Wastewater-Based Epidemiology for Community Monitoring of SARS-CoV-2: Progress and Challenges

Abstract

Wastewater-based epidemiology (WBE) is useful for the surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in communities, complementing clinical diagnostic testing of individuals. In this Review, we summarize recent progress and highlight remaining challenges in monitoring SARS-CoV-2 RNA in wastewater systems for community and environmental surveillance. Very low concentrations of viral particles and RNA present in the complicated wastewater and sewage sample matrix require efficient sample processing and sensitive detection. We discuss advantages and limitations of available methods for wastewater sample processing, including collection, separation, enrichment, RNA extraction, and purification. Efficient extraction of the viral RNA and removal of interfering sample matrices are critical to the subsequent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for sensitive detection of SARS-CoV-2 in wastewater. We emphasize the importance of implementing appropriate controls and method validation, which include the use of surrogate viruses for assessing extraction efficiency and normalization against measurable chemical and biological components in wastewater. Critical analysis of the published studies reveals imperative research needs for the development, validation, and standardization of robust and sensitive methods for quantitative detection of viral RNA and proteins in wastewater for WBE.

Spatial and Temporal Trends of SARS-CoV-2 RNA from Wastewater Treatment Plants over 6 Weeks in Cape Town, South Africa

“…Our results are consistent with these studies. In our study, untreated, influent wastewater samples were collected from the inlet of WWTPs, which other studies have demonstrated is the preferred sample type for investigating changes in SARS-CoV-2 RNA signals and correlations between clinical cases and hotspot identification [17,18].…”

The devil is in the details: emerging insights on the relevance of wastewater surveillance for SARS-CoV-2 to public health

“…However, the dominant focus of much of the research and discussions on the strengths and limitations of the PCR analysis methods, while clearly critical to the validity of WBS, can serve to oversimplify the reality that there are multiple steps involved in sample collection and preparation required to process and concentrate wastewater samples. Each of these steps can introduce variability, errors or uncertainty to the utility and ultimate interpretation at many points in the process, prior to running the ultimate PCR analysis (Kumblathan et al 2021).…”

Wastewater Based Surveillance of SARS-CoV-2: Challenges and Perspective from a Canadian Inter-laboratory Study

“…Other explanations include viral particle degradation, loss of viral particles during concentration, and RNA degradation during extraction ( Kantor et al, 2021 ). A recent review by Kumblathan et al (2021) outlined common methodologies used for WBS of SARS-CoV-2 and critically reviewed the challenges associated with WBS sampling, enrichment of viral particles, RNA extraction, and subsequent RNA detection. Enrichment methods include polyethylene glycol precipitation, aqueous two-phase partitioning, electronegative membranes, ultrafiltration and ultracentrifugation, and flocculation with a beef extract solution.…”

“…The laboratories in the Chik et al (2021) study used different standards for quantification, and anonymously reported whether an RNA or plasmid DNA standard was used. This is important to note that using plasmid DNA as a calibration standard as opposed to an RNA standard can result in two orders of magnitude variability, likely due to DNA being supercoiled during early stages of RT-qPCR ( Chik et al, 2021 ; Hou et al, 2010 ; Kumblathan et al, 2021 ). Regardless, in both the Chik et al (2021) and Pecson et al (2021) studies, laboratories were reporting relatively similar orders of magnitude of RNA copy numbers.…”

“…One potential method for precipitating SARS-CoV-2 from the solid phase is to use a beef extract solution ( Mlejnkova et al, 2020 ). The viral particles separated from the solid phase should be combined with the aqueous phase supernatant ( Kumblathan et al, 2021 ). Future studies should investigate each of the aqueous and solid phases individually to determine the relative abundance and partition characteristics of SARS-CoV-2 and surrogates in each phase.…”