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The coastal, unconfined aquifer at Huntington Beach, California contains saline groundwater (33 psu) adjacent to the water line, and a brackish groundwater (BGW) mixing zone (3-9 psu) approximately 50 m from the water line. According to salinity and water isotope analyses, the BGW composition varies spatially in the alongshore direction. Measurements obtained from two BGW wells indicate this water is a mixture of seawater and a freshwater end member (likely infiltrated runoff); results from a third more southerly well suggest the BGW is composed of seawater, freshwater, and water from the confined Talbert Aquifer. Saline groundwater, on the other hand, shares similar salinity and water isotopic composition with seawater. The saline groundwater is enriched with short- and long-lived radium isotopes relative to the surf zone, which in turn is enriched relative to waters further offshore. We derive eddy diffusion coefficients for the nearshore using 223Ra and 224Ra, and in conjunction with 226Ra activities, estimate a submarine groundwater discharge (SGD) rate between 4.2 and 8.9 L min-1m -1 of shoreline, respectively. Using analytical models, we estimate that between 74 and 100% of this discharge represents seawater than has been forced by tides and waves through the beach aquifer. Under one set of model assumptions, results indicate that a portion (up to 56%) of seawater pumped into the beach aquifer by waves and tides is not discharged suggesting salt water intrusion may be occurring. Because saline groundwater is enriched with dissolved inorganic nitrogen and soluble reactive phosphate relative to the coastal ocean, our results suggest that tidally and wave-driven seawater circulated through the beach aquifer represents an important mechanism for nutrient input to the nearshore environment in dry weather. Estimates of nutrient flux to the coastal ocean via SGD along 16-17 km of shoreline are equivalent to nutrient fluxes from two salt water wetlands near the study site during the dry season. © 2005 Elsevier Ltd. All rights reserved.

Elevated levels of enterococci bacteria, an indicator of fecal pollution, are routinely detected in the surf zone at Huntington State and City Beaches in southern California. A multidisciplinary study was carried out to identify sources of enterococci bacteria landward of the coastline. We find that enterococci bacteria are present at high concentrations in urban runoff, bird feces, marsh sediments, and on marine vegetation. Surprisingly, urban runoff appears to have relatively little impact on surf zone water quality because of the long time required for this water to travel from its source to the ocean. On the other hand, enterococci bacteria generated in a tidal saltwater marsh located near the beach significantly impact surf zone water quality. This study identifies a potential tradeoff between restoring coastal wetlands and protecting beach water quality and calls into question the use of ocean bathing water standards based on enterococci at locations near coastal wetlands.

Wastewater-based epidemiology, which seeks to assess disease occurrence in communities through measurements of infectious disease biomarkers in wastewater, may represent a valuable tool for understanding the occurrence of hepatitis A infections in communities. In this study, we measured concentrations of Hepatovirus A (HAV) RNA, in samples from 191 wastewater treatment plants spanning 40 US states and the District of Columbia from September 2023 to June 2024 and compared the measurements with traditional measures of disease occurrence. Nationally, 13.76% of the 21,079 wastewater samples were positive for HAV RNA, and both concentrations and positivity rates were associated with NNDSS hepatitis A case data nationally (Kendall rank correlation coefficient = 0.20, concentrations; and 0.33, positivity rate; both P < 0.05). We further demonstrated that higher rates of wastewater HAV detection were positively associated with socioeconomic indicators of vulnerability including homelessness and drug overdose deaths (both P < 0.0001). Areas with above average levels of homelessness were 48% more likely to have HAV wastewater detections, while areas with above average levels of drug overdose deaths were 14% more likely to have HAV wastewater detections. Using more granular case data, we present a case study in the state of Maine that reinforces these results and suggests a potential lead time for wastewater over clinical case detection and exposure events. The ability to detect HAV RNA in wastewater before clinical cases emerge could allow public health officials to implement targeted interventions like vaccination campaigns.IMPORTANCEDespite the existence of a highly effective vaccine for hepatitis A, outbreaks in vulnerable populations remain common. The disease can be asymptomatic or subclinical, and disproportionately impacts populations with inadequate access to healthcare, leading to a severe underestimation of the occurrence of this viral infection. This study investigates the potential for wastewater measurements of biomarkers of the causative agent of hepatitis A (HAV RNA) to provide insights into disease occurrence. Results highlight the potential for wastewater-based epidemiology to be a complementary tool to traditional surveillance for monitoring and controlling HAV transmission., Competing Interests: The authors declare no conflict of interest.

Wastewater-Based Epidemiology (WBE) has become a powerful tool for assessing disease occurrence in communities. This study investigates the coronavirus disease 2019 (COVID-19) epidemic in the United States during 2023-2024 using wastewater data from 189 wastewater treatment plants in 40 states and the District of Columbia. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pepper-mild mottle virus normalized SARS-CoV-2 RNA concentration data were compared with COVID-19 hospitalization admission data at both national and state levels. We further investigate temporal features in wastewater viral RNA abundance, with peak timing and cross-correlation lag analyses indicating that wastewater SARS-CoV-2 RNA concentrations precede hospitalization admissions by 2 to 12 days. Lastly, we demonstrate that wastewater treatment plant size has a significant effect on the variability of measured SARS-CoV-2 RNA concentrations. This study highlights the effectiveness of WBE as a non-invasive, timely and resource-efficient disease monitoring strategy, especially in the context of declining COVID-19 clinical reporting., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schenk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Wastewater surveillance for infectious agents has proved useful in identifying the circulation of viruses within populations. We investigated the presence and concentration of human immunodeficiency virus (HIV)-1 total nucleic acids (including both viral RNA and proviral DNA) in wastewater solids. We retrospectively measured HIV-1 nucleic acids in two samples per week for 26 months at two wastewater treatment plants serving populations with different prevalences of HIV infections in San Francisco and Santa Clara County, California, USA. We detected HIV nucleic acids in a majority of samples with concentrations ranging from non-detect to 3.9 × 10 5 cp/g ( N = 459 samples total). Concentrations of HIV-1 were significantly higher in samples from the wastewater treatment plant serving a population with a higher prevalence of people living with HIV than in the plant serving a population with a lower prevalence. The HIV-1 nucleic acids amplified were primarily DNA and thus represented proviral DNA shedding into wastewater. Additionally, we found that HIV-1 nucleic acid concentrations in wastewater solids were orders of magnitude higher than those in liquid wastewater indicating that the HIV-1 target preferentially sorbs to solids. Whether concentrations of HIV-1 in wastewater solids can be used to identify the number of incident cases remains unknown. Additional work on HIV-1 shedding from individuals with viremia and people living with HIV is needed to translate wastewater measurements into quantitative information on infections. Additional work may also be needed to document non-human sources of HIV-1 nucleic acids in wastewater., Importance: Human immunodeficiency virus (HIV)-1 has infected nearly 100 million people since it emerged in the 1980s. Antiretroviral therapy prevents transmission of HIV and also allows infected individuals to live healthy lives with normal life expectancy. Consequently, identifying unrecognized cases of HIV is of paramount importance. Since wastewater represents a composite biological sample from a community, it may provide valuable information on HIV-1 prevalence that can be used to inform HIV testing and outreach.

Norovirus is a leading cause of acute gastroenteritis and imposes a substantial disease burden. In California, USA, norovirus surveillance is limited. We evaluated correlations between wastewater norovirus concentrations and available public health surveillance data. Wastewater surveillance for norovirus genotype GII in California provided timely, localized, and actionable data for public health authorities.

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 (" E. coli ") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day -1 in the marine waters and -0.89 to -3.2 day -1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators., Competing Interests: The authors declare no conflict of interest.

We measured concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, influenza A and B viruses, respiratory syncytial virus, human metapneumovirus, enterovirus D68, human parainfluenza types 1, 2, 3, 4a, and 4b in aggregate, norovirus genotype II, rotavirus, Candida auris, hepatitis A virus, human adenovirus, mpox virus, H5 influenza A virus, and pepper mild mottle virus nucleic acids in wastewater solids prospectively at 191 wastewater treatment plants in 40 states across the United States plus Washington DC. Measurements were made two to seven times per week from 1 January 2022 to 30 June 2024, depending on wastewater treatment plant staff availability. Measurements were made using droplet digital (reverse-transcription-) polymerase chain reaction (ddRT-PCR) following best practices for making environmental molecular biology measurements. These data can be used to better understand disease occurrence in communities contributing to the wastewater., (© 2024. The Author(s).)

As part of the response to the highly pathogenic avian influenza A(H5N1) virus outbreak in U.S. cattle and poultry and the associated human cases, CDC and partners are monitoring influenza A virus levels and detection of the H5 subtype in wastewater. Among 48 states and the District of Columbia that performed influenza A testing of wastewater during May 12-July 13, 2024, a weekly average of 309 sites in 38 states had sufficient data for analysis, and 11 sites in four states reported high levels of influenza A virus. H5 subtype testing was conducted at 203 sites in 41 states, with H5 detections at 24 sites in nine states. For each detection or high level, CDC and state and local health departments evaluated data from other influenza surveillance systems and partnered with wastewater utilities and agriculture departments to investigate potential sources. Among the four states with high influenza A virus levels detected in wastewater, three states had corresponding evidence of human influenza activity from other influenza surveillance systems. Among the 24 sites with H5 detections, 15 identified animal sources within the sewershed or adjacent county, including eight milk-processing inputs. Data from these early investigations can help health officials optimize the use of wastewater surveillance during the upcoming respiratory illness season., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. John Anderson reports support from the Council of State and Territorial Epidemiologists (CSTE) for travel to the 2023 Advanced Molecular Detection Days and the 2024 Advanced Molecular Detection Academy. Alexandria B. Boehm reports institutional support from the Sergey Brin Family Foundation and grant support from the U.S. National Science Foundation for research coordination network for wastewater-based epidemiology and from the Sloan Foundation for public health partnerships and wastewater-based epidemiology, and membership on the state of California wastewater-based epidemiology committee of the State Water Board. Ruth Lynfield reports support from the Infectious Diseases Society of America, CSTE, the National Foundation for Infectious Diseases, and the American Academy of Pediatrics to attend state epidemiology meetings, ID Week, CSTE Board meetings and CSTE annual conference, National Foundation for Infectious Diseases meeting, and the Committee on Infectious Diseases meeting. Marlene K. Wolfe reports a subaward gift to Stanford University, a grant from the Rockefeller Foundation for implementation of wastewater-based epidemiology in Bangladesh and Ghana and a subaward from Ceres Nanosciences (subaward from National Institutes of Health funding) for wastewater monitoring research, and support from the American Society of Microbiology for an invited talk at a conference. No other potential conflicts of interest were disclosed.

Candida auris is an emerging, multidrug-resistant fungal pathogen that poses a significant public health threat in healthcare settings. Despite yearly clinical cases rapidly increasing from 77 to 8,131 in the last decade, surveillance data on its distribution and prevalence remain limited. We implemented a novel assay for C. auris detection on a nationwide scale prospectively from September 2023 to March 2024, analyzing a total of 13,842 samples from 190 wastewater treatment plants across 41 U.S. states. Assays were extensively validated through comparison to other known assays and internal controls. Of these 190 wastewater treatment plants, C. auris was detected in the wastewater solids of 65 of them (34.2%) with 1.45% of all samples having detectable levels of C. auris nucleic-acids. Detections varied seasonally, with 2.00% of samples positive in autumn vs 1.01% in winter ( P < 0.0001). The frequency of detection in wastewater was significantly associated with states having older populations ( P < 0.001), sewersheds containing more hospitals ( P < 0.0001), and sewersheds containing more nursing homes ( P < 0.001). These associations are in agreement with known C. auris epidemiology. This nationwide study demonstrates the viability of wastewater surveillance for C. auris surveillance and further highlights the value of wastewater surveillance when clinical testing is constrained., Importance: This study highlights the viability of wastewater surveillance when dealing with emerging pathogens. By leveraging an existing framework of wastewater surveillance, we reveal the widespread presence of C. auris in the United States. We further demonstrate that these wastewater detections are consistent with demographic factors relevant to C. auris epidemiology like age and number of hospitals or nursing homes. As C. auris and other pathogens continue to emerge, the low-cost and rapid nature of wastewater surveillance will provide public health officials with the information necessary to enact targeted prevention and control strategies., Competing Interests: D.D., X.-R.S.X., B.J.W., and B.S. are employees of Verily Life Sciences.

Respiratory syncytial virus (RSV) causes a large burden of respiratory illness globally. It has two subtypes, RSV A and RSV B, but little is known regarding the predominance of these subtypes during different seasons and their impact on morbidity and mortality. Using molecular methods, we quantified RSV A and RSV B RNA in wastewater solids across multiple seasons and metropolitan areas to gain insight into the predominance of RSV subtypes. We determined the predominant subtype for each group using the proportion of RSV A to total RSV (RSV A + RSV B) in each wastewater sample ( P A,WW ) and conducted a comparative analysis temporally, spatially, and against clinical specimens. A median P A,WW of 0.00 in the first season and 0.58 in the second season indicated a temporal shift in the predominant subtype. Spatially, while we observed dominance of the same subtype, P A,WW was higher in some areas ( P A,WW = 0.58-0.88). The same subtype predominated in wastewater and clinical samples, but clinical samples showed higher levels of RSV A (RSV A positivity in clinical samples = 0.79, median P A,WW = 0.58). These results suggest that wastewater, alongside clinical data, holds promise for enhanced subtype surveillance.IMPORTANCERespiratory syncytial virus (RSV) causes a large burden of respiratory illness globally. It has two subtypes, RSV A and RSV B, but little is known regarding the predominance of these subtypes during different seasons and their impact on morbidity and mortality. The study illustrates that information on subtype predominance can be gleaned from wastewater. As a biological composite sample from the entire contributing population, wastewater monitoring of RSV A and B can complement clinical surveillance of RSV., Competing Interests: The authors declare no conflict of interest.

Competing Interests: The authors declare no competing financial interest.

Background: Human norovirus (HuNoV) is a leading cause of disease globally, yet actual incidence is unknown. HuNoV infections are not reportable in the United States, and surveillance is limited to tracking severe illnesses or outbreaks. Wastewater monitoring for HuNoV has been done previously and results indicate it is present in wastewater influent and concentrations are associated with HuNoV infections in the communities contributing to wastewater. However, work has mostly been limited to monthly samples of liquid wastewater at one or a few wastewater treatment plants (WWTPs)., Objective: The objectives of this study are to investigate whether HuNoV GII preferentially adsorbs to wastewater solids, investigate concentrations of HuNoV GII in wastewater solids in wastewater treatment plants across the county, and explore how those relate to clinical measures of disease occurrence. In addition, we aim to develop and apply a mass-balance model that predicts the fraction of individuals shedding HuNoV in their stool based on measured concentrations in wastewater solids., Methods: We measured HuNoV GII RNA in matched wastewater solids and liquid influent in 7 samples from a WWTP. We also applied the HuNoV GII assay to measure viral RNA in over 6000 wastewater solids samples from 145 WWTPs from across the United States daily to three times per week for up to five months. Measurements were made using digital droplet RT-PCR., Results: HuNoV GII RNA preferentially adsorbs to wastewater solids where it is present at 1000 times the concentration in influent. Concentrations of HuNoV GII RNA correlate positively with clinical HuNoV positivity rates. Model output of the fraction of individuals shedding HuNoV is variable and uncertain, but consistent with indirect estimates of symptomatic HuNoV infections in the United States., Impact Statement: Illness caused by HuNoV is not reportable in the United States so there is limited data on disease occurrence. Wastewater monitoring can provide information about the community spread of HuNoV. Data from wastewater can be available within 24 h of sample receipt at a laboratory. Wastewater is agnostic to whether individuals seek medical care, are symptomatic, and the severity of illness. Knowledge gleaned from wastewater may be used by public health professionals to make recommendations on hand washing, surface disinfection, or other behaviors to reduce transmission of HuNoV, or medical doctors to inform clinical decision making., (© 2023. The Author(s).)

Enteric infections are important causes of morbidity and mortality, yet clinical surveillance is limited. Wastewater-based epidemiology (WBE) has been used to study community circulation of individual enteric viruses and panels of respiratory diseases, but there is limited work studying the concurrent circulation of a suite of important enteric viruses. A retrospective WBE study was carried out at two wastewater treatment plants located in California, United States. Using digital droplet polymerase chain reaction (PCR), we measured concentrations of human adenovirus group F, enteroviruses, norovirus genogroups I and II, and rotavirus nucleic acids in wastewater solids two times per week for 26 months ( n = 459 samples) between February 2021 and mid-April 2023. A novel probe-based PCR assay was developed and validated for adenovirus. We compared viral nucleic acid concentrations to positivity rates for viral infections from clinical specimens submitted to a local clinical laboratory to assess concordance between the data sets. We detected all viral targets in wastewater solids. At both wastewater treatment plants, human adenovirus group F and norovirus GII nucleic acids were detected at the highest concentrations (median concentrations greater than 10 5 copies/g), while rotavirus RNA was detected at the lowest concentrations (median on the order of 10 3 copies/g). Rotavirus, adenovirus group F, and norovirus nucleic acid concentrations were positively associated with clinical specimen positivity rates. Concentrations of tested viral nucleic acids exhibited complex associations with SARS-CoV-2 and other respiratory viral nucleic acids in wastewater, suggesting divergent transmission patterns.IMPORTANCEThis study provides evidence for the use of wastewater solids for the sensitive detection of enteric virus targets in wastewater-based epidemiology programs aimed to better understand the spread of enteric disease at a localized, community level without limitations associated with testing many individuals. Wastewater data can inform clinical, public health, and individual decision-making aimed to reduce the transmission of enteric disease., Competing Interests: B.H., D.D., and B.W. are employees of Verily Life Sciences, LLC.

Background: Quantifying contributions of environmental faecal contamination to child diarrhoea and growth faltering can illuminate causal mechanisms behind modest health benefits in recent water, sanitation, and hygiene (WASH) trials. We aimed to assess associations between environmental detection of enteropathogens and human or animal microbial source tracking markers (MSTM) and subsequent child health outcomes., Methods: In this individual participant data meta-analysis we searched we searched PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus for WASH intervention studies with a prospective design and concurrent control that measured enteropathogens or MSTM in environmental samples, or both, and subsequently measured enteric infections, diarrhoea, or height-for-age Z-scores (HAZ) in children younger than 5 years. We excluded studies that only measured faecal indicator bacteria. The initial search was done on Jan 19, 2021, and updated on March 22, 2023. One reviewer (AM) screened abstracts, and two independent reviewers (AM and RT) examined the full texts of short-listed articles. All included studies include at least one author that also contributed as an author to the present Article. Our primary outcomes were the 7-day prevalence of caregiver-reported diarrhoea and HAZ in children. For specific enteropathogens in the environment, primary outcomes also included subsequent child infection with the same pathogen ascertained by stool testing. We estimated associations using covariate-adjusted regressions and pooled estimates across studies., Findings: Data from nine published reports from five interventions studies, which included 8603 children (4302 girls and 4301 boys), were included in the meta-analysis. Environmental pathogen detection was associated with increased infection prevalence with the same pathogen and lower HAZ (ΔHAZ -0·09 [95% CI -0·17 to -0·01]) but not diarrhoea (prevalence ratio 1·22 [95% CI 0·95 to 1·58]), except during wet seasons. Detection of MSTM was not associated with diarrhoea (no pooled estimate) or HAZ (ΔHAZ -0·01 [-0·13 to 0·11] for human markers and ΔHAZ -0·02 [-0·24 to 0·21] for animal markers). Soil, children's hands, and stored drinking water were major transmission pathways., Interpretation: Our findings support a causal chain from pathogens in the environment to infection to growth faltering, indicating that the lack of WASH intervention effects on child growth might stem from insufficient reductions in environmental pathogen prevalence. Studies measuring enteropathogens in the environment should subsequently measure the same pathogens in stool to further examine theories of change between WASH, faecal contamination, and health. Given that environmental pathogen detection was predictive of infection, programmes targeting specific pathogens (eg, vaccinations and elimination efforts) can environmentally monitor the pathogens of interest for population-level surveillance instead of collecting individual biospecimens., Funding: The Bill & Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office., Competing Interests: Declaration of interests A portion of JK's salary is supported by an unrestricted donation to the London School of Hygiene & Tropical Medicine from Reckitt; this salary support is wholly unrelated to her role in the preparation of this manuscript. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Respiratory syncytial virus (RSV) is a leading cause of respiratory illness and hospitalization, but clinical surveillance detects only a minority of cases. Wastewater surveillance could determine the onset and extent of RSV circulation in the absence of sensitive case detection, but to date, studies of RSV in wastewater are few. We measured RSV RNA concentrations in wastewater solids from 176 sites during the 2022-2023 RSV season and compared those to publicly available RSV infection positivity and hospitalization rates. Concentrations ranged from undetectable to 10 7 copies per gram. RSV RNA concentration aggregated at state and national levels correlated with infection positivity and hospitalization rates. RSV season onset was determined using both wastewater and clinical positivity rates using independent algorithms for 14 states where both data were available at the start of the RSV season. In 4 of 14 states, wastewater and clinical surveillance identified RSV season onset during the same week; in 3 states, wastewater onset preceded clinical onset, and in 7 states, wastewater onset occurred after clinical onset. Wastewater concentrations generally peaked in the same week as hospitalization rates but after case positivity rates peaked. Differences in onset and peaks in wastewater versus clinical data may reflect inherent differences in the surveillance approaches., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

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.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs., Competing Interests: RUH is managing member of AquaVitas, LLC and founder of the ASU non-profit project OneWaterOneHealth operating in the same intellectual space. LPS is employed by Ceres Nanosciences.

Modeling the fate and transport of viruses and their genetic material in surface water is necessary to assess risks associated with contaminated surface waters and to inform environmental surveillance efforts. Temperature has been identified as a key variable affecting virus persistence in surface waters, but the effects of the presence of biological and inert particles and of their interaction with temperature have not been well characterized. We assessed these effects on the persistence of human norovirus (HuNoV) genotype II.4 purified from stool and MS2 in surface water. Raw or filter-sterilized creek water microcosms were inoculated and incubated in the dark at 10 °C, 15 °C, and 20 °C. HuNoV ( i.e. , genome segments and intact capsids) and MS2 ( i.e. , infectious MS2, genome segments, and intact capsids) concentrations were followed over 36 days. The range in positive, significant first-order decay rate constants for HuNoV in this study was 0.14 to 0.69 day -1 compared with 0.026 to 0.71 day -1 for that of MS2. Decay rate constants for HuNoV genome segments and infectious MS2 were largest in creek water that included biological and inert particles and incubated at higher temperatures. In addition, for HuNoV and MS2 incubated in raw or filter-sterilized creek water at 15 °C, capsid damage was not identified as a dominant inactivation mechanism. Environmental processes and events that affect surface water biological and inert particles, temperature, or both could lead to variable virus decay rate constants. Incorporating the effects of particles, temperature, and their interaction could enhance models of virus fate and transport in surface water.

Enveloped virus fate in the environment is not well understood; there are no quantitative data on sunlight inactivation of enveloped viruses in water. Herein, we measured the sunlight inactivation of two enveloped viruses (Phi6 and murine hepatitis virus, MHV) and a nonenveloped virus (MS2) over time in clear water with simulated sunlight exposure. We attenuated UV sunlight wavelengths using long-pass 50% cutoff filters at 280, 305, and 320 nm. With the lowest UV attenuation tested, all decay rate constants (corrected for UV light screening, k̂ ) were significantly different from dark controls; the MS2 k̂ was equal to 4.5 m 2 /MJ, compared to 16 m 2 /MJ for Phi6 and 52 m 2 /MJ for MHV. With the highest UV attenuation tested, only k̂ for MHV (6.1 m 2 /MJ) was different from the dark control. Results indicate that the two enveloped viruses decay faster than the nonenveloped virus studied, and k̂ are significantly impacted by UV attenuation. Differences in k̂ may be due to the presence of viral envelopes but may also be related to other differing intrinsic properties of the viruses, including genome length and composition. Reported k̂ values can inform strategies to reduce the risk from exposure to enveloped viruses in the environment.

Influenza A virus (IAV) causes significant morbidity and mortality in the United States and has pandemic potential. Identifying IAV epidemic patterns is essential to inform the timing of vaccinations and nonpharmaceutical interventions. In a prospective, longitudinal study design, we measured IAV RNA in wastewater settled solids at 163 wastewater treatment plants across 33 states to characterize the 2022-2023 influenza season at the state, health and human services (HHS) regional, and national scales. Influenza season onset, offset, duration, peak, and intensity using IAV RNA in wastewater were compared with those determined using laboratory-confirmed influenza hospitalization rates and outpatient visits for influenza-like illness (ILI). The onset for HHS regions as determined by IAV RNA in wastewater roughly corresponded with those determined using ILI when the annual geometric mean of IAV RNA concentration was used as a baseline (i.e., the threshold that triggers onset), although offsets between the two differed. IAV RNA in wastewater provided early warning of onset, compared to the ILI estimate, when the baseline was set at twice the limit of IAV RNA detection in wastewater. Peak when determined by IAV RNA in wastewater generally preceded peak determined by IAV hospitalization rate by 2 weeks or less. IAV RNA in wastewater settled solids is an IAV-specific indicator that can be used to augment clinical surveillance for seasonal influenza epidemic timing and intensity.

Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Martin Shafer reports serving on several Association of Public Health Laboratories’ advisory boards that support wastewater surveillance practice. Sandra L. McClellan reports service on the National Academies of Science, Engineering, and Medicine Community Wastewater-based Infectious Disease Surveillance Committee. Dagmara Antkiewicz reports institutional support from Epidemiology and Laboratory Capacity, Wastewater Surveillance 2020–2021 National Wastewater Surveillance System Budget Period 2, and Wisconsin Alumni Research Foundation COVID-19 Challenge; and uncompensated board membership on the Midwest Society of Environmental Toxicology and Chemistry. Alexandria B. Boehm reports institutional support from Sergey Brin Family Foundation, grants from the U.S. National Science Foundation, public health partnerships with the Sloan Foundation, and membership on the state of California wastewater-based epidemiology committee of the State Water Board. Bradley White reports contract support from Stanford University. Loren Hopkins reports uncompensated leadership of the National Academies for Science, Engineering, and Mathematics’ Committee on Wastewater. Marlene K. Wolfe reports subaward from a gift to Stanford University, grants for implementation of wastewater-based epidemiology in Bangladesh and Ghana from the Rockefeller Foundation, consulting fees from Verily related to the WastewaterSCAN program led by Stanford University, and conference attendance support from the American Society of Microbiology. Lauren Stadler reports support from the Houston Health Department, grants from the National Science Foundation, Jacobs Engineering Group, Dow Chemical Company, U.S. Department of Agriculture’s National Institute of Food and Agriculture, U.S.–Egypt Joint Science and Technology Fund, Colorado State University, and National Academies of Science, Engineering, and Medicine; and consulting fees from the Royal Society of Chemistry and State Analytics, LLC. No other potential conflicts of interest were disclosed.

Forecasting environmental hazards is critical in preventing or building resilience to their impacts on human communities and ecosystems. Environmental data science is an emerging field that can be harnessed for forecasting, yet more work is needed to develop methodologies that can leverage increasingly large and complex data sets for decision support. Here, we design a data-driven framework that can, for the first time, forecast bacterial standard exceedances at marine beaches with 3 days lead time. Using historical data sets collected at two California sites, we train nearly 400 forecast models using statistical and machine learning techniques and test forecasts against predictions from both a naive "persistence" model and a baseline nowcast model. Overall, forecast models are found to have similar sensitivities and specificities to the persistence model, but significantly higher areas under the ROC curve (a metric distinguishing a model's ability to effectively parse classes across decision thresholds), suggesting that forecasts can provide enhanced information beyond past observations alone. Forecast model performance at all lead times was similar to that of nowcast models. Together, results suggest that integrating the forecasting framework developed in this study into beach management programs can enable better public notification and aid in proactive pollution and health risk management.

In this retrospective study, we measured enterovirus D68 (EV-D68) genomic RNA in wastewater solids longitudinally at 2 California, USA, wastewater treatment plants twice per week for 26 months. EV-D68 RNA was undetectable except when concentrations increased from mid-July to mid-December 2022, which coincided with a peak in confirmed EV-D68 cases.

Despite the widespread adoption of wastewater surveillance, more research is needed to understand the fate and transport of viral genetic markers in wastewater. This information is essential for optimizing monitoring strategies and interpreting wastewater surveillance data. In this study, we examined the solid-liquid partitioning behavior of four viruses in wastewater: SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus (RV), and F+ coliphage/MS2. We used two approaches: (1) laboratory partitioning experiments using lab-grown viruses and (2) distribution experiments using endogenous viruses in raw wastewater. Partition experiments were conducted at 4 and 22 °C. Wastewater samples were spiked with varying concentrations of each virus, solids and liquids were separated via centrifugation, and viral RNA concentrations were quantified using reverse-transcription-digital droplet PCR (RT-ddPCR). For the distribution experiments, wastewater samples were collected from six wastewater treatment plants and processed without spiking exogenous viruses; viral RNA concentrations were measured in wastewater solids and liquids. In both experiments, RNA concentrations were higher in the solid fraction than the liquid fraction by approximately 3-4 orders of magnitude. Partition coefficients ( K F ) ranged from 2000-270,000 mL·g -1 across viruses and temperature conditions. Distribution coefficients ( K d ) were consistent with results from partitioning experiments. Further research is needed to understand how virus and wastewater characteristics might influence the partitioning of viral genetic markers in wastewater.

Wastewater-based epidemiology (WBE) emerged during the coronavirus disease 2019 (COVID-19) pandemic as a scalable and broadly applicable method for community-level monitoring of infectious disease burden. The lack of high-resolution fecal shedding data for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) limits our ability to link WBE measurements to disease burden. In this study, we present longitudinal, quantitative fecal shedding data for SARS-CoV-2 RNA, as well as for the commonly used fecal indicators pepper mild mottle virus (PMMoV) RNA and crAss-like phage (crAssphage) DNA. The shedding trajectories from 48 SARS-CoV-2-infected individuals suggest a highly individualized, dynamic course of SARS-CoV-2 RNA fecal shedding. Of the individuals that provided at least three stool samples spanning more than 14 days, 77% had one or more samples that tested positive for SARS-CoV-2 RNA. We detected PMMoV RNA in at least one sample from all individuals and in 96% (352/367) of samples overall. CrAssphage DNA was detected in at least one sample from 80% (38/48) of individuals and was detected in 48% (179/371) of all samples. The geometric mean concentrations of PMMoV and crAssphage in stool across all individuals were 8.7 × 10 4 and 1.4 × 10 4 gene copies/milligram-dry weight, respectively, and crAssphage shedding was more consistent for individuals than PMMoV shedding. These results provide us with a missing link needed to connect laboratory WBE results with mechanistic models, and this will aid in more accurate estimates of COVID-19 burden in sewersheds. Additionally, the PMMoV and crAssphage data are critical for evaluating their utility as fecal strength normalizing measures and for source-tracking applications. IMPORTANCE This research represents a critical step in the advancement of wastewater monitoring for public health. To date, mechanistic materials balance modeling of wastewater-based epidemiology has relied on SARS-CoV-2 fecal shedding estimates from small-scale clinical reports or meta-analyses of research using a wide range of analytical methodologies. Additionally, previous SARS-CoV-2 fecal shedding data have not contained sufficient methodological information for building accurate materials balance models. Like SARS-CoV-2, fecal shedding of PMMoV and crAssphage has been understudied to date. The data presented here provide externally valid and longitudinal fecal shedding data for SARS-CoV-2, PMMoV, and crAssphage which can be directly applied to WBE models and ultimately increase the utility of WBE., Competing Interests: The authors declare no conflict of interest.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers' sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples., Competing Interests: The authors declare a conflict of interest. A.N., B.J.F., M.K.W., and A.B.B., are co-inventors on a U.S. provisional patent application #63/387,657 that has been filed and relates to the methods presented in this manuscript. The other authors declare no competing interests.

Documenting the occurrence of viruses on fomites is crucial in determining the significance of fomite-mediated transmission and the potential use of fomites for environmental disease surveillance. We conducted a systematic review and meta-analysis to compile information on the occurrence of human viruses on fomites in the environment; we identified 134 peer-reviewed papers. We compiled sampling and measurement methods, results, quality control information, and whether virus data were compared with community health data from the papers. We conducted univariate and multivariate analyses to investigate if presence of virus on fomites was associated with virus type (enveloped, nonenveloped), sampling location (healthcare setting, nonhealthcare temporary setting, nonhealthcare nontemporary setting), and area of fomite swabbed (<50, 50-100, >100 cm 2 ). Across 275 data sets from the 134 papers, there was the most data available for Coronaviridae and from fomites at hospitals. Positivity rates, defined as the percent positive fomite samples, were low (median = 6%). Data were available on viruses from 16 different viral families, but data on viruses from 9 families had few ( n < 5) data sets. Many human virus families were not identified in this review (11 families). Less than 15% of the data sets reported virus concentrations in externally valid units (viruses per area of surface), and 16% provided a quantitative comparison between virus and health data. Virus type and area swabbed were significant predictors of virus presence on fomites, and the positivity rate of data sets collected from healthcare settings and nonhealthcare nontemporary settings (e.g., individual housing) were significantly higher than those collected in nonhealthcare temporary settings (e.g., restaurants). Data from this review indicates that viruses may be present on fomites, that fomite-mediated virus transmission may occur, and that fomites may provide information on circulation of infectious diseases in the community. However, more quantitative data on diverse viruses are needed, and method reporting needs significant improvements., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Wastewater monitoring can provide insights into respiratory disease occurrence in communities that contribute to the wastewater system. Using daily measurements of RNA of influenza A (IAV), respiratory syncytial virus (RSV), and human metapneumovirus (HMPV), as well as SARS-CoV-2 in wastewater solids from eight publicly owned treatment works in the Greater San Francisco Bay Area of California between July 2022 and early July 2023, we identify a "tripledemic" when concentrations of IAV, RSV, and SARS-CoV-2 peaked at approximately the same time. HMPV was also widely circulating. We designed novel hydrolysis probe RT-PCR assays for different IAV subtype markers to discern that the dominant circulating IAV subtype was H3N2. We show that wastewater data can be used to identify the onset and offset of wastewater disease occurrence events. This information can provide insight into disease epidemiology and timely, localized information to inform hospital staffing and clinical decision making to respond to circulating viruses. Whereas RSV and IAV wastewater events were mostly regionally coherent, HMPV events displayed localized occurrence patterns., Competing Interests: The authors declare the following competing financial interest(s): B.W., B.H., and D.D. are employees of Verily Life Sciences, LLC., (© 2023 The Authors. Published by American Chemical Society.)

Introduction: Over a third of the communities (39%) in the Central Valley of California, a richly diverse and important agricultural region, are classified as disadvantaged-with inadequate access to healthcare, lower socio-economic status, and higher exposure to air and water pollution. The majority of racial and ethnic minorities are also at higher risk of COVID-19 infection, hospitalization, and death according to the Centers for Disease Control and Prevention. Healthy Central Valley Together established a wastewater-based disease surveillance (WDS) program that aims to achieve greater health equity in the region through partnership with Central Valley communities and the Sewer Coronavirus Alert Network. WDS offers a cost-effective strategy to monitor trends in SARS-CoV-2 community infection rates., Methods: In this study, we evaluated correlations between public health and wastewater data (represented as SARS-CoV-2 target gene copies normalized by pepper mild mottle virus target gene copies) collected for three Central Valley communities over two periods of COVID-19 infection waves between October 2021 and September 2022. Public health data included clinical case counts at county and sewershed scales as well as COVID-19 hospitalization and intensive care unit admissions. Lag-adjusted hospitalization:wastewater ratios were also evaluated as a retrospective metric of disease severity and corollary to hospitalization:case ratios., Results: Consistent with other studies, strong correlations were found between wastewater and public health data. However, a significant reduction in case:wastewater ratios was observed for all three communities from the first to the second wave of infections, decreasing from an average of 4.7 ± 1.4 over the first infection wave to 0.8 ± 0.4 over the second., Discussion: The decline in case:wastewater ratios was likely due to reduced clinical testing availability and test seeking behavior, highlighting how WDS can fill data gaps associated with under-reporting of cases. Overall, the hospitalization:wastewater ratios remained more stable through the two waves of infections, averaging 0.5 ± 0.3 and 0.3 ± 0.4 over the first and second waves, respectively., Competing Interests: MG, AG, and JC are employed by Eurofins Environment Testing US. BW is employed by Verily Life Sciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kadonsky, Naughton, Susa, Olson, Singh, Daza-Torres, Montesinos-López, Garcia, Gafurova, Gushgari, Cosgrove, White, Boehm, Wolfe, Nuño and Bischel.)

Concentrations of SARS-CoV-2 RNA in wastewater settled solids from publicly owned treatment works (POTWs) historically correlated strongly with laboratory confirmed incident COVID-19 case data. With the increased availability of at-home antigen tests since late 2021 and early 2022, laboratory test availability and test seeking behavior has decreased. In the United States, the results from at-home antigen tests are not typically reportable to public health agencies and thus are not counted in case reports. As a result, the number of reported laboratory-confirmed incident COVID-19 cases has decreased dramatically, even during times of increased test positivity rates and wastewater concentrations of SARS-CoV-2 RNA. Herein, we tested whether the correlative relationship between wastewater concentrations of SARS-CoV-2 RNA and reported laboratory-confirmed COVID-19 incidence rate has changed since 1 May 2022, a point in time immediately before the onset of the BA.2/BA.5 surge, the first surge to begin after at-home antigen test availability was high in the region. We used daily data from three POTWs in the Greater San Francisco Bay Area of California, USA for the analysis. We found that although there is a significant positive association between wastewater measurements and incident rate data collected after 1 May 2022, the parameters describing the relationship are different than those describing the relationship between the data collected prior to 1 May 2022. If laboratory test seeking or availability continues to change, the relationship between wastewater and reported case data will continue to change. Our results suggest, assuming SARS-CoV-2 RNA shedding remains relatively stable among those infected with the virus as different variants emerge, that wastewater concentrations of SARS-CoV-2 RNA can be used to estimate COVID-19 cases as they would have been during the time when laboratory testing availability and test seeking behavior were at a high (here, before 1 May 2022) using the historical relationship between SARS-CoV-2 RNA and COVID-19 case data., Competing Interests: Bradley J. White, Dorothea Duong, and Bridgette Hughes are employees of Verily Life Sciences, LLC. There are no other competing interests., (©2023 Boehm et al.)

We measured concentrations of SARS-CoV-2, influenza A and B virus, respiratory syncytial virus (RSV), mpox virus, human metapneumovirus, norovirus GII, and pepper mild mottle virus nucleic acids in wastewater solids at twelve wastewater treatment plants in Central California, USA. Measurements were made daily for up to two years, depending on the wastewater treatment plant. Measurements were made using digital droplet (reverse-transcription-) polymerase chain reaction (RT-PCR) following best practices for making environmental molecular biology measurements. These data can be used to better understand disease occurrence in communities contributing to the wastewater., (© 2023. The Author(s).)

Fecal indicator bacteria (FIB) are monitored at beaches to assess water quality and associated health risk from recreational exposure. However, monitoring is generally conducted infrequently (i.e. weekly or less often), potentially leading to inaccurate assessment of water quality at a beach at the time of use. While some work has shown that FIB in marine environments can vary over short (e.g. subhourly) time scales, that work has been mainly focused on 'open' beaches. 'Enclosed' beaches-those that are partially barriered from exchange with offshore water and thus have different residence times and mixing dynamics in the nearshore environment-have been less studied. Here we present results from a high-frequency (once per 30 minutes) FIB sampling event conducted within a Central California, USA, harbor over 48 hours. FIB concentrations at this enclosed site were more variable at high-frequencies than what has been reported at open beach sites. Correlation and regression analyses showed FIB concentrations were most strongly associated with chlorophyll a concentration, turbidity, wind speed, and tide level. Results indicate the importance of measuring FIB concentrations and explanatory environmental parameters at appropriate temporal resolutions when conducting water quality monitoring or source tracking studies. Overall, this work highlights how high-frequency sampling can effectively provide information about water quality dynamics at beaches of interest., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Searcy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Concentrations of nucleic acids from a range of respiratory viruses in wastewater solids collected from wastewater treatment plants correlate to clinical data on disease occurrence in the community contributing to the wastewater. Viral nucleic acids enter wastewater from excretions deposited in toilets or drains. To relate measured concentrations in wastewater at a treatment plant to the number of community infections, viral nucleic-acid concentrations in human excretions are needed as inputs to a mass balance model. Here, we carried out a systematic review and meta-analysis to characterize the concentrations and presence of influenza A and B, respiratory syncytial virus, metapneumovirus, parainfluenza virus, rhinovirus, and seasonal coronaviruses in stool, urine, mucus, sputum, and saliva. We identified 220 data sets from 50 articles and reported viral concentrations and presence in these excretions. Data were unevenly distributed across virus type (with the most available for influenza) and excretion type (with the most available for respiratory excretions). Most articles only reported the presence or absence of the virus in a cross-sectional study design. There is a need for more concentration data, including longitudinal data, across all respiratory virus and excretion types. Such data would allow quantitatively linking virus wastewater concentrations to numbers of infected individuals.

Background: Respiratory disease is a major cause of morbidity and mortality; however, surveillance for circulating respiratory viruses is passive and biased. Wastewater-based epidemiology has been used to understand SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV) infection rates at a community level but has not been used to investigate other respiratory viruses. We aimed to use wastewater-based epidemiology to understand community viral respiratory infection occurrence., Methods: A retrospective wastewater-based epidemiology surveillance study was carried out at a large wastewater treatment plant located in California, USA. Using droplet digital RT-PCR, we measured RNA concentrations of influenza A and influenza B viruses, RSV A and RSV B, parainfluenza (1-4) viruses, rhinovirus, seasonal coronaviruses, and metapneumovirus in wastewater solids three times per week for 17 months (216 samples) between Feb 1, 2021, and June 21, 2022. Novel probe-based RT-PCR assays for non-influenza viral targets were developed and validated. We compared viral RNA concentrations to positivity rates for viral infections from clinical specimens submitted to California Sentinel Clinical Laboratories (sentinel laboratories) to assess concordance between the two datasets., Findings: We detected RNA from all tested viruses in wastewater solids. Human rhinovirus (median concentration 4300 [0-9500] copies per gram dry weight) and seasonal human coronaviruses (35 000 [17 000-56 000]) were found at the highest concentrations. Concentrations of viral RNA correlated significantly and positively with positivity rates of associated viral diseases from sentinel laboratories (tau 0·32-0·57, p<0·0009); the only exceptions were influenza B and RSV A, which were rarely detected in wastewater solids. Measurements from wastewater indicated coronavirus OC43 dominated the seasonal human coronavirus infections whereas parainfluenza 3 dominated among parainfluenza infections during the study period. Concentrations of all tested viral RNA decreased noticeably after the omicron BA.1 surge suggesting a connection between changes in human behaviour during the surge and transmission of all respiratory viruses., Interpretation: Wastewater-based epidemiology can be used to obtain information on circulation of respiratory viruses at a localised, community level without the need to test many individuals because a single sample of wastewater represents the entire contributing community. Results from wastewater can be available within 24 h of sample collection, generating real time information to inform public health responses, clinical decision making, and individual behaviour modifications., Funding: CDC Foundation., Competing Interests: Declaration of interests BH, DD, VC-H, AB, and BJW are employees of Verily Life Sciences. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: Handwashing is an important intervention which can reduce indirect disease transmission, however soap and water for handwashing purposes is not available in some low-resource regions. When handwashing with soap and water is not possible, individuals may use alternatives such as the Supertowel (a microfiber towel with an antimicrobial coating). Testing of viral inactivation as a result of antimicrobial treatment on the Supertowel, however, has been limited. The goal of this study is to provide information about the performance of the Supertowel's antimicrobial treatment against viruses, which will help inform the use of the towels as handwashing alternatives., Methods: We seeded the Supertowel and a regular microfiber towel with two bacteriophages (enveloped Phi6 and non-enveloped MS2) and monitored viral inactivation over time. Additionally, we assessed if temperature, humidity, whether the towel was initially wet or dry, or virus type had an effect on viral decay rate constants. Virus concentrations were measured repeatedly over 24 h., Results: We found that neither towel type (whether the towel was a Supertowel or a regular microfiber towel) nor humidity were significant variables in our model of decay rate constants ( P  = 0.06 and P  = 0.22, respectively). We found that the variables of temperature, whether towels were initially wet versus dry, and virus type were significantly different from 0, suggesting that these variables explained variance in the decay rate constant ( P  = 6.55 × 10 -13 , P  = 0.001, and P  < 2 × 10 -16 , respectively). Higher temperatures, dry towels, and enveloped viruses all resulted in increases in the decay rate constant., Conclusions: Viruses seeded onto a Supertowel decay similar to viruses seeded onto a regular towel indicating that the virucidal potential of the Supertowel is minimal., Competing Interests: The authors declare there are no competing interests., (©2023 Anderson et al.)

The emerging field of environmental DNA (eDNA) research lacks universal guidelines for ensuring data produced are FAIR-findable, accessible, interoperable, and reusable-despite growing awareness of the importance of such practices. In order to better understand these data usability challenges, we systematically reviewed 60 peer reviewed articles conducting a specific subset of eDNA research: metabarcoding studies in marine environments. For each article, we characterized approximately 90 features across several categories: general article attributes and topics, methodological choices, types of metadata included, and availability and storage of sequence data. Analyzing these characteristics, we identified several barriers to data accessibility, including a lack of common context and vocabulary across the articles, missing metadata, supplementary information limitations, and a concentration of both sample collection and analysis in the United States. While some of these barriers require significant effort to address, we also found many instances where small choices made by authors and journals could have an outsized influence on the discoverability and reusability of data. Promisingly, articles also showed consistency and creativity in data storage choices as well as a strong trend toward open access publishing. Our analysis underscores the need to think critically about data accessibility and usability as marine eDNA metabarcoding studies, and eDNA projects more broadly, continue to proliferate., Competing Interests: The authors declare there are no competing interests., (©2023 Shea et al.)

Background: Wastewater measurements of SARS-CoV-2 RNA have been extensively used to supplement clinical data on COVID-19. Most examples in the literature that describe wastewater monitoring for SARS-CoV-2 RNA use samples from wastewater treatment plants and individual buildings that serve as the primary residence of community members. However, wastewater surveillance can be an attractive supplement to clinical testing in K-12 schools where individuals only spend a portion of their time but interact with others in close proximity, increasing risk of potential transmission of disease., Methods: Wastewater samples were collected from two K-12 schools in California and divided into solid and liquid fractions to be processed for detection of SARS-CoV-2. The resulting detection rate in each wastewater fraction was compared to each other and the detection rate in pooled clinical specimens., Results: Most wastewater samples were positive for SARS-CoV-2 RNA when clinical testing was positive (75% for solid samples and 100% for liquid samples). Wastewater samples continued to test positive for SARS-CoV-2 RNA when clinical testing was negative or in absence of clinical testing (83% for both solid and liquid samples), indicating presence of infected individuals in the schools. Wastewater solids had a higher concentration of SARS-CoV-2 than wastewater liquids on an equivalent mass basis by three orders of magnitude., Competing Interests: The authors declare that they have no competing interests., (©2023 Kim and Boehm.)

Human norovirus (HuNoV) is an important cause of acute gastroenteritis and can be transmitted by water exposures, but its persistence in water is not well understood. Loss of HuNoV infectivity in surface water was compared with persistence of intact HuNoV capsids and genome segments. Surface water from a freshwater creek was filter-sterilized, inoculated with HuNoV (GII.4) purified from stool, and incubated at 15 or 20 °C. We measured HuNoV infectivity via the human intestinal enteroid system and HuNoV persistence via reverse transcription-quantitative polymerase chain reaction assays without (genome segment persistence) or with (intact viral capsid persistence) enzymatic pretreatment to digest naked RNA. For infectious HuNoV, results ranged from no significant decay to a decay rate constant ( "k ") of 2.2 day -1 . In one creek water sample, genome damage was likely a dominant inactivation mechanism. In other samples from the same creek, loss of HuNoV infectivity could not be attributed to genome damage or capsid cleavage. The range in k and the difference in the inactivation mechanism observed in water from the same site could not be explained, but variable constituents in the environmental matrix could have contributed. Thus, a single k may be insufficient for modeling virus inactivation in surface waters.

Background: Water, sanitation, and hygiene (WASH) improvements are promoted to reduce diarrhoea in low-income countries. However, trials from the past 5 years have found mixed effects of household-level and community-level WASH interventions on child health. Measuring pathogens and host-specific faecal markers in the environment can help investigate causal pathways between WASH and health by quantifying whether and by how much interventions reduce environmental exposure to enteric pathogens and faecal contamination from human and different animal sources. We aimed to assess the effects of WASH interventions on enteropathogens and microbial source tracking (MST) markers in environmental samples., Methods: We did a systematic review and individual participant data meta-analysis, which included searches from Jan 1, 2000, to Jan 5, 2023, from PubMed, Embase, CAB Direct Global Health, Agricultural and Environmental Science Database, Web of Science, and Scopus, of prospective studies with water, sanitation, or hygiene interventions and concurrent control group that measured pathogens or MST markers in environmental samples and measured child anthropometry, diarrhoea, or pathogen-specific infections. We used covariate-adjusted regression models with robust standard errors to estimate study-specific intervention effects and pooled effect estimates across studies using random-effects models., Findings: Few trials have measured the effect of sanitation interventions on pathogens and MST markers in the environment and they mostly focused on onsite sanitation. We extracted individual participant data on nine environmental assessments from five eligible trials. Environmental sampling included drinking water, hand rinses, soil, and flies. Interventions were consistently associated with reduced pathogen detection in the environment but effect estimates in most individual studies could not be distinguished from chance. Pooled across studies, we found a small reduction in the prevalence of any pathogen in any sample type (pooled prevalence ratio [PR] 0·94 [95% CI 0·90-0·99]). Interventions had no effect on the prevalence of MST markers from humans (pooled PR 1·00 [95% CI 0·88-1·13]) or animals (pooled PR 1·00 [95% CI 0·97-1·03])., Interpretation: The small effect of these sanitation interventions on pathogen detection and absence of effects on human or animal faecal markers are consistent with the small or null health effects previously reported in these trials. Our findings suggest that the basic sanitation interventions implemented in these studies did not contain human waste and did not adequately reduce exposure to enteropathogens in the environment., Funding: Bill and Melinda Gates Foundation and the UK Foreign and Commonwealth Development Office., Competing Interests: Declaration of interests JER reports that a portion of her salary is supported by an unrestricted donation to The London School of Hygiene & Tropical Medicine from Reckitt; the salary support is wholly unrelated to her role in the preparation of this manuscript. TC has interest in publicly traded companies. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Limited information is available on the decay rate of endogenous SARS-CoV-2 and pepper mild mottle virus (PMMoV) RNA in wastewater and primary settled solids, potentially limiting an understanding of how transit or holding times within wastewater infrastructure might impact RNA measurements and their relationship to community COVID-19 infections. In this study, primary settled solids samples were collected from two wastewater treatment plants in the San Francisco Bay Area. Samples were thoroughly mixed, aliquoted into subsamples, and stored at 4, 22, and 37 °C for 10 days. The concentrations of SARS-CoV-2 (N1 and N2 targets) and PMMoV RNA were measured using an RT-ddPCR. Limited decay (<1 log 10 reduction) was observed in the detection of viral RNA targets at all temperature conditions, suggesting that SARS-CoV-2 and PMMoV RNA can be highly persistent in solids. First-order decay rate constants ranged from 0.011 to 0.098 day -1 for SARS-CoV-2 RNA and from 0.010 to 0.091 day -1 for PMMoV RNA depending on the temperature conditions. A slower decay was observed for SARS-CoV-2 RNA in primary settled solids compared to previously reported decay in wastewater influent. Further research is needed to understand if solid content and wastewater characteristics might influence the persistence of viral RNA targets., Competing Interests: The authors declare the following competing financial interest(s): Dorothea Duong is an employee of Verily Life Sciences, LLC as indicated in the paper., (© 2022 The Authors. Published by American Chemical Society.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater settled solids correlate well with coronavirus disease 2019 (COVID-19) incidence rates (IRs). Here, we develop distributed lag models to estimate IRs using concentrations of SARS-CoV-2 RNA from wastewater solids and investigate the impact of sampling frequency on model performance. SARS-CoV-2 N gene and pepper mild mottle virus (PMMoV) RNA concentrations were measured daily at four wastewater treatment plants in California. Artificially reduced data sets were produced for each plant with sampling frequencies of once every 2, 3, 4, and 7 days. Sewershed-specific models that related daily N/PMMoV to IR were fit for each sampling frequency with data from mid-November 2020 through mid-July 2021, which included the period of time during which Delta emerged. Models were used to predict IRs during a subsequent out-of-sample time period. When sampling occurred at least once every 4 days, the in- and out-of-sample root-mean-square error changed by <7 cases/100 000 compared to daily sampling across sewersheds. This work illustrates that real-time, daily predictions of IR are possible with small errors, despite changes in circulating variants, when sampling frequency is once every 4 days or more. However, reduced sampling frequency may not serve other important wastewater surveillance use cases., Competing Interests: The authors declare the following competing financial interest(s): D.D., B.W., and B.H. are employees of Verily Life Sciences., (© 2022 The Authors. Published by American Chemical Society.)