ABSTRACT
Wastewater-based epidemiology (WBE) has been widely deployed during the COVID-19 pandemic, but with limited evaluation of the utility of discrete sampling for large sewersheds and low COVID-19 incidence. In this study, SARS-CoV-2 RNA was measured in 72 consecutive hourly influent grab samples collected at a wastewater treatment plant serving nearly 500000 residents when incidence was low (approximately 20 cases per 100000). We characterized diurnal variability and relationships between SARS-CoV-2 RNA detection and physicochemical covariates [flow rate, total ammonia nitrogen (TAN), and total solids (TS)]. The highest detection rate observed was 82% during the first peak flow, which occurred in the early afternoon (14:00). Higher detection rates were also observed when sampling above median TAN concentrations (71%;p < 0.01;median = 40.26 mg of NH4/L). SARS-CoV-2 RNA concentrations were weakly correlated with flow rate (Kendall's τ = 0.16;p < 0.01), TAN (τ = 0.19;p < 0.05), and TS (τ = 0.18;p < 0.01), suggesting generally low RNA sewer discharges as expected at low incidence. Our results elucidated sensible adjustments to maximize detection rates, including using multiple gene targets, collecting duplicate samples, and sampling during higher flow and TAN discharges. Optimizing the lower-incidence bounds of WBE can help assess its suitability for verifying COVID-19 reemergence or eradication. ©