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

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 – January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance: Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 – January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

We compared SARS-CoV-2 RNA concentrations in primary settled solids and raw wastewater samples matched in date to investigate the relationship between the two matrices.

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 - January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance: Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 - January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

In this study, the aging of culturable FIB and DNA representing genetic markers for Enterococcus spp. (ENT1A), general Bacteroides (GB3), and human-associated Bacteroides (HF183) in freshwater sediments was evaluated. Freshwater sediment was collected from four different sites within the upper and lower reach of the Topanga Creek Watershed and two additional comparator sites within the Santa Monica Bay, for a total of six sites. Untreated (ambient) and oven-dried (reduced microbiota) sediment was inoculated with 5% sewage and artificial freshwater. Microcosms were held for a 21-day period and sampled on day 0, 1, 3, 5, 7, 12, and 21. There were substantial differences in decay among the sediments tested, and decay rates were related to sediment characteristics. In the ambient sediments, smaller particle size and higher levels of organic matter and nutrients (nitrogen and phosphorus) were associated with increased persistence of the GB3 marker and culturable Escherichia coli (cEC) and enterococci (cENT). The HF183 marker exhibited decay rates of -0.50 to -0.96 day, which was 2-5 times faster in certain ambient sediments than decay of culturable FIB and the ENT1A and GB3 markers. The ENT1A and GB3 markers decayed at rates of between -0.07 and -0.28 and -0.10 to -0.44 day, and cEC and cENT decayed at rates of between -0.22 and -0.81 and -0.03 and -0.40 day, respectively. In the oven-dried sediments, increased persistence of all indicators and potential for limited growth of culturable FIB and the GB3 and ENT1A markers was observed. A simplified two-box model using the HF183 marker and cENT decay rates generated from the microcosm experiments was applied to two reaches within the Topanga Canyon watershed in order to provide context for the variability in decay rates observed. The model predicted lower ambient concentrations of enterococci in sediment in the upper (90 MPN g) versus lower Topanga watershed (530 MPN g) and low ambient levels of the HF183 marker (below the LLOQ) in sediments in both lower and upper watersheds. It is important to consider the variability in the persistence of genetic markers and FIB when evaluating indicators of fecal contamination in sediments, even within one watershed.