Persistence of endogenous SARS-CoV-2 and pepper mild mottle virus RNA in wastewater settled solids

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4{degrees}C, 22{degrees}C, and 37 {degrees}C for 10 days. The concentration of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA was measured using an RT-ddPCR. Limited decay (< 1 log10 reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 - 0.098 day-1 for SARS-CoV-2 RNA and 0.010 - 0.091 day-1 for PMMoV RNA, depending on temperature conditions. Slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets. SynopsisSARS-CoV-2 and PMMoV genomic RNA is highly stable in wastewater settled solids over 10 days at several environmentally relevant temperatures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/22268855v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a508forg.highwire.dtl.DTLVardef@19efc23org.highwire.dtl.DTLVardef@bb7748org.highwire.dtl.DTLVardef@194371e_HPS_FORMAT_FIGEXP M_FIG C_FIG

SARS-CoV-2 in wastewater settled solids is associated with COVID-19 cases in a large urban sewershed

Wastewater-based epidemiology (WBE) 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 pre-analytical 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, PMMoV) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlate positively and significantly with COVID-19 clinical 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.