Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance
Authors: Ahmed Warish , Simpson Stuart , Bertsch Paul , Bibby Kyle , Bivins Aaron , Blackall Linda , Bofill-Mas Silvia , Bosch Albert , Brandao Joao , Choi Phil , Ciesielski Mark , Donner Erica , D'Souza Nishita , Farnleitner Andreas , Gerrity Daniel , Gonzalez Raul , Griffith John , Gyawali Pradip , Haas Charles , Hamilton Kerry , Hapuarachchi Chanditha , Harwood Valerie , Haque Rehnuma , Jackson Greg , Khan Stuart , Khan Wesaal , Kitajima Masaaki , Korajkic Asja , La Rosa Giuseppina , Layton Blythe , Lipp Erin , McLellan Sandra , McMinn Brian , Medema Gertjan , Metcalfe Suzanne , Meijer Wim , Mueller Jochen , Murphy Heather , Naughton Colleen , Noble Rachel , Payyappat Sudhi , Petterson Susan , Pitkanen Tarja , Rajal Veronica , Reyneke Brandon , Roman Fernando , Rose Joan , Rusinol Marta , Sadowsky Michael , Sala-Comorera Laura , Setoh Yin Xiang , Sherchan Samendra , Sirikanchana Kwanrawee , Smith Wendy , Steele Joshua , Sabburg Rosalie , Symonds Erin , Thai Phong , Thomas Kevin , Tynan Josh , Toze Simon , Thompson Janelle , Whiteley Andy , Wong Judith , Sano Daisuke , Wuertz Stefan , Xagoraraki Irene , Zhang Qian , Zimmer-Faust Amity , Shanks Orin 
Published: April 2021

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&rsquo;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.