Environmental Science & Technology

Wastewater surveillance of respiratory pathogens can provide timely estimates of viral activity and disease trends in a population. Indoor air surveillance could be used similarly with some advantages but remains largely unvalidated at the community-scale. Here, an indoor air surveillance program was employed as part of public health environmental surveillance in Chicago, Illinois, USA. Ten air samplers were placed in healthcare and congregate living settings across the city. Weekly air samples were evaluated for influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2 over two respiratory virus seasons. Citywide, aggregated air sample positivity and viral load were closely correlated with local clinical case and wastewater surveillance data across all respiratory viruses. Virus trends in air data often preceded clinical and wastewater, although this varied across pathogens and respiratory virus seasons. Further, whole-genome sequencing of SARS-CoV-2 showed close correlation of variant proportions across all datasets. At the building-scale, air samples obtained from a single sampling device provided efficient respiratory virus surveillance, with well-correlated estimates of respiratory pathogens. These data demonstrate that air surveillance can provide accurate estimates of respiratory virus infections and variants at a building or community-scale, serving as an alternative or complementary tool for public health environmental surveillance.

Wastewater monitoring enables non-invasive, population-scale tracking of community infections independent of healthcare-seeking behavior and clinical diagnosis. Metagenomic sequencing extends this capability by enabling broad, pathogen-agnostic detection, genomic characterization, and identification of novel or unexpected threats. Here, we present data from CASPER (the Coalition for Agnostic Sequencing of Pathogens from Environmental Reservoirs), a U.S.-based wastewater metagenomic sequencing network designed for deep, untargeted pathogen monitoring at national scale. This release includes 1,206 samples collected between December 2023 and December 2025 from 27 sites across nine states, covering 13 million people. Deep sequencing (~1 billion read pairs per sample) generated 1.2 trillion read pairs (347 terabases), enabling detection of even rare taxa, with CASPER representing 66% of all untargeted wastewater sequencing data currently available on the NCBI Sequence Read Archive. Virus abundance trends correlate with nationwide wastewater PCR and clinical data for SARS-CoV-2, influenza A, and respiratory syncytial virus, while the pathogen-agnostic approach captures emerging threats, including avian influenza H5N1 during initial dairy cattle outbreaks, West Nile virus, and measles, among hundreds of viral taxa. As the largest publicly available untargeted wastewater sequencing dataset to date, CASPER provides a shared and growing resource for pathogen surveillance and microbial ecology.

The unprecedented widespread closing of buildings due to the COVID-19 pandemic has allowed water to stagnate in premise plumbing systems, creating conditions that may facilitate the growth of opportunistic pathogens. In this study, we flushed and collected samples from showers in buildings that had been unoccupied for approximately two months and quantified Legionella pneumophila using a commercial cultivation-based assay. In addition, all bacteria, Legionella spp., L. pneumophila, L. pneumophila serogroup 1, non-tuberculous mycobacteria (NTM), and Mycobacterium avium complex (MAC) were analyzed using quantitative PCR (qPCR). Despite low or negligible total chlorine in the stagnant pre-flush water samples, L. pneumophila were not detected by either method; Legionella spp., NTM, and MAC, however, were widespread. Using quantitative microbial risk assessment (QMRA), estimated risks of clinical illness from exposure to legionella and MAC via showering were generally low, but the risk of subclinical infection via Legionella spp. could exceed a 10-7 daily risk threshold if just a small fraction ([≥]0.1 %) of those legionellae detected by qPCR are highly infectious. Flushing cold and hot water lines rapidly restored a total chlorine (as chloramine) residual and decreased all bacterial gene targets to building inlet water levels within 30 min. Following flushing, the chlorine residual rapidly dissipated and bacterial gene targets rebounded, approaching pre-flush concentrations after 6 to 7 days of stagnation. These results suggest that stagnant water in premise plumbing may contain elevated levels of opportunistic pathogens; flushing, however, can rapidly improve water quality and reduce the health risk but the improvement will be short-lived if building disuse persists.

Exposure to ultrafine particles (UFP) is increasingly linked to adverse health outcomes. While nation-wide air monitoring networks in the United States do not measure UFP, small-scale measurements have revealed persistent patterns in urban UFP. This project maps hyperlocal UFP in a 12 km2 study area of a health effects study in Louisville, KY, through mobile measurements to elucidate the relationship between the urban landscape and UFP exposures. We measured UFP number concentration along all drivable streets ([~]340 km) during daytime and nighttime on both weekdays and weekends. After deconvoluting UFP levels to isolate local signals from neighborhood and urban signals, we fitted a land-use regression (LUR) model to explain differences in local UFP as a function of characteristics of the built and natural environment. Median UFP in the study domain was 6,850 #/cm3, which is comparable to urban background measured or estimated for other U.S. cities. UFP was higher during the weekend than on weekdays, potentially due to changes in local activity (e.g. increased restaurant hours) apparent at fine spatial scales. The final LUR model explained 61% of the spatial heterogeneity in log(UFP). Leave-one-area-out cross validation revealed overprediction in regions farther from highways and underprediction in regions with dense food service locations and major roads. This suggests that additional mobile measurements to capture longer-term, robust UFP may yield improved models.

Measurement of Enterococcus spp. levels with qPCR allows for same-day advisory notification of recreational water quality conditions, representing a major advance over traditional culture-based methods that require 18 or more hours to obtain results. In 2015, the United States Environmental Protection Agency released an Enterococcus qPCR protocol for recreational water quality testing. Over the past decade, there have been multiple advances in qPCR-based environmental testing, affording the opportunity to update the current methodology. A streamlined Enterococcus qPCR protocol is introduced that simplifies the mathematical model to estimate target sequence concentrations (TSC), reduces sample testing time by 20 min, incorporates a certified control material for standard curve generation, and introduces an inactivated E. faecalis whole cell DNA standard (WCDS) control material. A series of experiments were conducted 1) to compare results of the two Enterococcus qPCR protocols in analysis of marine, estuarine, and freshwater samples (n = 60), 2) to investigate alternative practices to adjust results due to potential water sample matrix interference, control material degradation, and/or analyst inconsistencies, and 3) to evaluate the performance, homogeneity, and stability of an inactivated E. faecalis cell preparation as a WCDS control material. Findings indicate a strong correlation between water sample mean log10 TSC per reaction results (R2 = 0.980) and 100% agreement in amplification and sample processing control tests. A Bayesian approach that accounts for uncertainty in qPCR measurements confirmed statistical equivalence for all water samples yielding paired measurements in the range of quantification, with 72.7% of samples exhibiting reduced error with the new streamlined protocol. Evaluation of three alternative practices to adjust for variation in Enterococcus qPCR measurements indicated no significant difference in water sample log10 TSC per reaction results with varying concentrations of treated sewage influent. Systematic testing of an inactivated WCDS control material yielded statistically equivalent performance compared to viable E. faecalis cell preparations. Homogeneity and stability experiments indicated that Enterococcus qPCR measurements of inactivated WCDS are reproducible across multiple preparations and that the material is stable at -20{degrees}C for at least 38 weeks. Together, experiments demonstrate that the streamlined protocol and alternative practices should make Enterococcus qPCR faster, easier to implement, safer, and more reproducible.

The COVID-19 pandemic has given rise to diverse approaches to track infections. The causative agent, SARS-CoV-2, is a fecally-shed RNA virus, and many groups have assayed wastewater for viral RNA fragments by quantitative reverse transcription polymerase chain reaction (qRT-PCR) as a proxy for COVID-19 prevalence in the community. Most groups report low levels of viral RNA that often skirt the methods theoretical limits of detection and quantitation. Here, we demonstrate the presence of SARS-CoV-2 structural proteins in wastewater using traditional immunoblotting and quantitate them from wastewater solids using an immuno-linked PCR method called Multiplex Paired-antibody Amplified Detection (MPAD). MPAD demonstrated facile detection of SARS-CoV-2 proteins compared with SARS-CoV-2 RNA via qRT-PCR in wastewater. In this longitudinal study, we corrected for stochastic variability inherent to wastewater-based epidemiology using multiple fecal content protein biomarkers. These normalized SARS-CoV-2 protein data correlated well with public health metrics. Our method of assaying SARS-CoV-2 protein from wastewater represents a promising and sensitive epidemiological tool to assess prevalence of fecally-shed pathogens in the community.

Wastewater based epidemiology (WBE) is a useful method to detect pathogen prevalence and may serve to effectively monitor diseases at a broad scale. WBE has been used throughout the COVID-19 pandemic to track localized and population-level disease burden through the quantification of SARS-CoV-2 RNA present in wastewater. Aside from case load estimation, WBE is being used to assay viral genomic diversity and the emergence of potential SARS-CoV-2 variants. Here, we present a study in which we sequenced RNA extracted from sewage influent samples obtained from eight wastewater treatment plants representing 16 million people in Southern California over April 2020 - August 2021. We sequenced SARS-CoV-2 with two methods: Illumina Respiratory Virus Enrichment and metatranscriptomic sequencing (N = 269), and QIAseq SARS-CoV-2 tiled amplicon sequencing (N = 95). We were able to classify SARS-CoV-2 reads into lineages and sublineages that approximated several named variants across a full year, and we identified a diversity of single nucleotide variants (SNVs) of which many are putatively novel SNVs, and SNVs of unknown potential function and prevalence. Through our retrospective study, we also show that several sublineages of SARS-CoV-2 were detected in wastewater up to several months before clinical detection, which may assist in the prediction of future Variants of Concern. Lastly, we show that sublineage diversity was similar between wastewater treatment plants across Southern California, and that diversity changed by sampling month indicating that WBE is effective across megaregions. As the COVID-19 pandemic moves to new phases, and additional SARS-CoV-2 variants emerge, the ongoing monitoring of wastewater is important to understand local and population-level dynamics of the virus. Our study shows the potential of WBE to detect SARS-CoV-2 variants throughout Southern Californias wastewater and track the diversity of viral SNVs and strains in urban and suburban locations. These results will aid in our ability to monitor the evolutionary potential of SARS-CoV-2 and help understand circulating SNVs to further combat COVID-19.

The ability to measure human sewage in environmental and wastewater samples is important to protect public health and natural water resources. A recent study reports the complete genome sequence of {Phi}B124-14, a bacteriophage capable of infecting a narrow subset of Bacteroides spp. closely associated with the human gut. To investigate the use of {Phi}B124-14 for fecal indicator and source identification applications, the entire genome was interrogated for potential human-associated genetic regions using a combination of bioinformatic and laboratory testing approaches. To assess bioinformatic predictions, 53 primer sets were subject to systematic testing using 100 fecal samples from ten animal sources, primary influent sewage from 36 geographical locations across the continental United States, and environmental surface waters with known human sewage impact. Based on candidate primer set end-point PCR analyses and next generation amplicon sequencing, two novel hydrolysis probe-based quantitative real-time PCR assays (qPCR), PS28 and PS30, were designed and evaluated. Both qPCR assays exhibited a sensitivity of 86.1%, a specificity of 100%, and successfully detected {Phi}B124-14-like genetic markers in sewage impacted environmental water samples. PS28 and PS30 performance was then compared to top performing DNA-based viral (CPQ_056 and CPQ_064) and bacterial (HF183/BacR287 and HumM2) human-associated qPCR assays. Findings indicate {Phi}B124-14 bacteriophage-like qPCR assays exhibit superior specificity, but markers consistently occur at lower concentrations in United States primary influent sewage samples. Furthermore, paired measurements of {Phi}B124-14 and crAssphage bacteriophage-like sequences in high volume (10 L) primary influent sewage samples (n = 38) indicate significant correlations ranging from r = 0.593 (p < 0.0001; PS30 versus CPQ_056) to r = 0.938 (p < 0.0001; PS28 versus PS30). A comparison of bacteriophage-like marker concentrations with cultured GB-124 in sewage samples showed no significant correlations (r [&le;] 0.215, p [&ge;] 0.183). Finally, novel qPCR methods for potential human sewage management applications and future research directions are discussed.

BackgroundPlasmids are major drivers of horizontal gene transfer (HGT), playing a central role in disseminating antimicrobial resistance in water resource recovery facilities (WRRFs). Most studies have focused on model or clinical plasmids in simplified settings, leaving their in situ dynamics in complex environmental communities poorly understood. To address this gap, we applied Hi-C metagenomic sequencing to systematically resolve plasmid-host associations and evaluate how environmental and operational factors influence plasmid persistence and host range across treatment stages and geographic regions in municipal WRRFs. ResultsWe identified 944 plasmid clusters across influent, activated sludge, and effluent samples from three WRRFs located in three U.S. states, revealing distinct plasmid distribution patterns and plasmid-host associations across facilities and treatment stages. While overall bacterial community composition remained relatively stable across treatment stages, plasmid-host interactions varied, indicating that environmental conditions and treatment processes influenced plasmid retention and transfer. Plasmids in influent and activated sludge exhibited broader host ranges relative to effluent, where, plasmids were associated with a narrower set of bacterial hosts, likely reflecting the impacts of disinfection. Notably, certain plasmids persisted across treatment stages but exhibited substantial shifts in their associated bacterial hosts. Key taxa such as Burkholderiaceae and Rhodocyclaceae remained abundant throughout, indicating that shifts in plasmid-host associations were not solely driven by community turnover, but also suggesting HGT. Supporting this, functional profiling of effluent plasmids revealed enrichment in conjugation-related genes and virulence factors, including oxidative stress resistance, which may facilitate plasmid persistence and dissemination. ConclusionsOur findings reveal that while wastewater microbial communities are diverse, plasmid-associated hosts are restricted to a few dominant bacterial families. Plasmid host range consistently narrows across treatment stages, reflecting the effectiveness of WRRFs in limiting plasmid persistence. However, shifts in host associations among highly similar plasmid clusters suggest potential HGT events. These results highlight the role of specific bacterial groups in plasmid dissemination and underscore the need for future studies to identify keystone taxa and mechanisms driving plasmid stability in complex microbial communities.

Digital PCR (dPCR) is increasingly used for SARS-CoV-2 wastewater surveillance due to its precision, absolute quantification, and reduced sensitivity to inhibition compared to quantitative PCR. Although the Bio-Rad ddPCR and QIAGEN QIAcuity dPCR platforms are widely adopted, their performance has not been directly compared for wastewater applications. We conducted a blinded comparison of these platforms using 95 archived wastewater influent samples from North Carolina collected in 2021-2022, spanning three orders of magnitude in SARS-CoV-2 concentration (1x103 to 5x105 copies L-1). Samples were stratified into low, medium, and high concentration bins and analyzed in triplicate for N1 and N2 gene targets and a bovine coronavirus processing control. Both platforms demonstrated statistically equivalent quantification across all targets, with mean differences [&le;]0.12 log copies L-1 (R2 > 0.93). Coefficients of variation were similar (3.96 - 7.61%), with no significant differences across concentration bins except for N2 in the low bin (difference: 0.87 percentage points). Measurement variability correlated strongly with wastewater treatment plant site (R2 = 0.89) rather than platform, indicating that sample matrix characteristics drive precision more than analytical platform. Process limits of detection ranged from 2,160-2,680 copies L-1 for Bio-Rad and 5,650-9,700 copies L-1 for QIAcuity for N1 and N2, respectively. The Bio-Rad platform processed samples 32% faster (305 vs. 435 minutes per 96 wells), while QIAcuity offered 29% lower consumables cost ($4.68 vs. $6.11 per well). These findings support the interchangeable use of both platforms for wastewater surveillance, with platform selection based on laboratory-specific operational needs. ImportanceAs wastewater-based epidemiology transitions from emergency response to sustained public health infrastructure, standardized molecular methods are essential for reliable data integration across surveillance networks. This study provides the first blinded comparison of two digital PCR platforms widely deployed for wastewater pathogen surveillance in the United States. We demonstrate quantitative equivalence between Bio-Rad ddPCR and QIAGEN QIAcuity platforms across three orders of magnitude in viral concentration, establishing that data from both platforms can be interpreted interchangeably for public health decision-making. This platform equivalence is critical as national surveillance systems aggregate data from diverse laboratories and as monitoring expands beyond SARS-CoV-2 to encompass additional respiratory viruses, antimicrobial resistance genes, and emerging pathogens. Our findings provide a methodological foundation for multi-platform surveillance networks and demonstrate that measurement variability is driven primarily by sample matrix characteristics rather than analytical platform choice.

Microplastics have emerged as a major environmental concern due to their widespread presence and potential health impacts. Growing water contamination has raised concerns about its presence in seafood consumed by humans. In this single-center, cross-sectional study, we collected a sample of 10 Pacific oysters sold locally in Northern California. We compared microplastic content with a prior study performed at the same location in 2015 (Rochman et al.). Anthropogenic debris was extracted from the whole tissue of shellfish using a 10% KOH solution and quantified under a dissecting microscope. Microplastics were found in all ten samples (compared with 4/12 samples in 2015), from three to greater than 40 particles per sample. These anthropogenic debris were primarily filaments from 0.2 to 2 mm long. Our study findings suggest that microplastics in bivalves sold for human consumption have increased in the past ten years, with potential human health concerns.

Wastewater, which contains everything from pathogens to pollutants, is a geospatially-and temporally-linked microbial fingerprint of a given population. As a result, it can be leveraged for monitoring multiple dimensions of public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (n=1,419 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County from 2020-2022. First, we used targeted amplicon sequencing (n=966) to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with clinical caseloads from University students (N = 1,503) and Miami-Dade County hospital patients (N = 3,939 patients), as well as an 8-day earlier detection of the Delta variant in wastewater vs. in patients. Additionally, in 453 metatranscriptomic samples, we demonstrate that different wastewater sampling locations have clinically and public-health-relevant microbiota that vary as a function of the size of the human population they represent. Through assembly, alignment-based, and phylogenetic approaches, we also detect multiple clinically important viruses (e.g., norovirus) and describe geospatial and temporal variation in microbial functional genes that indicate the presence of pollutants. Moreover, we found distinct profiles of antimicrobial resistance (AMR) genes and virulence factors across campus buildings, dorms, and hospitals, with hospital wastewater containing a significant increase in AMR abundance. Overall, this effort lays the groundwork for systematic characterization of wastewater to improve public health decision making and a broad platform to detect emerging pathogens.

BackgroundLiquefied petroleum gas (LPG) stoves have been promoted in low- and middle-income countries (LMICs) as a clean energy alternative to biomass burning cookstoves. ObjectiveWe sought to characterize kitchen area concentrations and personal exposures to nitrogen dioxide (NO2) within a randomized controlled trial in the Peruvian Andes. The intervention included the provision of an LPG stove and continuous fuel distribution with behavioral messaging to maximize compliance. MethodsWe measured 48-hour kitchen area NO2 concentrations at high temporal resolution in homes of 50 intervention participants and 50 control participants longitudinally within a biomass-to-LPG intervention trial. We also collected 48-hour mean personal exposures to NO2 among a subsample of 16 intervention and 9 control participants. We monitored LPG and biomass stove use continuously throughout the trial. ResultsIn 367 post-intervention 24-hour kitchen area samples of 96 participants homes, geometric mean (GM) highest hourly NO2 concentration was 138 ppb (geometric standard deviation [GSD] 2.1) in the LPG intervention group and 450 ppb (GSD 3.1) in the biomass control group. Post-intervention 24-hour mean NO2 concentrations were a GM of 43 ppb (GSD 1.7) in the intervention group and 77 ppb (GSD 2.0) in the control group. Kitchen area NO2 concentrations exceeded the WHO indoor hourly guideline an average of 1.3 hours per day among LPG intervention participants. GM 48-hour personal exposure to NO2 was 5 ppb (GSD 2.4) among 35 48-hour samples of 16 participants in the intervention group and 16 ppb (GSD 2.3) among 21 samples of 9 participants in the control group. DiscussionIn a biomass-to-LPG intervention trial in Peru, kitchen area NO2 concentrations were substantially lower within the LPG intervention group compared to the biomass-using control group. However, within the LPG intervention group, 69% of 24-hour kitchen area samples exceeded WHO indoor annual guidelines and 47% of samples exceeded WHO indoor hourly guidelines. Forty-eight-hour NO2 personal exposure was below WHO indoor annual guidelines for most participants in the LPG intervention group, and we did not measure personal exposure at high temporal resolution to assess exposure to cooking-related indoor concentration spikes. Further research is warranted to understand the potential health risks of LPG-related NO2 emissions and inform current campaigns which promote LPG as a clean-cooking option.

Surveillance of wastewater from healthcare facilities has the potential to identify the emergence of multidrug-resistant organisms (MDROs) of public health importance. Specifically, wastewater surveillance can provide sentinel surveillance of novel MDROs (e.g., emergence of Candida auris) in healthcare facilities and could help direct targeted prevention efforts and monitor longitudinal effects. Several knowledge gaps need to be addressed before wastewater surveillance can be used routinely for MDRO surveillance, including determining optimal approaches to sampling, processing, and testing wastewater for MDROs. To this end, we evaluated multiple methods for wastewater collection (passive, composite, and grab), concentration (nanoparticles, filtration, and centrifugation), and PCR quantification (real-time quantitative PCR vs. digital PCR) for C. auris and 5 carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like) twice weekly for 6 months at a long-term acute care hospital in Chicago, IL. We also tested the effects of different transport and sample storage conditions on PCR quantification. All genes were detected in facility wastewater, with blaKPC being the most consistently abundant. Experiments were done in triplicate with gene copy, variance, and number of detections between triplicates used to determine method efficacy. We found that passive samples processed immediately using a combination of centrifugation followed by bead-beating and dPCR provided the most reliable results for detecting MDROs. We also present the trade-offs of different approaches and use culture and metagenomics to elucidate clinical relevance. This study establishes a practical approach for wastewater surveillance as a potential tool for public health monitoring of MDRO burden in healthcare facilities.

Early detection of outbreaks and emerging pathogens is critical for public health and global biosecurity. Airports, as major international travel hubs with dense, enclosed populations, are high-risk settings for disease transmission and potential pathogen introduction. The U.S. Centers for Disease Control and Prevention, in collaboration with Ginkgo Biosecurity and the University of Wisconsin-Madison, implemented air monitoring for pathogen surveillance in congregate areas at four U.S. international airports. From October 2023 to August 2024, SARS-CoV-2 was detected by PCR in 98.3% of air samples and influenza A in 17.2%. These results correlated with positivity trends from other sample modalities, including aviation wastewater, traveler nasal swabs, and national clinical surveillance data. Targeted amplicon sequencing of SARS-CoV-2 from air samples correlated with contemporaneous lineages in wastewater collected and sequenced from the same airports. Metagenomic enrichment sequencing detected 30 viral species and recovered high-quality genomes for SARS-CoV-2, influenza, bocavirus, and seasonal coronaviruses. Together, these findings demonstrate that air sampling is a complementary surveillance modality to aviation wastewater for early pathogen detection at ports of entry.

Building closures related to the coronavirus disease (COVID-19) pandemic resulted in increased water stagnation in commercial building plumbing systems that heightened concerns related to the microbiological safety of drinking water post re-opening. The exact impact of extended periods of reduced water demand on water quality is currently unknown due to the unprecedented nature of widespread building closures. We analyzed 420 tap water samples over a period of six months, starting the month of phased reopening (i.e., June 2020), from sites at three commercial buildings that were subjected to reduced capacity due to COVID-19 social distancing policies and four occupied residential households. Direct and derived flow cytometric measures along with water chemistry characterization were used to evaluate changes in plumbing-associated microbial communities with extended periods of altered water demand. Our results indicate that prolonged building closures impacted microbial communities in commercial buildings as indicated by increases in microbial cell counts, encompassing greater proportion cells with high nucleic acids. While flushing reduced cell counts and increased disinfection residuals, the microbial community composition in commercial buildings were still distinct from those at residential households. Nonetheless, increased water demand post-reopening enhanced systematic recovery over a period of months, as microbial community fingerprints in commercial buildings converged with those in residential households. Overall, our findings suggest that sustained and gradual increases in water demand may play a more important role in the recovery of building plumbing-associated microbial communities as compared to short-term flushing, after extended periods of altered water demand that result in reduced flow volumes.

Viruses of concern for quantitative wastewater monitoring are usually selected as a result of an outbreak and subsequent detection in wastewater. However, targeted metagenomics could proactively identify viruses of concern when used as an initial screening tool. To evaluate the utility of targeted metagenomics for wastewater screening, we used ViroCap, a panel of probes designed to target all known vertebrate viruses. Untreated wastewater was collected from wastewater treatment plants (WWTPs) and building-level manholes associated with vulnerable populations in Houston, TX. We evaluated differences in vertebrate virus detection between WWTP and building-level samples, classified human viruses in wastewater, and performed phylogenetic analysis on astrovirus sequencing reads to evaluate targeted metagenomics for subspecies level classification. Vertebrate viruses varied widely across building-level samples. Rarely detected and abundant viruses were identified in WWTP and building-level samples, including enteric, respiratory, and bloodborne viruses. Furthermore, full length genomes were assembled from astrovirus reads and two human astrovirus serotypes were classified in wastewater samples. This study demonstrates the utility of targeted metagenomics as an initial screening step for public health surveillance. SynopsisThis work demonstrates the utility of targeted metagenomic shotgun sequencing to screen for human viruses in centralized and building-level wastewater samples. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/23287251v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@15c4fd8org.highwire.dtl.DTLVardef@17e2a19org.highwire.dtl.DTLVardef@1fe10b3org.highwire.dtl.DTLVardef@fa726f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths in the United States. Individual screening is typically done with either a clinical stool-based test or direct clinical examination such as a colonoscopy. Given the low compliance with current screening recommendations and the high morbidity and mortality observed in areas with health disparities, we consider whether population-based testing using human RNA biomarkers in wastewater might effectively track the presence of CRC at the neighborhood level might be feasible. Wastewater samples were collected from four clusters in Louisville, KY: three representing cancer hotspots and one serving as a control neighborhood for feasibility data. Three wastewater replicates were obtained from each cluster. Human RNA biomarkers were isolated, quantified, and their RNA concentration levels were compared to clinical correlates. All replicates showed detectable levels of human cancer-associated RNA, including CDH1, which is a colorectal neoplasia-associated biomarker. Among CRC cluster sewershed samples, 8 of 9 replicate samples (89%) had a ratio of CDH1/GAPDH >=1 while the control sewershed sample showed ratio <1 for 2 of 3 samples. These preliminary data indicate that human RNA biomarkers can be detected in pooled community wastewater samples. While we have successfully identified the presence of these markers, further investigation with additional samples and closer alignment with documented case activity is necessary.

Wastewater surveillance has been widely adopted since the COVID-19 pandemic, but non-sewered or onsite sanitation is a common form of sanitation in cities of low- and middle-income countries. Environmental surveillance in these settings requires expanding analyses beyond wastewater. We collected 81 soil samples adjacent to public waste bins inside the sewered and non-sewered areas of Maputo and a 150-meter-wide buffer zone between the two areas, as well as from subsistence farms near the wastewater treatment plant for comparison. We cultured Escherichia coli (E. coli) using the IDEXX Quanti-Tray/2000 system and determined the prevalence of 29 unique enteric pathogens via RT-qPCR on TaqMan array cards. E. coli concentrations were significantly higher (p<.001) in soils adjacent to public waste bins (mean = 5.05x105 per gram) compared to soils from farms (mean = 8.70x101 per gram). The mean number of unique pathogens was higher in soils from the non-sewered area (mean = 7.9, n=32) and the 150-meter buffer area (mean = 10.5, n=10) compared to the sewered area (mean = 4.6, n=20) and soils from farms (mean=3.8, n=19). Findings demonstrate that the presence of enteric pathogens in soils adjacent to public waste bins were associated with neighborhood sanitation infrastructure and may be a useful matrix for surveillance. In high-burden settings with poor sanitation, direct examination of soils and other environmental matrices are potentially scalable means of environmental pathogen surveillance to consider beyond conventional sampling matrices.

Although wastewater surveillance for assessing community health and well-being is now mainstream, most cities in low- and middle-income countries lack conventional wastewater services. In these settings, environmental surveillance beyond conventional wastewater offers the potential to inform public health responses, design interventions intended to reduce exposures, and to evaluate infection control programs. To explore these potential use cases, we measured pathogens, source-tracking markers, and fecal indicator bacteria in wastewater treatment plant (WWTP) influent and effluent, wastewater surface discharges, impacted river water, impacted soils, open drains, stormwater, and fecal sludges from onsite sanitation in Maputo, Mozambique. We detected a wide range of pathogens by multi-parallel RT-qPCR across all matrices, revealing a nuanced picture of pathogen flows in the city and suggesting the potential for exposures beyond those typically included in studies of sanitation and health. We developed a pooled regression model and observed lower pathogen concentrations in direct wastewater discharges (mean difference -1.4 log10 per liter, 95% CI: -1.7, -1.1), WWTP effluent (-0.97 log10, 95% CI: - 1.5, -0.47), water from open drains (-2.0 log10, 95% CI: -2.5, -1.6), impacted river water (-3.0 log10, 95% CI: -3.7, -2.4), and stormwater (-4.7 log10, 95% CI: -7.0, -3.3) compared to WWTP influent. We further observed that a one standard deviation increase in 7-day cumulative precipitation was associated with an increase in the pooled pathogen concentration in all matrices (0.11 log10, [0.04, 0.19]). Despite lower concentrations of pathogens in matrices compared to WWTP influent, frequent detection of pathogens indicates clear potential to use environmental pathogen surveillance to inform public health responses in cities lacking universal conventional wastewater, with a wide range of promising applications.