Wastewater-Based Surveillance Is an Effective Tool for Trending COVID-19 Prevalence in Communities: A Study of 10 Major Communities for 17 Months in Alberta.

The correlations between SARS-CoV-2 RNA levels in wastewater from 12 wastewater treatment plants and new COVID-19 cases in the corresponding sewersheds of 10 communities were studied over 17 months. The analysis from the longest continuous surveillance reported to date revealed that SARS-CoV-2 RNA levels correlated well with temporal changes of COVID-19 cases in each community. The strongest correlation was found during the third wave (r = 0.97) based on the population-weighted SARS-CoV-2 RNA levels in wastewater. Different correlations were observed (r from 0.51 to 0.86) in various sizes of communities. The population in the sewershed had no observed effects on the strength of the correlation. Fluctuation of SARS-CoV-2 RNA levels in wastewater mirrored increases and decreases of COVID-19 cases in the corresponding community. Since the viral shedding to sewers from all infected individuals is included, wastewater-based surveillance provides an unbiased and no-discriminate estimation of the prevalence of COVID-19 compared with clinical testing that was subject to testing-seeking behaviors and policy changes. Wastewater-based surveillance on SARS-CoV-2 represents a temporal trend of COVID-19 disease burden and is an effective and supplementary monitoring when the number of COVID-19 cases reaches detectable thresholds of SARS-CoV-2 RNA in wastewater of treatment facilities serving various sizes of populations.

Wastewater-Based Surveillance Is an Effective Tool for Trending COVID-19 Prevalence in Communities: A Study of 10 Major Communities for 17 Months in Alberta

The correlations between SARS-CoV-2 RNA levels in wastewater from 12 wastewater treatment plants and new COVID-19 cases in the corresponding sewersheds of 10 communities were studied over 17 months. The analysis from the longest continuous surveillance reported to date revealed that SARS-CoV-2 RNA levels correlated well with temporal changes of COVID-19 cases in each community. The strongest correlation was found during the third wave (r = 0.97) based on the population-weighted SARS-CoV-2 RNA levels in wastewater. Different correlations were observed (r from 0.51 to 0.86) in various sizes of communities. The population in the sewershed had no observed effects on the strength of the correlation. Fluctuation of SARS-CoV-2 RNA levels in wastewater mirrored increases and decreases of COVID-19 cases in the corresponding community. Since the viral shedding to sewers from all infected individuals is included, wastewater-based surveillance provides an unbiased and no-discriminate estimation of the prevalence of COVID-19 compared with clinical testing that was subject to testing–seeking behaviors and policy changes. Wastewater-based surveillance on SARS-CoV-2 represents a temporal trend of COVID-19 disease burden and is an effective and supplementary monitoring when the number of COVID-19 cases reaches detectable thresholds of SARS-CoV-2 RNA in wastewater of treatment facilities serving various sizes of populations. Fluctuation of SARS-CoV-2 RNA levels in wastewater reflects temporal trends of new COVID-19 cases in the community correspondingly.

Number of COVID-19 cases required in a population to detect SARS-CoV-2 RNA in wastewater in the province of Alberta, Canada: Sensitivity assessment.

With a unique and large size of testing results of 1,842 samples collected from 12 wastewater treatment plants (WWTP) for 14 months through from low to high prevalence of COVID-19, the sensitivity of RT-qPCR detection of SARS-CoV-2 RNA in wastewater that correspond to the communities was computed by using Probit analysis. This study determined the number of new COVID-19 cases per 100,000 population required to detect SARS-CoV-2 RNA in wastewater at defined probabilities and provided an evidence-based framework of wastewater-based epidemiology surveillance (WBE). Input data were positive and negative test results of SARS-CoV-2 RNA in wastewater samples and the corresponding new COVID-19 case rates per 100,000 population served by each WWTP. The analyses determined that RT-qPCR-based SARS-CoV-2 RNA detection threshold at 50%, 80% and 99% probability required a median of 8 (range: 4-19), 18 (9-43), and 38 (17-97) of new COVID-19 cases /100,000, respectively. Namely, the positive detection rate at 50%, 80% and 99% probability were 0.01%, 0.02%, and 0.04% averagely for new cases in the population. This study improves understanding of the performance of WBE SARS-CoV-2 RNA detection using the large datasets and prolonged study period. Estimated COVID-19 burden at a community level that would result in a positive detection of SARS-CoV-2 in wastewater is critical to support WBE application as a supplementary warning/monitoring system for COVID-19 prevention and control.

Validating and optimizing the method for molecular detection and quantification of SARS-CoV-2 in wastewater.

Wastewater surveillance of SARS-CoV-2 has become a promising tool to estimate population-level changes in community infections and the prevalence of COVID-19 disease. Although many studies have reported the detection and quantification of SARS-CoV-2 in wastewater, remarkable variation remains in the methodology. In this study, we validated a molecular testing method by concentrating viruses from wastewater using ultrafiltration and detecting SARS-CoV-2 using one-step RT-qPCR assay. The following parameters were optimized including sample storage condition, wastewater pH, RNA extraction and RT-qPCR assay by quantification of SARS-CoV-2 or spiked human coronavirus strain 229E (hCoV-229E). Wastewater samples stored at 4 °C after collection showed significantly enhanced detection of SARS-CoV-2 with approximately 2-3 PCR-cycle threshold (Ct) values less when compared to samples stored at -20 °C. Pre-adjustment of the wastewater pH to 9.6 to aid virus desorption followed by pH readjustment to neutral after solid removal significantly increased the recovery of spiked hCoV-229E. Of the five commercially available RNA isolation kits evaluated, the MagMAX-96 viral RNA isolation kit showed the best recovery of hCoV-229E (50.1 ± 20.1%). Compared with two-step RT-qPCR, one-step RT-qPCR improved sensitivity for SARS-CoV-2 detection. Salmon DNA was included for monitoring PCR inhibition and pepper mild mottle virus (PMMoV), a fecal indicator indigenous to wastewater, was used to normalize SARS-CoV-2 levels in wastewater. Our method for molecular detection of SARS-CoV-2 in wastewater provides a useful tool for public health surveillance of COVID-19.

SARS-CoV-2 surrogate (Phi6) environmental persistence within free-living amoebae.

The reported persistence of SARS-CoV-2 virions in aquatic environments highlights the need to better understand potential mechanisms that may prolong its dissemination. We evaluated the possibility that amoebae might serve as transport hosts by studying the interaction of the enveloped bacteriophage Phi6, as a potential surrogated along with one of the most common amoebae in engineered aquatic environments, Vermamoeba vermiformis. Using microscopy, imaging flow cytometry and bacteriophage cell culture, our results imply that the SARS-CoV-2 surrogate triggers amoebic mitochondria and induced apoptosis to promote viral persistence in trophozoites. Furthermore, virus-infected amoebae were still infectious after 2 months within FLA cysts. These results suggest that amoebae could contribute to the environmental persistence of SARS-CoV-2, including disinfection processes. In addition, amoebae could be a successful model system for understanding respiratory virus-eukaryotic biology at the cellular and molecular levels.