Highlights RT-ddPCR is more sensitive to inhibitors than RT-qPCR for primary clarified sludge Primary clarified sludge has elevated frequency of SARS-CoV-2 RNA detection Primary clarified sludge allows detection of RNA during low COVID-19 incidence PMMV normalization of RNA data reduces noise and increases precision PMMV normalization of RNA shows strongest correlation to epidemiological metrics

In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care units, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through measuring trends of viral RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 genes are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canada's national capital region, i.e., the City of Ottawa, ON (pop. = 1.1M) and the City of Gatineau, QC (pop. = 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentrations of RNA using both assays. RT-qPCR shows higher frequency of detection of N1 and N2 genes in PCS (92.7, 90.6%) as compared to PGS samples (79.2, 82.3%). Sampling of PCS may therefore be an effective approach for SARS-CoV-2 viral quantification, especially during periods of declining and low COVID-19 incidence in the community. The pepper mild mottle virus (PMMV) is determined to have a less variable RNA signal in PCS over a three month period for two WRRFs, regardless of environmental conditions, compared to Bacteroides 16S rRNA or human eukaryotic 18S rRNA, making PMMV a potentially useful biomarker for normalization of SARS-CoV-2 signal. PMMV-normalized PCS RNA signal from WRRFs of two cities correlated with the regional public health epidemiological metrics, identifying PCS normalized to a fecal indicator (PMMV) as a potentially effective tool for monitoring trends during decreasing and low-incidence of infection of SARS-Cov-2 in communities.

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Curtailing the Spring 2020 COVID-19 surge required sweeping and stringent interventions by governments across the world. Wastewater-based COVID-19 epidemiology programs have been initiated in many countries to provide public health agencies with a complementary disease tracking metric and facile surveillance tool. However, their efficacy in prospectively capturing resurgence following a period of low prevalence is unclear. In this study, the SARS-CoV-2 viral signal was measured in primary clarified sludge harvested every two days at the City of Ottawa’s water resource recovery facility during the summer of 2020, when clinical testing recorded daily percent positivity below 1%. In late July, increases of >400% in normalized SARS-CoV-2 RNA signal in wastewater were identified 48 hours prior to reported >300% increases in positive cases that were retrospectively attributed to community-acquired infections. During this resurgence period, SARS-CoV-2 RNA signal in wastewater preceded the reported >160% increase in community hospitalizations by approximately 96 hours. This study supports wastewater-based COVID-19 surveillance of populations in augmenting the efficacy of diagnostic testing, which can suffer from sampling biases or timely reporting as in the case of hospitalization census.

The COVID-19 pandemic has given rise to diverse approaches to track infections. The causative agent, SARS-CoV-2 is a fecally-shed RNA virus, and many groups have assayed wastewater for viral RNA fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as a proxy of COVID-19 prevalence in the community. Most groups report low levels of viral RNA that often skirt the methods theoretical limits of detection and quantitation. Here, we demonstrate the presence of SARS-CoV-2 structural proteins in wastewater using traditional immunoblotting and quantitate them from wastewater solids using an immuno-linked PCR method called Multiplex Paired-antibody Amplified Detection (MPAD). In this longitudinal study, we corrected for stochastic variability inherent to wastewater-based epidemiology using multiple fecal content protein biomarkers. These normalized SARS-CoV-2 protein data correlated well with public health metrics. Our method of assaying SARS-CoV-2 protein from wastewater represents a promising and sensitive epidemiological tool to assess prevalence of fecally-shed pathogens in the community.