Wastewater Surveillance for Norovirus, California, USA.

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.

Wastewater Surveillance for Influenza A Virus and H5 Subtype Concurrent with the Highly Pathogenic Avian Influenza A(H5N1) Virus Outbreak in Cattle and Poultry and Associated Human Cases - United States, May 12-July 13, 2024.

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.

Addressing Fecal Contamination in Rural Kenyan Households: The Roles of Environmental Interventions and Animal Ownership.

Combined water, sanitation, and handwashing (WSH) interventions could reduce fecal contamination along more transmission pathways than single interventions alone. We measured Escherichia coli levels in 3909 drinking water samples, 2691 child hand rinses, and 2422 toy ball rinses collected from households enrolled in a 2-year cluster-randomized controlled trial evaluating single and combined WSH interventions. Water treatment with chlorine reduced E. coli in drinking water. A combined WSH intervention improved water quality by the same magnitude but did not affect E. coli levels on hands or toys. One potential explanation for the limited impact of the sanitation intervention (upgraded latrines) is failure to address dog and livestock fecal contamination. Small ruminant (goat or sheep) ownership was associated with increased E. coli levels in stored water and on child hands. Cattle and poultry ownership was protective against child stunting, and domesticated animal ownership was not associated with child diarrhea. Our findings do not support restricting household animal ownership to prevent child diarrheal disease or stunting but do support calls for WSH infrastructure that can more effectively reduce household fecal contamination.

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.

The Tomato Brown Rugose Fruit Virus Movement Protein Gene Is a Novel Microbial Source Tracking Marker.

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.

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.

Respiratory virus concentrations in human excretions that contribute to wastewater: a systematic review and meta-analysis.

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.

Estimating Relative Abundance of 2 SARS-CoV-2 Variants through Wastewater Surveillance at 2 Large Metropolitan Sites, United States.

Monitoring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) is critical for public health management of coronavirus disease. Sequencing is resource-intensive and incompletely representative, and not all isolates can be sequenced. Because wastewater SARS-CoV-2 RNA concentrations correlate with coronavirus disease incidence in sewersheds, tracking VOCs through wastewater is appealing. We developed digital reverse transcription PCRs to monitor abundance of select mutations in Alpha and Delta VOCs in wastewater settled solids, applied these to July 2020-August 2021 samples from 2 large US metropolitan sewersheds, and compared results to estimates of VOC abundance from case isolate sequencing. Wastewater measurements tracked closely with case isolate estimates (Alpha, rp 0.82-0.88; Delta, rp 0.97). Mutations were detected in wastewater even at levels <5% of total SARS-CoV-2 RNA and in samples available 1-3 weeks before case isolate results. Wastewater variant monitoring should be strategically deployed to complement case isolate sequencing.

Detection of SARS-CoV-2 Variants Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in Wastewater Settled Solids Using Mutation-Specific Assays Is Associated with Regional Detection of Variants in Clinical Samples.

Changes in the circulation of SARS-CoV-2 variants of concern (VOCs) may require changes in the public health response to the COVID-19 pandemic, as they have the potential to evade vaccines and pharmaceutical interventions and may be more transmissive than other SARS-CoV-2 variants. As such, it is essential to track and prevent their spread in susceptible communities. We developed digital reverse transcription (RT)-PCR assays for mutations characteristic of VOCs and used them to quantify those mutations in samples of wastewater settled solids collected from a publicly owned treatment works (POTW) during different phases of the COVID-19 pandemic. Wastewater concentrations of single mutations characteristic of each VOC, normalized by the concentration of a conserved SARS-CoV-2 N gene, correlate with regional estimates of the proportion of clinical infections caused by each VOC. These results suggest that targeted RT-PCR assays can be used to detect variants circulating in communities and inform the public health response to the pandemic. IMPORTANCE Wastewater represents a pooled biological sample of the contributing community and thus a resource for assessing community health. Here, we show that emergence, spread, and disappearance of SARS-CoV-2 infections caused by variants of concern are reflected in the presence of variant genomic RNA in wastewater settled solids. This work highlights an important public health use case for wastewater.

SARS-CoV-2 RNA in Wastewater Settled Solids Is Associated with COVID-19 Cases in a Large Urban Sewershed.

Wastewater-based epidemiology may be useful for informing public health response to viral diseases like COVID-19 caused by SARS-CoV-2. We quantified SARS-CoV-2 RNA in wastewater influent and primary settled solids in two wastewater treatment plants to inform the preanalytical and analytical approaches and to assess whether influent or solids harbored more viral targets. The primary settled solids samples resulted in higher SARS-CoV-2 detection frequencies than the corresponding influent samples. Likewise, SARS-CoV-2 RNA was more readily detected in solids using one-step digital droplet (dd)RT-PCR than with two-step RT-QPCR and two-step ddRT-PCR, likely owing to reduced inhibition with the one-step ddRT-PCR assay. We subsequently analyzed a longitudinal time series of 89 settled solids samples from a single plant for SARS-CoV-2 RNA as well as coronavirus recovery (bovine coronavirus) and fecal strength (pepper mild mottle virus) controls. SARS-CoV-2 RNA targets N1 and N2 concentrations correlated positively and significantly with COVID-19 clinically confirmed case counts in the sewershed. Together, the results demonstrate that measuring SARS-CoV-2 RNA concentrations in settled solids may be a more sensitive approach than measuring SARS-CoV-2 in influent.

Effects of single and integrated water, sanitation, handwashing, and nutrition interventions on child soil-transmitted helminth and Giardia infections: A cluster-randomized controlled trial in rural Kenya.

BACKGROUND: Helminth and protozoan infections affect more than 1 billion children globally. Improving water quality, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration, while providing other quality of life benefits. METHODS AND FINDINGS: We enrolled geographic clusters of pregnant women in rural western Kenya into a cluster-randomized controlled trial (ClinicalTrials.gov NCT01704105) that tested 6 interventions: water treatment, improved sanitation, handwashing with soap, combined water treatment, sanitation, and handwashing (WSH), improved nutrition, and combined WSH and nutrition (WSHN). We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides, Trichuris trichiura, hookworm, and Giardia duodenalis among children born to the enrolled pregnant women (index children) and their older siblings. After 2 years of intervention exposure, we collected stool specimens from 9,077 total children aged 2 to 15 years in 622 clusters, including 2,346 children in an active control group (received household visits but no interventions), 1,117 in the water treatment arm, 1,160 in the sanitation arm, 1,141 in the handwashing arm, 1,064 in the WSH arm, 1,072 in the nutrition arm, and 1,177 in the WSHN arm. In the control group, 23% of children were infected with A. lumbricoides, 1% with T. trichiura, 2% with hookworm, and 39% with G. duodenalis. The analysis included 4,928 index children (median age in years: 2) and 4,149 older siblings (median age in years: 5); study households had an average of 5 people, <10% had electricity access, and >90% had dirt floors. Compared to the control group, Ascaris infection prevalence was lower in the water treatment arm (prevalence ratio [PR]: 0.82 [95% CI 0.67, 1.00], p = 0.056), the WSH arm (PR: 0.78 [95% CI 0.63, 0.96], p = 0.021), and the WSHN arm (PR: 0.78 [95% CI 0.64, 0.96], p = 0.017). We did not observe differences in Ascaris infection prevalence between the control group and the arms with the individual interventions sanitation (PR: 0.89 [95% CI 0.73, 1.08], p = 0.228), handwashing (PR: 0.89 [95% CI 0.73, 1.09], p = 0.277), or nutrition (PR: 86 [95% CI 0.71, 1.05], p = 0.148). Integrating nutrition with WSH did not provide additional benefit. Trichuris and hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reduced Giardia. Reanalysis of stool samples by quantitative polymerase chain reaction confirmed the reductions in Ascaris infections measured by microscopy in the WSH and WSHN groups. Trial limitations included imperfect uptake of targeted intervention behaviors, limited power to detect effects on rare parasite infections, and that it was not feasible to blind participants and sample collectors to treatment status. However, lab technicians and data analysts were blinded to treatment status. The trial was funded by the Bill & Melinda Gates Foundation and the United States Agency for International Development. CONCLUSIONS: Integration of improved water quality, sanitation, and handwashing could contribute to sustainable control strategies for Ascaris infections, particularly in similar settings with recent or ongoing deworming programs. Combining nutrition with WSH did not provide further benefits, and water treatment alone was similarly effective to integrated WSH. Our findings provide new evidence that drinking water should be given increased attention as a transmission pathway for Ascaris. TRIAL REGISTRATION: ClinicalTrials.gov NCT01704105.

Surface Cleaning and Disinfection: Efficacy Assessment of Four Chlorine Types Using Escherichia coli and the Ebola Surrogate Phi6.

In the 2014 West African Ebola outbreak, international organizations provided conflicting recommendations for disinfecting surfaces contaminated by uncontrolled patient spills. We compared the efficacy of four chlorine solutions (sodium hypochlorite, sodium dichloroisocyanurate, high-test hypochlorite, and generated hypochlorite) for disinfection of three surface types (stainless steel, heavy-duty tarp, and nitrile) with and without pre-cleaning practices (prewiping, covering, or both) and soil load. The test organisms were Escherichia coli and the Ebola surrogate Phi6. All tests achieved a minimum of 5.9 and 3.1 log removal in E. coli and Phi6, respectively. A 15 min exposure to 0.5% chlorine was sufficient to ensure <8 Phi6 plaque-forming unit (PFU)/cm2 in all tests. While chlorine types were equally efficacious with and without soil load, variation was seen by surface type. Wiping did not increase disinfection efficacy and is not recommended because it generates infectious waste. Covering spills decreased disinfection efficacy against E. coli on heavy-duty tarp but does prevent splashing, which is critical in Ebola contexts. Our results support the recommendation of a 15 min exposure to 0.5% chlorine, independently of chlorine type, surface, pre-cleaning practices, and organic matter, as an efficacious measure to interrupt disease transmission from uncontrolled spills in Ebola outbreaks.

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.

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.

Human pathogen nucleic acids in wastewater solids from 191 wastewater treatment plants in the United States.

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.

Prospective study of Candida auris nucleic acids in wastewater solids in 190 wastewater treatment plants in the United States suggests widespread occurrence.

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.