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

Abstract

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.

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada

“…Given the method of spiking and the relatively short duration between spiking and processing of samples during this study, it was recognized that spike-and-recovery approaches might not adequately represent the recovery of authentic in-situ SARS-CoV-2 that are likely to be transported predominantly via fecal matter particles in wastewater matrices. Indeed, the trace levels of SARS-CoV-2 RNA detected by participating laboratories that included or focused on the analysis of the solids fraction during this study supports other emerging reports of solids-associated behavior exhibited by in-situ SARS-CoV-2 in wastewater samples ( D'Aoust et al., 2020 ; Graham et al., 2020 ; Peccia et al., 2020 ). Phase partitioning of the target virus(es) can therefore influence apparent effectiveness of methods applied in this study.…”

“…While composite wastewater ( Medema et al., 2020a ) or samples of sludge from primary clarifiers ( D'Aoust et al., 2020 ; Graham et al., 2020 ; Peccia et al., 2020 ) collected over the course of a day might provide a better capture of the variation of in situ SARS-CoV-2 concentrations in wastewater, a composite sample was not needed for the purpose of the spike-and-recovery study intended to evaluate inter- and intra- laboratory variability of sample processing and analysis. A common raw wastewater grab sample, post-grit, was obtained from the Winnipeg Wastewater Treatment Plant on August 31, 2020 ( Table 1 ).…”

Catching a resurgence: Increase in SARS-CoV-2 viral RNA identified in wastewater 48 h before COVID-19 clinical tests and 96 h before hospitalizations

“…WBE has been applied at wastewater resource recovery facilities (WRRFs), with early studies demonstrating that viral signal is detected from both influent and primary clarified sludge, with recent studies identifying that at WRRFs, primary clarified sludge may provide more sensitive measurements than in influent ( D'Aoust et al, 2021 ; Graham et al, 2020 ; Peccia et al, 2020 ). Additionally, recent studies outlined that viral particles of SARS-CoV-2 in wastewater could in fact be predominantly solid particle-bound ( Canadian Water Network (CWN), 2020 ; Graham et al, 2020 ) due to the virus' lipid bilayer, which causes it to be lipophilic ( Schoeman and Fielding, 2019 ). Furthermore, it was observed in several studies that SARS-CoV-2 viral signal in wastewater could be utilized as a tool to track epidemiological metric trends ( Ahmed et al, 2020 ; Medema et al, 2020a ; Nemudryi et al, 2020 ; Thompson et al, 2020 ).…”

An insight into neurotoxic and toxicity of spike fragments SARS-CoV-2 by exposure environment: A threat to aquatic health?

“…So far, Sars-Cov-2 has been detected in several countries wastewaters of the American, European, and Asian continents, suggesting as mandatory the monitoring of the secondary transmission of the new coronavirus via wastewater (Liu et al 2020). On a more compelling perspective, strong evidence suggests that surveillance of primarily settled solids in wastewater through one-step ddPCR is a solid strategy to track the spread of Covid-19 disease transmission before the clinical cases break out in a particular location (Graham, 2020).…”