LAUSR

ABSTRACT Wastewater testing can be used to monitor SARS-CoV-2 infections in communities. Data from PCR-based wastewater testing are usually available to public health authorities within 5–7 days after excreta and other body fluids enter the sewer. While PCR-based methods can accurately detect and quantify SARS-CoV-2, sequencing-based methods are usually required to distinguish between variants, delaying the results and adding cost to the process. We developed and assessed a novel, customizable digital PCR (dPCR)-based genotyping method for SARS-CoV-2 variant detection in wastewater, which is more cost-effective, faster, and more accessible than sequencing. This approach was applied to more than 1,400 wastewater samples collected from six states between April 2023 and May 2024, with results displayed on a public dashboard alongside clinical data from the same period. The wastewater dPCR-based method effectively detected emerging variants, mirroring trends observed in clinical settings and in data on the Global Initiative on Sharing All Influenza Data (GISAID) platform; this method also provided early warning signals, as variants like EG.5 and FL were identified in wastewater before clinical detection. A subset of the wastewater samples was analyzed using both dPCR genotyping and sequencing, with good agreement between the two methods. The range of concordance for four different variants was between 62% and 98%. The successful development and implementation of this dPCR-based genotyping for wastewater samples demonstrates its cost-effectiveness and scalability. With the decline in clinical testing, wastewater surveillance becomes increasingly vital for monitoring SARS-CoV-2 variants and supplementing clinical surveillance efforts. IMPORTANCE As clinical specimens are collected and analyzed less for SARS-CoV-2, variant detection in wastewater provides a readily accessible and rich source of information on SARS-CoV-2 evolution. The detection of pathogen targets in wastewater samples using PCR assays is a sensitive, cost-effective way to monitor the levels of infectious diseases, like SARS-CoV-2, in a community. Unfortunately, because PCR-based methods are typically not used to distinguish between viral variants, most wastewater testing labs must rely on more expensive, time-consuming, and resource-intensive sequencing methods for these results. Building upon recent developments for variant detection using quantitative PCR, we developed and assessed a novel, customizable digital PCR-based genotyping method for SARS-CoV-2 variant detection in wastewater, which is more rapid, cost-effective, and accessible than sequencing. We characterize the method and offer insights for improvement in future implementations. As clinical specimens are collected and analyzed less for SARS-CoV-2, variant detection in wastewater provides a readily accessible and rich source of information on SARS-CoV-2 evolution. The detection of pathogen targets in wastewater samples using PCR assays is a sensitive, cost-effective way to monitor the levels of infectious diseases, like SARS-CoV-2, in a community. Unfortunately, because PCR-based methods are typically not used to distinguish between viral variants, most wastewater testing labs must rely on more expensive, time-consuming, and resource-intensive sequencing methods for these results. Building upon recent developments for variant detection using quantitative PCR, we developed and assessed a novel, customizable digital PCR-based genotyping method for SARS-CoV-2 variant detection in wastewater, which is more rapid, cost-effective, and accessible than sequencing. We characterize the method and offer insights for improvement in future implementations.