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

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

doi:10.1016/j.scitotenv.2021.149877

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

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

Research output: Contribution to journal › Review article › peer-review

130 Scopus citations

Abstract

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.

Original language	English (US)
Article number	149877
Journal	Science of the Total Environment
Volume	805
DOIs	https://doi.org/10.1016/j.scitotenv.2021.149877
State	Published - Jan 20 2022

Keywords

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

ASJC Scopus subject areas

Environmental Engineering
Environmental Chemistry
Waste Management and Disposal
Pollution

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10.1016/j.scitotenv.2021.149877

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Ahmed, W., Simpson, S. L., Bertsch, P. M., Bibby, K., Bivins, A., Blackall, L. L., Bofill-Mas, S., Bosch, A., Brandão, J., Choi, P. M., Ciesielski, M., Donner, E., D'Souza, N., Farnleitner, A. H., Gerrity, D., Gonzalez, R., Griffith, J. F., Gyawali, P., Haas, C. N., ... Shanks, O. C. (2022). Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance. Science of the Total Environment, 805, Article 149877. https://doi.org/10.1016/j.scitotenv.2021.149877

Ahmed, W, Simpson, SL, Bertsch, PM, Bibby, K, Bivins, A, Blackall, LL, Bofill-Mas, S, Bosch, A, Brandão, J, Choi, PM, Ciesielski, M, Donner, E, D'Souza, N, Farnleitner, AH, Gerrity, D, Gonzalez, R, Griffith, JF, Gyawali, P, Haas, CN, Hamilton, KA, Hapuarachchi, HC, Harwood, VJ, Haque, R, Jackson, G, Khan, SJ, Khan, W, Kitajima, M, Korajkic, A, La Rosa, G, Layton, BA, Lipp, E, McLellan, SL, McMinn, B, Medema, G, Metcalfe, S, Meijer, WG, Mueller, JF, Murphy, H, Naughton, CC, Noble, RT, Payyappat, S, Petterson, S, Pitkänen, T, Rajal, VB, Reyneke, B, Roman, FA, Rose, JB, Rusiñol, M, Sadowsky, MJ, Sala-Comorera, L, Setoh, YX, Sherchan, SP, Sirikanchana, K, Smith, W, Steele, JA, Subburg, R, Symonds, EM, Thai, P, Thomas, KV, Tynan, J, Toze, S, Thompson, J, Whiteley, AS, Wong, JCC, Sano, D, Wuertz, S, Xagoraraki, I, Zhang, Q, Zimmer-Faust, AG & Shanks, OC 2022, 'Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance', Science of the Total Environment, vol. 805, 149877. https://doi.org/10.1016/j.scitotenv.2021.149877

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

abstract = "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.",

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

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doi = "10.1016/j.scitotenv.2021.149877",

language = "English (US)",

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T1 - Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance

AU - Ahmed, Warish

AU - Simpson, Stuart L.

AU - Bertsch, Paul M.

AU - Bibby, Kyle

AU - Bivins, Aaron

AU - Blackall, Linda L.

AU - Bofill-Mas, Sílvia

AU - Bosch, Albert

AU - Brandão, João

AU - Choi, Phil M.

AU - Ciesielski, Mark

AU - Donner, Erica

AU - D'Souza, Nishita

AU - Farnleitner, Andreas H.

AU - Gerrity, Daniel

AU - Gonzalez, Raul

AU - Griffith, John F.

AU - Gyawali, Pradip

AU - Haas, Charles N.

AU - Hamilton, Kerry A.

AU - Hapuarachchi, Hapuarachchige Chanditha

AU - Harwood, Valerie J.

AU - Haque, Rehnuma

AU - Jackson, Greg

AU - Khan, Stuart J.

AU - Khan, Wesaal

AU - Kitajima, Masaaki

AU - Korajkic, Asja

AU - La Rosa, Giuseppina

AU - Layton, Blythe A.

AU - Lipp, Erin

AU - McLellan, Sandra L.

AU - McMinn, Brian

AU - Medema, Gertjan

AU - Metcalfe, Suzanne

AU - Meijer, Wim G.

AU - Mueller, Jochen F.

AU - Murphy, Heather

AU - Naughton, Coleen C.

AU - Noble, Rachel T.

AU - Payyappat, Sudhi

AU - Petterson, Susan

AU - Pitkänen, Tarja

AU - Rajal, Veronica B.

AU - Reyneke, Brandon

AU - Roman, Fernando A.

AU - Rose, Joan B.

AU - Rusiñol, Marta

AU - Sadowsky, Michael J.

AU - Sala-Comorera, Laura

AU - Setoh, Yin Xiang

AU - Sherchan, Samendra P.

AU - Sirikanchana, Kwanrawee

AU - Smith, Wendy

AU - Steele, Joshua A.

AU - Subburg, Rosalie

AU - Symonds, Erin M.

AU - Thai, Phong

AU - Thomas, Kevin V.

AU - Tynan, Josh

AU - Toze, Simon

AU - Thompson, Janelle

AU - Whiteley, Andy S.

AU - Wong, Judith Chui Ching

AU - Sano, Daisuke

AU - Wuertz, Stefan

AU - Xagoraraki, Irene

AU - Zhang, Qian

AU - Zimmer-Faust, Amity G.

AU - Shanks, Orin C.

PY - 2022/1/20

Y1 - 2022/1/20

N2 - 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.

AB - 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.

KW - COVID-19

KW - False negative

KW - False positive

KW - RT-PCR

KW - SARS-CoV-2

KW - Surveillance

KW - Wastewater

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DO - 10.1016/j.scitotenv.2021.149877

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

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