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, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections through measuring trends of RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 gene regions 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 gene regions in PCS (92.7, 90.6%, n = 6) as compared to PGS samples (79.2, 82.3%, n = 5). 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 (PMMoV) 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 18S rRNA, making PMMoV a potentially useful biomarker for normalization of SARS-CoV-2 signal. PMMoV-normalized PCS RNA signal from WRRFs of two cities correlated with the regional public health epidemiological metrics, identifying PCS normalized to a fecal indicator (PMMoV) as a potentially effective tool for monitoring trends during decreasing and low-incidence of infection of SARS-Cov-2 in communities.

Keywords: COVID-19; Primary clarified sludge; SARS-CoV-2; Solids; Virus; Wastewater.

Graphical abstract

Fig. 1

Location of the city of Ottawa and Gatineau WRRFs (GMP - Google Map Customizer).

Fig. 2

Flowchart showing sample work-up and processing for PGS and PCS, including RNA concentration, extraction and quantification.

Fig. 3

Comparison of copies per 100 µL of extracted RNA in RT-qPCR and RT-ddPCR for PGS (n=6) and PCS (n=5).

Fig. 4

Sensitivity of N1 and N2 RT-qPCR assays comparison between PCS and PGS samples. Significance between detections established using Chi-Squared test. Variance is shown with %CV (n=24).

Fig. 5

Variance of PCS normalization biomarkers (and SARS-CoV-2 for reference), (a) combined data set comprised of both cities samples, and (b) data set separated by city. Analysis of variance and maximum change in C_t (ΔC_t) (n=8).

Fig. 6

Ottawa WRRF trends of N1 and N2 SARS-CoV-2 viral copies with epidemiological metrics, (a) copies/L of PCS, (b) copies/d that was normalized by the mass flux through primary clarifier, (c) copies/copies of PMMoV that was normalized by PMMoV reference gene and (d) graph displaying the daily number of tests performed and the number of active COVID-19 cases/100K population.

Fig. 7

Gatineau WRRF trends of N1 and N2 SARS-CoV-2 viral copies with epidemiological metrics, (a) copies/L of PCS, (b) copies/d that was normalized by the mass flux through primary clarifier, (c) copies/copies of PMMoV that was normalized by PMMoV reference gene and (d) graph displaying the daily number of tests performed and the number of active COVID-19 cases/100K population.