Scaling of SARS-CoV‑2 RNA in Settled Solids from Multiple Wastewater Treatment Plants to Compare Incidence Rates of Laboratory-Confirmed COVID-19 in Their Sewersheds

Published and unpublished reports show that SARS-CoV-2 RNA in publicly owned treatment work (POTW) wastewater influent and solids is associated with new COVID-19 cases or incidence in associated sewersheds, but methods for comparing data collected from diverse POTWs to infer information about the relative incidence of laboratory-confirmed COVID-19 cases, and scaling to allow such comparisons, have not been previously established. Here, we show that SARS-CoV-2 N1 and N2 concentrations in solids normalized by concentrations of PMMoV RNA in solids can be used to compare incidence of laboratory confirmed new COVID-19 cases across POTWs. Using data collected at seven POTWs along the United States West Coast, Midwest, and East Coast serving ∼3% of the U.S. population (9 million people), we show that a 1 log change in N gene/PMMoV is associated with a 0.24 (range 0.19 to 0.29) log10 change in incidence of laboratory confirmed COVID-19. Scaling of N1 and N2 by PMMoV is consistent, conceptually, with a mass balance model relating SARS-CoV-2 RNA to the number of infected individuals shedding virus in their stool. This information should support the application of wastewater-based epidemiology to inform the response to the COVID-19 pandemic and potentially future viral pandemics.