Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

Ahmed, Warish; Simpson, Stuart L.; Bertsch, Paul M.; Bibby, Kyle; Bivins, Aaron; Blackall, Linda L.; Bofill-Mas, Sílvia; Bosch, Albert; Brandão, João; Choi, Phil M.; Ciesielski, Mark; Donner, Erica; D'Souza, Nishita; Farnleitner, Andreas H.; Gerrity, Daniel; Gonzalez, Raul; Griffith, John F.; Gyawali, Pradip; Haas, Charles N.; Hamilton, Kerry A.; Hapuarachchi, Hapuarachchige Chanditha; Harwood, Valerie J.; Haque, Rehnuma; Jackson, Greg; Khan, Stuart J.; Khan, Wesaal; Kitajima, Masaaki; Korajkic, Asja; La Rosa, Giuseppina; Layton, Blythe A.; Lipp, Erin; McLellan, Sandra; McMinn, Brian; Medema, Gertjan; Metcalfe, Suzanne; Meijer, Wim G.; Mueller, Jochen F.; Murphy, Heather; Naughton, Coleen C.; Noble, Rachel T.; Payyappat, Sudhi; Petterson, Susan; Pitkänen, Tarja; Rajal, Veronica B.; Reyneke, Brandon; Roman, Fernando A.; Rose, Joan B.; Rusiñol, Marta; Sadowsky, Michael J.; Sala-Comorera, Laura; Setoh, Yin Xiang; Sherchan, Samendra; Sirikanchana, Kwanrawee; Smith, Wendy; Steele, Joshua; Subburg, Rosalie; Symonds, Erin M.; Thai, Phong; Thomas, Kevin V.; Tynan, Josh; Toze, Simon; Thompson, Janelle; Whiteley, Andy S.; Wong, Judith Chui Ching; Sano, Daisuke; Wuertz, Stefan; Xagoraraki, Irene; Zhang, Qian; Zimmer-Faust, Amity G.; Shanks, Orin C.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering additional community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater can 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 increasing rapidly. However, there are no standardized protocols or 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 cause false-positive and false-negative errors in the surveillance of SARS-CoV-2, culminating in recommended 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 when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.