Persistence of Endogenous SARS-CoV-2 and Pepper Mild Mottle Virus RNA in Wastewater-Settled Solids

Abstract

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, 22, and 37 °C for 10 days. The concentrations of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA were measured using an RT-ddPCR. Limited decay (<1 log 10 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 to 0.098 day –1 for SARS-CoV-2 RNA and from 0.010 to 0.091 day –1 for PMMoV RNA depending on the temperature conditions. A 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.

Effect of Time and Temperature on SARS-CoV-2 in Municipal Wastewater Conveyance Systems

“…Temperature had the strongest effect on SARS-CoV-2 viral concentration (Kendall's tau, p < 0.05), significantly reducing observed concentration exposed to warmer conditions in JI (paired t-test, p = 0.02), and SS (paired t-test, p = 0.046). Temperature posing a significant effect on SARS-CoV-2 decay is consistent with other studies that indicate coronaviruses, and specifically SARS-CoV-2, are sensitive to warmer temperatures [15,[36][37][38][39] where decay rates for similar temperature windows ranged between 0.021-2.16 k/day [15,20,21,39]. Increasing temperatures reduce RNA stability, and make it more difficult for any virus to survive [40].…”

“…Specific parameters in the sewer collection system that have a known influence on viral RNA and are assumed to have an impact on SARS-CoV-2 detection in wastewater, include pH, temperature, organic matter, solids content, residence time in the sewer, sampling, and microbial antagonism [15][16][17]. While various studies have performed analysis on the decay of SARS-CoV-2 RNA in wastewater influent in a laboratory setting, they are often performed with surrogate enveloped virus spikes [15,[18][19][20][21][22] which have been shown to behave differently than authentic in situ SARS-CoV-2 found within the wastewater matrix [23,24]. A major barrier to performing in situ studies and comparing real wastewater systems is that the true input of SARS-CoV-2 virus from the population is unknown and likely different for individuals depending on the severity of disease, stage of infection, and other factors [25].…”

Wastewater monitoring of SARS-CoV-2 RNA at K-12 schools: comparison to pooled clinical testing data

“…Once at the laboratory, the sample was stored at 4 °C and analyzed within a week. Previous work indicated minimal degradation of viral RNA RT-PCR targets during this time frame ( Roldan-Hernandez et al, 2022 ; Yang et al, 2022 ). The autosamplers malfunctioned twice and no sample was available at School A on April 19 and at School B on April 22.…”

An efficient method to enhance recovery and detection of SARS-CoV-2 RNA in wastewater

“…Several studies have demonstrated that SARS-CoV-2 particles could be detected in 2-3 magnitude higher concentrations in solids and sludge of some wastewater samples than in the aqueous phase ( Peccia et al, 2020 ; D'Aoust et al 2021 ; Graham et al, 2021 ; Carillo-Reyes et al 2021 ; Westhaus et al 2021 ; Buonerba et al 2021 ). Roldan-Hernandez et al (2022) have shown persistence of SARS-CoV-2 in solids with a slower rate of decay than in the wastewater influent, further suggesting the importance of including the solid phase which can adsorb the viral particles and RNA. Our results suggest the need for a simple and efficient procedure for concentration of viral particles and RNA from the entire sample.…”