and following boosters of mRNA vaccines to individuals who experienced asymptomatic SARS-CoV-2 infection.

Reinfection with SARS-CoV-2 in a patient undergoing chemotherapy for lymphoma: Case report.

BACKGROUND: COVID-19 is usually a time-limited disease. However, prolonged infections and reinfections can occur among immunocompromised patients. It can be difficult to distinguish a prolonged infection from a new one, especially when reinfection occurs early. METHODS: We report the case of a 57-year-old man infected with SARS-CoV-2 while undergoing chemotherapy for follicular lymphoma. He experienced prolonged symptomatic infection for 3 months despite a 5-day course of remdesivir and eventually deteriorated and died. RESULTS: Viral genome sequencing showed that his final deterioration was most likely due to reinfection. Serologic studies confirmed that the patient did not seroconvert. CONCLUSIONS: This case report highlights that reinfection can occur rapidly (62-67 d) among immunocompromised patients after a prolonged disease. We provide substantial proof of prolonged infection through repeated nucleic acid amplification tests and positive viral culture at day 56 of the disease course, and we put forward evidence of reinfection with viral genome sequencing.

HISTORIQUE: La COVID-19 est généralement une maladie limitée dans le temps. Toutefois, des infections et réinfections prolongées peuvent survenir chez des patients immunodéprimés. Il peut être difficile de distinguer une infection prolongée d'une nouvelle infection, particulièrement lorsque la réinfection se produit rapidement. MÉTHODOLOGIE: Les auteurs rendent compte du cas d'un homme de 57 ans infecté par le SRAS-CoV-2 alors qu'il était sous chimiothérapie pour soigner un lymphome folliculaire. Il a souffert d'une infection symptomatique prolongée de trois mois, malgré un traitement de cinq jours au remdésivir. Son état s'est finalement détérioré et il est décédé. RÉSULTATS: Le séquençage du génome viral a démontré que la détérioration finale de son état a probablement été causée par une réinfection. Les études sérologiques ont confirmé qu'il n'avait pas présenté de séroconversion. CONCLUSIONS: Le présent rapport de cas établit la possibilité d'une réinfection rapide (au bout de 62 à 67 jours) chez les patients immunodéprimés après une longue maladie. Les auteurs fournissent des preuves substantielles d'une infection prolongée par des tests répétés d'amplification des acides nucléiques et par des cultures virales positives au 56e jour de l'évolution de la maladie, et ils présentent des preuves de réinfection grâce au séquençage du génome viral.

Evaluation of the performance of multiple immunoassay diagnostic platforms on the National Microbiology Laboratory SARS-CoV-2 National Serology Panel.

BACKGROUND: Serological assays designed to detect SARS-CoV-2 antibodies are being used in serological surveys and other specialized applications. As a result, and to ensure that the outcomes of serological testing meet high quality standards, evaluations are required to assess the performance of these assays and the proficiency of laboratories performing them. METHODS: A panel of 60 plasma/serum samples from blood donors who had reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections and 21 SARS-CoV-2 negative samples were secured and distributed to interested laboratories within Canada (n = 30) and the United States (n = 1). Participating laboratories were asked to provide details on the diagnostic assays used, the platforms the assays were performed on, and the results obtained for each panel sample. Laboratories were blinded with respect to the expected outcomes. RESULTS: The performance of the different assays evaluated was excellent, with the high-throughput platforms of Roche, Ortho, and Siemens demonstrating 100% sensitivity. Most other high-throughput platforms had sensitivities of >93%, with the exception of the IgG assay using the Abbott ARCHITECT which had an average sensitivity of only 87%. The majority of the high-throughput platforms also demonstrated very good specificities (>97%). CONCLUSION: This proficiency study demonstrates that most of the SARS-CoV-2 serological assays utilized by provincial public health or hospital laboratories in Canada have acceptable sensitivity and excellent specificity.

HISTORIQUE: Les dosages sérologiques conçus pour dépister les anticorps anti-SRAS-CoV-2 sont utilisés dans les études sérologiques et d'autres applications spécialisées. Par conséquent, et pour s'assurer que leurs résultats respectent des normes de qualité, il faut procéder à des évaluations de leur performance et de la compétence des laboratoires à les effectuer. MÉTHODOLOGIE: Les chercheurs ont obtenu une batterie de 60 prélèvements de plasma et de sérum chez des donneurs dont l'amplification en chaîne par polymérase après transcription inverse (RT-PCR) avait confirmé des infections par le SRAS-CoV-2 et de 21 prélèvements dont les résultats étaient négatifs au SRAS-CoV-2 et les ont distribués aux laboratoires intéressés du Canada (n = 30) et des États-Unis (n = 1). Ils ont invité les laboratoires participants à fournir de l'information détaillée sur les dosages diagnostiques utilisés, les plateformes sur lesquelles les dosages étaient exécutés et les résultats obtenus pour chaque échantillon. Les chercheurs ont demandé aux laboratoires participants de fournir de l'information détaillée sur les dosages diagnostiques utilisés, les plateformes sur lesquelles les dosages ont été effectués, et les résultats obtenus à l'égard de chaque échantillon. Les laboratoires ont mené les études à l'insu des résultats escomptés. RÉSULTATS: Les divers dosages avaient une excellente exécution, les plateformes à haut débit de Roche, d'Ortho et de Siemens démontrant une sensibilité de 100 %. La plupart des autres plateformes à haut débit avaient des sensibilités de plus de 93 %, à l'exception des dosages des IgG faisant appel à l'analyseur ARCHITECT d'Abbott, dont la sensibilité moyenne était de seulement 87 %. La majorité des plateformes à haut débit avaient également une très bonne spécificité (plus de 97 %). CONCLUSION: La présente étude de compétence démontre que la plupart des dosages sérologiques du SRAS-CoV-2 évalués dans des laboratoires sanitaires provinciaux ou les laboratoires hospitaliers du Canada possèdent une sensibilité acceptable et une excellente spécificité.

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance.

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 µM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.

The REinfection in COVID-19 Estimation of Risk (RECOVER) study: Reinfection and serology dynamics in a cohort of Canadian healthcare workers.

BACKGROUND: Understanding the immune response to natural infection by SARS-CoV-2 is key to pandemic management, especially in the current context of emerging variants. Uncertainty remains regarding the efficacy and duration of natural immunity against reinfection. METHODS: We conducted an observational prospective cohort study in Canadian healthcare workers (HCWs) with a history of PCR-confirmed SARS-CoV-2 infection to (i) measure the average incidence rate of reinfection and (ii) describe the serological immune response to the primary infection. RESULTS: Our cohort comprised 569 HCWs; median duration of individual follow-up was 371 days. We detected six cases of reinfection in absence of vaccination between August 21, 2020, and March 1, 2022, for a reinfection incidence rate of 4.0 per 100 person-years. Median duration of seropositivity was 415 days in symptomatics at primary infection compared with 213 days in asymptomatics (p < 0.0001). Other characteristics associated with prolonged seropositivity for IgG against the spike protein included age over 55 years, obesity, and non-Caucasian ethnicity. CONCLUSIONS: Among unvaccinated healthcare workers, reinfection with SARS-CoV-2 following a primary infection remained rare.

Influenza A(H1N1)pdm09 Virus but Not Respiratory Syncytial Virus Interferes with SARS-CoV-2 Replication during Sequential Infections in Human Nasal Epithelial Cells.

The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.

Seroprevalence of SARS-CoV-2 Antibodies Among Children in School and Day Care in Montreal, Canada.

Importance: Quebec prioritized in-person learning after the first wave of the COVID-19 pandemic, with school closures being implemented temporarily in selected schools or in hot-spot areas. Quebec's decision to keep most schools open was controversial, especially in Montreal, which was the epicenter of Canada's first and second waves; therefore, understanding the extent to which children were infected with SARS-CoV-2 provides important information for decisions about school closures. Objective: To estimate the seroprevalence of SARS-CoV-2 antibodies in children and teenagers in 4 neighborhoods of Montreal, Canada. Design, Setting, and Participants: This cohort study (the Enfants et COVID-19: Étude de séroprévalence [EnCORE] study) enrolled a convenience sample of children aged 2 to 17 years between October 22, 2020, and March 22, 2021, in Montreal, Canada. Exposures: Potential exposure to SARS-CoV-2. Main Outcomes and Measures: The main outcome was seroprevalence of SARS-CoV-2 antibodies, collected using dried blood spots (DBSs) and analyzed with a research-based enzyme-linked immunosorbent assay (ELISA). Parents also completed an online questionnaire that included questions on self-reported COVID-19 symptoms and tests, along with sociodemographic questions. Results: This study included 1632 participants who provided a DBS sample from 30 day cares, 22 primary schools, and 11 secondary schools. The mean (SD) age of the children who provided a DBS sample was 9.0 (4.4) years; 801 (49%) were female individuals, with 354 participants (22%) from day cares, 725 (44%) from primary schools, and 553 (34%) from secondary schools. Most parents had at least a bachelor's degree (1228 [75%]), and 210 (13%) self-identified as being a racial or ethnic minority. The mean seroprevalence was 5.8% (95% CI, 4.6%-7.0%) but increased over time from 3.2% (95% CI, 0.7%-5.8%) in October to November 2020 to 8.4% (95% CI, 4.4%-12.4%) in March to April 2021. Of the 95 children with positive SARS-CoV-2 antibody results, 78 (82%) were not tested or tested negative with reverse transcription-polymerase chain reaction (RT-PCR) testing, and all experienced mild (49 [52%]) or no clinical symptoms (46 [48%]). The children of parents who self-identified as belonging to a racial and ethnic minority group were more likely to be seropositive compared with children of White parents (adjusted seroprevalence ratio, 1.9; 95% CI, 1.1-2.6). Conclusions and Relevance: These results provide a benchmark of the seroprevalence status in Canadian children. The findings suggest that there was more transmission occurring in children compared with what was being detected by RT-PCR, although children experienced few or mild symptoms. It will be important to continue monitoring the serological status of children, particularly in the context of new COVID-19 variants of concern and in the absence of mass vaccination campaigns targeting young children.

Seroprevalence of anti-SARS-CoV-2 antibodies among school and daycare children and personnel: protocol for a cohort study in Montreal, Canada.

INTRODUCTION: Further evidence is needed to understand the contribution of schools and daycares for the spread of COVID-19 in the context of diverse transmission dynamics and continually evolving public health interventions. The Enfants et COVID-19: Étude de séroprévalence (EnCORE) study will estimate the seroprevalence and seroconversion of SARS-CoV-2 among school and daycare children and personnel. In addition, the study will examine associations between seroprevalence and sociodemographic characteristics and reported COVID-19 symptoms and tests, and investigates changes in health, lifestyle and well-being outcomes. METHODS AND ANALYSIS: This study includes children and personnel from 62 schools and daycares in four neighbourhoods in Montreal, Canada. All children aged 2-17 years attending one of the participating schools or daycares and their parents are invited to participate, as well as a sample of personnel members. Participants respond to brief questionnaires and provide blood samples, collected via dried blood spot, at baseline (October 2020-March 2021) and follow-up (May-June 2021). Questionnaires include sociodemographic and household characteristics, reported COVID-19 symptoms and tests, potential COVID-19 risk factors and prevention efforts and health and lifestyle information. Logistic regression using generalised estimating equations will be used to estimate seroprevalence and seroconversion, accounting for school-level clustering. ETHICS AND DISSEMINATION: This study was approved by the research ethics boards of the Université de Montréal (CERSES) and the Centre Hospitalier Universitaire Sainte-Justine. Results will contribute to our knowledge about SARS-CoV-2 transmission in schools and daycares and will be made available to study participants and their families, school and public health decision-makers and the research community.