Improved recovery of SARS-CoV-2 from wastewater through application of RNA and DNA stabilising agents.

Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.

Wastewater surveillance for SARS-CoV-2: Lessons learnt from recent studies to define future applications.

Wastewater-based epidemiology (WBE) is successful in the detection of the spread of SARS-CoV-2. This review examines the methods used and results of recent studies on the quantification of SARS-CoV-2 in wastewater. WBE becomes essential, especially with virus transmission path uncertainty, limitations on the number of clinical tests that could be conducted, and a relatively long period for infected people to show symptoms. Wastewater surveillance was used to show the effect of lockdown on the virus spread. A WBE framework tailored for SARS-CoV-2 that incorporates lessons learnt from the reviewed studies was developed. Results of the review helped outline challenges facing the detection of SARS-CoV-2 in wastewater samples. A comparison between the various studies with regards to sample concentration and virus quantification was conducted. Five different primers sets were used for qPCR quantification; however, due to limited data availability, there is no consensus on the most sensitive primer. Correlating the slope of the relationship between the number of gene copies vs. the cumulative number of infections normalized to the total population served with the average new cases, suggests that qPCR results could help estimating the number of new infections. The correlation is improved when a lag period was introduced to account for asymptomatic infections. Based on lessons learnt from recent studies, it is recommended that future applications should consider the following: 1) ensuring occupational safety in managing sewage collection and processing, 2) evaluating the effectiveness of greywater disinfection, 3) measuring viral RNA decay due to biological and chemical activities during collection and treatment, 4) assessing the effectiveness of digital PCR, and 5) conducting large scale international studies that follow standardized protocols.