Twelve Distinct Laboratory Methods Used to Measure SARS-CoV-2 in Wastewaters throughout a Three-Year Ontario-Wide, Canada Study: Impact on Public Health Interpretation of Disease Incidence

Abstract Wastewater and environmental monitoring (WEM) was a critical public health surveillance tool for SARS-CoV-2 surveillance during the COVID-19 Pandemic. However, substantial methodological heterogeneity across laboratories continues to challenge the interpretation and thus compromise the actionability of resulting WEM measurements. This study quantifies interlaboratory concordance in SARS-CoV-2 WEM measurements using influent wastewater samples collected between September 2021 and January 2024 at a single wastewater treatment facility within the Ontario Wastewater Surveillance Initiative, analyzed independently by 12 laboratories using their routine methods. In the absence of a known true viral concentration, interlaboratory WEM measurements were evaluated against a facility-specific longitudinal benchmark derived from routine surveillance at the source facility and correlated to clinical surveillance metrics. Concordance was assessed across four WEM measurement units commonly used in practice: SARS-CoV-2 copies/mL, SARS-CoV-2 copies/copies of PMMoV, and their standardized counterpart wastewater viral activity level (WVAL) units of WVAL-standardized SARS-CoV-2 copies/mL and WVAL-standardized SARS-CoV-2 copies/copies of PMMoV. Measurements in each unit were analyzed using complementary analytical frameworks, including categorical concordance metrics, principal component analysis, and linear mixed-effects modelling. Across the study period, interlaboratory measurements consistently captured benchmark temporal dynamics, including major peaks and periods of low activity, but showed substantial variation in magnitude and public-health interpretation across laboratory methods. Concordance was strongest during epidemiological extremes and deteriorated during transitional periods, increasing the risk of misclassification with potentially implications for public health decision-making. To explore potential laboratory methodological drivers of agreement, associations between the benchmark concordance and the laboratory-specific concentration, extraction, and RT-qPCR analytical steps were assessed using Fisher’s exact tests, alongside extracted-mass threshold analyses. No single methodological factor showed a statistically significant association with benchmark concordance in this study; however, several parameters, including RNA template volume, total RT-qPCR reaction volume, and extracted mass of analyzed settled solids, may warrant further investigation in future studies.