Development and use of a method based on the anti-N reactivity of longitudinal samples to better estimate SARS-CoV-2 seroprevalence in a vaccinated population

Background Emerging evidence suggests that COVID-19 vaccination decreases the sensitivity of anti-nucleocapsid (N) serologies, making them less reliable to assess recently-acquired infections. We therefore developed and tested a new approach based on the ratio of the anti-N absorbance of longitudinal samples to overcome this limitation. Methods Previously vaccinated repeat plasma donors provided at least one pre-infection (reference) and one post-infection (test) sample. All samples were tested using an in-house anti-N ELISA. Seropositivity was determined based on the ratio between the anti-N absorbance of the test and reference samples. The ratio approach was tested in a real-world setting during three cross-sectional serosurveys carried out among plasma donors in Québec, Canada. Results Using a cut-off ratio of 1.5, the approach had a sensitivity of 95.2% among the 248 previously vaccinated and infected donors compared with 63.3% for the conventional approach. When tested in a real-world setting, the ratio-based approach yielded an adjusted seroprevalence of 27.4% (95% confidence interval [CI]=23.8%-30.9%) at the latest time point considered, compared to 15.1% (95% CI=12.2%-18.0%) for the conventional approach. Conclusions This article describes a new and highly-sensitive approach that captures a significantly greater proportion of vaccinated individuals with a recent history of SARS-CoV-2 infection. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Ministère de la Santé et des Services Sociaux (MSSS) du Québec and the Public Health Agency of Canada (through the Reference Group on the Surveillance of vaccines and the COVID-19 Immunity Task Force). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The project was approved by the Hema-Quebec Research Ethics Board (REB # B6-2021-003 and # 2021-020). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript.