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.

Determining the Economically Optimum Metaphylactic Strategy for Cattle Cohorts of Varied Demographic Characteristics.

Metaphylactic antibiotic use in feeder cattle is a common practice to control respiratory disease. Antimicrobial stewardship is important to ensure continued efficacy and to protect animal welfare. The objective of this study is to identify characteristics of cohorts of cattle that had not received metaphylaxis that would have benefited economically from the use of metaphylaxis. Cohorts (n = 12,785; 2,206,338 head) from 13 feedlots that did not receive metaphylaxis were modeled using an economic model to estimate net returns for three metaphylactic options. Logistic regression models with covariates for entry weight, sex, average daily weight gain, number of animals per cohort, and days on feed, with feedlot as a random effect, were used to determine the model-adjusted probability of cohorts benefiting economically from metaphylaxis. Most (72%) cohorts in this data set that had not received metaphylaxis at arrival would not economically benefit from metaphylaxis. Sex, entry weight category, number of cattle in the cohort, and average daily weight gain were associated with the likelihood of benefitting economically from metaphylaxis. The results illustrated that cattle cohort demographics influenced the probability that cohorts would benefit economically from metaphylaxis and the type of metaphylaxis utilized, and integrating this information has the potential to influence the metaphylaxis decision.

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.

Expansion of wastewater-based disease surveillance to improve health equity in California's Central Valley: sequential shifts in case-to-wastewater and hospitalization-to-wastewater ratios.

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.

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.

Dating the origin and spread of specialization on human hosts in Aedes aegypti mosquitoes.

The globally invasive mosquito subspecies Aedes aegypti aegypti is an effective vector of human arboviruses, in part because it specializes in biting humans and breeding in human habitats. Recent work suggests that specialization first arose as an adaptation to long, hot dry seasons in the West African Sahel, where Ae. aegypti relies on human-stored water for breeding. Here, we use whole-genome cross-coalescent analysis to date the emergence of human-specialist populationsand thus further probe the climate hypothesis. Importantly, we take advantage of the known migration of specialists out of Africa during the Atlantic Slave Trade to calibrate the coalescent clock and thus obtain a more precise estimate of the older evolutionary event than would otherwise be possible. We find that human-specialist mosquitoes diverged rapidly from ecological generalists approximately 5000 years ago, at the end of the African Humid Period-a time when the Sahara dried and water stored by humans became a uniquely stable, aquatic niche in the Sahel. We also use population genomic analyses to date a previously observed influx of human-specialist alleles into major West African cities. The characteristic length of tracts of human-specialist ancestry present on a generalist genetic background in Kumasi and Ouagadougou suggests the change in behavior occurred during rapid urbanization over the last 20-40 years. Taken together, we show that the timing and ecological context of two previously observed shifts towards human biting in Ae. aegypti differ; climate was likely the original driver, but urbanization has become increasingly important in recent decades.

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.

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.

Multi-Year Mass-Trapping With Autocidal Gravid Ovitraps has Limited Influence on Insecticide Susceptibility in Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

Mass-trapping has been used to control outbreaks of Aedes aegypti (Linnaeus) (Diptera: Culicidae) in Puerto Rico since 2011. We investigated the effect of multi-year, insecticide-free mass trapping had on the insecticide susceptibility profile of Ae. aegypti. Eggs collected in southern Puerto Rico were used to generate F1 populations that were tested for susceptibility to permethrin, sumethrin, bifenthrin, deltamethrin, and malathion according to CDC bottle bioassays protocols. All populations of Ae. aegypti were resistant to the synthetic pyrethroids and mosquitoes from two locations were partially resistant to malathion. Population genetic analysis, using a double digest restriction sites associated DNA sequencing (ddRADseq) approach, indicated a large amount of migration between study sites effectively homogenizing the mosquito populations. Mass-trapping using noninsecticidal autocidal gravid ovitraps did not restore susceptibility to five active ingredients that are found in commercial insecticides. Migration between communities was high and would have brought outside alleles, including resistant alleles to the treatment communities. Further investigation suggests that household use of commercially available insecticide products may continue to select for resistance in absence of public health space spraying of insecticides.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.