Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study
Rigorous method development and validation to detect and quantify SARS-CoV-2 RNA in wastewater has led to important advances in community disease surveillance using quantitative molecular biology tools. Despite this progress, agreement on standardized workflows for this important public health objec...
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Frontiers Media S.A.
2025-06-01
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| author | Janine McCalder Janine McCalder Jangwoo Lee Jangwoo Lee Judy Qiu Qiaozhi Li Linnet Immaraj Nicole Acosta María A. Bautista Melissa Wilson Barbara Waddell Kristine Du Aito Ueno Rhonda Clark Rhonda Clark Alexander Krusina Danielle A. Southern Tyler Williamson Tyler Williamson Chloe Papparis Paul Montesclaros Lance Non September Stefani Gail Visser Puja Pradhan Norma Ruecker John Conly John Conly John Conly John Conly John Conly John Conly Steve Hrudey Kevin Frankowski Bonita Lee Bonita Lee Michael D. Parkins Michael D. Parkins Michael D. Parkins Xiaoli Pang Xiaoli Pang Casey R. J. Hubert |
| author_facet | Janine McCalder Janine McCalder Jangwoo Lee Jangwoo Lee Judy Qiu Qiaozhi Li Linnet Immaraj Nicole Acosta María A. Bautista Melissa Wilson Barbara Waddell Kristine Du Aito Ueno Rhonda Clark Rhonda Clark Alexander Krusina Danielle A. Southern Tyler Williamson Tyler Williamson Chloe Papparis Paul Montesclaros Lance Non September Stefani Gail Visser Puja Pradhan Norma Ruecker John Conly John Conly John Conly John Conly John Conly John Conly Steve Hrudey Kevin Frankowski Bonita Lee Bonita Lee Michael D. Parkins Michael D. Parkins Michael D. Parkins Xiaoli Pang Xiaoli Pang Casey R. J. Hubert |
| author_sort | Janine McCalder |
| collection | DOAJ |
| description | Rigorous method development and validation to detect and quantify SARS-CoV-2 RNA in wastewater has led to important advances in community disease surveillance using quantitative molecular biology tools. Despite this progress, agreement on standardized workflows for this important public health objective has been elusive. Multiple studies have compared different protocols but have been limited by short periods of observation or low numbers of test sites. Here we compare results from two parallel workflows for wastewater processing and quantifying SARS-CoV-2 gene targets from five wastewater treatment plants in three large cities in Alberta, Canada for up to 29-months. In total 1,482 wastewater samples were processed using either affinity columns followed by RT-qPCR with DNA-based standards or using ultrafiltration followed by RT-qPCR with RNA-based standards. Results from either workflow correlated well with each other, and with 5-day rolling averages of clinically diagnosed COVID-19 cases (i.e., in the early part of the 29-month study period when clinical testing was performed routinely). This highlights that different workflows both effectively and reliably monitored SARS-CoV-2 trends in wastewater. Parallel quantification of pepper mild mottle virus genomes and normalization were inconsistent between the two workflows, suggesting that normalization strategies may require adjustment for different wastewater processing protocols. Freezing wastewater samples diminished measured SARS-CoV-2 RNA levels significantly, whereas short term sample storage at +4°C gave consistent results. Overall, this work demonstrates that different workflows can deliver similarly effective wastewater-based surveillance for community COVID-19 burden. As this emerging technology is used more routinely, investigators should prioritize consistent application of a given workflow to a high-quality standard over time, whereas focusing on all testing programs adopting identical workflows and methods may be unnecessary. |
| format | Article |
| id | doaj-art-26e1bea830a44983bac1e97ae399c77c |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-26e1bea830a44983bac1e97ae399c77c2025-08-20T03:21:39ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-06-011610.3389/fmicb.2025.15478311547831Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal studyJanine McCalder0Janine McCalder1Jangwoo Lee2Jangwoo Lee3Judy Qiu4Qiaozhi Li5Linnet Immaraj6Nicole Acosta7María A. Bautista8Melissa Wilson9Barbara Waddell10Kristine Du11Aito Ueno12Rhonda Clark13Rhonda Clark14Alexander Krusina15Danielle A. Southern16Tyler Williamson17Tyler Williamson18Chloe Papparis19Paul Montesclaros20Lance Non21September Stefani22Gail Visser23Puja Pradhan24Norma Ruecker25John Conly26John Conly27John Conly28John Conly29John Conly30John Conly31Steve Hrudey32Kevin Frankowski33Bonita Lee34Bonita Lee35Michael D. Parkins36Michael D. Parkins37Michael D. Parkins38Xiaoli Pang39Xiaoli Pang40Casey R. J. Hubert41Geomicrobiology Group, Department of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaGeomicrobiology Group, Department of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, CanadaSchool of Public Health, University of Alberta, Edmonton, AB, CanadaSchool of Public Health, University of Alberta, Edmonton, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaGeomicrobiology Group, Department of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaGeomicrobiology Group, Department of Biological Sciences, University of Calgary, Calgary, AB, CanadaAdvancing Canadian Water Assets, University of Calgary, Calgary, AB, CanadaDepartment of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaDepartment of Community Health Sciences, University of Calgary, Calgary, AB, CanadaO’Brien Institute for Public Health, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, CanadaDepartment of Biological Sciences, University of Calgary, Calgary, AB, Canada0Water Quality Services, Calgary, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, CanadaO’Brien Institute for Public Health, University of Calgary, Calgary, AB, Canada1Department of Pathology and Laboratory Medicine, University of Calgary, Calgary, AB, Canada2Infection Prevention and Control, Alberta Health Services, Calgary, AB, Canada3Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada4Analytical and Environmental Toxicology, University of Alberta, Edmonton, AB, CanadaAdvancing Canadian Water Assets, University of Calgary, Calgary, AB, Canada5Department of Pediatrics, University of Alberta, Edmonton, AB, Canada6Women and Children’s Health Research Institute; Li Ka Shing Institute of Virology, Edmonton, AB, CanadaDepartment of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Medicine, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada3Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, CanadaDepartment of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada7Alberta Precision Laboratories, Public Health Laboratory; Li Ka Shing Institute of Virology, Alberta Health Services, Edmonton, AB, CanadaGeomicrobiology Group, Department of Biological Sciences, University of Calgary, Calgary, AB, CanadaRigorous method development and validation to detect and quantify SARS-CoV-2 RNA in wastewater has led to important advances in community disease surveillance using quantitative molecular biology tools. Despite this progress, agreement on standardized workflows for this important public health objective has been elusive. Multiple studies have compared different protocols but have been limited by short periods of observation or low numbers of test sites. Here we compare results from two parallel workflows for wastewater processing and quantifying SARS-CoV-2 gene targets from five wastewater treatment plants in three large cities in Alberta, Canada for up to 29-months. In total 1,482 wastewater samples were processed using either affinity columns followed by RT-qPCR with DNA-based standards or using ultrafiltration followed by RT-qPCR with RNA-based standards. Results from either workflow correlated well with each other, and with 5-day rolling averages of clinically diagnosed COVID-19 cases (i.e., in the early part of the 29-month study period when clinical testing was performed routinely). This highlights that different workflows both effectively and reliably monitored SARS-CoV-2 trends in wastewater. Parallel quantification of pepper mild mottle virus genomes and normalization were inconsistent between the two workflows, suggesting that normalization strategies may require adjustment for different wastewater processing protocols. Freezing wastewater samples diminished measured SARS-CoV-2 RNA levels significantly, whereas short term sample storage at +4°C gave consistent results. Overall, this work demonstrates that different workflows can deliver similarly effective wastewater-based surveillance for community COVID-19 burden. As this emerging technology is used more routinely, investigators should prioritize consistent application of a given workflow to a high-quality standard over time, whereas focusing on all testing programs adopting identical workflows and methods may be unnecessary.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1547831/fullSARS-CoV-2wastewaterwastewater-based epidemiologyconcentration methoddirect extractionmethod comparison |
| spellingShingle | Janine McCalder Janine McCalder Jangwoo Lee Jangwoo Lee Judy Qiu Qiaozhi Li Linnet Immaraj Nicole Acosta María A. Bautista Melissa Wilson Barbara Waddell Kristine Du Aito Ueno Rhonda Clark Rhonda Clark Alexander Krusina Danielle A. Southern Tyler Williamson Tyler Williamson Chloe Papparis Paul Montesclaros Lance Non September Stefani Gail Visser Puja Pradhan Norma Ruecker John Conly John Conly John Conly John Conly John Conly John Conly Steve Hrudey Kevin Frankowski Bonita Lee Bonita Lee Michael D. Parkins Michael D. Parkins Michael D. Parkins Xiaoli Pang Xiaoli Pang Casey R. J. Hubert Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study Frontiers in Microbiology SARS-CoV-2 wastewater wastewater-based epidemiology concentration method direct extraction method comparison |
| title | Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study |
| title_full | Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study |
| title_fullStr | Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study |
| title_full_unstemmed | Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study |
| title_short | Consistent trends from different methods for monitoring SARS-CoV-2 in urban wastewater during a 29-month longitudinal study |
| title_sort | consistent trends from different methods for monitoring sars cov 2 in urban wastewater during a 29 month longitudinal study |
| topic | SARS-CoV-2 wastewater wastewater-based epidemiology concentration method direct extraction method comparison |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1547831/full |
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