Diurnal Variability of SARS-CoV-2 RNA Concentrations in Hourly Grab Samples of Wastewater Influent during Low COVID-19 Incidence.

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 500 000 residents when incidence was low (approximately 20 cases per 100 000). 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.

SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.