Spatial and temporal variation in respiratory syncytial virus (RSV) subtype RNA in wastewater and relation to clinical specimens.

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 (PA,WW) and conducted a comparative analysis temporally, spatially, and against clinical specimens. A median PA,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, PA,WW was higher in some areas (PA,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 PA,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.

Observations of Respiratory Syncytial Virus (RSV) Nucleic Acids in Wastewater Solids Across the United States in the 2022-2023 Season: Relationships with RSV Infection Positivity and Hospitalization Rates.

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 107 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.

Persistence of human respiratory viral RNA in wastewater-settled solids.

Wastewater-based epidemiology has emerged as a valuable tool for monitoring respiratory viral diseases within communities by analyzing concentrations of viral nucleic-acids in wastewater. However, little is known about the fate of respiratory virus nucleic-acids in wastewater. Two important fate processes that may modulate their concentrations in wastewater as they move from household drains to the point of collection include sorption or partitioning to wastewater solids and degradation. This study investigated the decay kinetics of genomic nucleic-acids of seven human respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, human rhinovirus (HRV), and influenza A virus (IAV), as well as pepper mild mottle virus (PMMoV) in wastewater solids. Viruses (except for PMMoV) were spiked into wastewater solids and their concentrations were followed for 50 days at three different temperatures (4°C, 22°C, and 37°C). Viral genomic RNA decayed following first-order kinetics with decay rate constants k from 0 to 0.219 per day. Decay rate constants k were not different from 0 for all targets in solids incubated at 4°C; k values were largest at 37°C and at this temperature, k values were similar across nucleic-acid targets. Regardless of temperature, there was limited viral RNA decay, with an estimated 0% to 20% reduction, over the typical residence times of sewage in the piped systems between input and collection point (<1 day). The k values reported herein can be used directly in fate and transport models to inform the interpretation of measurements made during wastewater surveillance.IMPORTANCEUnderstanding whether or not the RNA targets quantified for wastewater-based epidemiology (WBE) efforts decay during transport between drains and the point of sample collection is critical for data interpretation. Here we show limited decay of viral RNA targets typically measured for respiratory disease WBE.

Persistence and free chlorine disinfection of human coronaviruses and their surrogates in water.

The coronavirus disease 2019 pandemic illustrates the importance of understanding the behavior and control of human pathogenic viruses in the environment. Exposure via water (drinking, bathing, and recreation) is a known route of transmission of viruses to humans, but the literature is relatively void of studies on the persistence of many viruses, especially coronaviruses, in water and their susceptibility to chlorine disinfection. To fill that knowledge gap, we evaluated the persistence and free chlorine disinfection of human coronavirus OC43 (HCoV-OC43) and its surrogates, murine hepatitis virus (MHV) and porcine transmissible gastroenteritis virus (TGEV), in drinking water and laboratory buffer using cell culture methods. The decay rate constants of human coronavirus and its surrogates in water varied, depending on virus and water matrix. In drinking water without disinfectant addition, MHV showed the largest decay rate constant (estimate ± standard error, 2.25 ± 0.09 day-1) followed by HCoV-OC43 (0.99 ± 0.12 day-1) and TGEV (0.65 ± 0.06 day-1), while in phosphate buffer without disinfectant addition, HCoV-OC43 (0.51 ± 0.10 day-1) had a larger decay rate constant than MHV (0.28 ± 0.03 day-1) and TGEV (0.24 ± 0.02 day-1). Upon free chlorine disinfection, the inactivation rates of coronaviruses were independent of free chlorine concentration and were not affected by water matrix, though they still varied between viruses. TGEV showed the highest susceptibility to free chlorine disinfection with the inactivation rate constant of 113.50 ± 7.50 mg-1 min-1 L, followed by MHV (81.33 ± 4.90 mg-1 min-1 L) and HCoV-OC43 (59.42 ± 4.41 mg-1 min-1 L). IMPORTANCE: This study addresses an important knowledge gap on enveloped virus persistence and disinfection in water. Results have immediate practical applications for shaping evidence-based water policies, particularly in the development of disinfection strategies for pathogenic virus control.

A retrospective longitudinal study of adenovirus group F, norovirus GI and GII, rotavirus, and enterovirus nucleic acids in wastewater solids at two wastewater treatment plants: solid-liquid partitioning and relation to clinical testing data.

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 105 copies/g), while rotavirus RNA was detected at the lowest concentrations (median on the order of 103 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.

Is detection of enteropathogens and human or animal faecal markers in the environment associated with subsequent child enteric infections and growth: an individual participant data meta-analysis.

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.

Temperature and particles interact to affect human norovirus and MS2 persistence in surface water.

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.

Wastewater surveillance for bacterial targets: current challenges and future goals.

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.

Sunlight Inactivation of Enveloped Viruses in Clear 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 m2/MJ, compared to 16 m2/MJ for Phi6 and 52 m2/MJ for MHV. With the highest UV attenuation tested, only k̂ for MHV (6.1 m2/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.

United States Influenza 2022-2023 Season Characteristics as Inferred from Wastewater Solids, Influenza Hospitalization, and Syndromic Data.

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.

Trends of Enterovirus D68 Concentrations in Wastewater, California, USA, February 2021-April 2023.

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.

Occurrence of Human Viruses on Fomites in the Environment: A Systematic Review and Meta-analysis.

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 cm2). 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.

Adsorption of Respiratory Syncytial Virus, Rhinovirus, SARS-CoV-2, and F+ Bacteriophage MS2 RNA onto Wastewater Solids from Raw Wastewater.

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 (KF) ranged from 2000-270,000 mL·g-1 across viruses and temperature conditions. Distribution coefficients (Kd) 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.