PLOS Water

BACKGROUND: Integrated WASH interventions are essential for improving public health by increasing access to safe water, sanitation, and hygiene services. This study evaluates their impact on water access and household knowledge, attitudes, and practices (KAP) in rural communities by comparing intervention and non-intervention areas. METHODS: A cross-sectional survey was conducted in May 2025 across six kebele administrations (three intervention and three control). Data were collected from 396 households with children under five using structured questionnaires, with equal representation from both groups. Descriptive analysis was applied to compare outcomes. RESULTS: Children in intervention areas experienced significantly lower diarrhea rates (2.5% vs. 34.9%). Households also showed improved health behaviors, including higher rates of facility births (88.9% vs. 63.6%), breastfeeding (98% vs. 89.9%), and vaccination (78.8% vs. 59.1%). Access to safe water improved markedly: all intervention households used protected sources, spent less time collecting water (13.9 vs. 55.8 minutes), and consumed more water daily (20.6 vs. 10.5 liters). Safely managed water services reached 59.6% compared to just 1% in control areas. Sanitation and hygiene practices were also better, with higher latrine access (95% vs. 78.3%), reduced open defecation (23.2% vs. 52%), and increased handwashing with soap (48.5% vs. 12.1%). Knowledge, attitudes, and practices were significantly stronger in intervention communities. CONCLUSION: Integrated WASH interventions significantly improve water access, hygiene practices, and child health outcomes. Sustaining these benefits requires continued investment in infrastructure, community awareness, and behavior change programs. KEY WORDS: Water, sanitation and hygiene, KAP, rural Ethiopia

Background: Beaches are popular summertime destinations in Canada. However, they can be affected by specific fecal pollution sources, increasing the risk of recreational water illness. Objectives: This study was conducted to determine the risks of acute gastrointestinal illness (AGI) among Canadian beachgoers and to evaluate the influence of different fecal indicator bacteria (FIB) and other water quality measures on assessing these risks. Methods: In a prospective cohort design, beachgoers were recruited at sites across Canada from 2023 to 2025. Sociodemographic characteristics and exposures were determined through an on-site survey, with a 7-day follow-up survey to determine risks of AGI. Bayesian mixed-effects logistic regression models were fitted to evaluate the effects of an ordinal water contact variable (no contact, minimal contact, body immersion, and swallowed water) on the incident risk of AGI, with an interaction included for water quality indicators. The levels of six FIB and water quality measures were assessed: Escherichia coli, enterococci DNA, three microbial source tracking DNA markers (human HF183/BacR287, human mitochondria, seagull Gull4), and turbidity. Results: A total of 4085 participants were recruited, with 67.6% completing the follow-up survey. The overall incident risk of AGI was 2.6%. Both swallowing water and body immersion increased AGI risks compared to no water contact: median of 20 excess cases (95% Credible Interval [CrI]: 4, 64) and 5 excess cases (95% CrI: 1, 19) of AGI predicted per 1000 beachgoers, respectively. Escherichia coli and seagull DNA marker levels were associated with AGI among those who had water contact, particularly among those who reported swallowing water. Discussion: While the overall burden of AGI due to beach water contact in Canada was low, increased risks are associated with E. coli levels particularly among those who swallow water. This could be related to fecal contamination from seagulls. However, there is substantial uncertainty in the predicted effect sizes.

Soil is an important reservoir for antimicrobial resistance (AMR) and increasingly recognized as a pathogen transmission pathway. While studies have detected AMR in soil in various settings, dominant contributors to domestic soil contamination with antimicrobial-resistant organisms in low-income countries remain unidentified. We conducted a cross-sectional study with 237 households in southern Malawi, specifically peri-urban Bangwe near Blantyre, to identify factors associated with the abundance of cefotaxime-resistant E. coli in yard soil. Enumerators employed structured surveys and sampled 30 cm2 of yard soil per household. We used IDEXX Quanti-Tray/2000 with Colilert-18 and cefotaxime supplement to enumerate the most probable number (MPN) of cefotaxime-resistant E. coli per dry gram of soil. We conducted multivariable regression to assess associations between the abundance of cefotaxime-resistant E. coli and household sanitation, animal ownership and management, child health and antibiotic use, and weather. Of 228 soil samples, 68% harbored cefotaxime-resistant E. coli at a mean of 0.90 log10-MPN/dry gram. Compared to households without animals, households had approximately 0.50-log lower mean cefotaxime-resistant E. coli abundance in soil if animals were enclosed at night and 0.50-log higher abundance if they were not (p-values<0.005). Additionally, samples had approximately 0.70-log lower mean cefotaxime-resistant E. coli abundance if soil was dry at the time of collection and if it came from a household in the top wealth quintile (p-values<0.005). Daytime animal confinement, household sanitation, child health, antibiotic use, rainfall, temperature and ambient humidity were not associated with cefotaxime-resistant E. coli abundance. Findings suggest that animal husbandry and soil moisture had stronger associations with cefotaxime-resistant E. coli in soil compared to sanitation or antibiotic use. These results underscore the importance of a One Health approach and the relevance of domestic animals and environmental factors to AMR in soil. Studies should quantify soilborne AMR exposure and evaluate associations with specific animal management/enclosure practices.

Research background. Foodborne diseases (FBDs) remain a pressing global public health issue, with courier-based food delivery systems increasingly recognized as potential contamination pathways. In Zambia, despite the Food Safety Act No. 7 of 2019, limited evidence exists on microbial risks in courier-mediated food transport. This study was conducted to assess pathogenic contamination in food carriers used by courier bikers in Lusaka during the 2025/2026 cholera outbreak response. Experimental approach. An analytical cross sectional design was employed. Ninety three food carriers (bags, cooler boxes, and metal containers) were randomly sampled from courier bikers. Swabs from internal surfaces were processed within 24 hours using standard microbiological culture and biochemical identification methods. Statistical analyses (Chi square tests, Pearson correlations, and logistic regression) were applied to determine associations between contamination and operational factors. Results and conclusions. Microbial contamination was detected in 69% of carriers. The most common pathogens were Escherichia coli (30%), coagulase negative Staphylococcus (24%), and Staphylococcus aureus (18%), with additional isolates including Gram-positive bacilli (11%) and Klebsiella pneumoniae (8%). Logistic regression identified cleaning frequency as the strongest predictor of contamination, with infrequent cleaning associated with significantly higher odds ratios (26.5 to 94.7, p < .05). Carrier type also influenced contamination risk, while years in service and certification status were not significant. The findings highlight that inadequate cleaning practices and carrier design are primary drivers of microbiological risks in courier based food delivery systems. Novelty and scientific contribution. This study provides the first empirical evidence of microbial contamination in courier food carriers in Lusaka, Zambia. It underscores the urgent need for strengthened hygiene protocols and routine sanitation enforcement to protect consumers from foodborne pathogens and antimicrobial resistance. The work contributes novel insights into food safety risks in emerging delivery systems, with implications for policy, public health interventions, and consumer protection in Zambia and beyond.

Introduction Access to safe water, sanitation, and hygiene (WASH) in schools is critical for child health, learning, and gender equity. In Kenya, the Kenya School Health Policy and the Basic Education Act outline standards for school WASH; however, implementation remains uneven due to inadequate infrastructure, weak inter-sectoral coordination, and limited financing. This study aimed to identify priority components for strengthening school WASH implementation and generate policy-relevant recommendations based on expert consensus in Uasin Gishu County, Kenya. Methods and Results A Delphi technique consisting of two iterative rounds was used to reach expert consensus. In Round 1, 20 purposively selected experts including head teachers, county education officials, public health officers, water and public works officers, and NGO representatives participated in key informant interviews. Emergent themes informed development of a structured Round 2 questionnaire administered through CommCare online app. Quantitative data were analyzed using descriptive statistics (means, standard deviations, percentage agreement), while qualitative responses underwent thematic coding using NVivo 12. Experts reached strong consensus on essential components required for strengthening school WASH implementation. Key priorities included clear governance structures, designated budget lines, inclusive infrastructure, menstrual hygiene management (MHM), curriculum integration, sustained capacity building, and systematic monitoring. Multi-sectoral collaboration and recognition of best-performing schools were also emphasized as important motivators for compliance and sustainability. Equity considerations particularly the need for disability-friendly facilities and school-community outreach were highlighted as critical. Agreement levels ranged from 74% to 100%, with most items scoring mean values between 4.5 and 4.8 on a 5-point Likert scale, indicating strong consensus among experts. Conclusion strengthening implementation of school WASH in Kenya requires coordinated governance, predictable funding, reliable water systems, inclusive sanitation, strengthened MHM, and consistent monitoring beyond infrastructure investment alone. Integrating these expert-validated priorities within existing national policies offers a practical pathway to improving learner health, reducing absenteeism especially among girls and promoting equitable educational outcomes.

Respiratory infections caused by bacterial pathogens like Mycobacterium tuberculosis and Bordetella pertussis have increased since the COVID 19 pandemic, yet clinical surveillance of both suffers from underreporting and delayed diagnoses. Wastewater monitoring is a valuable public health surveillance tool that can help fill gaps in clinical data yet has rarely been applied to respiratory bacterial pathogens despite evidence of bacterial shedding via excretion types that enter wastewater. In this study, we investigated the possibility for wastewater monitoring of two bacterial respiratory diseases, tuberculosis and pertussis, using two case studies of wastewater monitoring for M. tuberculosis and B. pertussis. We retrospectively measured concentrations of these pathogens in wastewater samples collected longitudinally from communities with and without known outbreaks of these diseases. We designed and validated a novel B. pertussis specific assay for the NAD(P) gene; B. pertussis nucleic acids were detected sporadically in wastewater during an identified outbreak. We used a highly specific, established assay for M. tuberculosis nucleic acids, and found low concentrations of the marker in wastewater that were lag-correlated with clinical incidence rates 5 weeks later. Findings support the potential of wastewater monitoring for M. tuberculosis and B. pertussis to enable identification of communities with outbreaks of tuberculosis and pertussis and provide early warning for tuberculosis.

Background SARS-CoV-2 genomic surveillance data remain limited in most low and middle-income countries (LMICs), resulting in significant gaps in the understanding of variant circulation and evolution. Wastewater-based epidemiology (WBE) presents a non-invasive, cost-effective, and population-representative surveillance approach that can complement clinical testing, particularly in densely populated urban informal settlements with limited healthcare access. This study aimed to pilot wastewater-based genomic surveillance as a multifaceted public health tool in Kenya. Methods A prospective study was conducted using wastewater samples collected from two WHO-validated environmental surveillance sites -- Eastleigh A (Kamukunji sub-county) and Mathare (Starehe sub-county) -- in Nairobi, Kenya, between December 2022 and October 2023. A total of 272 samples were collected using Moore swabs at a frequency of two to three times per week. Samples were concentrated using Nanotrap(R) Magnetic Virus Particles, and nucleic acid was extracted using the Qiagen QIAamp Viral RNA Mini Kit. SARS-CoV-2 was detected using RT-PCR (TaqPath COVID-19 CE-IVD RT-PCR Kit). Library preparation for whole-genome sequencing was performed using the Illumina COVIDSeq kit, and sequencing was conducted on the Illumina MiSeq platform. Bioinformatic analysis was performed using Terra.bio and RStudio, and phylogenetic analysis included sequences abstracted from GISAID. Results Of 272 samples, 238 (87.5%) tested positive with a cycle threshold (Ct) value of less than 36. Genomic analysis of 181 sequences identified Omicron as the predominant circulating variant, detected in 59% of samples. Other variants included XBB (16%), XBB.2.3(10%), XBB.1.9.X (5%), and additional minor variants. These findings were concordant with clinical sequencing data from Kenya over the same period. Conclusions Wastewater-based genomic surveillance reliably reflected SARS-CoV-2 variant trends observed in clinical data. This approach provides early signals of variant emergence and evolution, offering a cost-effective complement to clinical surveillance in resource-limited settings.

Methods to concentrate wastewater samples are essential for sensitive environmental surveillance of infectious diseases. We defined six main principles used to concentrate viral pathogens in wastewater and performed a systematic review and meta-analysis of their performance. PubMed and Web of Science were searched on 31 January 2025 using terms wastewater, sewage, concentration methods and wastewater surveillance. We included all studies comparing [&ge;]2 concentration methods for virus detection. Our search identified 49 eligible studies published since 2013 across seven continents. We ranked the performance of evaluated methods in each study and generated an overall performance metric for each method principle by virus group (enveloped vs. non-enveloped) using Plackett-Luce analysis. Precipitation and filtration methods were the most studied, while magnetic bead-based and centrifugation were least studied. Magnetic bead-based methods were more effective for concentrating enveloped viruses (63% of pairwise comparisons), whereas flocculation performed better for non-enveloped viruses (60%). However, no single method strongly dominated and method rankings were variable between studies. This study provides evidence-based guidance for selecting wastewater concentration methods to support environmental surveillance of viral pathogens.

BackgroundUrogenital schistosomiasis is a major cause of preventable morbidity, primarily in rural, resource-limited regions. After decades of mass drug administration, changing epidemiologic landscapes, and ongoing resource limitations, test-and-treat models may be necessary to meet elimination goals. However, diagnostic capacity remains centralized and laboratory-dependent, and community-led, contextually adapted implementation strategies remain poorly defined. This study describes the accuracy and feasibility of a low-cost diagnostic toolkit and explores community-integrated implementation models. Methodology/Principal FindingsThis mixed-methods study enrolled 418 participants from five endemic sites near Oyan River Dam, Ogun State, Nigeria in July 2025. Urine samples underwent parallel analysis by community health extension workers utilizing the toolkit and by laboratory technicians using standard microscopy. The toolkit consisted of a reusable urine filtration device paired with a under-$2 paper microscope. Semi-structured interviews with community health extension workers and key informants were analyzed using the Consolidated Framework for Implementation Research. Prevalence was 27.5% (115/418). Community health extension workers demonstrated progressive improvement in diagnostic accuracy across five sequential communities (n=237), rising from 52.5% (95% CI 37.5-67.1) to 92.1% (79.2-97.3) over eight study days (Cochran-Armitage Z=3.08, p=0.002). Specificity improved from 53.6% to 96.3% (Z=3.00, p=0.003), final sensitivity reached 81.8% (52.3-94.9), and final Cohens kappa reached 0.803. In the hands of laboratory scientists, Foldscope microscopy achieved 91.0% sensitivity and 99.3% specificity. Conclusions/SignificanceCommunity-led diagnostic task-shifting for urogenital schistosomiasis control is accurate, feasible, and implementation-ready. Consolidated Framework for Implementation Research-guided analysis demonstrated strong end-user acceptability, with local ownership, collaboration, and trust-building as key implementation facilitators. This approach addresses diagnostic gaps in resource-limited endemic settings with relevance to other community health worker-led strategies. Author SummarySchistosomiasis is a parasitic infection that spreads through contact with freshwater and often goes undetected and untreated for years. Most common in sub-Saharan Africa, the disease damages the bladder and genitourinary tract, increasing risk of infertility, bladder cancer, and HIV transmission. It is most prevalent in rural communities where the snail intermediate host thrives in local water sources used daily for fishing, farming, and bathing. One such area is the Oyan River in Nigeria. Here, we found that barriers to diagnosis and treatment of the illness include distance and transportation. In this study, community health workers diagnosed their neighbors and community members using a low-cost toolkit: a <$2 / 2700 microscope, called the Foldscope paired with a small steel filter card we designed, called the SchistoFilter.. We enrolled 418 people across five villages along the Oyan River in Nigeria and trained eight community health workers to use this toolkit at the point of care. By the fifth community visite, they reached 92.1% accuracy. The study team interviewed community health workers and government officials to contextualize this approach, and they were enthusiastic: The tools can be used with confidence, the training is feasible, and what is most needed is a reliable supply chain and supportive oversight.

BackgroundThe rapid growth of the worlds urban population has contributed to the expansion of informal urban settlements in many cities across the world. In these settings, lack of safe sanitation combined with high population density and poverty contributes to heightened health risks for often vulnerable populations. The aim of this study was to evaluate the effect of a shared, onsite sanitation intervention on the nutritional status of children in Maputo, Mozambique. MethodsThe Maputo Sanitation (MapSan) trial was a controlled before-and-after study to evaluate the effect of a shared, onsite sanitation intervention on child health in Maputo, Mozambique. Here, we report the effects on childhood stunting, wasting and underweight, and height-for-age, weight-for-height and weight-for-age z-scores. Children were enrolled aged 1-48 months at baseline and outcomes were measured before and 12 and 24 months after the intervention, with concurrent measurement among children in a comparable control arm. The primary analysis was intention-to-treat. The trial was registered at ClinicalTrials.gov, number NCT02362932. ResultsWe enrolled 757 and 852 children in the intervention and control groups respectively. There was no evidence for an effect of the intervention on any outcome at 12 or 24 months of follow-up except for wasting where there was very weak evidence for an effect (adjusted prevalence ratio: 0.497; 95% CI: 0.22-1.11; p=0.09). In two exploratory analyses - one including only those children born into compounds post-intervention and a second excluding children in control compounds which had independently improved their sanitation facilities during follow-up - we found that stunting increased in the intervention group whilst wasting decreased. ConclusionsThis study contributes to the growing evidence on the role of sanitation in shaping child health outcomes in informal urban settlements. We found no evidence for an effect on stunting and weak evidence for an effect on wasting. More research is needed to understand how sanitation can reduce childhood undernutrition in complex urban environments.

This cross-sectional study assessed hygienic practices, microbial contamination, and associated factors among street food vendors in Mymensingh City, Bangladesh, from August 2024 to February 2025. Using purposive sampling, 300 vendors were evaluated through structured questionnaires, observational assessments, and laboratory analysis of food samples (n = 300) for bacterial load (log-transformed Total Viable Count, log_TVC). Results revealed that 87.33% (95% CI: 83.6-91.1) of vendors practiced poor hygiene: 90.7% (95% CI: 87.4-94.0) did not cover food, 7% (95% CI: 4.1-9.9) used disinfectants, and 81.00% (95% CI: 76.6-85.4) reused ingredients beyond one day. Knowledge gaps were stark, with 89.7% (95% CI: 86.3-93.1%) demonstrating insufficient basic hygiene knowledge and 90.7% (95% CI: 87.4-94.0%) lacking understanding of hygienic food processing. Education significantly influenced practices; secondary-educated vendors exhibited markedly better hygiene awareness (65.0% vs. 2.89% in uneducated groups). Bacterial loads varied across food types, with Velpuri showing the highest mean log_TVC (8.00, 95% CI: 7.65-8.35) and fast food the lowest (7.69, 95% CI: 7.34-8.03). Significant correlations emerged between hygiene knowledge and practices: vendors aware of foodborne diseases were more likely to use gloves (Spearman's r = 0.199, p = 0.001) and cover food (r = 0.118, p = 0.041). Challenges included financial constraints (62.25% [95% CI: 56.1-68.4] among uneducated vendors), knowledge gaps, and time limitations. The study underscores systemic issues in street food safety, driven by low education and economic barriers. Interventions targeting vendor education, affordable hygiene solutions, and stricter enforcement of ingredient freshness protocols are urgently needed to mitigate foodborne disease risks in urban Bangladesh.

Mass gatherings pose a concern for public health because they are associated with dense crowds, increased social interaction, and travel, all of which can facilitate the rapid transmission of infectious diseases. Wastewater and environmental surveillance (WES) were used for pathogen monitoring during the 2024 NFL Annual Player Selection Meeting (the Draft) in Detroit, MI, an event that drew an estimated 775,000 attendees. Wastewater and environmental samples were queried for respiratory viruses and clinically relevant antimicrobial resistance genes (ARG). WES did not detect an increase in the concentration of monitored respiratory viruses (SARS-CoV-2, IAV, IBV, and RSV) associated with the 2024 NFL Draft. In contrast, WES detected a transient increase in carbapenemase targets in wastewater, primarily driven by a fourfold increase in blaOXA-48. Resistome structure in wastewater was dominated by sampling site characteristics rather than changes associated with the event. The Draft weekend coincided with rainfall-driven combined sewer overflow (CSO), potentially allowing the dissemination of ARG to the environment. In surface waters receiving wastewater effluent, an increase in detection frequency and normalized concentrations for multiple ARG were observed following the Draft. WES provided an overview of pathogen prevalence before, during, and after a large-scale gathering, showing how it can warn of emerging health risks in near real time.

The widespread adoption of energy-saving bulbs like light-emitting diodes and compact fluorescent lamps in Zambia has raised significant environmental and public health issues because some of these bulbs contain dangerous materials like mercury. This study sought to evaluate households' understanding and disposal practices of used and damaged energy-saving bulbs in Lusaka, Zambia's Mtendere Compound. A cross-sectional design was used, with structured questionnaires distributed to a randomly chosen sample of households. The research showed that, although most participants were aware of the energy efficiency advantages of these bulbs, they had little understanding of their possible health risks and safe disposal procedures. The majority of households reported throwing away broken and used bulbs with their regular household trash, while only a small percentage followed the suggested disposal procedures. Environmental contamination and heightened health risks are exacerbated by a lack of awareness and inadequate municipal waste management systems for hazardous household waste. The research advocates for improved public education initiatives, the creation of specific collection sites for dangerous waste, and the formulation of explicit national regulations and policies for the handling of discarded and damaged energy-saving bulbs. In rapidly urbanizing areas like Mtendere, tackling these issues is essential for protecting public health and advancing environmental sustainability. Key Words: Knowledge, Practices, Waste Disposal, and Mercury coated bulbs

Wastewater surveillance (WWS) is established as a vital tool for monitoring polio and SARS-CoV-2 with potential to improve surveillance for many other infectious diseases. This study evaluated the feasibility of detecting measles virus (MeV) RNA in wastewater as part of a national WS preparedness trial in Brisbane, Australia, from March to June 2025. Composite and passive sampling methods were employed in parallel at three wastewater treatment plants serving populations between 230,000 and 584,000. Nucleic acids were extracted and analyzed using RT-qPCR targeting MeV N and M genes to distinguish wild-type and vaccine strains. MeV RNA were detected in both 24-hour composite and passive samples on May 26 to 27, 2025 from the largest catchment of 584,000 which also included an international airport. No measles cases were reported in this city or region within 4 weeks of the WS detections. These were confirmed as vaccine-derived measles virus (MeVV) strain via specific RT-qPCR assay. Extraction recoveries varied (11.5% to 70.5%), with passive sampling showing higher efficiency. This is the first report of use of passive samples for detection of MeV. These findings are consistent with other studies reporting WWS results of both MeVV genotype A and wild type genotype B and/or D. It demonstrates the potential for sensitive MeV WWS with rapid differentiation of MeVV from wild type MeV shedding, including in airport transport hubs and with different sample types. Use of WWS could strengthen measles surveillance by enabling rapid detection of MeV RNA and supporting outbreak preparedness and response. This requires optimised methods which are specific to or differentiate wild-type MeV from MeVV. Furthermore, the successful detection of MeV using passive sampling in this study highlights its potential for deployment in diverse global contexts which may include non-sewered settings.

Mobile laboratories (MLs), whether vehicle mounted or portable, provide a versatile platform for on-site wastewater and environmental surveillance (WES) of pathogens, particularly in remote locations with limited laboratory infrastructure. However, molecular workflows intended for ML deployment require careful optimization to account for locally available equipment, consumables, infrastructure, workforce capacity, and operational constraints. In this study, we optimized an integrated ML workflow combining Oxford Nanopore Technologies (ONT) for shotgun metagenomics, multiplex metabarcoding for community level microbial analysis, and Biomeme based qPCR for targeted pathogen analysis. To further explore the potential of metagenomics for resistome assessment, we evaluated two whole metagenome enrichment approaches for their ability to improve detection of antimicrobial resistance genes. We introduce and validate a novel ONT based strategy for multiplexed sequencing small subunit (SSU) rRNA amplicon sequencing, enabling simultaneous profiling of bacteria, archaea, and microeukaryotes in complex microbial communities with multiplex metabarcoding. Sample pretreatment and nucleic acid (NA) extraction in this ML workflow were optimized using a combination chemical mechanical lysis approach followed by magnetic bead based NA purification. Workflow performance was verified using a mock community (ZymoBIOMICS Microbial Community Standard, Zymo Research, USA) and wastewater samples spiked with inactivated Mpox virus (MPXV), demonstrating accurate taxonomic representation and sensitive MPXV detection. Comparison with a commercial ZymoBIO bead beating kit for sediment sample showed ML NA extraction performed comparably. The time efficient multiplex metabarcoding workflow enabled simultaneous profiling of bacterial, archaeal, and eukaryotic diversity and produced results more concordant with qPCR based pathogen detection than the REPLIg Cell Whole Genome Amplification (WGA) & Whole Transcriptome amplification (WTA). The protocol for Mpox virus genome characterization was successfully validated for whole genome sequencing (WES) based detection and incorporated into the standard ML workflow. Across both high and low biomass environmental matrices, the Multiple Displacement Amplification (MDA) based metagenomic workflow, combined with the ML NA extraction procedure, reliably reproduced the expected composition of the Microbial Community Standard. Collectively, the integration of ONT technology with MDA metagenomics and mobile qPCR workflows provides an effective One Health approach for pathogen surveillance and outbreak response across heterogeneous environmental settings, which was later further enhanced by an offline bioinformatic and visualization pipeline enabling near real time detection of pathogens and AMR thus early risk assessment.

Wastewater and environmental monitoring (WEM) was a critical public health surveillance tool for SARS-CoV-2 surveillance during the COVID-19 Pandemic. However, substantial methodological heterogeneity across laboratories continues to challenge the interpretation and thus compromise the actionability of resulting WEM measurements. This study quantifies interlaboratory concordance in SARS-CoV-2 WEM measurements using influent wastewater samples collected between September 2021 and January 2024 at a single wastewater treatment facility within the Ontario Wastewater Surveillance Initiative, analyzed independently by 12 laboratories using their routine methods. In the absence of a known true viral concentration, interlaboratory WEM measurements were evaluated against a facility-specific longitudinal benchmark derived from routine surveillance at the source facility and correlated to clinical surveillance metrics. Concordance was assessed across four WEM measurement units commonly used in practice: SARS-CoV-2 copies/mL, SARS-CoV-2 copies/copies of PMMoV, and their standardized counterpart wastewater viral activity level (WVAL) units of WVAL-standardized SARS-CoV-2 copies/mL and WVAL-standardized SARS-CoV-2 copies/copies of PMMoV. Measurements in each unit were analyzed using complementary analytical frameworks, including categorical concordance metrics, principal component analysis, and linear mixed-effects modelling. Across the study period, interlaboratory measurements consistently captured benchmark temporal dynamics, including major peaks and periods of low activity, but showed substantial variation in magnitude and public-health interpretation across laboratory methods. Concordance was strongest during epidemiological extremes and deteriorated during transitional periods, increasing the risk of misclassification with potentially implications for public health decision-making. To explore potential laboratory methodological drivers of agreement, associations between the benchmark concordance and the laboratory-specific concentration, extraction, and RT-qPCR analytical steps were assessed using Fishers exact tests, alongside extracted-mass threshold analyses. No single methodological factor showed a statistically significant association with benchmark concordance in this study; however, several parameters, including RNA template volume, total RT-qPCR reaction volume, and extracted mass of analyzed settled solids, may warrant further investigation in future studies.

Background Informal food supply chains in regions affected by conflict present significant risks associated with the transmission of foodborne pathogens and the development of antimicrobial resistance (AMR). The objective of this research is to investigate the prevalence and AMR patterns of Salmonella spp., Shigella spp., and Listeria monocytogenes in poultry feeds and ready-to-eat (RTE) foods available for sale in Maiduguri, located in northeastern Nigeria. Methods A cross-sectional survey was conducted involving the collection of 120 poultry feed samples and approximately 251 samples of RTE foods, with 120 samples specifically designated for the analysis of Listeria. Pathogen isolation was performed using standardized microbiological protocols (ISO/FDA-BAM), and the assessment of AMR was conducted utilizing the disk diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines for Shigella (n=5), Salmonella (n=15), and Listeria (n=3) isolates. Prevalence rates were calculated with 95% confidence intervals derived from the Wilson score method. Results The study revealed a prevalence of Salmonella spp. at 10.0% (95% CI 5.8-16.8) in poultry feeds, with a maximum occurrence of 25.0% identified in the Monday Market, contrasting with a prevalence of 2.3% (0.8-6.7) reported in RTE foods, particularly in Bulumkutu fruits and meats from the Custom Market. Shigella spp. was identified in 3.33% (1.3-8.1) of feed samples and in 0.76% (0.1-4.2) of RTE foods, specifically within pineapple. L. monocytogenes was detected in 2.5% (0.8-7.1) of RTE foods, exclusively sourced from Baga Road market, including meat and sala sour milk. The isolates of Salmonella obtained from feeds demonstrated high levels of multidrug resistance (100% to tetracycline and 83.33% to fluoroquinolones), while Salmonella from RTE foods maintained resistance to tetracycline but exhibited a loss of resistance to fluoroquinolones. Notably, L. monocytogenes exhibited 100% resistance to both fluoroquinolones and cephalosporins while remaining sensitive to tetracycline. The presence of coliforms indicated inadequate hygiene conditions in the feeds, with 60.0% of samples showing contamination. Conclusion This initial study, which encompasses multiple pathogens within the conflict-affected region of Maiduguri, reveals significant upstream contamination of Salmonella in poultry feeds. It delineates the risks associated with ready-to-eat (RTE) foods and highlights the exclusive presence of L. monocytogenes in RTE products. Furthermore, the study indicates divergence in antimicrobial resistance (AMR) based on the food matrix. The findings underscore the urgent need for enhanced One Health surveillance, the implementation of improved hygiene practices in feed production, and the regulation of antimicrobial usage in informal poultry systems. Such measures are essential to effectively mitigate foodborne threats and the emergence of antimicrobial resistance in similarly underserved regions.

Invasive round goby Neogobius melanostomus have advanced eastward through the state of New York and provinces of Ontario and Quebec over the past two decades and are approaching Lake Champlain, one of the largest lakes in North America. This manuscript describes international efforts to monitor round goby populations during 2021-2025 on (a) the southern approach to Lake Champlain via the Hudson River and Champlain Canal, and (b) the northern approach to Lake Champlain via the Saint Lawrence River and Richelieu River. Monitoring utilized environmental DNA (eDNA), backpack electrofishing, beach seining, benthic trawling, and viral hemorrhagic septicemia virus (VHSV) testing. In the Champlain Canal, round goby were captured as far north as the downstream side of the C1 dam (97 kilometers [km] from Lake Champlain) while eDNA detections occurred as far north as the upstream side of the C2 dam (90 km from Lake Champlain). In the Richelieu River, round goby were captured as far south as Saint-Marc-sur-Richelieu (82 km from Lake Champlain) while the southern-most eDNA detections occurred near the Canadian side of the international border (4 km from Lake Champlain). Water temperature influenced habitat usage of round goby in the Champlain Canal, with catch rates in near-shore areas declining at < 10 {degrees}C. All VHSV test results were non-detections at the mouth of the Richelieu River, while one positive and two inconclusive results occurred along the Champlain Canal. Together, these data have informed multiple mitigation measures and have implications for management of aquatic invasive species across North America.

BackgroundCholera outbreaks remain a major public-health challenge in sub-Saharan Africa, where diagnostic capacity is limited and clinical case definitions are non-specific and reply heavily on syndromic diagnosis. Rapid identification of Vibrio cholerae is critical, yet cholera-suspected diarrhoea can have multiple infectious causes not captured by targeted diagnostics. MethodsWe evaluated a mobile, culture-independent metagenomic sequencing workflow for on-site detection of gastrointestinal pathogens directly from faecal samples in Burundi. The offline workflow combined long-read ONT sequencing with rapid, laptop-based taxonomic and antimicrobial resistance (AMR) screening and was deployed across a health centre, a district hospital, and a refugee transit camp. The frontline and real-time results were verified using both conventional culturing and in-depth bioinformatic analyses. ResultsV. cholerae signals were only detected in a subset of suspected cholera cases, while many samples were dominated by alternative bacterial taxa, most frequently Escherichia coli. V. cholerae abundance correlated strongly with detection of the cholera toxin phage CTX{varphi}, supporting differentiation between toxigenic signal and background exposure. AMR genes were detected across samples, providing early situational insight into resistance determinants among gastrointestinal bacteria. ConclusionsMobile, offline metagenomic sequencing enables rapid frontline characterization of gastrointestinal disease, especially cholera-suspected, in resource-limited settings and complements existing diagnostics by improving etiological resolution and outbreak response. Author SummaryCholera remains a major cause of severe diarrhoeal disease in many low-resource settings, where diagnosis often relies on symptoms and limited laboratory testing. However, patients suspected of cholera can be infected by a wide range of other pathogens that are not detected by standard diagnostics. In this study, we evaluated a portable, sequencing-based approach that allows direct identification of pathogens from stool samples at the point of care, without the need for laboratory infrastructure, internet access, or culture. Using this approach in multiple settings in Burundi, including a health centre, hospital, and refugee camp, we found a subset of suspected cholera cases were associated with Vibrio cholerae. Other cases were also dominated by other bacteria, particularly Escherichia coli. We also detected antimicrobial resistance genes across samples, providing additional information relevant for treatment and surveillance. Our findings demonstrate that mobile metagenomic sequencing can improve the identification of disease causes directly in outbreak settings and help distinguish true cholera cases from other gastrointestinal infections. This approach has the potential to strengthen outbreak response, improve patient management, and support more accurate disease surveillance in resource-limited environments.

BackgroundRising global temperatures and eutrophication are increasing the intensity and frequency of cyanobacterial harmful algal blooms that release toxins including microcystin-LR (MC-LR). MC-LR inhibits protein phosphatases in the human liver and brain, but its accumulation in the placenta is unclear. Placental transporter expression varies across pregnancy and is influenced by physiological cues, such as low oxygen concentrations which activate HIF1A, and trophoblast cell fusion forming syncytiotrophoblasts that engage CREB-driven transcription. This study examined whether MC-LR accumulates in placental cells, which transporters mediate uptake, and how these transporters are regulated by HIF1A and CREB. MethodsIntracellular accumulation of MC-LR (0.1-10 {micro}M, 3 hour) was measured in human cytotrophoblasts (JAR, BeWo) and extravillous trophoblasts (HTR-8/SVneo) by western blotting for MC-LR-adducted proteins. Organic anion transporting polypeptide (OATP) involvement was tested using cyclosporin A (10 {micro}M), an OATP inhibitor, before exposure to the OATP substrate or MC-LR. Cells were also cultured under 3%, 8%, or 20% O2 to induce hypoxic responses or treated with forskolin (a potent intracellular cAMP inducer) to stimulate cell fusion before MC-LR exposure. ResultsMC-LR accumulated in all three placenta cell lines in a concentration-dependent manner. Cyclosporin A reduced MC-LR uptake by 57% in JAR cells, confirming OATP-mediated transport. Low O2 increased OATP4A1 expression and function but reduced protein phosphatase expression, decreasing MC-LR-bound proteins by 52-72%. Forskolin increased OATP4A1 expression and enhanced MC-LR uptake >2.5-fold. ConclusionMC-LR enters placental trophoblasts via active OATP transport, likely OATP4A1, and uptake increases under hypoxia and trophoblast fusion.