Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance
Creators: name: Bosch, A.
name: Noble, R.T.
name: Khan, W.
name: Medema, G.
name: McMinn, B.
name: Harwood, V.J.
name: Thompson, J.
name: Bibby, K.
name: Sala-Comorera, L.
name: Bivins, A.
name: Subburg, R.
name: Roman, F.A., Jr
name: Simpson, S.L.
name: Rajal, V.B.
name: Gyawali, P.
name: La Rosa, G.
name: Griffith, J.F.
name: Meijer, W.G.
name: Khan, S.J.
name: Gerrity, D.
name: Gonzalez, R.
name: Toze, S.
name: Sadowsky, M.J.
name: McLellan, S.L.
name: Hapuarachchi, H.C.
name: D&apos;Souza, N.
name: Zimmer-Faust, A.G.
name: Tynan, J.
name: Bertsch, P.M.
name: Hamilton, K.A.
name: Zhang, Q.
name: Wuertz, S.
name: Rusiñol, M.
name: Pitkänen, T.
name: Steele, J.A.
name: Korajkic, A.
name: Kitajima, M.
name: Petterson, S.
name: Metcalfe, S.
name: Ciesielski, M.
name: Farnleitner, A.H.
name: Naughton, C.C.
name: Lipp, E.
name: Donner, E.
name: Layton, B.A.
name: Ahmed, W.
name: Thomas, K.V.
name: Xagoraraki, I.
name: Sano, D.
name: Rose, J.B.
name: Murphy, H.
name: Smith, W.
name: Sirikanchana, K.
name: Bofill-Mas, S.
name: Brandão, J.
name: Choi, P.M.
name: Shanks, O.C.
name: Payyappat, S.
name: Thai, P.
name: Symonds, E.M.
name: Jackson, G.
name: Mueller, J.F.
name: Wong, J.C.C.
name: Haque, R.
name: Whiteley, A.S.
name: Blackall, L.L.
name: Setoh, Y.X.
name: Sherchan, S.P.
name: Haas, C.N.
name: Reyneke, B.
Publisher: name: Elsevier B.V.
Year: 2022

description: Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially 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 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR 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.