Wastewater-based surveillance of COVID-19 and removal of SARS-CoV-2 RNA across a major wastewater treatment plant in San Antonio, Texas

Wastewater-based epidemiology (WBE) targeting SARS-CoV-2 RNA detection in municipal wastewater is considered a valuable tool for COVID-19 surveillance in a community. However, the persistence and removal of SARS-CoV-2 RNA in wastewater treatment plants (WWTPs) have not been well investigated. This study is aimed at detecting SARS-CoV-2 RNA in wastewater to correlate viral concentrations with clinical COVID-19 cases in the sewershed and determine whether the SARS-CoV-2 genetic material is detectable after treatment. Raw influent, primary effluent (after primary clarification), secondary effluent (after activated sludge treatment), and final effluent (after chlorination) samples were collected two times a week from the largest WWTP in San Antonio (Texas) during April to November 2021 and analyzed for SARS-CoV-2 RNA (N1 and N2 genes) concentrations using the reverse transcription droplet digital polymerase chain reaction (RT-ddPCR). SARS-CoV-2 RNA was detected in 98.5% (n = 34 weeks) of the raw influent samples and anticipated the trends of the COVID-19 outbreak. Furthermore, a higher correlation between viral concentrations and COVID-19 cases was observed for two days a week sampling frequency (rho = 0.75, p <0.001) than one day per week (rho = 0.60, p <0.001). Despite the high SARS-CoV-2 RNA concentrations in raw sewage, a significant amount of viral RNA was removed at primary and secondary clarifiers (removal efficiencies were 54% and 94%, respectively) and was undetectable in final effluents. These results demonstrate the performance of the WWTP in reducing the SARS-CoV-2 RNA concentration and further highlight the role of tertiary treatment and chlorination in eliminating SARS-CoV-2 RNA in receiving waters.