key: cord-0919063-dh5n9340
authors: Tang, Xuyang; Sharma, Abha; Pasic, Maria; Brown, Patrick; Colwill, Karen; Gelband, Hellen; Birnboim, H. Chaim; Nagelkerke, Nico; Bogoch, Isaac I.; Bansal, Aiyush; Newcombe, Leslie; Slater, Justin; Rodriguez, Peter S.; Huang, Guowen; Fu, Sze Hang; Meh, Catherine; Wu, Daphne C.; Kaul, Rupert; Langlois, Marc-André; Morawski, Ed; Hollander, Andy; Eliopoulos, Demetre; Aloi, Benjamin; Lam, Teresa; Abe, Kento T.; Rathod, Bhavisha; Fazel-Zarandi, Mahya; Wang, Jenny; Iskilova, Mariam; Pasculescu, Adrian; Caldwell, Lauren; Barrios-Rodiles, Miriam; Mohammed-Ali, Zahraa; Vas, Nandita; Santhanam, Divya Raman; Cho, Eo Rin; Qu, Kathleen; Jha, Shreya; Jha, Vedika; Suraweera, Wilson; Malhotra, Varsha; Mastali, Kathy; Wen, Richard; Sinha, Samir; Reid, Angus; Gingras, Anne-Claude; Chakraborty, Pranesh; Slutsky, Arthur S.; Jha, Prabhat
title: Assessment of SARS-CoV-2 Seropositivity During the First and Second Viral Waves in 2020 and 2021 Among Canadian Adults
date: 2022-02-16
journal: JAMA Netw Open
DOI: 10.1001/jamanetworkopen.2021.46798
sha: 171b7837ea38d2d2b95a4a51c2a9f7245664b151
doc_id: 919063
cord_uid: dh5n9340

IMPORTANCE: The incidence of infection during SARS-CoV-2 viral waves, the factors associated with infection, and the durability of antibody responses to infection among Canadian adults remain undocumented. OBJECTIVE: To assess the cumulative incidence of SARS-CoV-2 infection during the first 2 viral waves in Canada by measuring seropositivity among adults. DESIGN, SETTING, AND PARTICIPANTS: The Action to Beat Coronavirus study conducted 2 rounds of an online survey about COVID-19 experience and analyzed immunoglobulin G levels based on participant-collected dried blood spots (DBS) to assess the cumulative incidence of SARS-CoV-2 infection during the first and second viral waves in Canada. A sample of 19 994 Canadian adults (aged ≥18 years) was recruited from established members of the Angus Reid Forum, a public polling organization. The study comprised 2 phases (phase 1 from May 1 to September 30, 2020, and phase 2 from December 1, 2020, to March 31, 2021) that generally corresponded to the first (April 1 to July 31, 2020) and second (October 1, 2020, to March 1, 2021) viral waves. MAIN OUTCOMES AND MEASURES: SARS-CoV-2 immunoglobulin G seropositivity (using a chemiluminescence assay) by major geographic and demographic variables and correlation with COVID-19 symptom reporting. RESULTS: Among 19 994 adults who completed the online questionnaire in phase 1, the mean (SD) age was 50.9 (15.4) years, and 10 522 participants (51.9%) were female; 2948 participants (14.5%) had self-identified racial and ethnic minority group status, and 1578 participants (8.2%) were self-identified Indigenous Canadians. Among participants in phase 1, 8967 had DBS testing. In phase 2, 14 621 adults completed online questionnaires, and 7102 of those had DBS testing. Of 19 994 adults who completed the online survey in phase 1, fewer had an educational level of some college or less (4747 individuals [33.1%]) compared with the general population in Canada (45.0%). Survey respondents were otherwise representative of the general population, including in prevalence of known risk factors associated with SARS-CoV-2 infection. The cumulative incidence of SARS-CoV-2 infection among unvaccinated adults increased from 1.9% in phase 1 to 6.5% in phase 2. The seropositivity pattern was demographically and geographically heterogeneous during phase 1 but more homogeneous by phase 2 (with a cumulative incidence ranging from 6.4% to 7.0% in most regions). The exception was the Atlantic region, in which cumulative incidence reached only 3.3% (odds ratio [OR] vs Ontario, 0.46; 95% CI, 0.21-1.02). A total of 47 of 188 adults (25.3%) reporting COVID-19 symptoms during phase 2 were seropositive, and the OR of seropositivity for COVID-19 symptoms was 6.15 (95% CI, 2.02-18.69). In phase 2, 94 of 444 seropositive adults (22.2%) reported having no symptoms. Of 134 seropositive adults in phase 1 who were retested in phase 2, 111 individuals (81.8%) remained seropositive. Participants who had a history of diabetes (OR, 0.58; 95% CI, 0.38-0.90) had lower odds of having detectable antibodies in phase 2. CONCLUSIONS AND RELEVANCE: The Action to Beat Coronavirus study found that the incidence of SARS-CoV-2 infection in Canada was modest until March 2021, and this incidence was lower than the levels of population immunity required to substantially reduce transmission of the virus. Ongoing vaccination efforts remain central to reducing viral transmission and mortality. Assessment of future infection-induced and vaccine-induced immunity is practicable through the use of serial online surveys and participant-collected DBS.

Given the concerns about rising COVID cases during a second wave, we conducted a spatial analysis of PCR-confirmed COVID cases in the 93 public health units in Canada, using data up to July 2020. A Poisson spatial regression had PCR-confirmed case counts as the response variable, an age-sex adjusted expected count as an 'offset', and predictor variables of lung cancer incidence (as a proxy for smoking exposure, a risk factor for severe COVID), unemployment, and proportion of visible minorities. A spatial random effect term, where each unit has conditional dependence with each of its neighbors, was added to account for spatial clustering. This analysis identified 17 health units at notably higher risk of COVID incidence (eFigure 1), which had 28.5% visible minorities, twice the 14.5% rate of visible minorities for the whole of Canada.

For Phase I, antibodies were eluted from a 4.7 mm punch in 77 µL of PBS + 0.1% Tween (PBS-T) and 1% Triton X-100. In Phase 2, to ensure sufficient eluate to test three antigens, antibodies were eluted in 99 µL. Punches were incubated in elution buffer for 4 hours with gentle shaking (150 RPM) at room temperature or overnight at 4 o C. The samples were then centrifuged at 1000 g for 30 seconds.

Automated chemiluminescent ELISA assays were performed as previously described on a ThermoFisher Scientific F7 robotic platform 1 with a few modifications. Briefly, LUMITRAC 600 high-binding white polystyrene 384-well microplates (Greiner Bio-One #781074, VWR #82051-268) were pre-coated overnight with 10 µL /well of antigen (50 ng spike (SmT1), 20 ng RBD and 7 ng nucleocapsid, all supplied by the National Research Council of Canada (NRC)). After washing (all washes were 4 times with 100 µL PBS-T), wells were blocked for 1 hour in 80 µL 5% Blocker BLOTTO (ThermoFisher Scientific, #37530) and then washed. 10 µL of sample (2.5 µL of DBS eluate diluted in 1% final Blocker BLOTTO in PBS-T) was added to each well and incubated for 2 hours at room temperature. After washing, 10 µL of a human anti-IgG fused to HRP (IgG#5, supplied by NRC, final of 0.9 ng/well) diluted in 1% final Blocker BLOTTO in PBS-T was added to each well followed by a 1-hour incubation at room temperature. After 4 washes, 10 µL of SuperSignal ELISA pico chemiluminescent substrate (diluted 1:4 in MilliQ distilled H20) was added to each well and incubated for 5-8 min at room temperature. Chemiluminescence was read on an EnVision (Perkin Elmer) plate reader at 100 ms/well using an ultra-sensitive detector.

Each 384-well assay plate included replicates of a standard reference curve of a human anti-spike IgG antibody (VHH72 supplied by NRC) 2 or an anti-nucleocapsid IgG antibody (Genscript, #A02039), positive and negative master mixes of pooled serum samples, human IgG negative control (Sigma, #I4506), and blanks as controls. Negative and/or positive DBS controls (defined using plasma serology results) were included in 4 runs in phase 1 and in 9 runs in phase 2.

For Phase 1, blank values were subtracted from all raw reads (counts per second) and the values were expressed as a ratio of the 0.0156 µg/mL point of the standard curve of VHH72. The stricter threshold for positives (0.39) was determined by calculating the mean plus 3 standard deviations of negative control DBS samples (n=5 samples tested 12 times in total, seronegativity defined by plasma serology -pilot studies with samples acquired locally or through the NML established that eluents from engineered blood spots yielded results highly correlated to those of liquid plasma; data not shown and preprint 3 ). The lenient threshold for samples (0.27) was calculated as >3SD from the presumed negative distribution of the Ab-C samples. Samples with a relative ratio of > 0.05 and < 0.3 were selected as presumed negatives, and the mean plus 3 standard deviations of these samples was used for the cut-off. Data were analyzed and plots were generated in R Version 4.0. 1. 4 For Phase 2, raw sample reads (counts per second) were expressed as a ratio to a blank-adjusted midpoint of the relevant reference curve (0.0156 µg/mL point for VHH72 and 0.0625 µg/mL for anti-nucleocapsid). A panel of 187 DBS samples supplied by the National Microbiology Lab of Canada (90 negative and 97 positive samples tested in duplicate) was used to establish a stricter cut off corresponding to a 1% false positive rate using receiver operating characteristic (ROC) curve analysis for each of the three antigens. 5 At these cut offs, the sensitivity values are 0.98 for spike and RBD and 0.92 for nucleocapsid. The lenient threshold for samples for the spike antigen was calculated using 3SD from the mean of the log distribution of the presumed negative distribution of the Ab-C samples in phase 1. Density distributions of the log of the relative ratios were separated using the mixtool function normalmixEM of the mixtools R package based on expectation minimization of only two subpopulations assumed to have a Gaussian distribution. normalmixEM initialization `mean` and the `standard deviation` parameters were set based on the visual estimation from the density distribution of the log of the relative ratio. The initially assumed proportion between the two subpopulations was set to 50%. Density distributions were plotted using default R parameters (bandwidth: bw.nrd0 that "implements a rule-of-thumb for choosing the bandwidth of a Gaussian kernel density estimator"). eFigure 3 shows the reproducibility of the standard dilution curves of VHH72 (in µg/mL) across the different runs (run 1 to run 6) in Phase 1. The reproducibility in signal (blank-adjusted counts per second) between runs in the linear range and in the more dilute concentrations is high. More variation in signal is observed for the less dilute concentrations outside of the linear range. The red dotted line for the spike antigen is the 3SD cut off from mean of presumed negatives from Phase 1. The 1% FPR and presumed negative cutoff for phase 2 were more similar for RBD and NP than for spike. Hence, for spike we opted to use a lenient cut off (phase 1 0.34 cut off) to capture more positives. 

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in patients with COVID-19

A simple protein-based surrogate neutralization assay for SARS-CoV-2

Dried blood spot specimens for SARS-CoV-2 antibody testing: A multi-site, multi-assay comparison

RStudio: Integrated Development for R. Version 1.3.959. RStudio Team

Made-in-Canada" serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination. medRxiv. Preprint posted online