SARS-CoV‑2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed

Wastewater-based epidemiology may
be useful for informing public
health response to viral diseases like COVID-19 caused by SARS-CoV-2.
We quantified SARS-CoV-2 RNA in wastewater influent and primary settled
solids in two wastewater treatment plants to inform the preanalytical
and analytical approaches and to assess whether influent or solids
harbored more viral targets. The primary settled solids samples resulted
in higher SARS-CoV-2 detection frequencies than the corresponding
influent samples. Likewise, SARS-CoV-2 RNA was more readily detected
in solids using one-step digital droplet (dd)­RT-PCR than with two-step
RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition
with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal
time series of 89 settled solids samples from a single plant for SARS-CoV-2
RNA as well as coronavirus recovery (bovine coronavirus) and fecal
strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets
N1 and N2 concentrations correlated positively and significantly with
COVID-19 clinically confirmed case counts in the sewershed. Together,
the results demonstrate that measuring SARS-CoV-2 RNA concentrations
in settled solids may be a more sensitive approach than measuring
SARS-CoV-2 in influent.