Evaluation of sampling frequency and normalization of SARS-CoV-2 wastewater concentrations for capturing COVID-19 burdens in the community

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Wastewater surveillance for SARS-CoV-2 provides an approach for assessing the infection burden across a city. For these data to be useful for public health, measurement variability and the relationship to case data need to be established. We measured SARS-CoV-2 RNA concentrations in the influent of twelve wastewater treatment plants from August 2020 to January 2021. Replicate samples demonstrated that N1 gene target concentrations varied by ±21% between technical replicate filters and by ±14% between duplicate assays. COVID-19 cases were correlated significantly (rho≥0.70) to wastewater SARS-CoV-2 RNA concentrations for seven plants, including large and small cities. SARS-CoV-2 data normalized to flow improved correlations to reported COVID-19 cases for some plants, but normalizing to a spiked recovery control (BCoV) or a fecal marker (PMMoV or HF183) generally reduced correlations. High frequency sampling demonstrated that a minimum of two samples collected per week was needed to maintain accuracy in trend analysis. We found a significantly different ratio of COVID-19 cases to SARS-CoV-2 loads in one of three large communities, suggesting a higher rate of undiagnosed cases. These data demonstrate that SARS-CoV-2 wastewater surveillance can provide a useful community-wide metric to assess the course of the COVID-19 pandemic.