Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Abstract

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.

A novel passive sampling approach for SARS-CoV-2 in wastewater in a Canadian province with low prevalence of COVID-19

“…Further, composite sampling may not be a reliable monitoring approach in areas of low COVID-19 prevalence as composite samples can dilute SARS-CoV-2 signals. 19,20 Alternatively, sampling upstream of WWTPs at sewershed pump stations, manholes, or institution-level sites (e.g., commercial properties, airports, university campuses, etc.) can target specific areas of interest.…”

Operationalizing a routine wastewater monitoring laboratory for SARS-CoV-2

“…Additionally, the pipeline presented here allowed meta-analysis of the data, revealing the variability present in each step of sample processing. While some amount of variation is due to lower quality or technical error, some appears to be inherent methodological variation or variation in the wastewater itself 27,28 . In the future, modeling the contributions of these variables to overall noise in the data could provide better confidence in the results.…”

“…The development of a robust quality assurance plan is critical for any laboratory monitoring SARS-CoV-2 in wastewater. Others have documented the controls and standards required for WBE 25,28 . Our newly developed quality scoring framework and code represent one possible model for communicating data quality to partners in public health.…”

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

“…Wastewater based surveillance (WBS) of SARS-CoV-2 RNA is gaining traction because of its many advantages over individual testing, particularly the cost-effectiveness of a relatively unbiased pooled sample and the ability to detect virus shed from infected asymptomatic or pre-symptomatic individuals (Bivins et al 2020, Hart & Halden 2020, Kitajima et al 2020, Ahmed et al 2021). WBS also yields information several days and almost two weeks faster than it takes to collate individual testing and hospitalization records, respectively (Nemudryi et al 2020).…”

“…However, disentangling confounded method effects from such large studies is challenging. There have been a few controlled experiments looking at specific methodological choices, such as concentration techniques (Ahmed et al 2020b, Torii et al 2020, La Rosa et al 2021, LaTurner et al 2021, Whitney et al 2021) and processing platform (Feng et al 2021, Cieselski et al 2021, Ahmed et al 2021). Here we expand upon those efforts by conducting controlled experiments to examine a range of sample processing choices (Fig.…”