Adequacy of serial self-performed SARS-CoV-2 rapid antigen-detection testing for longitudinal mass screening in the workplace (preprint)

Importance: Longitudinal mass testing using rapid antigen detection tests (RADT) for serial screening of asymptomatic persons has been proposed for preventing SARS-CoV-2 community transmission. The feasibility of this strategy relies on implementation of accurate self-performed RADT testing where people live, work, or attend school. Objective: To quantify the adequacy of serial self-performed SARS-CoV-2 RADT testing in the workplace, in terms of the frequency of correct execution of procedural steps and accurate interpretation of the range of possible RADT results. We compared results using the instructions provided by the manufacturer to those with modified instructions that were informed by the most frequent or most critical errors we observed. Design: Repeated cross-sectional, diagnostic accuracy study performed prospectively in the field. Setting: Businesses in Montreal, Quebec, Canada, with at least 2 active cases of SARS-CoV-2 infection. Participants: Untrained, asymptomatic persons in their workplace, not meeting Public Health quarantine criteria. Exposures: A Modified Quick Reference Guide compared to the original manufacturer's instructions. Main Outcome(s) and Measure(s): The difference in the proportions of correctly performed procedural steps, and the difference in proportions of correctly interpreted RADT proficiency panel results. The secondary outcome, among subjects with two self-testing visits, compared the second to the first self-test visit using the same measures. Results: Overall, 1892 tests were performed among 647 subjects. For self-test visit 1, significantly better accuracy in test interpretation was observed using the Modified Quick Reference Guide for weak positive (55.6% vs. 12.3%; 43.3 percentage point improvement, 95% confidence interval [CI] 33.0%-53.8%), positive (89.6% vs. 51.5%; 38.1% difference, 95%CI 28.5%-47.5%), strong positive (95.6% vs. 84.0%; 11.6% improvement, 95%CI 6.8%-16.3%) and invalid (87.3% vs. 77.3%; 10.0% improvement, 95%CI 3.8%-16.3%) tests. Use of the modified guide was associated with smaller, statistically significant, improvements on self-test visit 2. For procedural steps identified as critical for the validity of test results, adherence to procedural testing steps did not differ meaningfully according to instructions provided or reader experience. Conclusions and Relevance: Longitudinal mass RADT testing for SARS-CoV-2 can be accurately self-performed in an intended-use setting; this work provides evidence for how to optimise performance.

Specificity of SARS-CoV-2 antibody-detection assays against S and N protein among pre-COVID-19 sera from patients with protozoan and helminth parasitic infections (preprint)

BackgroundWe aimed to assess the specificity of SARS-CoV-2 antibody detection assays among people with known tissue-borne parasitic infections. MethodsWe tested three SARS-CoV-2 antibody-detection assays (cPass SARS-CoV-2 Neutralization Antibody Detection Kit, Abbott SARS-CoV-2 IgG assay, and STANDARD Q COVID-19 IgM/IgG Combo Rapid Test) among 559 pre-COVID-19 sera. ResultsThe specificity of assays was 95-98% overall. However, lower specificity was observed among sera from patients with protozoan infections of the reticuloendothelial system, such as human African trypanosomiasis (Abbott Architect; 88% [95%CI 75-95]), visceral leishmaniasis (SD RDT IgG; 80% [95%CI 30-99]), and from patients with recent malaria from a holoendemic area of Senegal (ranging from 91% for Abbott Architect and SD RDT IgM to 98-99% for cPass and SD RDT IgG). For specimens from patients with evidence of past or present helminth infection overall, test specificity estimates were all [≥] 96%. Sera collected from patients clinically suspected of parasitic infections that tested negative for these infections yielded a specificity of 98-100%. The majority (>85%) of false-positive results were positive by only one assay. ConclusionsThe specificity of SARS-CoV-2 serological assays among sera from patients with tissue-borne parasitic infections was below the threshold required for decisions about individual patient care. Specificity is markedly increased by the use of confirmatory testing with a second assay. Finally, the SD RDT IgG proved similarly specific to laboratory-based assays and provides an option in low-resource settings when detection of anti-SARS-CoV-2 IgG is indicated.

Systematic Testing for SARS-CoV-2 Infection Among Essential Workers in Montreal, Canada: A Prospective Observational and Cost Assessment Study (preprint)

BACKGROUNDEssential workers are at increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We did a prospective study to estimate the yield, acceptability, and costs of workplace-based systematic SARS-CoV-2 testing of asymptomatic essential workers. METHODSWe recruited non-healthcare essential businesses, in Montreal, Canada. Mobile teams, composed of two non-healthcare professionals each, visited businesses. Consenting, asymptomatic employees provided saline gargle specimens under supervision. Mobile team members self-sampled weekly. Specimens were analyzed using reverse-transcription polymerase chain reaction (RT-PCR). If an outbreak was detected ([≥]2 positives), we retested all initially negative participants. We did logistic regression for factors associated with a positive test. We estimated costs ($CAD) of this strategy. RESULTSFrom 27 January to 12 March 2021, 69 essential businesses were visited. Of an estimated 2348 employees onsite, 2128 (90.6%) participated. Across 2626 tests, 53 (2.0%) were positive. Self-reported non-Caucasian ethnicity (aOR 3.7, 95% CI: 1.4-9.9) and a negative SARS-CoV-2 test before the study (0.4, 0.2-0.8) were positively and negatively associated with a positive test, respectively. Five businesses--3 manufacturing/supplier and 2 meat processing-- were experiencing an outbreak. At these businesses, 40 (4.4%) of 917 participants were positive on the initial test. We repeated testing at three of these businesses over 2-3 weeks: 8/350 (2.3%) were positive on the second test, and zero were positive on the third and fourth test (148 tests); no employer reported new positives to 26 March 2021. In all other businesses, 1211 participants were tested once--5 (0.4%) were positive at three childcare enterprises, one grocery store, and one manufacturing/supplier. Per person, RT-PCR costs were $34.00 and all other costs $8.67. No mobile team member tested positive. INTERPRETATIONOnsite sampling of essential workers with saline gargle is safe, acceptable, and inexpensive. Repeat testing appeared to eliminate outbreaks. Systematic testing should be considered part of SARS-CoV-2 preventive efforts.

Evaluation of a Commercial Culture-free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison with an Anti-RBD ELISA Assay (preprint)

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.

Global seroprevalence of SARS-CoV-2 antibodies: a systematic review and meta-analysis (preprint)

BackgroundStudies reporting estimates of the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have rapidly emerged. We aimed to synthesize seroprevalence data to better estimate the burden of SARS-CoV-2 infection, identify high-risk groups, and inform public health decision making. MethodsIn this systematic review and meta-analysis, we searched publication databases, preprint servers, and grey literature sources for seroepidemiological study reports, from January 1, 2020 to August 28, 2020. We included studies that reported a sample size, study date, location, and seroprevalence estimate. Estimates were corrected for imperfect test accuracy with Bayesian measurement error models. We conducted meta-analysis to identify demographic differences in the prevalence of SARS-CoV-2 antibodies, and meta-regression to identify study-level factors associated with seroprevalence. We compared region-specific seroprevalence data to confirmed cumulative incidence. PROSPERO: CRD42020183634. FindingsWe identified 338 seroprevalence studies including 2.3 million participants in 50 countries. Seroprevalence was low in the general population (median 3.2%, IQR 1.0-6.4%) and slightly higher in at-risk populations (median 5.4%, IQR 1.5-18.4%). Median seroprevalence varied by WHO Global Burden of Disease region (p < 0.01), from 1.0% in Southeast Asia, East Asia and Oceania to 18.8% in South Asia. National studies had lower seroprevalence estimates than local (p = 0.02) studies. Compared to White persons, Black persons (prevalence ratio [RR] 2.34, 95% CI 1.60-3.43) and Asian persons (RR 1.56, 95% CI 1.22-2.01) were more likely to be seropositive. Seroprevalence was higher among people ages 18-64 compared to 65 and over (RR 1.26, 95% CI 1.04-1.52). Health care workers had a 1.74x (95% CI: 1.18-2.58) higher risk compared to the general population. There was no difference in seroprevalence between sexes. There were 123 studies (36%) at low or moderate risk of bias. Seroprevalence estimates from national studies were median 11.9 (IQR 8.0 - 16.6) times higher than the corresponding SARS-CoV-2 cumulative incidence. InterpretationMost of the population remains susceptible to SARS-CoV-2 infection. Public health measures must be improved to protect disproportionately affected groups, including non-White people and adults. Measures taken in SE Asia, E Asia and Oceania, and Latin America and Caribbean may have been more effective in controlling virus transmission than measures taken in other regions. FundingPublic Health Agency of Canada through the COVID-19 Immunity Task Force.

Sequential ER stress and inflammatory responses are induced by SARS-CoV-2 ORF3 through ERphagy (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have resulted in a number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, diseases and therapy remains limited, underlining the urgency of fundamental studies and drug development. Studies have shown that induction of autophagy and hijacking of autophagic machinery are essential for infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3 as an inducer of autophagy and revealed that ORF3 localizes to the ER and induces FAM134B-related ERphagy through the HMGB1-Beclin1 pathway. As a consequence, ORF3 induces ER stress and inflammatory responses through ERphagy and sensitizes cells to ER stress-induced cell death, suggesting that SARS-CoV-2 ORF3 hijacks ERphagy and then harms ER homeostasis to induce inflammatory responses through excessive ER stress. These findings reveal a sequential induction of ERphagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and provide therapeutic potential for ERphagy and ER stress-related drugs for COVID-19 treatment and prevention. ImportanceSARS-CoV-2 infection and replication require autophagosome-like double-membrane vacuoles. Inhibition of autophagy suppresses viral replication, indicating the essential role of autophagy in SARS-CoV-2 infection. However, how SARS-CoV-2 hijacks autophagy and the function of autophagy in the disease progression remain unknown. Here, we reveal that SARS-CoV-2 ORF3 induces ERphagy and consequently induces ER stress to trigger acute inflammatory responses and enhance sensitivity to ER stress-induced apoptosis. Our studies uncover ERphagy-induced inflammatory responses during SARS-CoV-2 infection and provide a promising therapeutic approach for treating SARS-CoV-2 infection and inflammatory responses in COVID-19 by manipulating autophagy and ER stress.

Single-cell Transcriptome of Bronchoalveolar Lavage Fluid Reveals Dynamic Change of Macrophages During SARS-CoV-2 Infection in Ferrets (preprint)

Although the profile of immune cells changes during the natural course of SARS-CoV-2 inflection in human patients, few studies have used a longitudinal approach to reveal their dynamic features. Here, we performed single-cell RNA sequencing of bronchoalveolar lavage fluid cells longitudinally obtained from SARS-CoV-2-infected ferrets. Landscape analysis of the lung immune microenvironment showed dynamic changes in cell proportions and characteristics in uninfected control, at 2 days post-infection (dpi) (early stage of SARS-CoV-2 infection with peak viral titer), and 5 dpi (resolution phase). NK cells and CD8+ T cells exhibited activated subclusters with interferon-stimulated features, which were peaked at 2 dpi. Intriguingly, macrophages were classified into 10 distinct subpopulations, and their relative proportions changed over the time. We observed prominent transcriptome changes among monocyte-derived infiltrating macrophages and differentiated M1/M2 macrophages, especially at 2 dpi. Moreover, trajectory analysis revealed gene expression changes from monocyte-derived infiltrating macrophages toward M1 or M2 macrophages and identified the distinct macrophage subpopulation that had rapidly undergone SARS-CoV-2-mediated activation of inflammatory responses. Finally, we found that different spectrums of M1 or M2 macrophages showed distinct patterns of gene modules downregulated by immune-modulatory drugs. Overall, these results elucidate fundamental aspects of the immune response dynamics provoked by SARS-CoV-2 infection.

Generation and Characterization of recombinant SARS-CoV-2 expressing reporter genes (preprint)

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab), but no vaccines, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. ImportanceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

Replicating bacterium-vectored vaccine expressing SARS-CoV-2 Membrane and Nucleocapsid proteins protects against severe COVID-19 disease in hamsters (preprint)

An inexpensive readily manufactured COVID-19 vaccine that protects against severe disease is needed to combat the pandemic. We have employed the LVS {Delta}capB vector platform, previously used successfully to generate potent vaccines against the Select Agents of tularemia, anthrax, plague, and melioidosis, to generate a COVID-19 vaccine. The LVS {Delta}capB vector, a replicating intracellular bacterium, is a highly attenuated derivative of a tularemia vaccine (LVS) previously administered to millions of people. We generated vaccines expressing SARS-CoV-2 structural proteins and evaluated them for efficacy in the golden Syrian hamster, which develops severe COVID-19 disease. Hamsters immunized intradermally or intranasally with a vaccine co-expressing the Membrane (M) and Nucleocapsid (N) proteins, then challenged 5-weeks later with a high dose of SARS-CoV-2, were protected against severe weight loss and lung pathology and had reduced viral loads in the oropharynx and lungs. Protection by the vaccine, which induces murine N-specific interferon-gamma secreting T cells, was highly correlated with pre-challenge serum anti-N TH1-biased IgG. This potent vaccine against severe COVID-19 should be safe and easily manufactured, stored, and distributed, and given the high homology between MN proteins of SARS-CoV and SARS-CoV-2, has potential as a universal vaccine against the SARS subset of pandemic causing {beta}-coronaviruses.