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.

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.

Human norovirus (HuNoV) GII RNA in wastewater solids at 145 United States wastewater treatment plants: comparison to positivity rates of clinical specimens and modeled estimates of HuNoV GII shedders.

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.

More than a Tripledemic: Influenza A Virus, Respiratory Syncytial Virus, SARS-CoV-2, and Human Metapneumovirus in Wastewater during Winter 2022-2023.

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.

Divergence of wastewater SARS-CoV-2 and reported laboratory-confirmed COVID-19 incident case data coincident with wide-spread availability of at-home COVID-19 antigen tests.

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.

Human viral nucleic acids concentrations in wastewater solids from Central and Coastal California USA.

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.

Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study.

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.

Persistence of Endogenous SARS-CoV-2 and Pepper Mild Mottle Virus RNA in Wastewater-Settled Solids.

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 log10 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.

SARS-CoV-2 RNA Wastewater Settled Solids Surveillance Frequency and Impact on Predicted COVID-19 Incidence Using a Distributed Lag Model.

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.

Regional Replacement of SARS-CoV-2 Variant Omicron BA.1 with BA.2 as Observed through Wastewater Surveillance.

Greater knowledge of circulating SARS-CoV-2 variants can inform pandemic response, vaccine development, disease epidemiology, and use of monoclonal antibody treatments. We developed custom assays targeting characteristic mutations in SARS-CoV-2 variants Omicron BA.1 and BA.2 and confirmed their sensitivity and specificity in silico and in vitro. We then applied these assays to daily wastewater solid samples from eight publicly owned treatment works in the greater Bay Area of California, United States, over four months to obtain a spatially and temporally intensive data set. We documented regional replacement of BA.1 with BA.2 in agreement with, and ahead of, clinical sequencing data. This study highlights the utility of wastewater surveillance for real-time tracking of SARS-CoV-2 sublineage circulation. The results suggest that concerted efforts to design RT-PCR assays that target variant and variant sublineage characteristic mutations for wide-scale wastewater monitoring implementation will be informative for pandemic response.

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

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.

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.

A kinetic isotope effect and isotope exchange study of the nonenzymatic and the equine serum butyrylcholinesterase-catalyzed thioester hydrolysis.

Formylthiocholine (FTC) was synthesized and found to be a substrate for nonenzymatic and butyrylcholinesterase (BChE)-catalyzed hydrolysis. Solvent (D2O) and secondary formyl-H kinetic isotope effects (KIEs) were measured by an NMR spectroscopic method. The solvent (D2O) KIEs are (D2O)k = 0.20 in 200 mM HCl, (D2O)k = 0.81 in 50 mM HCl, and (D2O)k = 4.2 in pure water. The formyl-H KIEs are (D)k = 0.80 in 200 mM HCl, (D)k = 0.77 in 50 mM HCl, (D)k = 0.75 in pure water, (D)k = 0.88 in 50 mM NaOH, and (D)(V/K) = 0.89 in the BChE-catalyzed hydrolysis in MES buffer at pH 6.8. Positional isotope exchange experiments showed no detectable exchange of (18)O into the carbonyl oxygen of FTC or the product, formate, under any of the above conditions. Solvent nucleophile-O KIEs were determined to be (18)k = 0.9917 under neutral conditions, (18)k = 1.0290 (water nucleophile) or (18)k = 0.989 (hydroxide nucleophile) under alkaline conditions, and (18)(V/K) = 0.9925 for BChE catalysis. The acidic, neutral, and BChE-catalyzed reactions are explained in terms of a stepwise mechanism with tetrahedral intermediates. Evidence for a change to a direct displacement mechanism under alkaline conditions is presented.