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Background: Positive correlations have been reported between wastewater SARS-CoV-2 concentrations and a community's burden of infection, disease or both. However, previous studies mostly compared wastewater to clinical case counts or nonrepresentative convenience samples, limiting their quantitative potential. Objectives: This study examined whether wastewater SARS-CoV-2 concentrations could provide better estimations for SARS-CoV-2 community prevalence than reported cases of COVID-19. In addition, this study tested whether wastewater-based epidemiology methods could identify neighborhood-level COVID-19 hotspots and SARS-CoV-2 variants. Methods: Community SARS-CoV-2 prevalence was estimated from eight randomized door-to-door nasal swab sampling events in six Oregon communities of disparate size, location, and demography over a 10-month period. Simultaneously, wastewater SARS-CoV-2 concentrations were quantified at each community's wastewater treatment plant and from 22 Newport, Oregon, neighborhoods. SARS-CoV-2 RNA was sequenced from all positive wastewater and nasal swab samples. Clinically reported case counts were obtained from the Oregon Health Authority. Results: Estimated community SARS-CoV-2 prevalence ranged from 8 to 1,687/10,000 persons. Community wastewater SARS- CoV-2 concentrations ranged from 2.9 to 5.1 [log.sub.10] gene copies per liter. Wastewater SARS-CoV-2 concentrations were more highly correlated (Pearson's r = 0.96; [R.sup.2] = 0.91) with community prevalence than were clinically reported cases of COVID-19 (Pearson's r = 0.85; [R.sup.2] = 0.73). Monte Carlo simulations indicated that wastewater SARS-CoV-2 concentrations were significantly better than clinically reported cases at estimating prevalence (p < 0.05). In addition, wastewater analyses determined neighborhood-level COVID-19 hot spots and identified SARS-CoV-2 variants (B.1 and B.1.399) at the neighborhood and city scales. Discussion: The greater reliability of wastewater SARS-CoV-2 concentrations over clinically reported case counts was likely due to systematic biases that affect reported case counts, including variations in access to testing and underreporting of asymptomatic cases. With these advantages, combined with scalability and low costs, wastewater-based epidemiology can be a key component in public health surveillance of COVID-19 and other communicable infections. https://doi.org/10.1289/EHP10289, Introduction Wastewater-based epidemiology (WBE) has emerged as an effective and sensitive approach for monitoring COVID-19 presence in a community through the detection of the novel coronavirus (SARS-CoV-2) shed by infected [...]

Microbial source tracking and a mass balance approach were used to identify sources of fecal indicator bacteria (FIB) in the Hanalei River, Kaua'i, Hawai'i. Historically, concentrations enterococci and Clostridium perfringens were significantly higher during storm flows compared to non-storm flows in the Hanalei River, and correlated to total suspended solids in the river. During targeted dry weather studies, the Hanalei River bed sediments and streambank soils were documented to harbor E. coli, enterococci, and the human- and pig-specific fecal markers in Bacteroidales, suggesting that sediments and soils may be potential sources of these microorganisms to the Hanalei river. The human-specific marker in Bacteroidales was four times as likely to be detected in sediment and soil samples as in water samples. Furthermore, the occurrence of host-specific source tracking markers is indicative that a portion of FIB present in the Hanalei River are of fecal origin. A mass balance approach was used to explore causes of observed FIB loadings and losses along different reaches of the river. Resuspension or deposition of FIB-laden river sediments cannot account for changes in E. coli and enterococci concentrations along the river during dry weather. Additionally, losses due to bacterial inactivation were insignificant. Groundwater and ditches draining agricultural and urban lands were shown to provide sufficient FIB fluxes to account for the observed loads along some river reaches. The presence of the human-specific Bacteroidales marker in the river water, sediments and adjacent soils, as well as the presence of the human enterovirus marker in the water, suggests that there is widespread human fecal contamination in the Hanalei River that is likely a result of nearby wastewater disposal systems. [ABSTRACT FROM AUTHOR]

Fecal indicator bacteria (FIB) are nearly ubiquitous in California (CA) beach sands. Sands were collected from 55 beaches along the CA coast. Ninety-one percent of the beaches had detectable enterococci (ENT) while 62% had detectable E. coli (EC) in their sands. The presence of a putative bacterial source (such as a river), the degree of wave shelter, and surrounding land use explained a significant (p < 0.05) fraction of the variation in both ENT and EC densities between beaches. Sand characteristics including moisture content, organic carbon, and percent fines, significantly (p < 0.05) influenced only EC densities in beach sand. We assayed 34 of 163 sand samples for salmonellae, but did not detect this bacterial pathogen. The potential for FIB to be transported from the sand to sea was investigated at a single wave-sheltered beach with high densities of ENT in beach sand: Lovers Point, CA (LP). We collected samples of exposed and submerged sands as well as water over a 24 h period in order to compare the disappearance or appearance of ENT in sand and the water column. Exposed sands had significantly higher densities of ENT than submerged sands with the highest densities located near the high tide line. Water column ENT densities began low, increased sharply during the first flood tide and slowly decreased over the remainder of the study. During the first flood tide, the number of ENT that entered the water column was nearly equivalent to the number of ENT lost from exposed sands when they were submerged by seawater. The decrease in nearshore ENT concentrations after the initial influx can be explained by ENT die-off and dilution with clean ocean water. While some ENT in the water and sand at LP might be of human origin because they were positive for the esp gene, others lacked the esp gene and were therefore equivocal with respect to their origin. Follow-up sampling at LP revealed the presence of the human specific Bacteroides marker in water and sand. [ABSTRACT FROM AUTHOR]

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods. [ABSTRACT FROM AUTHOR]

Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training. [ABSTRACT FROM AUTHOR]

Multiple studies worldwide have confirmed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected in wastewater. However, there is a lack of data directly comparing the wastewater SARS-CoV-2 RNA concentration with the prevalence of coronavirus disease 2019 (COVID-19) in individuals living in sewershed areas. Here, we correlate wastewater SARS-CoV-2 signals with SARS-CoV-2 positivity rates in symptomatic and asymptomatic individuals and compare positivity rates in two underserved communities in Portland, Oregon to those reported in greater Multnomah County. 403 individuals were recruited via two COVID-19 testing sites over a period of 16 weeks. The weekly SARS-CoV-2 positivity rate in our cohort ranged from 0 to 21.7% and trended higher than symptomatic positivity rates reported by Multnomah County (1.9-8.7%). Among the 362 individuals who reported symptom status, 76 were symptomatic and 286 were asymptomatic. COVID-19 was detected in 35 participants: 24 symptomatic, 9 asymptomatic, and 2 of unknown symptomatology. Wastewater testing yielded 0.33-149.9 viral RNA genomic copies/L/person and paralleled community COVID-19 positive test rates. In conclusion, wastewater sampling accurately identified increased SARS-CoV-2 within a community. Importantly, the rate of SARS-CoV-2 positivity in underserved areas is higher than positivity rates within the County as a whole, suggesting a disproportionate burden of SARS-CoV-2 in these communities., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)