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However, developing and implementing the methods underpinning these programmes in regions with prolonged periods of low community transmission has proven challenging. In Victoria, Australia, wastewater surveillance provided early warning of unknown community infections and informed timely public health decisions to limit their spread when case numbers were low. To achieve this, we developed a methodological approach sensitive to extremely low viral loads and could readily identify false positives within short turnaround times. Here, we describe the successful development, implementation, and evaluation of analytic methods using Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) and amplicon sequencing in tandem with CRISPR DETECTR in an ongoing, large-scale surveillance programme to detect SARS-CoV-2 in wastewater in Victoria, Australia. Our study covers ten months, from July 2020 to April 2021, and includes all state-wide health districts and prolonged periods with no known, active community cases among the \u223c6.7 million population.<\/jats:p>","DOI":"10.2166\/wh.2025.164","type":"journal-article","created":{"date-parts":[[2025,2,4]],"date-time":"2025-02-04T13:35:36Z","timestamp":1738676136000},"page":"89-99","update-policy":"https:\/\/doi.org\/10.2166\/iwapcrossmarkpolicypage","source":"Crossref","is-referenced-by-count":2,"title":["Multi-tiered strategy for large-scale wastewater detection of SARS-CoV-2 in low-case settings provides confidence for public health actions"],"prefix":"10.2166","volume":"23","author":[{"given":"Aaron","family":"Jex","sequence":"first","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"},{"name":"b Faculty of Science, The University of Melbourne, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6376-2630","authenticated-orcid":false,"given":"Nijoy","family":"John","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"},{"name":"b Faculty of Science, The University of Melbourne, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"McCarthy","sequence":"additional","affiliation":[{"name":"c Environmental and Public Health Microbiology Laboratory (EPHM Lab), Department of Civil Engineering, Monash University, Clayton, Victoria 3800, Australia"},{"name":"d School of Civil and Environmental Engineering, Queensland University of Technology, Brisbane 4000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven","family":"Myers","sequence":"additional","affiliation":[{"name":"e Department of Microbiology, Australian Laboratory Services, Scoresby, Victoria 3179, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natacha","family":"Begue","sequence":"additional","affiliation":[{"name":"e Department of Microbiology, Australian Laboratory Services, Scoresby, Victoria 3179, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christelle","family":"Schang","sequence":"additional","affiliation":[{"name":"c Environmental and Public Health Microbiology Laboratory (EPHM Lab), Department of Civil Engineering, Monash University, Clayton, Victoria 3800, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shane P.","family":"Usher","sequence":"additional","affiliation":[{"name":"f Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Louise","family":"Baker","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christine","family":"Kaucner","sequence":"additional","affiliation":[{"name":"e Department of Microbiology, Australian Laboratory Services, Scoresby, Victoria 3179, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul","family":"Monis","sequence":"additional","affiliation":[{"name":"g Environment and Wastewater, Australian Water Quality Centre, SA Water, Adelaide, South Australia 5000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leon M.","family":"Hartman","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brianna","family":"Flynn","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James E.","family":"Merrett","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Lister","sequence":"additional","affiliation":[{"name":"h Department of Health, State Government of Victoria, Melbourne, Victoria 3000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marco","family":"Herold","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"},{"name":"i School of Cancer Medicine, Olivia Newton-John Cancer Research Institute, La Trobe University, Heidelberg, Victoria 3084, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrew","family":"Kueh","sequence":"additional","affiliation":[{"name":"a Infection and Global Health, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia"},{"name":"i School of Cancer Medicine, Olivia Newton-John Cancer Research Institute, La Trobe University, Heidelberg, Victoria 3084, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ngai Ning","family":"Cheng","sequence":"additional","affiliation":[{"name":"g Environment and Wastewater, Australian Water Quality Centre, SA Water, Adelaide, South Australia 5000, Australia"},{"name":"h Department of Health, State Government of Victoria, Melbourne, Victoria 3000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jackson","family":"Nolan","sequence":"additional","affiliation":[{"name":"h Department of Health, State Government of Victoria, Melbourne, Victoria 3000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Leon","family":"Caly","sequence":"additional","affiliation":[{"name":"j Victorian Infectious Diseases Reference Laboratory, Peter Doherty Institute for Infection and Immunity, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julian","family":"Druce","sequence":"additional","affiliation":[{"name":"j Victorian Infectious Diseases Reference Laboratory, Peter Doherty Institute for Infection and Immunity, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruce","family":"Thorley","sequence":"additional","affiliation":[{"name":"j Victorian Infectious Diseases Reference Laboratory, Peter Doherty Institute for Infection and Immunity, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter J.","family":"Scales","sequence":"additional","affiliation":[{"name":"f Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jonathan","family":"Schmidt","sequence":"additional","affiliation":[{"name":"k South East Water Corporation, Frankston, Victoria 3199, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Suzie","family":"Sarkis","sequence":"additional","affiliation":[{"name":"h Department of Health, State Government of Victoria, Melbourne, Victoria 3000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicholas D.","family":"Crosbie","sequence":"additional","affiliation":[{"name":"l Recycled Water and Integrated Water Management, Melbourne Water Corporation, Docklands, Victoria 3001, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachael","family":"Poon","sequence":"additional","affiliation":[{"name":"h Department of Health, State Government of Victoria, Melbourne, Victoria 3000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monica","family":"Nolan","sequence":"additional","affiliation":[{"name":"h Department of Health, State Government of Victoria, Melbourne, Victoria 3000, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"945","published-online":{"date-parts":[[2025,1,21]]},"reference":[{"key":"2025022714114399400_JWH-D-24-00164C1","doi-asserted-by":"crossref","first-page":"139960","DOI":"10.1016\/j.scitotenv.2020.139960","article-title":"Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater","volume":"739","year":"2020","journal-title":"Sci. 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Dis."},{"key":"2025022714114399400_JWH-D-24-00164C5","unstructured":"Bannert\n            M.\n          , ThoeniS. & BisingerS. (2022) tstools: A Time Series Toolbox for Official Statistics. Available at: https:\/\/CRAN.R-project.org\/package=tstools."},{"key":"2025022714114399400_JWH-D-24-00164C6","doi-asserted-by":"crossref","first-page":"147469","DOI":"10.1016\/j.scitotenv.2021.147469","article-title":"Epidemiological evaluation of sewage surveillance as a tool to detect the presence of COVID-19 cases in a low case load setting","volume":"786","year":"2021","journal-title":"Sci. Total Environ."},{"key":"2025022714114399400_JWH-D-24-00164C7","doi-asserted-by":"crossref","first-page":"120804","DOI":"10.1016\/j.watres.2023.120804","article-title":"Utility of wastewater genomic surveillance compared to clinical surveillance to track the spread of the SARS-CoV-2 Omicron variant across England","volume":"247","year":"2023","journal-title":"Water Res."},{"issue":"Pt 4","key":"2025022714114399400_JWH-D-24-00164C8","first-page":"150816","article-title":"SARS-CoV-2 RNA in urban wastewater samples to monitor the COVID-19 pandemic in Lombardy, Italy (March\u2013June 2020)","volume":"806","year":"2022","journal-title":"Sci. Total Environ."},{"key":"2025022714114399400_JWH-D-24-00164C9","doi-asserted-by":"crossref","first-page":"101947","DOI":"10.1016\/j.jwpe.2021.101947","article-title":"Approaches applied to detect SARS-CoV-2 in wastewater and perspectives post-COVID-19","volume":"40","year":"2021","journal-title":"J. Water Proc. 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Total Environ."},{"issue":"1","key":"2025022714114399400_JWH-D-24-00164C16","doi-asserted-by":"crossref","first-page":"17870","DOI":"10.1038\/s41598-023-44500-0","article-title":"Regional reemergence of a SARS-CoV-2 Delta lineage amid an Omicron wave detected by wastewater sequencing","volume":"13","year":"2023","journal-title":"Sci. Rep."},{"issue":"7","key":"2025022714114399400_JWH-D-24-00164C17","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1038\/s41551-021-00760-7","article-title":"CRISPR-based diagnostics","volume":"5","year":"2021","journal-title":"Nat. Biomed. 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