Several treatment plants were sampled for influent, primary clarifier sludge, return activated sludge (RAS), and anaerobically digested sludge throughout nine weeks during the summer of the COVID-19 pandemic. Primary clarifier sludge had a significantly higher number of SARS-CoV-2 gene copy number per liter (GC/L) than other sludge samples, within a range from 1.0 × 10 5 to 1.0 × 10 6 GC/L. Gene copy numbers in raw influent significantly correlated with gene copy numbers in RAS in Silver Creek ( p -value = 0.007, R 2 = 0.681) and East Canyon (p-value = 0.009, R 2 = 0.775) WRFs; both of which lack primary clarifiers or industrial pretreatment processes. This data indicates that SARS-CoV-2 gene copies tend to partition into primary clarifier sludges, at which point a significant portion of them are removed through sedimentation. Furthermore, it was found that East Canyon WRF gene copy numbers in influent were a significant predictor of daily cases ( p -value = 0.0322, R 2 = 0.561), and gene copy numbers in RAS were a significant predictor of weekly cases (p-value = 0.0597, R 2 = 0.449). However, gene copy numbers found in primary sludge samples from other plants significantly predicted the number of COVID-19 cases for the following week ( t = 2.279) and the week after that ( t = 2.122) respectively. These data indicate that SARS-CoV-2 extracted from WRF biosolids may better suit epidemiological monitoring that exhibits a time lag. It also supports the observation that primary sludge removes a significant portion of SARS-CoV-2 marker genes. In its absence, RAS can also be used to predict the number of COVID-19 cases due to direct flow through from influent. This research represents the first of its kind to thoroughly examine SARS-CoV-2 gene copy numbers in biosolids throughout the wastewater treatment process and the relationship between primary, return activated, and anaerobically digested sludge and reported positive COVID-19 cases.

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