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

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.