ONE ASSAY TO TEST THEM ALL: COMPARING MULTIPLEX ASSAYS FOR EXPANSION OF RESPIRATORY VIRUS SURVEILLANCE (preprint)

Background: Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Methods: Clinical performance of XpertXpress SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPathTM COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChekTM SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. Results: The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1-2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97% and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. Conclusion: TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhancediagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections.

Robust and Functional Immunity up to 9 months after SARS-CoV-2 infection: a Southeast Asian longitudinal cohort (preprint)

Assessing the duration of humoral and cellular immunity remains key to overcome the current SARS-CoV-2 pandemic, especially in understudied populations in least developed countries. Sixty-four Cambodian individuals with laboratory-confirmed infection with asymptomatic or mild/moderate clinical presentation were evaluated for humoral immune response to the viral spike protein and antibody effector functions during acute phase of infection and at 6-9 months follow-up. Antigen-specific B cells, CD4+ and CD8+ T cells were characterized, and T cells were interrogated for functionality at late convalescence. Anti-spike (S) antibody titers decreased over time, but effector functions mediated by S-specific antibodies remained stable. S- and nucleocapsid (N)-specific B cells could be detected in late convalescence in the activated memory B cell compartment and are mostly IgG+. CD4+ and CD8+ T cell immunity was maintained to S and membrane (M) protein. Asymptomatic infection resulted in decreased ADCC and frequency of SARS-CoV-2-specific CD4+ T cells at late convalescence. Whereas anti-S antibodies correlated with S-specific B cells, there was no correlation between T cell response and humoral immunity. Hence, all aspects of a protective immune response are maintained up to nine months after SARS-CoV-2 infection in the absence of re-infection. One sentence summaryFunctional immune memory to SARS-CoV-2, consisting of polyfunctional antibodies, memory B cells and memory T cells are maintained up to nine months in a South-East Asian cohort in the absence of re-infection.

Serological reconstruction of COVID-19 epidemics through analysis of antibody kinetics to SARS-CoV-2 proteins (preprint)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a complex antibody response that varies by orders of magnitude between individuals and over time. Waning antibody levels lead to reduced sensitivity of serological diagnostic tests over time. This undermines the utility of serological surveillance as the SARS-CoV-2 pandemic progresses into its second year. Here we develop a multiplex serological test for measuring antibodies of three isotypes (IgG, IgM, IgA) to five SARS-CoV-2 antigens (Spike (S), receptor binding domain (RBD), Nucleocapsid (N), Spike subunit 2, Membrane-Envelope fusion) and the Spike proteins of four seasonal coronaviruses. We measure antibody responses in several cohorts of French and Irish hospitalized patients and healthcare workers followed for up to eleven months after symptom onset. The data are analysed with a mathematical model of antibody kinetics to quantify the duration of antibody responses accounting for inter-individual variation. One year after symptoms, we estimate that 36% (95% range: 11%, 94%) of anti-S IgG remains, 31% (9%, 89%) anti-RBD IgG remains, and 7% (1%, 31%) anti-N IgG remains. Antibodies of the IgM isotype waned more rapidly, with 9% (2%, 32%) anti-RBD IgM remaining after one year. Antibodies of the IgA isotype also waned rapidly, with 10% (3%, 38%) anti-RBD IgA remaining after one year. Quantitative measurements of antibody responses were used to train machine learning algorithms for classification of previous infection and estimation of time since infection. The resulting diagnostic test classified previous infections with 99% specificity and 98% (95% confidence interval: 94%, 99%) sensitivity, with no evidence for declining sensitivity over the time scale considered. The diagnostic test also provided accurate classification of time since infection into intervals of 0 - 3 months, 3 - 6 months, and 6 - 12 months. Finally, we present a computational method for serological reconstruction of past SARS-CoV-2 transmission using the data from this test when applied to samples from a single cross-sectional sero-prevalence survey.

Assessment of Inactivation Procedures for SARS-CoV-2 (preprint)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), presents a challenge to laboratorians and healthcare workers around the world. Handling of biological samples from individuals infected with the SARS-CoV-2 virus requires strict biosafety and biosecurity measures. Within the laboratory, non-propagative work with samples containing the virus requires, at minimum, Biosafety Level-2 (BSL-2) techniques and facilities. Therefore, handling of SARS-CoV-2 samples remains a major concern in areas and conditions where biosafety and biosecurity for specimen handling is difficult to maintain, such as in rural laboratories or austere field testing sites. Inactivation through physical or chemical means can reduce the risk of handling live virus and increase testing ability worldwide. Herein we assess several chemical and physical inactivation techniques employed against SARS-CoV-2 isolates from Cambodian COVID-19 patients. This data demonstrates that all chemical (AVL, inactivating sample buffer and formaldehyde) and heat treatment (56{degrees}C and 98{degrees}C) methods tested completely inactivated viral loads of up to 5 log10.