Evaluation of Sampling, Analysis, and Normalization Methods for SARS-CoV-2 Concentrations in Wastewater to Assess COVID-19 Burdens in Wisconsin Communities

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SARS-CoV-2, wastewater, RT-ddPCR, bovine coronavirus, pepper mild mottle virus, HF183, COVID-19 cases, COVID-19


Wastewater surveillance for SARS-CoV-2 provides an approach for assessing the infection burden across a sewer service area. For these data to be useful for public health, measurement variability and the relationship to case data need to be established. We determined SARS-CoV-2 RNA concentrations in the influent of 12 wastewater treatment plants from August 2020 to January 2021. Technical replicates for N1 gene concentrations showed a relative standard deviation of 24%, suggesting it is possible to track relatively small (∼30%) changes in SARS-CoV-2 concentrations over time. COVID-19 cases were correlated significantly (ρ ≥ 0.70) to wastewater SARS-CoV-2 RNA concentrations across large and small service areas, with weaker relationships (ρ ≥ 0.59) in two communities. SARS-CoV-2 concentrations normalized to per capita slightly improved correlations to COVID-19 incidence, but normalizing to a spiked recovery control (BCoV) or a fecal marker (PMMoV or HF183) reduced correlations for a number of plants. Daily sampling demonstrated that a minimum of two samples collected per week were needed to maintain accuracy in trend analysis. The differences in the strength of SARS-CoV-2 relationships to COVID-19 incidence and the effect of normalization on these data among communities demonstrate that rigorous validation should be performed at individual sites where wastewater surveillance programs are implemented.