Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low COVID-19 incidence and prevalence

Abstract

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.

Uncertainties in estimating SARS-CoV-2 prevalence by wastewater-based epidemiology

“…In addition, recent studies have also reported the presence of SARS-CoV-2 RNA in wastewater solids and sludge samples collected at various locations of the WWTP [70] , [71] , [72] . Higher concentrations of SARS-CoV-2 RNA were detected in sludge samples [71] , sediments of wastewater [73] and wastewater solids collected from the primary settler [72] , [74] , comparing to wastewater influent samples.…”

“…A recent study found that using PMMoV alone was sufficient to inform of relative recovery and to normalize between samples without adding external surrogate viruses in wastewater [21] . In addition, for WBE studies using sludge or solids from wastewater, PMMoV also showed superior performance in reducing background noise that possibly associated with the collection and transport of the samples along with the RNA concentration, extraction, and detection steps, over gut resident HF183 Bacteroides and 18S rRNA that is expressed in human cells [70] . CrAssphage, a human-specific gut-associated bacteriophage, was thought to be potentially a more specific biomarker due to its high detection sensitivity and abundance in wastewater [101] .…”

Wastewater-Based Epidemiology (WBE) and Viral Detection in Polluted Surface Water: A Valuable Tool for COVID-19 Surveillance—A Brief Review

“…The findings also evidenced that pepper mild mottle virus (PMMV) RNA can be a reliable normalization biomarker, to control noise associated with variances in daily operations, sampling, storage, processing and sample analysis. At last, after normalization of the SARS-CoV-2 signal, a significant correlation was observed between viral RNA gene copies/L and epidemiological data, considering daily new cases, active COVID-19 cases in the last fourteen days, and the percent of daily-testing positives for COVID-19 [ 52 ].…”

Predictive power of SARS-CoV-2 wastewater surveillance for diverse populations across a large geographical range

“…A goal of this study was to provide communities with a readily interpretable picture of changes in viral prevalence through WBE monitoring. Most commonly, wastewater viral load has been reported as qPCR CT or as the logarithm of genome copies per liter of wastewater 1,2,6,7,9,[23][24][25][26][27] . While those measures hold meaning for experts, they can be opaque to the communities and officials who use the information.…”

“…WBE has the potential to be especially helpful in localities where access to individual testing is difficult due to limited or sparsely distributed resources. Several studies this year have already demonstrated the realized and potential advantages of WBE for monitoring levels of in communities [1][2][3][4] . In addition to tracking spread, WBE can provide early warning signs of outbreaks and display the efficacy of public health interventions similar to what has been done with polio or herpes viruses 5 .…”