Increasing incidence of antibiotic resistance in clinical and environmental settings calls for increased scalability in their surveillance. Current screening technologies are limited by the number of samples and genes that can easily be screened. We demonstrate here digital multiplex ligation assay (dMLA) as a low-cost targeted genomic detection workflow capable of highly-parallel screening of bacterial isolates for multiple target gene regions simultaneously. Here, dMLA is used for simultaneous detection of 1187 β-lactamase-encoding genes, including extended spectrum β-lactamase (ESBL) genes, in 74 bacterial isolates. We demonstrate dMLA as a light-weight and cost-efficient workflow which provides a highly scalable tool for antimicrobial resistance surveillance and is also adaptable to genetic screening applications beyond antibiotic resistance.

Antimicrobial resistance (AMR) is a growing public health challenge that is expected to disproportionately burden lower- and middle-income countries (LMICs) in the coming decades. Although the contributions of human and veterinary antibiotic misuse to this crisis are well-recognized, environmental transmission (via water, soil or food contaminated with human and animal faeces) has been given less attention as a global driver of AMR, especially in urban informal settlements in LMICs - commonly known as 'shanty towns' or 'slums'. These settlements may be unique hotspots for environmental AMR transmission given: (1) the high density of humans, livestock and vermin living in close proximity; (2) frequent antibiotic misuse; and (3) insufficient drinking water, drainage and sanitation infrastructure. Here, we highlight the need for strategies to disrupt environmental AMR transmission in urban informal settlements. We propose that water and waste infrastructure improvements tailored to these settings should be evaluated for their effectiveness in limiting environmental AMR dissemination, lowering the community-level burden of antimicrobial-resistant infections and preventing antibiotic misuse. We also suggest that additional research is directed towards developing economic and legal incentives for evaluating and implementing water and waste infrastructure in these settings. Given that almost 90% of urban population growth will occur in regions predicted to be most burdened by the AMR crisis, there is an urgent need to build effective, evidence-based policies that could influence massive investments in the built urban environment in LMICs over the next few decades.

Escherichia coli is present in multiple hosts and environmental compartments as a normal inhabitant, temporary or persistent colonizer, and as a pathogen. Transmission of E. coli between hosts and with the environment is considered to occur more often in areas with poor sanitation. We performed whole-genome comparative analyses on 60 E. coli isolates from soils and fecal sources (cattle, chickens, and humans) in households in rural Bangladesh. Isolates from household soils were in multiple branches of the reconstructed phylogeny, intermixed with isolates from fecal sources. Pairwise differences between all strain pairs were large (minimum, 189 single nucleotide polymorphisms [SNPs]), suggesting high diversity and heterogeneous origins of the isolates. The presence of multiple virulence and antibiotic resistance genes is indicative of the risk that E. coli from soil and feces represent for the transmission of variants that pose potential harm to people. Analysis of the accessory genomes of the Bangladeshi E. coli relative to E. coli genomes available in NCBI identified a common pool of accessory genes shared among E. coli isolates in this geographic area. Together, these findings indicate that in rural Bangladesh, a high level of E. coli in soil is likely driven by contributions from multiple and diverse E. coli sources (human and animal) that share an accessory gene pool relatively unique to previously published E. coli genomes. Thus, interventions to reduce environmental pathogen or antimicrobial resistance transmission should adopt integrated One Health approaches that consider heterogeneous origins and high diversity to improve effectiveness and reduce prevalence and transmission.

Background: Infectious diseases can be transmitted via fomites (contaminated surfaces/objects); disinfection can interrupt this transmission route. However, disinfection guidelines for low-resource outbreak settings are inconsistent and not evidence-based.
Methods: A systematic review of surface disinfection efficacy studies was conducted to inform low-resource outbreak guideline development. Due to variation in experimental procedures, outcomes were synthesized in a narrative summary focusing on chlorine-based disinfection against seven pathogens with potential to produce outbreaks in low-resource settings (Mycobacterium tuberculosis, Vibrio cholerae, Salmonella spp., hepatitis A virus, rotavirus, norovirus, Ebola virus).
Results: Data were extracted from 89 laboratory studies and made available, including 20 studies on relevant pathogens used in combination with surrogate data to determine minimum target concentration × time ("CT") factors. Stainless steel (68%) and chlorine-based disinfectants (56%) were most commonly tested. No consistent trend was seen in the influence of chlorine concentration and exposure time on disinfection efficacy. Disinfectant application mode; soil load; and surface type were frequently identified as influential factors in included studies.
Conclusions: This review highlights that surface disinfection efficacy estimates are strongly influenced by each study's experimental conditions. We therefore recommend laboratory testing to be followed by field-based testing/monitoring to ensure effectiveness is achieved in situ.

Infectious disease transmission is frequently mediated by the environment, where people's movements through and interactions with the environment dictate risks of infection and/or illness. Capturing these interactions, and quantifying their importance, offers important insights into effective interventions. In this study, we capture high time-resolution activity data for twenty-five Vietnamese farmers during collection and land application of human excreta for agriculture. Although human excreta use improves productivity, the use increases risks of enteric infections for both farmers and end users. In our study, the activity data are integrated with environmental microbial sampling data into a stochastic-mechanistic simulation of E. coli contamination on hands and E. coli ingested. Results from the study include frequent and variable contact rates for farmers' hands (from 34 to 1344 objects contacted per hour per hand), including highly variable hand-to-mouth contact rates (from 0 to 9 contacts per hour per hand). The frequency of hand-to-mouth contacts was substantially lower than the widely-used frequency previously reported for U.S. Office Workers. Environmental microbial contamination data highlighted ubiquitous E. coli contamination in the environment, including excreta, hands, toilet pit, handheld tools, soils, surfaces, and water. Results from the simulation suggest dynamic changes in E. coli contamination on hands, and wide variation in hand contamination and E. coli ingested amongst the farmers studied. Sensitivity analysis suggests that E. coli contamination on hands and ingested doses are most influenced by contamination of handheld tools, excreta, and the toilet pit as well as by frequency of hand-to-mouth contacts. The study findings are especially relevant given the context: no farmers reported adequate storage time of human excreta, and personal protective mask availability did not prevent hand-to-mouth contacts. Integrating high time-resolution activity data into exposure assessments highlights variation in exposures amongst farmers, and offers greater insight into effective interventions and their potential impacts.

Escherichia coli pathotypes (i.e., enteropathogenic and enterotoxigenic) have been identified among the pathogens most responsible for moderate-to-severe diarrhea in low- and middle-income countries (LMICs). Pathogenic E. coli are transmitted from infected human or animal feces to new susceptible hosts via environmental reservoirs such as hands, water, and soil. Commensal E. coli, which includes nonpathogenic E. coli strains, are widely used as fecal bacteria indicator, with their presence associated with increased likelihood of enteric pathogens and/or diarrheal disease. In this study, we investigated E. coli contamination in environmental reservoirs within households (N = 142) in high-population density communities of Harare, Zimbabwe. We further assessed the interconnectedness of the environmental compartments by investigating associations between, and household-level risk factors for, E. coli contamination. From the data we collected, the source and risk factors for E. coli contamination are not readily apparent. One notable exception is the presence of running tap water on the household plot, which is associated with significantly less E. coli contamination of drinking water, handwashing water, and hands after handwashing. In addition, E. coli levels on hands after washing are significantly associated with handwashing water contamination, hand contamination before washing, and diarrhea incidence. Finally, we observed that animal ownership increases E. coli contamination in soil, and E. coli in soil are correlated with contamination on hands before washing. This study highlights the complexity of E. coli contamination in household environment within LMICs. More, larger, studies are needed to better identify sources and exposure pathways of E. coli—and enteric pathogens generally—to identify effective interventions.

Quantitative data on human-environment interactions are needed to fully understand infectious disease transmission processes and conduct accurate risk assessments. Interaction events occur during an individual’s movement through, and contact with, the environment, and can be quantified using diverse methodologies. Methods that utilize videography, coupled with specialized software, can provide a permanent record of events, collect detailed interactions in high resolution, be reviewed for accuracy, capture events difficult to observe in real-time, and gather multiple concurrent phenomena. In the accompanying video, the use of specialized software to capture human-environment interactions for human exposure and disease transmission is highlighted. Use of videography, combined with specialized software, allows for the collection of accurate quantitative representations of human-environment interactions in high resolution. Two specialized programs include the Virtual Timing Device for the Personal Computer, which collects sequential microlevel activity time series of contact events and interactions, and LiveTrak, which is optimized to facilitate annotation of events in real-time. Opportunities to annotate behaviors at high resolution using these tools are promising, permitting detailed records that can be summarized to gain information on infectious disease transmission and incorporated into more complex models of human exposure and risk.

Noch immer sind die Quellen und Übertragungswege von E. coli-Kontaminationen nicht ganz verstanden. Um Antworten auf offene Fragen zu finden, wurden im Rahmen eines Forschungsprojekts 142 Haushalte in der simbabwischen Hauptstadt Harare auf das Vorhandensein von E. coli untersucht. Zudem wurden Haushaltsmerkmale gesammelt. Dadurch erhoffte sich das Forschungsteam, signifikante Zusammenhänge aufzuzeigen.

As more countries engage in water reuse, either intended or de facto, there is an urgent need to more comprehensively evaluate resulting environmental and public health concerns. While antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are increasingly coming under the spotlight, as emerging contaminants, existing water reuse regulations and guidelines do not adequately address these concerns. This perspectives paper seeks to frame the various challenges that need to be resolved to identify meaningful and realistic target types and levels of antibiotic resistance benchmarks for water reuse. First, there is the need for standardized and agreed-upon methodologies to identify and quantify ARB and ARGs. Second, even if methodologies are available, identifying which ARB and ARGs to monitor that would best relate to the occurrence of disease burden remains unknown. Third, a framework tailored to assessing the risks associated with ARB and ARGs during reuse is urgently needed. Fourth, similar to protecting drinking water sources, strategies to prevent dissemination of ARB and ARGs via wastewater treatment and reuse are required to ensure that appropriate barriers are emplaced. Finally, current wastewater treatment technologies could benefit from modification or retrofit to more effectively remove ARB and ARGs while also producing a high quality product for water and resource recovery. This perspectives paper highlights the need to consider ARB and ARGs when evaluating the overall safety aspects of water reuse and ways by which this may be accomplished.

Understanding virus transfer between liquid and skin is necessary to estimate transmission during water-related activities. Here, we modeled virus transfer from liquid-to-skin and skin-to-liquid. We performed human subject studies using three bacteriophages as pathogenic virus surrogates: nonenveloped MS2 and Qβ and enveloped Φ6. Our study shows that transfer from liquid-to-skin is describable by a single model based on (1) virus concentration and (2) volume of liquid remaining on skin. Contact times (0.1–30 min), and virus species had little-to-no influence on virus transfer. Likewise, liquid conditions (pH 6–9, ionic strength 10–550 mM) had no influence on transfer as shown for MS2. The model accounts for both, virus adsorbed onto the skin, and virus in the liquid retained on skin. In comparison, virus transfer from skin-to-liquid was influenced by the wetness of the skin and by liquid type (water, saliva). 90 ± 19% of the virus inoculated on the skin are transferred to the water when the skin remains wet compared to 30 ± 17% when the skin is dry. The transfer from skin-to-liquid was 41% higher when the recipient liquid was water as compared with saliva. This study quantifies virus transfer between liquid and skin and guides risk assessments of water-related activities.

Background: Consistent domestic hand hygiene can reduce diarrhea-related morbidity and mortality and the spread of other communicable diseases. However, it remains uncertain which technique of handwashing is most effective and practicable during everyday life. The goal of this study is to determine how the handwashing technique, as performed in the daily life by the participants of this case study in Harare, Zimbabwe, influences microbial handwashing effectiveness.
Methods: Handwashing technique of 173 primary caregivers was observed in their homes and hand rinse samples were collected before and after handwashing. Samples were analyzed for Escherichia coli and total coliform concentrations. Generalized linear models were used to predict fecal hand contamination after washing from observed handwashing technique.
Results: Cleaning under fingernails, scrubbing the fingertips, using soap, and drying hands through rubbing on clothes or a clean towel statistically significantly reduced E coli contamination of hands after washing. Tap use, scrubbing fingertips, and rubbing hands on clothes to dry them statistically significantly reduced total coliform contamination.
Conclusions: Recommendations for effective and practicable domestic handwashing in Harare, Zimbabwe, should include performing specific handscrubbing steps (ie, cleaning under the fingernails and rubbing the fingertips), and soap and tap use. This calls for further research to develop behavior change interventions that explicitly promote effective handwashing technique at critical times.

Every year, more than half a million children die due to diarrheal diseases. Recent studies have identified the most important etiologies of diarrheal disease are enterotoxigenic and enteropathogenic E. coli, Shigella spp., rotavirus, norovirus and Cryptosporidium spp. These etiologies are unsurprisingly characterized by a combination of high shedding, high infectivity, and transmissibility through multiple environmental reservoirs. The relative importance of the transmission routes is likely site-specific. So the impact of interventions, which typically target only one or two environmental reservoirs, is likely also site-specific. The factors influencing the transmission routes most important for diarrheal disease are complex, including – at a minimum – etiology of endemic disease; and water, sanitation, and hygiene infrastructure and practices. The site-specific nature – and complexity of transmission – helps explain the observed variation in impacts of water, sanitation, and hygiene interventions. It may also render efforts to estimate or quantify global means for interventions' impacts irrelevant. The theme of this Perspective is that greater reductions in diarrheal disease transmission in LMICs can be achieved by designing interventions to interrupt the most important environmental transmission pathways. Intervention choice should be informed by site-specific conditions, most notably: diarrheal etiology and existing water, sanitation, and hygiene infrastructure and practices. The theme is discussed through the lens of the characteristics of the most important diarrheal diseases (shedding, infectivity, growth, and persistence) and the general characteristics of environmental reservoirs (exposure pathways and fecal contamination). The discussion highlights when interventions – and combinations of interventions – will be most effective at reducing diarrheal disease burden.

Ribavirin is a pharmaceutical antiviral used for the treatment of RNA virus infections including norovirus, hepatitis C virus, hepatitis E virus, Lassa virus, respiratory syncytial virus, and rhinovirus. Despite the drug's history and documented efficacy, the antiviral mechanism of Ribavirin remains unclear. Mechanisms proposed include depletion of the intracellular GTP pool, immunomodulatory effects, induction of error catastrophe, inhibition of viral polymerase activity, and/or inhibition of viral capping. In the present study, we leveraged deep sequencing data to demonstrate that Ribavirin increases murine norovirus (MNV-1) viral diversity. By serial passaging MNV-1 in RAW 264.7 cells for twenty generations in the presence of Ribavirin, we demonstrated statistically significant increases in both the number of unique haplotypes and the average pairwise difference (APD). Based on statistically significant differences in the probability of nucleotide mutations based on Roche 454 sequencing, we also demonstrated that single nucleotide substitutions are increased in the presence of Ribavirin. Finally, we demonstrated Ribavirin's impact on statistically significantly reducing the relative proportion of the dominant sequence within the quasispecies.

Access to water and sanitation are important determinants of behavioral responses to hygiene and sanitation interventions. We estimated cluster-specific water access and sanitation coverage to inform a constrained randomization technique in the SHINE trial. Technicians and engineers inspected all public access water sources to ascertain seasonality, function, and geospatial coordinates. Households and water sources were mapped using open-source geospatial software. The distance from each household to the nearest perennial, functional, protected water source was calculated, and for each cluster, the median distance and the proportion of households within 500 m and >1500 m of such a water source. Cluster-specific sanitation coverage was ascertained using a random sample of 13 households per cluster. These parameters were included as covariates in randomization to optimize balance in water and sanitation access across treatment arms at the start of the trial. The observed high variability between clusters in both parameters suggests that constraining on these factors was needed to reduce risk of bias.

The increased awareness of the role of environmental matrices in enteric disease transmission has resulted in the need for rapid, field-based methods for fecal indicator bacteria and pathogen detection. Evidence of the specificity of β-glucuronidase-based assays for detection of Escherichia coli from environmental matrices relevant to enteric pathogen transmission in developing countries, such as hands, soils, and surfaces, is limited. In this study, we quantify the false-positive rate of a β-glucuronidase-based E. coli detection assay (Colilert) for two environmental reservoirs in Bangladeshi households (hands and soils) and three fecal composite sources (cattle, chicken, and humans). We investigate whether or not the isolation source of E. coli influences phenotypic and genotypic characteristics. Phenotypic characteristics include results of biochemical assays provided by the API-20E test; genotypic characteristics include the Clermont phylogroup and the presence of enteric and/or environmental indicator genes sfmH, rfaI, and fucK. Our findings demonstrate no statistically significant difference in the false-positive rate of Colilert for environmental compared to enteric samples. E. coli isolates from all source types are genetically diverse, representing six of the seven phylogroups, and there is no difference in relative frequency of phylogroups between enteric and environmental samples. We conclude that Colilert, and likely other β-glucuronidase-based assays, is appropriate for detection of E. coli on hands and in soils with low false-positive rates. Furthermore, E. coli isolated from hands and soils in Bangladeshi households are diverse and indistinguishable from cattle, chicken, and human fecal isolates, using traditional biochemical assays and phylogrouping.

Understanding the excretion and treatment of human waste (feces and urine) in low and middle income countries (LMICs) is necessary to design appropriate waste management strategies. However, excretion and treatment are often difficult to quantify due to decentralization of excreta management. We address this gap by developing a mechanistic, stochastic model to characterize phosphorus, nitrogen, biochemical oxygen demand (BOD), and fecal coliform pollution from human excreta for 108 LMICs. The model estimates excretion and treatment given three scenarios: 1) use of existing sanitation systems, 2) use of World Health Organization-defined "improved sanitation", and 3) use of best available technologies. Our model estimates that more than 10⁹ kg/yr each of phosphorus, nitrogen and BOD are produced. Of this, 22(19–27)%, 11(7–15)%, 17(10–23)%, and 35(22–47)% (mean and 95% range) BOD, nitrogen, phosphorus, and fecal coliforms, respectively, are removed by existing sanitation systems. Our model estimates that upgrading to "improved sanitation" increases mean removal slightly to between 17 and 53%. Under the best available technology scenario, only approximately 60–80% of pollutants are treated. To reduce impact of nutrient and microbial pollution on human and environmental health, improvements in both access to adequate sanitation and sanitation treatment efficiency are needed.

Diarrheal diseases are a leading cause of under-five mortality and morbidity in sub-Saharan Africa. Quantitative exposure modeling provides opportunities to investigate the relative importance of fecal-oral transmission routes (e.g. hands, water, food) responsible for diarrheal disease. Modeling, however, requires accurate descriptions of individuals’ interactions with the environment (i.e., activity data). Such activity data are largely lacking for people in low-income settings. In the present study, we collected activity data and microbiological sampling data to develop a quantitative microbial exposure model for two female caretakers in peri-urban Tanzania. Activity data were combined with microbiological data of contacted surfaces and fomites (e.g. broom handle, soil, clothing) to develop example exposure profiles describing second-by-second estimates of fecal indicator bacteria (E. coli and enterococci) concentrations on the caretaker’s hands. The study demonstrates the application and utility of video activity data to quantify exposure factors for people in low-income countries and apply these factors to understand fecal contamination exposure pathways. This study provides both a methodological approach for the design and implementation of larger studies, and preliminary data suggesting contacts with dirt and sand may be important mechanisms of hand contamination. Increasing the scale of activity data collection and modeling to investigate individual-level exposure profiles within target populations for specific exposure scenarios would provide opportunities to identify the relative importance of fecal-oral disease transmission routes.