Comparison of gene targets and sampling regimes for SARS-CoV-2 quantification for wastewater epidemiology in UK prisons.

Prisons are high-risk settings for infectious disease transmission, due to their enclosed and semi-enclosed environments. The proximity between prisoners and staff, and the diversity of prisons reduces the effectiveness of non-pharmaceutical interventions, such as social distancing. Therefore, alternative health monitoring methods, such as wastewater-based epidemiology (WBE), are needed to track pathogens, including SARS-CoV-2. This pilot study assessed WBE to quantify SARS-CoV-2 prevalence in prison wastewater to determine its utility within a health protection system for residents. The study analysed 266 samples from six prisons in England over a 12-week period for nucleoprotein 1 (N1 gene) and envelope protein (E gene) using quantitative reverse transcriptase-polymerase chain reaction. Both gene assays successfully detected SARS-CoV-2 fragments in wastewater samples, with both genes significantly correlating with COVID-19 case numbers across the prisons (p < 0.01). However, in 25% of the SARS-positive samples, only one gene target was detected, suggesting that both genes be used to reduce false-negative results. No significant differences were observed between 14- and 2-h composite samples, although 2-h samples showed greater signal variance. Population normalisation did not improve correlations between the N1 and E genes and COVID-19 case data. Overall, WBE shows considerable promise for health protection in prison settings.

Trapping and bypassing of suspended particulate matter, particulate nutrients and faecal indicator organisms in the river-estuary transition zone of a shallow macrotidal estuary.

Hydrodynamic controls of the transport of suspended particulate matter (SPM), particulate nutrients and faecal indicator organisms (FIOs) in the river-estuary transition zone (RETZ) of a shallow macrotidal estuary were studied on tidal and seasonal timescales. The RETZ was found to be a conduit for SPM rather than a zone of particle accumulation during spring tides, with complex size fractionation and biogeochemical exchanges of particulate nutrient/FIO compositions. The downstream RETZ was characterised by flood-dominant currents, but with ebb-dominant turbulence due to the suppression of flood tide turbulence by salinity stratification created by lateral convergence; this produced a net seaward mass transport of SPM. Without lateral convergence in the narrower upstream RETZ, flood-dominant currents and flood-dominant turbulence were experienced. Hence the RETZ exported SPM landwards from its upper end and seawards from its lower end - a process observed throughout the year during spring tides and low-to-mean river flows. During neap tides when SPM concentrations were reduced, the RETZ became a zone of minor particle accumulation as its lower end imported SPM landwards from the estuary and its upper end imported SPM seawards from the river. During a river flood event, net SPM flux was significantly increased and was seawards throughout the RETZ. SPM mass concentration and carbon, nitrogen, phosphorous, and FIO concentrations peaked due to local resuspension and advection of an ephemeral estuarine turbidity maximum (ETM). The ETM formed on the advancing flood tide due to entrainment of material from intertidal flats. Flocculation and settling occurred at high slack water. The ETM was reconstituted by entrainment on the ebb and was composed of larger flocs than on the flood. Particulate nutrients and FIOs were associated with flocs in the 10-200 µm range but not with smaller or larger flocs. SPM concentrations in the resuspension component and ETM exceeded microbial water quality standards, emphasising the need for monitoring practices that consider tidal dynamics. The results from this study showing periodic SPM export from, rather than prolonged accumulation in, the RETZ and the influence of particle size fractionation on biogeochemical fluxes in the RETZ, are likely to be transferable to many other embayment-type estuaries on macrotidal coasts.

Wastewater-based epidemiology for surveillance of infectious diseases in healthcare settings.

PURPOSE OF REVIEW: Wastewater-based surveillance (WBS) (epidemiology) using near-source sampling (NSS) in large buildings, hospitals and care homes is reviewed covering three main areas: state-of-the-art WBS, benefits/opportunities NSS has for hospital infection control systems and new insights from hospital wastewater surveillance and policy implications. RECENT FINDINGS: Wastewater provides aggregate, anonymous sources of data where the spatial resolution can be linked to populations being served. In hospitals, clear links established between wastewater RNA-fragments signal to nosocomial COVID-19 cases/outbreaks. Detecting other targets from hospital wastewater such as antimicrobial resistance markers is considered a substantial opportunity for this technology. Other clinically relevant infections, that is influenza and monkeypox, can be perceived, and sub-variant resolution to target public health response in near real time to benefit hospital infection control. WBS can reduce hospitals' clinical testing requirements, as diagnostic costs are aggregated into fewer samples while still detecting single cases. SUMMARY: WBS using NSS can inform infectious disease monitoring earlier, faster and cheaper than conventional monitoring. Routine sampling using wastewater provides a platform for risk-based sampling and enables smarter allocation of resources. Finally, hospital wastewater can be used for the benefit of the wastewater surveillance field as a promising source to monitor emerging threats and resolve longstanding questions on faecal shedding. Hospital monitoring in low-income settings is considered a priority for future research.

Wastewater monitoring for detection of public health markers during the COVID-19 pandemic: Near-source monitoring of schools in England over an academic year.

BACKGROUND: Schools are high-risk settings for infectious disease transmission. Wastewater monitoring for infectious diseases has been used to identify and mitigate outbreaks in many near-source settings during the COVID-19 pandemic, including universities and hospitals but less is known about the technology when applied for school health protection. This study aimed to implement a wastewater surveillance system to detect SARS-CoV-2 and other public health markers from wastewater in schools in England. METHODS: A total of 855 wastewater samples were collected from 16 schools (10 primary, 5 secondary and 1 post-16 and further education) over 10 months of school term time. Wastewater was analysed for SARS-CoV-2 genomic copies of N1 and E genes by RT-qPCR. A subset of wastewater samples was sent for genomic sequencing, enabling determination of the presence of SARS-CoV-2 and emergence of variant(s) contributing to COVID-19 infections within schools. In total, >280 microbial pathogens and >1200 AMR genes were screened using RT-qPCR and metagenomics to consider the utility of these additional targets to further inform on health threats within the schools. RESULTS: We report on wastewater-based surveillance for COVID-19 within English primary, secondary and further education schools over a full academic year (October 2020 to July 2021). The highest positivity rate (80.4%) was observed in the week commencing 30th November 2020 during the emergence of the Alpha variant, indicating most schools contained people who were shedding the virus. There was high SARS-CoV-2 amplicon concentration (up to 9.2x106 GC/L) detected over the summer term (8th June - 6th July 2021) during Delta variant prevalence. The summer increase of SARS-CoV-2 in school wastewater was reflected in age-specific clinical COVID-19 cases. Alpha variant and Delta variant were identified in the wastewater by sequencing of samples collected from December to March and June to July, respectively. Lead/lag analysis between SARS-CoV-2 concentrations in school and WWTP data sets show a maximum correlation between the two-time series when school data are lagged by two weeks. Furthermore, wastewater sample enrichment coupled with metagenomic sequencing and rapid informatics enabled the detection of other clinically relevant viral and bacterial pathogens and AMR. CONCLUSIONS: Passive wastewater monitoring surveillance in schools can identify cases of COVID-19. Samples can be sequenced to monitor for emerging and current variants of concern at the resolution of school catchments. Wastewater based monitoring for SARS-CoV-2 is a useful tool for SARS-CoV-2 passive surveillance and could be applied for case identification and containment, and mitigation in schools and other congregate settings with high risks of transmission. Wastewater monitoring enables public health authorities to develop targeted prevention and education programmes for hygiene measures within undertested communities across a broad range of use cases.

Inhibitory mechanisms on dry anaerobic digestion: Ammonia, hydrogen and propionic acid relationship.

Inhibitory pathways in dry anaerobic digestion are still understudied and current knowledge on wet processes cannot be easily transferred. This study forced instability in pilot-scale digesters by operating at short retention times (40 and 33 days) in order to understand inhibition pathways over long term operation (145 days). The first sign of inhibition at elevated total ammonia concentrations (8 g/l) was a headspace hydrogen level over the thermodynamic limit for propionic degradation, causing propionic accumulation. The combined inhibitory effect of propionic and ammonia accumulation resulted in further increased hydrogen partial pressures and n-butyric accumulation. The relative abundance of Methanosarcina increased while that of Methanoculleus decreased as digestion deteriorated. It was hypothesized that high ammonia, total solids and organic loading rate inhibited syntrophic acetate oxidisers, increasing their doubling time and resulting in its wash out, which in turn inhibited hydrogenotrophic methanogenesis and shifted the predominant methanogenic pathway towards acetoclastic methanogenesis at free ammonia over 1.5 g/l. C/N increases to 25 and 29 reduced inhibitors accumulation but did not avoid inhibition or the washout of syntrophic acetate oxidising bacteria.

Multifunctional Heterogeneous Ion-Exchange Membranes for Ion and Microbe Removal in Low-Salinity Water.

Here, multifunctional heterogeneous ion-exchange metal nanocomposite membranes were prepared for surface water desalination and bacterial inactivation under low-pressure (0.05 MPa) filtration conditions. Ultrafiltration (UF) heterogeneous ion exchange membranes (IEMs) were modified with different concentrations of AgNO3 and CuSO4 solutions using the intermatrix synthesis (IMS) technique to produce metal nanocomposite membranes. Scanning electron microscopy (SEM) images revealed that the metal nanoparticles (MNPs) (Ag and Cu) were uniformly distributed on the surface and the interior of the nanocomposite membranes. With increasing metal precursor solution concentration (0.01 to 0.05 mol·L-1), the metal content of Ag and Cu nanocomposite membranes increased from 0.020 to 0.084 mg·cm-2 and from 0.031 to 0.218 m·cm-2 respectively. Results showed that the hydrodynamic diameter diameters of Ag and Cu nanoparticles (NPs) increased from 62.42 to 121.10 nm and from 54.2 to 125.7 nm respectively, as the metal precursor concentration loaded increased. The leaching of metals from metal nanocomposite membranes was measured in a dead-end filtration system, and the highest leaching concentration levels were 8.72 ppb and 5.32 ppb for Ag and Cu, respectively. The salt rejection studies indicated that ionic selectivity was improved with increasing metal content. Bacterial filtration showed higher antibacterial activity for metal nanocomposite membranes, reaching 3.6 log bacterial inactivation.

Molecular insights informing factors affecting low temperature anaerobic applications: Diversity, collated core microbiomes and complexity stability relationships in LCFA-fed systems.

Fats, oil and grease, and their hydrolyzed counterparts-long chain fatty acids (LCFA) make up a large fraction of numerous wastewaters and are challenging to degrade anaerobically, more so, in low temperature anaerobic digestion (LtAD) systems. Herein, we perform a comparative analysis of publicly available Illumina 16S rRNA datasets generated from LCFA-degrading anaerobic microbiomes at low temperatures (10 and 20 °C) to comprehend the factors affecting microbial community dynamics. The various factors considered were the inoculum, substrate and operational characteristics, the reactor operation mode and reactor configuration, and the type of nucleic acid sequenced. We found that LCFA-degrading anaerobic microbiomes were differentiated primarily by inoculum characteristics (inoculum source and morphology) in comparison to the other factors tested. Inoculum characteristics prominently shaped the species richness, species evenness and beta-diversity patterns in the microbiomes even after long term operation of continuous reactors up to 150 days, implying the choice of inoculum needs careful consideration. The generalised additive models represented through beta diversity contour plots revealed that psychrophilic bacteria RBG-13-54-9 from family Anaerolineae, and taxa WCHB1-41 and Williamwhitmania were highly abundant in LCFA-fed microbial niches, suggesting their role in anaerobic treatment of LCFAs at low temperatures of 10-20 °C. Overall, we showed that the following bacterial genera: uncultured Propionibacteriaceae, Longilinea, Christensenellaceae R7 group, Lactivibrio, candidatus Caldatribacterium, Aminicenantales, Syntrophus, Syntrophomonas, Smithella, RBG-13-54-9, WCHB1-41, Trichococcus, Proteiniclasticum, SBR1031, Lutibacter and Lentimicrobium have prominent roles in LtAD of LCFA-rich wastewaters at 10-20 °C. This study provides molecular insights of anaerobic LCFA degradation under low temperatures from collated datasets and will aid in improving LtAD systems for treating LCFA-rich wastewaters.

Detection and characterization of bioaerosol emissions from wastewater treatment plants: Challenges and opportunities.

Rapid population growth and urbanization process have led to increasing demand for wastewater treatment capacity resulting in a non-negligible increase of wastewater treatment plants (WWTPs) in several cities around the world. Bioaerosol emissions from WWTPs may pose adverse health risks to the sewage workers and nearby residents, which raises increasing public health concerns. However, there are still significant knowledge gaps on the interplay between process-based bioaerosol characteristics and exposures and the quantification of health risk which limit our ability to design effective risk assessment and management strategies. This review provides a critical overview of the existing knowledge of bioaerosol emissions from WWTPs including their nature, magnitude and size distribution, and highlights the shortcoming associated with existing sampling and analysis methods. The recent advancements made for rapid detection of bioaerosols are then discussed, especially the emerging real time detection methods to highlight the directions for future research needs to advance the knowledge on bioaerosol emissions from WWTPs.

First insights into the prokaryotic community structure of Lake Cote, Costa Rica: Influence on nutrient cycling.

Prokaryotic diversity in lakes has been studied for many years mainly focusing on community structure and how the bacterial assemblages are driven by physicochemical conditions such as temperature, oxygen, and nutrients. However, little is known about how the composition and function of the prokaryotic community changes upon lake stratification. To elucidate this, we studied Lake Cote in Costa Rica determining prokaryotic diversity and community structure in conjunction with physicochemistry along vertical gradients during stratification and mixing periods. Of the parameters measured, ammonium, oxygen, and temperature, in that order, were the main determinants driving the variability in the prokaryotic community structure of the lake. Distinct stratification of Lake Cote occurred (March 2018) and the community diversity was compared to a period of complete mixing (March 2019). The microbial community analysis indicated that stratification significantly altered the bacterial composition in the epi-meta- and hypolimnion. During stratification, the Deltaproteobacteria, Chloroflexi, Bacteroidetes, Nitrospirae, and Euryarchaeota were dominant in the hypolimnion yet largely absent in surface layers. Among these taxa, strict or facultative anaerobic bacteria were likely contributing to the lake nitrogen biogeochemical cycling, consistent with measurements of inorganic nitrogen measurements and microbial functional abundance predictions. In general, during both sampling events, a higher abundance of Alphaproteobacteria, Betaproteobacteria, Actinobacteria, and Cyanobacteria was found in the oxygenated layers. Lake Cote had a unique bacterial diversity, with 80% of Amplicon Sequence Variant (ASV) recovered similar to unclassified/uncultured strains and exhibits archetypal shallow lake physicochemical but not microbial fluctuations worthy of further investigation. This study provides an example of lake hydrodynamics impacts to microbial community and their function in Central American lakes with implications for other shallow, upland, and oligotrophic lake systems.

Dissemination of metaldehyde catabolic pathways is driven by mobile genetic elements in Proteobacteria.

Bioremediation of metaldehyde from drinking water using metaldehyde-degrading strains has recently emerged as a promising alternative. Whole-genome sequencing was used to obtain full genomes for metaldehyde degraders Acinetobacter calcoaceticus E1 and Sphingobium CMET-H. For the former, the genetic context of the metaldehyde-degrading genes had not been explored, while for the latter, none of the degrading genes themselves had been identified. In A. calcoaceticus E1, IS91 and IS6-family insertion sequences (ISs) were found surrounding the metaldehyde-degrading gene cluster located in plasmid pAME76. This cluster was located in closely-related plasmids and associated to identical ISs in most metaldehyde-degrading ß- and γ-Proteobacteria, indicating horizontal gene transfer (HGT). For Sphingobium CMET-H, sequence analysis suggested a phytanoyl-CoA family oxygenase as a metaldehyde-degrading gene candidate due to its close homology to a previously identified metaldehyde-degrading gene known as mahX. Heterologous gene expression in Escherichia coli alongside degradation tests verified its functional significance and the degrading gene homolog was henceforth called mahS. It was found that mahS is hosted within the conjugative plasmid pSM1 and its genetic context suggested a crossover between the metaldehyde and acetoin degradation pathways. Here, specific replicons and ISs responsible for maintaining and dispersing metaldehyde-degrading genes in α, ß and γ-Proteobacteria through HGT were identified and described. In addition, a homologous gene implicated in the first step of metaldehyde utilisation in an α-Proteobacteria was uncovered. Insights into specific steps of this possible degradation pathway are provided.

Coliphages as viral indicators of sanitary significance for drinking water.

Coliphages are virus that infect coliform bacteria and are used in aquatic systems for risk assessment for human enteric viruses. This mini-review appraises the types and sources of coliphage and their fate and behavior in source waters and engineered drinking water treatment systems. Somatic (cell wall infection) and F+ (male specific) coliphages are abundant in drinking water sources and are used as indicators of fecal contamination. Coliphage abundances do not consistently correlate to human enteric virus abundance, but they suitably reflect the risks of exposure to human enteric viruses. Coliphages have highly variable surface characteristics with respect to morphology, size, charge, isoelectric point, and hydrophobicity which together interact to govern partitioning and removal characteristics during water treatment. The groups somatic and F+ coliphages are valuable for investigating the virus elimination during water treatment steps and as indicators for viral water quality assessment. Strain level analyses (e.g., Qß or GA-like) provide more information about specific sources of viral pollution but are impractical for routine monitoring. Consistent links between rapid online monitoring tools (e.g., turbidity, particle counters, and flow cytometry) and phages in drinking water have yet to be established but are recommended as a future area of research activity. This could enable the real-time monitoring of virus and improve the process understanding during transient operational events. Exciting future prospects for the use of coliphages in aquatic microbiology are also discussed based on current scientific evidence and practical needs.

Resin-Loaded Heterogeneous Polyether Sulfone Ion Exchange Membranes for Saline Groundwater Treatment.

Arid areas often contain brackish groundwater that has a salinity exceeding 500 mg/L. This poses several challenges to the users of the water such as a salty taste and damage to household appliances. Desalination can be one of the key solutions to significantly lower the salinity and solute content of the water. However, the technology requires high energy inputs as well as managing waste products. This paper presents the fabrication of ultrafiltration heterogeneous ion exchange membranes for brackish groundwater treatment. Scanning electron microscopy (SEM) images showed a relatively uniform resin particle distribution within the polymer matrix. The mean roughness of the cation exchange membrane (CEM) and anion exchange membrane (AEM) surfaces increased from 42.12 to 317.25 and 68.56 to 295.95 nm, respectively, when resin loading was increased from 1 to 3.5 wt %. Contact angle measures suggested a more hydrophilic surface (86.13 to 76.26° and 88.10 to 74.47° for CEM and AEM, respectively) was achieved with greater resin loading rates. The ion exchange capacity (IEC) of the prepared membranes was assessed using synthetic groundwater in a dead-end filtration system and removal efficiency of K+, Mg2+, and Ca2+ were 56.0, 93.5, and 85.4%, respectively, for CEM with the highest resin loading. Additionally, the anion, NO3- and SO42- removal efficiency was 84.2% and 52.4%, respectively, for the AEM with the highest resin loading. This work demonstrates that the prepared ultrafiltration heterogeneous ion exchange membranes have potential for selective removal for of ions by ion exchange, under filtration conditions at low pressure of 0.05 MPa.

Monitoring occurrence of SARS-CoV-2 in school populations: A wastewater-based approach.

Clinical testing of children in schools is challenging, with economic implications limiting its frequent use as a monitoring tool of the risks assumed by children and staff during the COVID-19 pandemic. Here, a wastewater-based epidemiology approach has been used to monitor 16 schools (10 primary, 5 secondary and 1 post-16 and further education) in England. A total of 296 samples over 9 weeks have been analysed for N1 and E genes using qPCR methods. Of the samples returned, 47.3% were positive for one or both genes with a detection frequency in line with the respective local community. WBE offers a low cost, non-invasive approach for supplementing clinical testing and can provide longitudinal insights that are impractical with traditional clinical testing.

Probe-based qPCR assay enables the rapid and specific detection of bacterial degrading genes for the pesticide metaldehyde in soil.

Metaldehyde, a molluscicide pesticide, has been identified as a pollutant of concern due to its repeated detection in drinking water, thereby generating numerous compliance failures for water utilities. Biological degradation potential for metaldehyde is widespread in soils, occurring at different rates, but to date, no molecular methods for its assessment have been reported. Here, three genes belonging to a shared metaldehyde-degrading gene cluster present in bacteria were used as candidates for development of a quantitative PCR (qPCR) assay for assessing the metaldehyde-degrading potential in soil. Screening of gene targets, primer pairs and optimization of reaction conditions led to the development of a sensitive and specific probe-based qPCR method for quantifying the mahY metaldehyde-degrading gene from soil. The technique was tested across 8 soils with different compositions and origins. The degrading pathway was detected in 4/8 soils, in which a higher number of gene copies correlated with periods of greater metaldehyde removal. Additionally, swift elimination of the pesticide was observed in soils with an elevated initial number of mahY gene copies. The gene cluster was not detected in other soils, even though metaldehyde removal occurred, indicating that other biological degrading pathways are also important in nature. The method described here is the first one available to estimate the microbial metaldehyde degradation potential and activity in soils, and can also be used to detect degrading microorganisms in systems such as sand filters for water purification or to monitor degrading strains in engineered processes.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.

Achieving drinking water compliance levels for metaldehyde with an acclimated sand bioreactor.

Metaldehyde removal was delivered to below the 0.1 µg L-1 regulatory concentration in a laboratory scale continuous upflow fluidised sand bioreactor that had undergone acclimation through selective enrichment for metaldehyde degradation. This is the first reported case of successful continuous flow biological treatment of metaldehyde from real drinking water sources treating environmentally realistic metaldehyde concentrations. The impact of the acclimation process was impermanent, with the duration of effective treatment directly related to the elevated concentration of metaldehyde used during the enrichment process. The efficacy of the approach was demonstrated in continuous flow columns at both laboratory and pilot scale enabling degradation rates of between 0.1 and 0.2 mg L-1 h-1. Future work needs to focus on optimisation of the sand bioreactor and the acclimation process to ensure viability and feasibility of the approach at full scale.

Risk factors and transmission pathways associated with infant Campylobacter spp. prevalence and malnutrition: A formative study in rural Ethiopia.

Early infection from enteropathogens is recognised as both a cause and effect of infant malnutrition. Specifically, evidence demonstrates associations between growth shortfalls and Campylobacter infection, endemic across low-income settings, with poultry a major source. Whilst improvements in water, sanitation and hygiene (WASH) should reduce pathogen transmission, interventions show inconsistent effects on infant health. This cross-sectional, formative study aimed to understand relationships between infant Campylobacter prevalence, malnutrition and associated risk factors, including domestic animal husbandry practices, in rural Ethiopia. Thirty-five households were visited in Sidama zone, Southern Nations, Nationalities and Peoples' region. Infant and poultry faeces and domestic floor surfaces (total = 102) were analysed for presumptive Campylobacter spp. using selective culture. Infant anthropometry and diarrhoeal prevalence, WASH facilities and animal husbandry data were collected. Of the infants, 14.3% were wasted, 31.4% stunted and 31.4% had recent diarrhoea. Presumptive Campylobacter spp. was isolated from 48.6% of infant, 68.6% of poultry and 65.6% of floor surface samples. Compared to non-wasted infants, wasted infants had an increased odds ratio (OR) of 1.41 for a Campylobacter-positive stool and 1.81 for diarrhoea. Positive infant stools showed a significant relationship with wasting (p = 0.026) but not stunting. Significant risk factors for a positive stool included keeping animals inside (p = 0.027, OR 3.5), owning cattle (p = 0.018, OR 6.5) and positive poultry faeces (p<0.001, OR 1.34). Positive floor samples showed a significant correlation with positive infant (p = 0.023), and positive poultry (p = 0.013, OR 2.68) stools. Ownership of improved WASH facilities was not correlated with lower odds of positive stools. This formative study shows a high prevalence of infants positive for Campylobacter in households with free-range animals. Findings reaffirm contaminated floors as an important pathway to infant pathogen ingestion and suggest that simply upgrading household WASH facilities will not reduce infection without addressing the burden of contamination from animals, alongside adequate separation in the home.