Preprint Review Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 17 April 2021 / Approved: 19 April 2021 / Online: 19 April 2021 (13:08:13 CEST)

How to cite: Ahmed, W.; Simpson, S.; Bertsch, P.; Bibby, K.; Bivins, A.; Blackall, L.; Bofill-Mas, S.; Bosch, A.; Brandao, J.; Choi, P.; Ciesielski, M.; Donner, E.; D'Souza, N.; Farnleitner, A.; Gerrity, D.; Gonzalez, R.; Griffith, J.; Gyawali, P.; Haas, C.; Hamilton, K.; Hapuarachchi, C.; Harwood, V.; Haque, R.; Jackson, G.; Khan, S.; Khan, W.; Kitajima, M.; Korajkic, A.; La Rosa, G.; Layton, B.; Lipp, E.; McLellan, S.; McMinn, B.; Medema, G.; Metcalfe, S.; Meijer, W.; Mueller, J.; Murphy, H.; Naughton, C.; Noble, R.; Payyappat, S.; Petterson, S.; Pitkanen, T.; Rajal, V.; Reyneke, B.; Roman, F.; Rose, J.; Rusinol, M.; Sadowsky, M.; Sala-Comorera, L.; Setoh, Y.X.; Sherchan, S.; Sirikanchana, K.; Smith, W.; Steele, J.; Sabburg, R.; Symonds, E.; Thai, P.; Thomas, K.; Tynan, J.; Toze, S.; Thompson, J.; Whiteley, A.; Wong, J.; Sano, D.; Wuertz, S.; Xagoraraki, I.; Zhang, Q.; Zimmer-Faust, A.; Shanks, O. Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance. Preprints 2021, 2021040481 (doi: 10.20944/preprints202104.0481.v1). Ahmed, W.; Simpson, S.; Bertsch, P.; Bibby, K.; Bivins, A.; Blackall, L.; Bofill-Mas, S.; Bosch, A.; Brandao, J.; Choi, P.; Ciesielski, M.; Donner, E.; D'Souza, N.; Farnleitner, A.; Gerrity, D.; Gonzalez, R.; Griffith, J.; Gyawali, P.; Haas, C.; Hamilton, K.; Hapuarachchi, C.; Harwood, V.; Haque, R.; Jackson, G.; Khan, S.; Khan, W.; Kitajima, M.; Korajkic, A.; La Rosa, G.; Layton, B.; Lipp, E.; McLellan, S.; McMinn, B.; Medema, G.; Metcalfe, S.; Meijer, W.; Mueller, J.; Murphy, H.; Naughton, C.; Noble, R.; Payyappat, S.; Petterson, S.; Pitkanen, T.; Rajal, V.; Reyneke, B.; Roman, F.; Rose, J.; Rusinol, M.; Sadowsky, M.; Sala-Comorera, L.; Setoh, Y.X.; Sherchan, S.; Sirikanchana, K.; Smith, W.; Steele, J.; Sabburg, R.; Symonds, E.; Thai, P.; Thomas, K.; Tynan, J.; Toze, S.; Thompson, J.; Whiteley, A.; Wong, J.; Sano, D.; Wuertz, S.; Xagoraraki, I.; Zhang, Q.; Zimmer-Faust, A.; Shanks, O. Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance. Preprints 2021, 2021040481 (doi: 10.20944/preprints202104.0481.v1).

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.

Subject Areas

COVID-19; SARS-CoV-2; Wastewater; Surveillance; False-positive; False-negative; RT-PCR

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