De facto reuse (DFR) refers to the incidental or unintentional incorporation of treated wastewater into natural water bodies used as a source of drinking water. Increasing recognition of this practice has highlighted a potential risk of human exposure to various chemicals and pathogens originating from wastewater. In this study, quantitative microbial risk assessment (QMRA) was used to determine the infection risks associated with norovirus, adenovirus, enterovirus, Cryptosporidium, and Giardia for DFR in Southern Nevada (i.e., Lake Mead). Scenarios included three lake levels to encompass current (329 m) and possible scenarios associated with continued drought conditions (312 m and 297 m). Starting with observed raw wastewater pathogen concentrations at local wastewater treatment plants, risks were estimated after accounting for facility-specific wastewater treatment trains, discharge-specific dilution and decay in the environmental buffers (based on hydrodynamic modeling), and drinking water treatment. Log reduction values (LRVs) for wastewater treatment were also calibrated to observed Cryptosporidium concentrations in the environment to characterize 'gaps' in crediting (LRVgap = 1.97). For the baseline lake level, the median cumulative risk of gastrointestinal infection from all pathogens was 10-4.59 infections per person per year, with Cryptosporidium as the primary driver of risk. Risks increased significantly for the lower lake elevations but still satisfied the annual risk benchmark of 10-4. The impacts of seasonality were also studied for norovirus, indicating increased risks during fall and spring. Overall, this study demonstrates that the current design and operation of the Southern Nevada DFR system is protective of public health with respect to enteric pathogen exposure, even if the current Colorado River Basin drought continues or worsens.

We sequenced 53 Legionella pneumophila isolates from Southern Nevada groundwater, an understudied source despite its use for drinking water. Genomes averaged 3.48 Mbp, and 25 carried plasmids. These data provide a resource for identifying genomic features distinguishing environmental strains from those associated with disease outbreak.

ABSTRACT The IDEXX Legiolert test has been implemented for the quantification of Legionella pneumophila in a variety of potable and non-potable water matrices. For example, through coordination with the state regulatory agency, the Southern Nevada Water Authority (SNWA) has implemented treatment strategies and corresponding L. pneumophila monitoring (via Legiolert) to ensure groundwater quality and public health protection. Because of its specificity to L. pneumophila , confirmation of a positive Legiolert test is often assumed to be unnecessary, although high false positive rates have previously been reported for Legiolert testing of groundwater systems. This study evaluated four confirmation methodologies on liquid media harvested from Legiolert-positive trays: latex agglutination, MALDI-MS, rapid qPCR for the mip and srkA genes, and BCYE plating (±L-cysteine). With the exception of BCYE, these confirmation methods provide results in <24 h, allowing for rapid regulatory and/or public health response. Of 108 presumptive positive analyses of groundwater samples collected in Southern Nevada, only two were ultimately determined to be false positive Legiolert results, although one prompted the shutdown of the corresponding groundwater well. qPCR of the mip gene had the highest L. pneumophila confirmation rate (96%), followed by MALDI-MS (92%), latex agglutination (88%), and BCYE (±L-cysteine) (88%). Although Legiolert alone proved to be effective and relatively accurate in detecting L. pneumophila , confirmation testing yielded valuable supporting information, including serogroup determination and false positive identification ( Stenotrophomonas maltophilia ). Collectively, these data highlight the benefits and drawbacks of various L. pneumophila confirmation methodologies for reporting and decision-making by SNWA and other drinking water utilities. IMPORTANCE In the United States, Legionella pneumophila is the leading cause of drinking water-associated illnesses, hospitalization, and deaths. It is the causative agent of Legionnaires’ disease and the less severe Pontiac Fever. As awareness of L. pneumophila risks increases and monitoring plans are implemented, it is imperative that laboratory analysts, practitioners, and decision-makers understand the limitations of available methods and the value of confirmation in increasing data confidence and informing appropriate actions. Groundwater is commonly used as a drinking water source for public and private systems and generally has less stringent treatment requirements than other sources. Studies have described groundwater as an environmental reservoir for L. pneumophila , but there have also been reports of high false positive rates when monitoring groundwater, particularly when using IDEXX Legiolert. Therefore, the results from this study provide critical knowledge to those monitoring L. pneumophila and using the data for regulatory compliance and/or operational decision-making.

This study demonstrates the use of wastewater monitoring for opioids and other high-risk substances on a university campus, intended to inform public health surveillance and response efforts for youth and transitional age youth. Over a 14-week campaign, we collected 54 grab samples from three campus manholes, including one that isolated flows from a student housing complex, and analyzed them for 24 parent compounds and metabolites using LC-MS/MS. Heroin and its metabolite 6-acetylmorphine were among several opioid-related detections, illustrating how campus wastewater monitoring can capture actionable use events for targeted public health interventions. Fentanyl, norfentanyl, and xylazine were consistently below their method reporting limits across all 54 samples, consistent with recent declines in national overdose and wastewater datasets. The highest detection frequencies were associated with methamphetamine (22-50%), amphetamine (39-83%), and a THC metabolite (50-100%). This study also highlights the implications of sample type (grab vs. composite) and day of week for wastewater-based epidemiology (WBE). Considering that the use of opioid-related WBE as an actionable public health surveillance tool is still relatively new, additional case studies are needed to explore the potential of this emerging tool and increase confidence in deploying public health interventions in response to wastewater data.

There is ongoing debate regarding the necessity of excess pathogen treatment to address potential public health risks from direct potable reuse (DPR) schemes. One approach in quantitative microbial risk assessment (QMRA) has been to incorporate conservative treatment failure scenarios, reflecting the assumption that DPR systems may have shorter response times to treatment failures than indirect potable reuse (IPR) systems. In this study, deterministic and stochastic QMRA approaches were used to determine minimum log reduction value (LRV) requirements for potable reuse schemes, explicitly accounting for process failures of varying magnitude, duration, and frequency while meeting health-based targets specified by World Health Organization (WHO) and Australian guidelines. Evaluation of multiple failure scenarios demonstrates a direct and quantifiable link between selected failure assumptions and the resulting excess pathogen treatment requirements. The results highlight that failure assumption selection represents a critical risk management decision that should be considered holistically within a risk-based regulatory framework. This work also provides insight into how excess pathogen removal contributes to system robustness by offering protection against a range of treatment failure conditions.

Coagulation, flocculation, and sedimentation (CFS) is widely applied as a combined unit process in the treatment of drinking water, wastewater, and recycled water; however, virus reduction through CFS has not been sufficiently characterized to assign pathogen log reduction value (LRV) credits. This study collected data through a systematic review that yielded over 1000 LRVs from 43 manuscripts covering 46 viruses to characterize virus reduction through CFS. The results demonstrate that CFS is effective at reducing viruses, with 68% of virus LRVs greater than 1. A mixed-effects model was used to identify potential mechanisms of virus reduction with ferric and aluminum coagulants, as well as factors associated with variability in performance. Key insights from the model show that virus reduction is: (1) improved at lower pH, similar to natural organic matter (NOM) reduction, (2) lower in treatment of secondary effluent than in drinking water treatment, (3) virus-dependent, and (4) dependent on virus enumeration methods, with lower LRVs observed for molecular techniques. These findings demonstrate the potential for CFS to provide consistent and explainable virus reduction, potentially establishing a foundation for regulatory crediting in potable reuse applications. Future crediting frameworks will need to account for the factors impacting performance to accurately quantify and assign credit for virus reduction.

Background:Prior studies involving music festivals (including on-site surveys, drug checking, and wastewater-based analyses) suggest that recreational substance use is common among festivalgoers, though preferred substances and usage may be region- and event-specific. However, multiyear, event-specific wastewater surveillance data in the United States are limited. Objective:This study aimed to characterize concentrations of high-risk substances (HRS) and their metabolites in wastewater surrounding an annual electronic dance music (EDM) festival in Las Vegas, Nevada, USA. Using a progressive sampling approach over 3 consecutive years (2023-2025), we assessed trends and examined comparisons by sample type, day, and location to inform situational awareness and future public health surveillance efforts and risk mitigation strategies. Methods:Wastewater samples were collected from utility access points at the festival site (2024-2025), off-site in the surrounding community (2023-2024), and at the downstream wastewater treatment plant (WWTP; 2023-2025) during the festival week and weekend. For comparison, samples from a nonfestival weekend were collected off-site and at the WWTP in 2023. Target analytes consisted of 24 parent compounds and metabolites, including stimulants, opioids, and sedatives, which were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with isotope dilution. Analyte selection prioritized substances associated with overdose risk and with prior festival-related surveillance. Results:Cocaine (and 3 of 4 measured metabolites), methamphetamine, amphetamine, 11-nor-9-carboxy-Δ-9-tetrahydrocannabinol (THC-COOH), and tramadol were detected in 100% of festival weekend samples; 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) was detected (at concentrations of up to nearly 2 mg/L) in all but 1 festival site sample collected prior to the start of the event in 2025. Festival-associated signals were most pronounced for stimulant-related analytes, which were also detected in WWTP and off-site samples during the festival. Compared with nonfestival weeks, MDMA concentrations were approximately 10 to 30 times higher at off-site locations and nearly 100 times higher at the downstream WWTP during the festival. Opioid-related concentrations were comparable at the WWTP and the festival site, although there were notable detections of norfentanyl and relatively high concentrations of tramadol in some festival site samples. MDMA- and cocaine-related analyte concentrations peaked during the final festival days in 2024 and 2025; the 2023 samples highlighted the presence of HRS in local wastewater during nonfestival weekends. Conclusions:Event-focused, multiyear wastewater surveillance detected stimulants and psychoactive substances, opioids, and Δ-9-tetrahydrocannabinol (THC) associated with an annual EDM festival weekend. Findings are consistent with previous wastewater-based studies of music and dance festivals, with MDMA concentrations among the highest reported in similar surveillance efforts. Codetection of opioid- and stimulant-related analytes suggests varied usage preferences, polysubstance use, and/or unintentional adulteration (eg, lacing). These findings demonstrate the value of wastewater monitoring as a complementary, population-level tool to support public health preparedness, harm-reduction strategies, and attendee safety during large-scale events. These data also inform wastewater utilities about expected influent composition during special events, with relevance for treatment operations and water reuse applications.

The global use of reclaimed (or recycled) water for irrigation in agriculture presents a sustainable solution to water scarcity and regional saltwater intrusion control by augmenting freshwater supplies. Reclaimed water typically consists of municipal wastewater effluent that has been treated with a combination of physical, biological, and/or chemical processes to a water quality that is consistent with the intended beneficial use. In the US and globally, the use of reclaimed water for certain food crop irrigation uses raises potential food safety concerns, in part because of the lack of federal regulations specific to the irrigation of produce (i.e., fruits and vegetables commonly consumed as raw) with reclaimed water. In the US, the consequent reliance on nonuniform state requirements complicated public health perspectives. Furthermore, there is still uncertainty related to the efficacy of wastewater treatment as it relates to constituents of emerging concerns (CECs), including pathogens such as Cyclospora cayetanensis, and the toxicological relevance of the ever-expanding and evolving list of chemical hazards. Collectively, these issues hinder the practical adoption of reclaimed water by interested agricultural stakeholders and provoke dissent by regional communities. This review aims to examine the current status of reclaimed water use for produce crop irrigation, focusing on state regulations, microbial criteria, operational characteristics, efficacy of treatment processes in mitigating microbial hazards, and associated studies evaluating the degree to which current reclaimed water standards adequately protect public health. Arguably, research is still needed to evaluate the efficacy of various treatment trains against the more recalcitrant pathogens, such as C. cayetanensis, and to understand when this hazard may pose potential contamination risks.

The rapid evolution of antifungal resistance in Candidozyma auris (formerly Candida auris) presents significant challenges for conventional public health surveillance methods, particularly in detecting emergent and highly transmissible drug-resistant variants. Here, using wastewater-based epidemiology tools initially developed during the COVID-19 pandemic, we implement a high-resolution, facility-level early warning system to monitor C. auris infections and resistance patterns. Our evaluation across Southern Nevada demonstrates that upstream sewage monitoring at healthcare facilities provides significant sensitivity (p < 0.001) compared to wastewater treatment plant sampling. By combining amplicon sequencing and MALDI-TOF mass spectrometry, we identify clinically-relevant, resistance-associated variants in wastewater samples, while whole-genome sequencing reveals >90% genomic concordance between 443 wastewater-derived genomes and 2945 clinical isolates. We also detect previously unreported subclades and resistance mutations, including FKS1 Phe635Leu and co-occurring ERG11/FKS1 variants in wastewater samples up to nearly five months before their appearance in clinical settings. Transcriptomic profiling of drug-resistant isolates under antifungal and stress conditions identifies previously uncharacterized adaptation mechanisms, including differential regulation of ribosomal assembly pathways and cell cycle checkpoints. These findings highlight how wastewater intelligence can enhance traditional public health approaches for early detection and monitoring of C. auris outbreaks and antifungal resistance.

Contaminants in municipal drinking water, specifically total trihalomethanes (TTHMs), are mutagenic. Exposures to TTHMs have been associated with urothelial carcinoma (UC) in both people and dogs. Mutations in the BRAF gene and other genomic copy number aberrations detected in voided urothelial cells with commercial CADET BRAF and BRAF-PLUS tests have also been associated with the presence of UC in dogs. However, it is not known whether these urothelial mutations can be linked to TTHM exposures in dogs. The objectives of this ecological study were to compare the incidence of detected urinary BRAF mutations or genomic copy number aberrations in dogs residing in a city with relatively high drinking water TTHMs (Las Vegas, NV) to a city with significantly lower TTHMs (Reno, NV), and to expand the study group to include BRAF and BRAF-PLUS test results and municipal drinking water TTHM concentrations by zip code across the United States. Dogs living in Las Vegas had a higher relative risk of urothelial mutations compared to dogs living in Reno (RR 2.52; 95% CI, 1.77-3.38; p < 0.0001). However, this risk could not be attributed to higher municipal drinking water TTHMs. Across the United States, the population-adjusted incidence of urothelial mutations in voided urine was not associated with municipal water TTHMs, but was instead associated in cross-sectional analyses with age, neutered status, higher regional test submission rates and previously reported high-risk breeds for UC (Scottish terriers, West Highland white terriers, Shetland sheepdogs, beagles and wirehaired fox terriers). The higher RR for a positive BRAF or BRAF-PLUS test in Las Vegas versus Reno could be solely due to higher test submission rates (2.97 versus 0.97 per 1000 dogs) but could also reflect other environmental exposures not considered in this study.

Secondary biological wastewater treatment is a core component of potable reuse treatment trains, but it is often uncredited for virus attenuation. This study evaluated virus log reduction values (LRVs) at two full-scale water resource recovery facilities in Southern Nevada: one with conventional activated sludge and another with lagoon treatment. Four human enteric viruses and four fecal indicator viruses were quantified using molecular assays; culture assays quantified F-specific and somatic coliphages. Median LRVs were low (< 1.0) for plant viruses and generally higher (> 1.0) for adenovirus and crAssphage, consistent with predictions from a solids partitioning model. The fifth percentile LRVs that often drive regulatory determinations were < 0.5 for norovirus GI/GII. For conventional activated sludge, nucleic acid decay was a significant contributor to the molecular LRVs, whereas culturable coliphage data (median LRV = 2.5) highlighted solids attachment and subsequent physical removal as a dominant mechanism. In contrast, lagoon treatment sometimes achieved LRVs > 4.0 for culturable coliphages, primarily due to temperature-dependent inactivation. Based on the collective insight from recent studies, adoption of a broad virus LRV crediting framework for conventional activated sludge systems in potable reuse applications will require better alignment between molecular and culture methods and a deeper understanding of virus attenuation mechanisms.

Genome sequencing from wastewater enables accurate and cost-effective identification of SARS-CoV-2 variants. However, existing computational pipelines have limitations in detecting emerging variants not yet characterized in humans. Here, we present an unsupervised learning approach that clusters co-varying and time-evolving mutation patterns to identify SARS-CoV-2 variants. To build our model, we sequence 3659 wastewater samples collected over two years from urban and rural locations in Southern Nevada. We then develop a multivariate independent component analysis (ICA)-based pipeline to transform mutation frequencies into independent sources. These data-driven time-evolving and co-varying sources are compared to 8810 SARS-CoV-2 clinical genomes from Nevadans. Our method accurately detects the Delta variant in late 2021, Omicron variants in 2022, and emerging recombinant XBB variants in 2023. Our approach also reveals the spatial and temporal dynamics of variants in both urban and rural regions; achieves earlier detection of most variants compared to other computational tools; and uncovers unique co-varying mutation patterns not associated with any known variant. The multivariate nature of our pipeline boosts statistical power and supports accurate early detection of SARS-CoV-2 variants. This feature offers a unique opportunity to detect emerging variants and pathogens, even in the absence of clinical testing.

As the population of persons experiencing homelessness (PEHs) continues to grow in urban areas, including Las Vegas in Southern Nevada, it becomes increasingly important to quantify and mitigate health risks encountered by this vulnerable population. This study examines exposure risks from Campylobacter, a leading cause of bacterial gastroenteritis, among unsheltered PEHs who utilize untreated water from Las Vegas' flood control tunnels and washes for personal hygiene and cleaning. Utilizing quantitative microbial risk assessment (QMRA), we evaluated three exposure scenarios: direct bathing, toothbrushing, and hand-to-mouth contact following hand submersion. The median probability of illness for a single toothbrushing (5 %) or bathing event (5 %) exceeded the adopted risk threshold of 3.2 %, with estimated median annual risks reaching 36 % under monthly exposure frequencies. High concentrations of Campylobacter in these urban waterways underscore the urgent need for targeted interventions, including improved access to clean water and sanitation facilities, to mitigate the health risks faced by PEHs. This study provides a step forward in understanding and addressing the risks of interacting with water in urban waterways, including flood control infrastructure.

Regulatory frameworks for potable reuse often include stringent log reduction value (LRV) targets for viruses and protozoa. To reliably protect public health while also considering the sustainability of advanced water treatment, it is critically important to accurately and rapidly assess pathogen removal and avoid under-crediting of any unit process in a potable reuse treatment train. This study systematically evaluates secondary biological wastewater treatment across solids retention times (SRTs) ranging from 2 to 20 days and uses culture and molecular methods to characterize attenuation of 12 viruses, including fecal indicators, surrogate bacteriophages, and enteric pathogens. This study also proposes a mechanistic model for physical removal based on a solids partitioning/adsorption framework incorporating mixed liquor suspended solids (MLSS) concentrations. Mean sample-specific partitioning coefficients (Kd) ranged from 2.1 log10 mL/g for cucumber green mottle mosaic virus (CGMMV) to 3.8 log10 mL/g for phiX174. Kd values were strongly correlated with LRVs, although the resulting adsorption models (sample-specific, linear, and Freundlich) could not fully explain the large variability in virus removal across all conditions. LRVs of 1-3 were observed for all viruses, but the 5th percentile LRVs that drive regulatory determinations were often <0.5. This study proposes several approaches for awarding implicit LRVs of ≥1 using quantitative microbial risk assessment (QMRA) coupled with observed secondary treatment performance or secondary effluent concentrations.

Objectives:This study explored expanded traveler- and tourism-focused wastewater monitoring in Las Vegas, Nevada, USA to complement community SARS-CoV-2 surveillance. Methods:Wastewater samples were collected November 2023 to July 2024 from the largest community-scale wastewater treatment plant in Southern Nevada, USA (N = 112 samples) and two upstream utility access holes (i.e. manholes), isolating an international airport (N = 68 samples) and a commercial area with high-density bars and nightclubs (N = 30-33 samples). Polymerase chain reaction-based methods quantified RNA concentrations of SARS-CoV-2 and pepper mild mottle virus; whole genome sequencing characterized SARS-CoV-2 variants (N = 83 qualifying samples). Results:SARS-CoV-2 concentrations exhibited concordance between liquids- and solids-based approaches. Similar trends were observed between methods and sampling locations; however, select manhole-level findings suggested potentially divergent COVID-19 infection profiles relative to residents. Whole genome sequencing also demonstrated similarities across sampling locations, although airport samples facilitated the identification of SARS-CoV-2 variants that either failed to spread locally (EG.6, JN.1.11) or preceded detection at the wastewater treatment plant (JN.1.7, KP.3). Conclusions:These findings offer new insight into the operationalization of broader traveler- and tourism-focused wastewater monitoring, which may capture SARS-CoV-2 concentration spikes and genomic profiles in high-tourism/nightlife areas that community-scale sampling might otherwise miss.

Temporal variability and methodological differences in data normalization, among other factors, complicate effective trend analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wastewater surveillance data and its alignment with coronavirus disease 2019 (COVID-19) clinical outcomes. As there is no consensus approach for these analyses yet, this study explored the use of piecewise linear trend analysis (joinpoint regression) to identify significant trends and trend turning points in SARS-CoV-2 RNA wastewater concentrations (normalized and non-normalized) and corresponding COVID-19 case rates in the greater Las Vegas metropolitan area (Nevada, USA) from mid-2020 to April 2023. The analysis period was stratified into three distinct phases based on temporal changes in testing protocols, vaccination availability, SARS-CoV-2 variant prevalence, and public health interventions. While other statistical methodologies may require fewer parameter specifications, joinpoint regression provided an interpretable framework for characterization and comparison of trends and trend turning points, revealing sewershed-specific variations in trend magnitude and timing that also aligned with known variant-driven waves. Week-level trend agreement corroborated previous findings demonstrating a close relationship between SARS-CoV-2 wastewater surveillance data and COVID-19 outcomes. These findings guide future applications of advanced statistical methodologies and support the continued integration of wastewater-based epidemiology as a complementary approach to traditional COVID-19 surveillance systems.

Utility research programs can provide solutions to a wide range of challenges and help utilities prepare for the future. Utilities are uniquely positioned to blend fundamental and applied research. Core research themes pursued by the Southern Nevada Water Authority include regulatory compliance, infrastructure protection, and contaminants of emerging concern.

Effective biofilm recovery is essential when characterizing microbial communities on granular filter media in water treatment applications. This study evaluated (1) DNA extraction kit modifications to maximize DNA recovery and (2) low frequency sonication (40 kHz) durations to maximize recovery of viable cells for flow cytometry (FCM) considering filter media type (anthracite and granular activated carbon [GAC]), media age, and filter runtime. Both DNA and intact cell concentrations were greater for new GAC (< 12 months of full-scale operation) and anthracite compared to old GAC (> 20 years). Biomass yields for old GAC were improved by using a less aggressive bead-beating protocol (for DNA) and reduced sonication duration (for intact cells). Finally, collecting media at the beginning of a filter run (i.e., post-backwash) improved biomass recovery compared to sampling after long filter runtimes. Further study of carbon attrition during extraction is recommended.

This review identified 30 studies from 1992–2024 that performed quantitative microbial risk assessments on potable reuse and compared individual assumptions, summarized influential parameters, and analyzed results.