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Custer randomized trials are often used to study large-scale public health interventions. In large trials, even small improvements in statistical efficiency can have profound impacts on the required sample size and cost. Pair matched randomization is one strategy with potential to increase trial efficiency, but to our knowledge there have been no empirical evaluations of pair-matching in large-scale, epidemiologic field trials. Location integrates many socio-demographic and environmental characteristics into a single feature. Here, we show that geographic pair-matching leads to substantial gains in statistical efficiency for 14 child health outcomes that span growth, development, and infectious disease through a re-analysis of two large-scale trials of nutritional and environmental interventions in Bangladesh and Kenya. We estimate relative efficiencies ≥ 1.1 for all outcomes assessed and relative efficiencies regularly exceed 2.0, meaning an unmatched trial would have needed to enroll at least twice as many clusters to achieve the same level of precision as the geographically pair-matched design. We also show that geographically pair-matched designs enable estimation of fine-scale, spatially varying effect heterogeneity under minimal assumptions. Our results demonstrate broad, substantial benefits of geographic pair-matching in large-scale, cluster randomized trials.

BackgroundUnderstanding pathogen-specific relationships with climate is crucial to informing interventions under climate change.MethodsWe matched spatiotemporal temperature, precipitation, surface water, and humidity data to data from a trial in rural Bangladesh that measured diarrhea and enteropathogen prevalence in children 0-2 years from 2012-2016. We fit generalized additive models and estimated percent changes in prevalence using projected precipitation under Shared Socio-Economic pathways describing sustainable development (SSP1), middle of the road (SSP2), and fossil fuel development (SSP5) scenarios.FindingsAn increase from 15°C to 30°C in weekly average temperature was associated with 5.0% higher diarrhea, 6.4% higher Norovirus, and 13.0% higher STEC prevalence. Above-median precipitation was associated with 1.27-fold (95% CI 0.99, 1.61) higher diarrhea; higher Cryptosporidium, tEPEC, ST-ETEC, STEC, Shigella, EAEC, Campylobacter, Aeromonas, and Adenovirus 40/41; and lower aEPEC, Giardia, Sapovirus, and Norovirus prevalence. Other associations were weak or null. Compared to the study period, diarrhea prevalence was similar under SSP1 (7%), 3.4% (2.7%, 4.3%) higher under SSP2, and 5.7% (4.4%, 7.0%) higher under SSP5. Prevalence of pathogens responsible for a large share of moderate-to-severe diarrhea in this setting (Shigella, Aeromonas) were 13-20% higher under SSP2 and SSP5.InterpretationHigher temperatures and precipitation were associated with higher prevalence of diarrhea and multiple enteropathogens; higher precipitation was associated with lower prevalence of some enteric viruses. Under likely climate change scenarios, we projected increased prevalence of diarrhea and enteropathogens responsible for clinical illness. Our findings inform pathogen-specific adaptation and mitigation strategies and priorities for vaccine development.FundingBill & Melinda Gates Foundation, National Institute of Allergy and Infectious Diseases, National Heart, Lung, And Blood Institute, Stanford University School of Medicine, Chan Zuckerberg BiohubResearch in ContextEvidence before this studyWe searched Google Scholar and Scopus for studies published from January 1, 2000 to present using the following three queries: 1) child; and diarrhea OR “enteric infection”; and meteorological OR environmental OR “surface water” OR “standing water”; and risk AND/OR factors; 2) Climate AND change AND project* AND diarrhea OR diarrhoea; 3) climate AND change AND project* OR model AND enter* AND infect* AND E. coli. Studies generally focused on individual risk factors for diarrhea transmission or enteric infection, with an emphasis on temperature and precipitation. Studies found that higher temperatures were associated with higher incidence of bacterial diarrhea and lower incidence of viral diarrhea; few studies have investigated associations between temperature and parasitic diarrhea. Heavy rainfall, particularly after dry periods, was associated with higher diarrhea prevalence, though heavy rainfall during rainy seasons was found to protect against diarrhea incidence. Similarly, flooding of surface water and shallow wells was also associated with higher diarrhea prevalence. Very few studies investigated associations between diarrhea or enteropathogen carriage and surface water presence, or humidity. A recent individual participant meta-analysis of studies in 19 low- and middle-income countries found that higher precipitation was associated with a small decrease in enterotoxigenic E. coli (ETEC) and Campylobacter spp. prevalence and no difference in Shigella, Cryptosporidium, or Giardia, or enteric virus prevalence. Weekly average temperature increases of 10-40° C within the study period were associated with higher risk of Campylobacter, ETEC, Shigella, Cryptosporidium, Giardia, and adenovirus, and lower risk of sapovirus and rotavirus, and generally, associations were stronger. Higher humidity was associated with higher risk of enteric bacterial infections and lower risk of enteric virus infection. A small number of studies have projected diarrhea under climate change in low- and middle-income countries. Studies have estimated a 15-20% increase in global diarrhea risk in 2040-2069 relative to 1961-1990, up to 21% increase in diarrhea incidence in northern India from 2013 to the 2040s, 3-10% increase in diarrhea cases in the Gaza Strip associated with 1.5° to 2°C increases in temperature, and an 8% increase in diarrhea burden by 2050 in Nepal. One study estimated an additional 1,625,073 ETEC diarrhea cases in Bangladesh from 2046-2065 due to climate change. No studies estimated changes in the presence of multiple enteropathogens under possible climate change scenarios.Added value of this studyThis study matched remote sensing data on precipitation, temperature, surface water, and humidity to data on diarrhea and enteropathogen carriage in children under 2 years from a trial in rural Bangladesh. We fit flexible models to investigate potentially non-linear relationships between each climate and environmental risk factor and each health outcome. In addition, we predicted the prevalence of each outcome under three possible climate change scenarios. Our predictions leveraged the spatiotemporal distributions of both climate precipitation projections and diarrhea and enteropathogen carriage in the trial. To our knowledge, this is the first study to predict prevalence under climate change scenarios for both diarrhea and multiple enteropathogens in a rural, low-resource south Asian setting. Our study sheds light on how climate change may impact diarrhea and enteropathogen carriage in Bangladesh, which is highly vulnerable to climate change due to its low altitude and seasonal flooding.Implications of all the available evidenceWe found that both higher temperatures and precipitation were associated with higher diarrhea prevalence, consistent with prior studies. Compared to prior studies, which have primarily found associations between diarrhea and heavy rainfall following a dry period, we found that moderate levels of weekly precipitation were associated with higher diarrhea prevalence. Relationships between climatic and environmental variables and enteropathogen carriage varied by taxa, as other studies have found, but for some pathogens and risk factors, we observed associations in different directions than prior studies. Under middle of the road and fossil fuel-based development climate change scenarios, we projected increased diarrhea prevalence and taxa-specific changes in enteropathogen prevalence. Our findings suggest that climate change may exacerbate diarrhea burden in rural Bangladesh in the absence of sustainable development and inform prioritization of pathogen-specific mitigation and/or adaptation interventions (e.g., vaccines) for young children in rural, South Asian settings under climate change.

BackgroundClimate change may influence the effectiveness of environmental interventions. We investigated if climate and environment modified the effect of low-cost, point-of-use water, sanitation, and handwashing (WASH) interventions on diarrhea and predicted intervention effectiveness under climate change scenarios.MethodsWe analyzed data from a cluster-randomized trial in rural Bangladesh that measured diarrhea prevalence in children 0-2 years from 2012-2016. We matched remote sensing data on temperature, precipitation, humidity, and surface water to households by location and measurement date. We estimated prevalence ratios (PR) for WASH interventions vs. control stratified by environmental factors using generative additive models and targeted maximum likelihood estimation. We estimated intervention effects under predicted precipitation in the study region in 2050 for climate change scenarios from different Shared Socioeconomic Pathways (SSPs).FindingsWASH interventions more effectively prevented diarrhea under higher levels of total precipitation in the previous week and when there was heavy rain in the previous week (heavy rainfall PR = 0.38, 95% CI 0.23-0.62 vs. no heavy rainfall PR = 0.77, 0.60-0.98). We did not detect substantial effect modification by other environmental variables. WASH intervention effectiveness increased under most climate change scenarios; in a fossil-fueled development scenario (SSP5), the PR was 0.46 (0.44-0.48) compared to 0.67 (0.65-0.68) in the study.InterpretationWASH interventions had the strongest effect on diarrhea under higher precipitation, and effectiveness may increase under climate change without sustainable development. WASH interventions may improve population resilience to climate-related health risks.FundingBill & Melinda Gates Foundation, National Institute of Allergy and Infectious Diseases, National Heart, Lung, And Blood InstituteResearch in Context PanelEvidence before this studyWe searched Google Scholar using the search terms “sanitation” OR “hygiene” OR “WASH” OR “water quality”; and “heterogen*” or “effect modif*”; and “climate” OR “environmental”; and “diarrhea” OR “enteric infection”; and “risk” AND/OR “factors”. In general, the effect modification of sanitation and hygiene on diarrhea or enteropathogen infection by climate and environment is not well studied. One study in Ecuador investigated different relationships between rainfall, diarrhea, and unimproved sanitation and water sources. They found that poor sanitation was most strongly associated with elevated diarrhea after precipitation minimums, whereas poor water sources were most strongly associated with elevated diarrhea after precipitation maximums. One study in Rwanda also found that high levels of runoff were protective against diarrhea only in households with unimproved toilets. In Bangladesh, one study found that access to tubewells was most effective at reducing childhood diarrhea in non-flood controlled areas. No prior studies have projected changes in WASH effectiveness under different climate change scenarios.Added value of this studyTo our knowledge, this is the first study to assess differences in WASH intervention effectiveness for multiple environmental variables and the first to predict future intervention effectiveness under different climate change scenarios. We spatiotemporally matched individual-level data from a trial in rural Bangladesh to remote sensing data on temperature, precipitation, humidity, and surface water presence. We compared the effectiveness of WASH interventions to prevent diarrhea under different levels of potential environmental effect modifiers. We then used climate models with high spatiotemporal resolution to predict changes in intervention effectiveness under different climate change scenarios.Implications of all the available evidenceWe found that WASH interventions were more effective under increasing precipitation and following heavy rain (over 80th percentile) events. Prior studies have found that diarrhea tends to increase with rainfall. Several studies have documented that precipitation-associated increases in diarrhea occurred primarily after heavy rain that followed dry periods, but we found that WASH intervention effectiveness in preventing diarrhea was higher even at moderate precipitation levels that were not necessarily preceded by a dry period. Under climate change scenarios with higher precipitation, we projected that WASH interventions may become more effective. In settings similar to rural Bangladesh, our findings suggest that regions that are expected to experience greater rainfall should be prioritized for intervention installation, promotion, and maintenance and that these activities might be most impactful during the rainy season.

Background We evaluated the impact of low-cost water, sanitation, and handwashing (WSH) and child nutrition interventions on enteropathogen carriage in the WASH Benefits cluster-randomized controlled trial in rural Bangladesh. Methods We analyzed 1411 routine fecal samples from children 14 ± 2 months old in the WSH (n = 369), nutrition counseling plus lipid-based nutrient supplement (n = 353), nutrition plus WSH (n = 360), and control (n = 329) arms for 34 enteropathogens using quantitative polymerase chain reaction. Outcomes included the number of co-occurring pathogens; cumulative quantity of 4 stunting-associated pathogens; and prevalence and quantity of individual pathogens. Masked analysis was by intention-to-treat. Results Three hundred twenty-six (99.1%) control children had 1 or more enteropathogens detected (mean, 3.8 ± 1.8). Children receiving WSH interventions had lower prevalence and quantity of individual viruses than controls (prevalence difference for norovirus: –11% [95% confidence interval {CI}, –5% to –17%]; sapovirus: –9% [95% CI, –3% to –15%]; and adenovirus 40/41: –9% [95% CI, –2% to –15%]). There was no difference in bacteria, parasites, or cumulative quantity of stunting-associated pathogens between controls and any intervention arm. Conclusions WSH interventions were associated with fewer enteric viruses in children aged 14 months. Different strategies are needed to reduce enteric bacteria and parasites at this critical young age.

ImportanceA regulated stress response is essential for healthy trajectories, but the integrated effects of early childhood environmental and nutritional interventions on stress physiology are unknown.ObjectiveTo assess the effects of a combined nutritional, water, sanitation, and handwashing intervention on physiological stress response, oxidative stress, and DNA methylation.Design, Setting, and ParticipantsIn a trial in rural Bangladesh, we randomized geographical clusters of pregnant women and their in-utero children into either the combined nutritional, water, sanitation, and handwashing intervention or the control group. Physiological stress response, oxidative stress, and methylation levels of 757 children were measured at ages one and two years. Analysis was intention-to-treat.InterventionsThe intervention group received combined nutritional counseling and lipid-based nutrient supplements, chlorinated drinking water, upgraded sanitation, and handwashing with soap (N+WSH). The control group did not receive interventions.Main Outcomes and MeasuresWe measured four isomers of urinary F2-isoprostanes [iPF(2α)-III; 2,3-dinor-iPF(2α)-III; iPF(2α)-VI; 8,12-iso-iPF(2α)-VI] at year one. At year two, we measured pre- and post-stressor concentrations of salivary alpha-amylase and cortisol, overall methylation of the glucocorticoid receptor (NR3C1) exon 1F promoter including methylation levels at the nerve growth factor-inducible protein A (NGFI-A) binding site, mean arterial pressure, and resting heart rate.ResultsChildren in the N+WSH group had lower levels of F2-isoprostanes compared to controls (difference -0.16 to -0.19 log ng/mg of creatinine, PPP=0.023) in the N+WSH group. Children in the N+WSH group exhibited decreased logit-transformed methylation of the NGFI-A transcription factor binding site (−0.04; 95% CI, -0.08 to 0; P=0.037).Conclusions and RelevanceA nutritional, water, sanitation, and handwashing intervention reduced oxidative stress, enhanced hypothalamic-pituitary-adrenocortical axis activity, and reduced methylation levels in a transcription factor binding site of the glucocorticoid receptor gene. A targeted environmental and nutritional intervention affected the set point, reactivity, and regulation of the physiological stress system in early childhood, which may have implications for long-term health and developmental trajectories.Trial RegistrationClinicalTrials.govNCT01590095Key PointsQuestionDo intensive nutritional and environmental interventions alter physiological stress response, oxidative stress levels, and epigenetic programming during the first two years of life?FindingsIn this cluster randomized clinical trial of 757 children, a combined nutritional, drinking water, sanitation, and handwashing intervention significantly reduced oxidative stress, enhanced the cortisol response, and reduced methylation levels in a transcription factor binding site of the glucocorticoid receptor gene.MeaningThe integrated nutritional, drinking water, sanitation, and handwashing intervention enhanced adaptive responses of the physiological stress system in early childhood, which may have implications for long-term health and developmental trajectories.

While studies have speculated that immune function may play a role in how water, sanitation, hygiene, and nutritional (N+WSH) interventions may individually impact child growth and development, the combined effects of these interventions on immune system development are unknown. Here, we report on a trial in rural Bangladesh, where we cluster-randomized pregnant women into control and N+WSH arms. Among the birth cohort, we quantified plasma IL-1β, IL-6, TNF-α, IL-2, IL-12p70, IFN-γ, IL-4, IL-5, IL-13, IL-17A, IL-21, IL-10, and GM-CSF at ages 14 and 28 months. Cytokine ratios were included as prespecified outcomes to examine the net inflammatory environment. We assessed 704 children. After one year, TNF-α/IL-10, IL-12/IL-10, and IL-17A/IL-10 ratios were lower in the intervention group compared to the control group (mean difference: -0.12 to -0.19, p

Background:Previously, we demonstrated that a water, sanitation, handwashing, and nutritional intervention improved linear growth and was unexpectedly associated with shortened childhood telomere length (TL) (Lin et al., 2017). Here, we assessed the association between TL and growth.Methods:We measured relative TL in whole blood from 713 children. We reported differences between the 10th percentile and 90th percentile of TL or change in TL distribution using generalized additive models, adjusted for potential confounders.Results:In cross-sectional analyses, long TL was associated with a higher length-for-age Z score at age 1 year (0.23 SD adjusted difference in length-for-age Z score [95% CI 0.05, 0.42; FDR-corrected p-value = 0.01]). TL was not associated with other outcomes.Conclusions:Consistent with the metabolic telomere attrition hypothesis, our previous trial findings support an adaptive role for telomere attrition, whereby active TL regulation is employed as a strategy to address ‘emergency states’ with increased energy requirements such as rapid growth during the first year of life. Although short periods of active telomere attrition may be essential to promote growth, this study suggests that a longer overall initial TL setting in the first 2 years of life could signal increased resilience against future telomere erosion events and healthy growth trajectories.Funding:Funded by the Bill and Melinda Gates Foundation.Clinical trial number:NCT01590095

BACKGROUND The widespread and unrestricted use of antibiotics has led to the emergence and spread of antibiotic-resistant bacteria (ARB), antibiotic-resistance genes (ARGs), and antibiotic residues in the environment. Conventional wastewater treatment plants (WWTPs) are not designed for effective and adequate removal of ARB, ARGs, and antibiotic residues, and therefore, they play an important role in the dissemination of antimicrobial resistance (AMR) in the natural environment. OBJECTIVE We will conduct a systematic review to determine the most effective treatment strategies for the removal of ARB, ARGs, and antibiotic residues from the treated effluent disposed into the environment from WWTPs that receive municipal, hospital, and domestic discharge. METHODS We will search the MEDLINE, EMBASE, Web of Science, World Health Organization Global Index Medicus, and ProQuest Environmental Science Collection databases for full-text peer-reviewed journal articles published between January 2001 and December 2020. We will select only articles published in the English language. We will include studies that measured (1) the presence, concentration, and removal rate of ARB/ARGs going from WWTP influent to effluent, (2) the presence, concentration, and types of antibiotics in the effluent, and (3) the possible selection of ARB in the effluent after undergoing treatment processes in WWTPs. At least two independent reviewers will extract data and perform risk of bias assessment. An acceptable or narrative synthesis method will be followed to synthesize the data and present descriptive characteristics of the included studies in a tabular form. The study has been approved by the Ethics Review Board at the International Centre for Diarrhoeal Disease Research, Bangladesh (protocol number: PR-20113). RESULTS This protocol outlines our proposed methodology for conducting a systematic review. Our results will provide an update to the existing literature by searching additional databases. CONCLUSIONS Findings from our systematic review will inform the planning of proper treatment methods that can effectively reduce the levels of ARB, ARGs, and residual antibiotics in effluent, thus lowering the risk of the environmental spread of AMR and its further transmission to humans and animals. INTERNATIONAL REGISTERED REPORT PRR1-10.2196/33365

Author(s): Wessells, K Ryan; Arnold, Charles D; Stewart, Christine P; Prado, Elizabeth L; Abbeddou, Souheila; Adu-Afarwuah, Seth; Arnold, Benjamin F; Ashorn, Per; Ashorn, Ulla; Becquey, Elodie; Brown, Kenneth H; Byrd, Kendra A; Campbell, Rebecca K; Christian, Parul; Fernald, Lia CH; Fan, Yue-Mei; Galasso, Emanuela; Hess, Sonja Y; Huybregts, Lieven; Jorgensen, Josh M; Kiprotich, Marion; Kortekangas, Emma; Lartey, Anna; Le Port, Agnes; Leroy, Jef L; Lin, Audrie; Maleta, Kenneth; Matias, Susana L; Mbuya, Mduduzi NN; Mridha, Malay K; Mutasa, Kuda; Naser, Abu Mohd; Paul, Rina R; Okronipa, Harriet; Ouedraogo, Jean-Bosco; Pickering, Amy J; Rahman, Mahbubur; Schulze, Kerry; Smith, Laura E; Weber, Ann M; Zongrone, Amanda; Dewey, Kathryn G | Abstract: ABSTRACTBackgroundSmall-quantity lipid-based nutrient supplements (SQ-LNS) have been shown to reduce the prevalence of anemia and iron deficiency among infants and young children, but effects on other micronutrients are less well known. Identifying subgroups who may experience greater benefits from SQ-LNS, or who are more likely to respond to the intervention, may facilitate the development of public health policies and programs.ObjectiveOur objective was to identify study-level and individual-level modifiers of the effect of SQ-LNS on child hematological and micronutrient status outcomes.MethodsWe conducted a two-stage meta-analysis of individual participant data from 13 randomized controlled trials of SQ-LNS provided to children 6 to 24 months of age in low- and middle-income countries (n = 15,946). Outcomes were hemoglobin (Hb), inflammation-adjusted plasma ferritin, soluble transferrin receptor, zinc, retinol and retinol binding protein (RBP), and erythrocyte zinc protoporphyrin, and respective dichotomous outcomes indicative of anemia and micronutrient deficiency. We generated study-specific estimates of SQ-LNS vs. control, including main effects and subgroup estimates for individual-level effect modifiers, and pooled the estimates using fixed-effects models. We used random effects meta-regression to examine potential study-level effect modifiers.ResultsProvision of SQ-LNS decreased the prevalence of anemia (Hb l 110 g/L) by 16% (relative reduction), iron deficiency (plasma ferritin l 12 µg/L) by 56% and iron deficiency anemia (IDA; Hb l 110 g/L and plasma ferritin l 12 µg/L) by 64%. We observed positive effects of SQ-LNS on hematological and iron status outcomes within all subgroups of the study-level and individual-level effect modifiers, but effects were larger in certain subgroups. For example, effects of SQ-LNS on anemia and iron status were greater in trials that provided SQ-LNS for g 12 months and provided 9 mg/d vs. l 9 mg iron/d, and among later-born (vs. first-born) children. There was no effect of SQ-LNS on plasma zinc or retinol, but there was a 7% increase in plasma RBP and a 56% reduction in vitamin A deficiency (RBP l 0.70 µmol/L), with little evidence of effect modification by individual-level characteristics.ConclusionsSQ-LNS provided to infants and young children 6-24 months of age can substantially reduce the prevalence of anemia, iron deficiency, and IDA across a range of individual, population and study design characteristics. Policy-makers and program planners should consider SQ-LNS within intervention packages to prevent anemia and iron deficiency. This study was registered at www.crd.york.ac.uk/PROSPERO as CRD42020156663.

BackgroundMultiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmitted via several pathways through human, animal, and environmental reservoirs. Individual quantitative PCR (qPCR) assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan Array Card (TAC) that allows simultaneous detection of multiple enteropathogens has only previously been validated in human clinical samples.MethodsHere, we performed a comprehensive double-blinded comparison of the performance of a custom TAC relative to standard qPCR for the detection of eight enteric targets, by using spiked samples, wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji.FindingsBoth methods exhibited high and comparable specificity (TAC: 100%, qPCR: 94%), sensitivity (TAC: 92%; qPCR: 100%), and quantitation accuracy (TAC: 91%; qPCR: 99%) in non-inhibited sample matrices. PCR inhibitors substantially impacted detection via TAC, though this issue was alleviated by 10-fold sample dilution. Among samples from informal settlements, the two techniques were comparable for detection (89% agreement) and quantitation (R2= 0.82). The TAC additionally included 38 other targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR.InterpretationOverall, the two techniques produce comparable results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel that enables a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs in turn allows better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load.FundingWellcome TrustOur Planet, Our Healthprogram [OPOH grant 205222/Z/16/Z].

Summary Background Multiple bacteria, viruses, protists, and helminths cause enteric infections that greatly impact human health and wellbeing. These enteropathogens are transmited via several pathways through human, animal, and environmental reservoirs. Individual qPCR assays have been extensively used to detect enteropathogens within these types of samples, whereas the TaqMan array card (TAC), which allows simultaneous detection of multiple enteropathogens, has only previously been validated in human clinical samples. Methods In this methodological comparison study, we compared the performance of a custom 48-singleplex TAC relative to standard qPCR. We established the sensitivity and specificity of each method for the detection of eight enteric targets, by using spiked samples with varying levels of PCR inhibition. We then tested the prevalence and abundance of pathogens in wastewater from Melbourne (Australia), and human, animal, and environmental samples from informal settlements in Suva, Fiji using both TAC and qPCR. Findings Both methods exhibited similarly high specificity (TAC 100%, qPCR 94%), sensitivity (TAC 92%, qPCR 100%), and quantitation accuracy (TAC 91%, qPCR 99%) in non-inhibited sample matrices with spiked gene fragments. PCR inhibitors substantially affected detection via TAC, though this issue was alleviated by ten-fold sample dilution. Among samples from informal settlements, the two techniques performed similarly for detection (89% agreement) and quantitation (R2 0·82) for the eight enteropathogen targets. The TAC additionally included 38 other enteric targets, enabling detection of diverse faecal pathogens and extensive environmental contamination that would be prohibitively labour intensive to assay by standard qPCR. Interpretation The two techniques produced similar results across diverse sample types, with qPCR prioritising greater sensitivity and quantitation accuracy, and TAC trading small reductions in these for a cost-effective larger enteropathogen panel enabling a greater number of enteric pathogens to be analysed concurrently, which is beneficial given the abundance and variety of enteric pathogens in environments such as urban informal settlements. The ability to monitor multiple enteric pathogens across diverse reservoirs could allow better resolution of pathogen exposure pathways, and the design and monitoring of interventions to reduce pathogen load. Funding Wellcome Trust Our Planet, Our Health programme.

Poor nutrition and hand hygiene are risk factors for acute respiratory infections (ARIs). Safe drinking water and sanitation can reduce exposure to pathogens and encourage healthy immune responses, reducing the risk of ARIs. Within a trial assessing impacts of water, sanitation, and handwashing (WASH), and nutritional interventions, we evaluated effects on ARIs. The WASH Benefits cluster-randomized trial enrolled pregnant women from Kenyan villages and evaluated health outcomes in children born to enrolled mothers 1 and 2 years after intervention delivery. Geographically adjacent clusters were block-randomized into a passive control (no promotional visits), a double-sized active control (monthly visits to measure mid–upper arm circumference), and six intervention groups: chlorinated drinking water (W), improved sanitation (S), handwashing with soap (H), combined WSH, improved nutrition (N) through counseling and lipid-based nutrient supplementation (LNS), and combined WSHN. The main outcome was the prevalence of ARI symptoms (cough, panting, wheezing, or difficulty breathing) in children younger than 3 years. Masking participants was not possible. Analyses were intention-to-treat. Between November 2012 and May 2014, 702 clusters were enrolled, including 6,960 (year 1) and 7,088 (year 2) children with ARI data. The cluster-level intra-cluster correlation coefficient for ARIs was 0.026 across both years. Water, sanitation, and handwashing interventions with behavior change messaging did not reduce ARIs. Nutrition counseling and LNS modestly reduced ARI symptoms compared with controls in year 1 [prevalence ratio (PR): 0.87, 95% confidence interval (CI): 0.77–0.99], but no effect in the combined WSHN group weakens this finding.

Malaria is a leading cause of morbidity and mortality among children under five years of age, with most cases occurring in Sub-Saharan Africa. Children in this age group in Africa are at greatest risk worldwide for developmental deficits. There are research gaps in quantifying the risks of mild malaria cases, understanding the pathways linking malaria infection and poor child development, and evaluating the impact of malaria on the development of children under five years. We analyzed the association between malaria infection and gross motor, communication, and personal social development in 592 children age 24 months in rural, western Kenya as part of the WASH Benefits environmental enteric dysfunction sub-study. Eighteen percent of children had malaria, 20% were at risk for gross motor delay, 21% were at risk for communication delay, and 23% were at risk for personal social delay. Having a positive malaria test was associated with increased risk for gross motor, communication, and personal social delay while adjusting for child characteristics, household demographics, study cluster, and intervention treatment arm. Mediation analyses suggested that anemia was a significant mediator in the pathway between malaria infection and risk for gross motor, communication, and personal social development delays. The proportion of the total effect of malaria on the risk of developmental delay that is mediated by anemia across the subscales was small (ranging from 9% of the effect on gross motor development to 16% of the effect on communication development mediated by anemia). Overall, malaria may be associated with short-term developmental delays during a vulnerable period of early life. Therefore, preventative malaria measures and immediate treatment are imperative for children&rsquo, s optimal development, particularly in light of projections of continued high malaria transmission in Kenya and Africa.

BackgroundWe hypothesized that drinking water, sanitation, handwashing (WSH), and nutritional interventions would improve environmental enteric dysfunction (EED), a potential contributor to stunting.MethodsWithin a subsample of a cluster-randomized, controlled trial in rural Bangladesh, we enrolled pregnant women in 4 arms: control, WSH, child nutrition counseling plus lipid-based nutrient supplements (N), and nutrition plus WSH (N+WSH). Among the birth cohort, we measured biomarkers of gut inflammation (myeloperoxidase, neopterin), permeability (alpha-1-antitrypsin, lactulose, mannitol), and repair (regenerating gene 1β) at median ages 3, 14, and 28 months. Analysis was intention-to-treat.ResultsWe assessed 1512 children. At age 3 months, compared to controls, neopterin was reduced by nutrition (-0.21 log nmol/L; 95% confidence interval [CI], -.37, -.05) and N+WSH (-0.20 log nmol/L; 95% CI, -.34, -.06) interventions; similar reductions were observed at 14 months. At 3 months, all interventions reduced lactulose and mannitol (-0.60 to -0.69 log mmol/L). At 28 months, myeloperoxidase was elevated in the WSH and nutrition arms (0.23-0.27 log ng/mL) and lactulose was higher in the WSH arm (0.30 log mmol/L; 95% CI, .07, .53).ConclusionsReductions in permeability and inflammation at ages 3 and 14 months suggest that the interventions promoted healthy intestinal maturation; however, by 28 months, the WSH and nutrition arms showed elevated EED biomarkers. These results underscore the importance of developing a better understanding of EED pathophysiology and targeting interventions early in childhood, when they are likely to have the largest benefit to intestinal health.Clinical trials registrationNCT01590095.

Fresh produce, when consumed raw, can be a source of exposure to antimicrobial residues, antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) of clinical importance. This review aims to determine: (1) the presence and abundance of antimicrobial residues, ARB and ARGs in fresh agricultural products sold in retail markets and consumed raw; (2) associated health risks in humans; and (3) pathways through which fresh produce becomes contaminated with ARB/ARGs. We searched the Ovid Medline, Web of Science and Hinari databases as well as grey literature, and identified 40 articles for inclusion. All studies investigated the occurrence of multidrug-resistant bacteria, and ten studies focused on ARGs in fresh produce, while none investigated antimicrobial residues. The most commonly observed ARB were E. coli (42.5%) followed by Klebsiella spp. (22.5%), and Salmonella spp. (20%), mainly detected on lettuce. Twenty-five articles mentioned health risks from consuming fresh produce but none quantified the risk. About half of the articles stated produce contamination occurred during pre- and post-harvest processes. Our review indicates that good agricultural and manufacturing practices, behavioural change communication and awareness-raising programs are required for all stakeholders along the food production and consumption supply chain to prevent ARB/ARG exposure through produce.

BackgroundSoil-transmitted helminths (STH) and Giardia duodenalis are responsible for a large burden of disease globally. In low-resource settings, household finished floors (e.g., concrete floors) may reduce transmission of STH and G. duodenalis.MethodsIn a prospective cohort of children nested within two randomised trials in rural Bangladesh and Kenya, we estimated associations between household finished flooring and STH and G. duodenalis prevalence. In 2015-2016, we collected stool samples from children aged 2-16 years in rural Bangladesh and Kenya. We detected STH infection using qPCR (Bangladesh N=2,800; Kenya N=3,094) and detected G. duodenalis using qPCR in Bangladesh (N=6,894) and ELISA in Kenya (N=8,899). We estimated adjusted prevalence ratios (aPRs) using log-linear models adjusted for potential confounders.FindingsAt enrolment, 10% of households in Bangladesh and 5% in Kenya had finished floors. In both countries, household finished flooring was associated with lower Ascaris lumbricoides prevalence (Bangladesh aPR: 0.33, 95% CI 0.14, 0.78; Kenya aPR: 0.62, 95% CI 0.39, 0.98) and any STH (Bangladesh aPR: 0.73, 95% CI 0.52, 1.01; Kenya aPR: 0.57, 95% CI 0.37, 0.88). Household finished floors were also associated with lower Necator americanus prevalence in Bangladesh (aPR: 0.52, 95% CI 0.29, 0.94) and G. duodenalis prevalence in both countries (Bangladesh aPR: 0.78, 95% CI 0.64, 0.95; Kenya: aPR: 0.82, 95% CI 0.70, 0.97).InterpretationIn low-resource settings, living in households with finished floors over a two-year period was associated with lower prevalence of G. duodenalis and certain STH in children.FundingBill & Melinda Gates Foundation grant OPPGD759

Background The Revitalizing Informal Settlements and their Environments (RISE) programme aims to assess the health, wellbeing, and ecological impacts of a water-sensitive-cities approach to improving urban informal settlements. Incorporating water-cycle management and green technology sanitation strategies, we aim to reduce flood risk and improve sanitation and waste water treatment leading to cleaner and healthier environments. Here we present the initial design pre-intervention for evaluation in the first 12 settlements in Makassar, Indonesia. Methods Initial environmental, and wellbeing and human health assessments were implemented starting in October, 2018, in 12 settlements in Makassar, Indonesia. Ecological assessments include measuring of temperature and humidity via iButtons installed in select homes, recording of bio-acoustic to measure biodiversity within settlement boundaries, and trapping disease vectors quarterly. Implemented evaluation of environmental contamination includes sampling water and soil sources for total coliforms as well as collecting soil via bootsocks by walking predefined transects. Human assessment includes an annual baseline survey of all settlement households, assessing self-reported symptoms, health-care system utilisation, and subjective wellbeing. Additionally, children younger than 5 years are surveyed quarterly for caregiver reported symptoms of diarrhoea and febrile illness, blood samples and anthropometry are being collected annually, and faeces samples are requested quarterly. Findings Ecological assessments have provided more than a million temperature data points. 21 000 mosquitos have been captured and identified. A total of 114 water samples, 84 bootsocks, and 91 soil samples have been collected, with sampling prior to and during the wet season. We have identified over 600 households within the 12 settlements. Health assessments of children under the age of 5 years have revealed 282 children with collection of 234 faeces samples and 188 blood samples. Interpretation We have successfully implemented baseline ecological and human health and wellbeing assessment tools in all 12 settlements, which will allow for the evaluation of water-sensitive-cities approach in RISE programme. Funding The Wellcome Trust (Our Planet, Our Health grant 205222/Z/16/Z ).

Author(s): Pickering, Amy; Swarthout, Jenna; Mureithi, MaryAnne; Mboya, John; Arnold, Benjamin; Wolfe, Marlene; Dentz, Holly; Lin, Audrie; Arnold, Charles; Rao, Gouthami; Stewart, Christine; Ram, Pavani; Clasen, Thomas; Colford, John; Null, Clair | Abstract: Combined water, sanitation, and handwashing (WSH) interventions have the potential to reduce fecal pathogens along more transmission pathways than single interventions alone. We measured Escherichia coli levels in 3909 drinking water samples, 2691 child hand rinses, and 2422 toy ball rinses collected from households enrolled in a two-year cluster-randomized controlled trial evaluating single and combined WSH interventions. Water treatment alone reduced E. coli in drinking water, while a combined WSH intervention improved water quality by the same magnitude but did not affect levels of fecal indicator bacteria on child hands or toy balls. The failure of the WSH interventions to reduce E. coli along important child exposure pathways is consistent with the lack of a protective effect from the interventions on child diarrhea or child growth during the trial. Our results have important implications for WSH program design; the sanitation and handwashing interventions implemented in this trial should not be expected to reduce child exposure to fecal contamination in other similar settings.

Background Helminth and protozoan infections affect more than 1 billion children globally. Improving water quality, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration, while providing other quality of life benefits. Methods and findings We enrolled geographic clusters of pregnant women in rural western Kenya into a cluster-randomized controlled trial (ClinicalTrials.gov NCT01704105) that tested 6 interventions: water treatment, improved sanitation, handwashing with soap, combined water treatment, sanitation, and handwashing (WSH), improved nutrition, and combined WSH and nutrition (WSHN). We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides, Trichuris trichiura, hookworm, and Giardia duodenalis among children born to the enrolled pregnant women (index children) and their older siblings. After 2 years of intervention exposure, we collected stool specimens from 9,077 total children aged 2 to 15 years in 622 clusters, including 2,346 children in an active control group (received household visits but no interventions), 1,117 in the water treatment arm, 1,160 in the sanitation arm, 1,141 in the handwashing arm, 1,064 in the WSH arm, 1,072 in the nutrition arm, and 1,177 in the WSHN arm. In the control group, 23% of children were infected with A. lumbricoides, 1% with T. trichiura, 2% with hookworm, and 39% with G. duodenalis. The analysis included 4,928 index children (median age in years: 2) and 4,149 older siblings (median age in years: 5); study households had an average of 5 people, 90% had dirt floors. Compared to the control group, Ascaris infection prevalence was lower in the water treatment arm (prevalence ratio [PR]: 0.82 [95% CI 0.67, 1.00], p = 0.056), the WSH arm (PR: 0.78 [95% CI 0.63, 0.96], p = 0.021), and the WSHN arm (PR: 0.78 [95% CI 0.64, 0.96], p = 0.017). We did not observe differences in Ascaris infection prevalence between the control group and the arms with the individual interventions sanitation (PR: 0.89 [95% CI 0.73, 1.08], p = 0.228), handwashing (PR: 0.89 [95% CI 0.73, 1.09], p = 0.277), or nutrition (PR: 86 [95% CI 0.71, 1.05], p = 0.148). Integrating nutrition with WSH did not provide additional benefit. Trichuris and hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reduced Giardia. Reanalysis of stool samples by quantitative polymerase chain reaction confirmed the reductions in Ascaris infections measured by microscopy in the WSH and WSHN groups. Trial limitations included imperfect uptake of targeted intervention behaviors, limited power to detect effects on rare parasite infections, and that it was not feasible to blind participants and sample collectors to treatment status. However, lab technicians and data analysts were blinded to treatment status. The trial was funded by the Bill & Melinda Gates Foundation and the United States Agency for International Development. Conclusions Integration of improved water quality, sanitation, and handwashing could contribute to sustainable control strategies for Ascaris infections, particularly in similar settings with recent or ongoing deworming programs. Combining nutrition with WSH did not provide further benefits, and water treatment alone was similarly effective to integrated WSH. Our findings provide new evidence that drinking water should be given increased attention as a transmission pathway for Ascaris. Trial registration ClinicalTrials.gov NCT01704105., Interventions for improved water and hygiene in pregnant women in Kenya resulted in fewer Ascaris infections in newborns, Amy Pickering and colleagues reveal., Author summary Why was this study done? Intestinal worm and protozoan infections affect >1 billion children and are associated with growth faltering and impaired cognitive development. High reinfection rates can prevent mass drug administration programs from eliminating transmission. Improved water quality, sanitation, handwashing, and nutrition could interrupt environmental transmission of parasites, but few trials evaluating these interventions have measured parasite infections as an outcome. What did the researchers do and find? The authors conducted a randomized controlled trial among a birth cohort to test if single and combined improved drinking water quality, sanitation, handwashing, and nutrition interventions can reduce intestinal worm and Giardia infections. The authors measured parasite infections after 2 years of intervention exposure. The authors demonstrated that water treatment alone and integrated water, sanitation, and handwashing interventions can sustainably reduce roundworm (Ascaris) infection prevalence among young children in Kenya. Improved nutrition did not enhance the effectiveness of the water, sanitation, and handwashing interventions, and none of the interventions reduced Giardia. What do these findings mean? Improving water quality, sanitation, and handwashing concurrently in the household environment can protect children from infection with Ascaris. The study also provides evidence that water treatment alone may provide a similar level of protection against Ascaris infection, suggesting that combining water, sanitation, and handwashing interventions does not yield greater health benefits than implementing single interventions. Treating drinking water is a relatively unexplored strategy for controlling intestinal worm infections, and drinking water should be given increased attention as an ingestion exposure pathway for Ascaris eggs.

Background Soil transmitted helminths (STH) infect >1.5 billion people. Mass drug administration (MDA) effectively reduces infection; however, there is evidence for rapid reinfection and risk of potential drug resistance. We conducted a randomized controlled trial in Bangladesh (WASH Benefits, NCT01590095) to assess whether water, sanitation, hygiene and nutrition interventions, alone and combined, reduce STH in a setting with ongoing MDA. Methodology/Principal findings In 2012–2013, we randomized 720 clusters of 5551 pregnant women into water treatment, sanitation, handwashing, combined water+sanitation+handwashing (WSH), nutrition, nutrition+WSH (N+WSH) or control arms. In 2015–2016, we enrolled 7795 children, aged 2–12 years, of 4102 available women for STH follow-up and collected stool from 7187. We enumerated STH infections with Kato-Katz. We estimated intention-to-treat intervention effects on infection prevalence and intensity. Participants and field staff were not blinded; laboratory technicians and data analysts were blinded. Prevalence among controls was 36.8% for A. lumbricoides, 9.2% for hookworm and 7.5% for T. trichiura. Most infections were low-intensity. Compared to controls, the water intervention reduced hookworm by 31% (prevalence ratio [PR] = 0.69 (0.50,0.95), prevalence difference [PD] = -2.83 (-5.16,-0.50)) but did not affect other STH. Sanitation improvements reduced T. trichiura by 29% (PR = 0.71 (0.52,0.98), PD = -2.17 (-4.03,-0.38)), had a similar borderline effect on hookworm and no effect on A. lumbricoides. Handwashing and nutrition interventions did not reduce any STH. WSH and N+WSH reduced hookworm prevalence by 29–33% (WSH: PR = 0.71 (0.52,0.99), PD = -2.63 (-4.95,-0.31); N+WSH: PR = 0.67 (0.50,0.91), PD = -3.00 (-5.14,-0.85)) and marginally reduced A. lumbricoides. Effects on infection intensity were similar. Conclusions/Significance In a low-intensity infection setting with MDA, we found modest but sustained hookworm reduction from water treatment and combined WSH interventions. Impacts were more pronounced on STH species with short vs. long-term environmental survival. Our findings suggest possible waterborne transmission for hookworm. Water treatment and sanitation improvements can augment MDA to interrupt STH transmission. Trial registration NCT01590095., Author summary Soil-transmitted helminths (STH) are associated with a large disease burden worldwide. Mass administration of deworming drugs for preventive chemotherapy is the cornerstone of global strategy for STH control, but treated individuals often rapidly become reinfected, and there is also concern about emerging drug resistance. Interventions to treat drinking water, wash hands at critical times and isolate human feces from the environment through improved sanitation could reduce STH transmission by reducing the spread of ova from the feces of infected individuals into the environment and subsequently to new hosts. Nutrition improvements could reduce host susceptibility to infection. Existing evidence on the effect of these interventions on STH is scarce. In a setting with ongoing mass drug administration, we assessed the effect of individual and combined water, sanitation, handwashing and nutrition interventions on STH infection in children. Approximately 2.5 years after the initiation of interventions, we found reductions in STH infection from water treatment and sanitation interventions; there was no reduction from the handwashing and nutrition interventions. While the reductions were modest in magnitude compared to cure rates achieved by deworming drugs, they indicated sustained reduction in environmental transmission. The reductions were more pronounced for STH species that do not have long-term environmental reservoirs. These findings suggest that water treatment and sanitation interventions can augment mass drug administration programs in striving toward elimination of STH.

Author(s): Williams, AM; Chantry, CJ; Young, SL; Achando, BS; Allen, LH; Arnold, BF; Colford, JM; Dentz, HN; Hampel, D; Kiprotich, MC; Lin, A; Null, CA; Nyambane, GM; Shahab-Ferdows, S; Stewart, CP | Abstract: © 2016 American Society for Nutrition. Background: Breast milk vitamin B-12 concentration may be inadequate in regions in which animal-source food consumption is low or infrequent. Vitamin B-12 deficiency causes megaloblastic anemia and impairs growth and development in children. Objective: We measured vitamin B-12 in breast milk and examined its associations with household hunger, recent animalsource food consumption, and vitamin B-12 intake. Methods: In a cross-sectional substudy nested within a cluster-randomized trial assessing water, sanitation, hygiene, and nutrition interventions in Kenya, we sampled 286 women 1-6 mo postpartum. Mothers hand-expressed breast milk 1 min into a feeding after 90 min observed nonbreastfeeding. The Household Hunger Scale was used to measure hunger, food intake in the previous week was measured with the use of a food-frequency questionnaire (FFQ), and vitamin B-12 intake was estimated by using 24-h dietary recall. An animal-source food score was based on 10 items from the FFQ (range: 0-70). Breast milk vitamin B-12 concentration was measured with the use of a solid-phase competitive chemiluminescent enzyme immunoassay and was modeled with linear regression. Generalized estimating equations were used to account for correlated observations at the cluster level. Results: Median (IQR) vitamin B-12 intake was 1.5 μg/d (0.3, 9.7 mg/d), and 60% of women consumed l 2.4 μg/d, the estimated average requirement during lactation. Median (IQR) breast milk vitamin B-12 concentration was 113 pmol/L (61, 199 pmol/L); 89% had concentrations l 310 pmol/L, the estimated adequate concentration. Moderate or severe hunger prevalence was 27%; the animal-source food score ranged from 0 to 30 item-d/wk. Hunger and recent animal-source food and vitamin B-12 intake were not associated with breast milk vitamin B-12 concentrations. Maternal age was negatively associated with breast milk vitamin B-12 concentrations. Conclusion: Most lactating Kenyan women consumed less than the estimated average requirement of vitamin B-12 and had low breast milk vitamin B-12 concentrations. We recommend interventions that improve vitamin B-12 intake in lactating Kenyan women to foster maternal health and child development.

Author(s): Byrd, Kendra A; Williams, Thomas N; Lin, Audrie; Pickering, Amy J; Arnold, Benjamin F; Arnold, Charles D; Kiprotich, Marion; Dentz, Holly N; Njenga, Sammy M; Rao, Gouthami; Colford, John M; Null, Clair; Stewart, Christine P | Abstract: BackgroundThe relation between subclinical hemoglobinopathies and concentrations of the iron-regulatory hormone hepcidin is not well characterized.ObjectiveWe investigated the relation of hepcidin concentration with hemoglobinopathies among young children in Kenya.MethodsWe quantified serum hepcidin and ferritin in 435 Kenyan children aged 14-20 mo in a subsample of the Water, Sanitation, and Handwashing (WASH) Benefits Trial. Blood samples were genotyped for α+-thalassemia and for sickle cell disorder. Hepcidin was compared across sickle cell and α+-thalassemia genotypes separately by using generalized linear models, and children who were normozygous for both conditions were also compared with those who had either of these conditions. In the association between hepcidin and ferritin, we assessed effect modification by genotype.ResultsIn this population, we found that 16.2% had sickle cell trait and 0.2% had sickle cell disorder, whereas 40.0% were heterozygous for α+-thalassemia and 8.2% were homozygous. Hepcidin concentration did not differ by genotype, but effect modification was found by genotype in the association between hepcidin and ferritin (Pnln0.1). Among normozygous sickle cell children (HbAA), there was an association between hepcidin and ferritin (βn=n0.92; 95% CI: 0.72, 1.10). However, among those with sickle cell trait (HbAS), the association was no longer significant (βn=n0.31; 95% CI: -0.04, 0.66). Similarly, among children who were normozygous (αα/αα) or heterozygous (-α/αα) for α+-thalassemia, hepcidin and ferritin were significantly associated [βn=n0.94 (95% CI: 0.68, 1.20) and βn=n0.77 (95% CI: 0.51, 1.03), respectively]; however, in children who were homozygous for α+-thalassemia (-α/-α), there was no longer a significant association (βn=n0.45; 95% CI: -0.10, 1.00).ConclusionHepcidin was not associated with hemoglobin genotype, but there may be a difference in the way hepcidin responds to iron status among those with either sickle cell trait or homozygous α+-thalassemia in young Kenyan children. This trial was registered at clinicaltrials.gov as NCT01704105.

Background.Helminth and protozoan infections affect >1 billion children globally. Improved water, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration (MDA), while providing other quality of life benefits.Methods and Findings.We enrolled geographic clusters of pregnant women into a cluster-randomized controlled trial that tested six interventions: disinfecting drinking water(W), improved sanitation(S), handwashing with soap(H), combined WSH, improved nutrition(N), and combined WSHN. We assessed intervention effects on parasite infections by measuringAscaris lumbricoides,Trichuris trichiura, hookworm, andGiardia duodenalisamong individual children born to enrolled mothers and their older siblings (ClinicalTrials.govNCT01704105). We collected stool specimens from 9077 total children in 622 clusters, including 2346 children in control, 1117 in water, 1160 in sanitation, 1141 in handwashing, 1064 in WSH, 1072 in nutrition, and 1177 in WSHN. In the control group, 23% of children were infected withAscaris lumbricoides, 1% withTrichuris trichuria, 2% with hookworm and 39% withGiardia duodenalis. After two years of intervention exposure,Ascarisinfection prevalence was 18% lower in the water treatment arm (95% confidence interval (CI) 0%, 33%), 22% lower in the WSH arm (CI 4%, 37%), and 22% lower in the WSHN arm (CI 4%, 36%) compared to control. Individual sanitation, handwashing, and nutrition did not significantly reduceAscarisinfection on their own, and integrating nutrition with WSH did not provide additional benefit.Trichurisand hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reducedGiardia. Reanalysis of stool samples by quantitative polymerase chain reaction (qPCR) confirmed the reductions inAscarisinfections measured by microscopy in the WSH and WSHN groups. Lab technicians and data analysts were blinded to treatment assignment, but participants and sample collectors were not blinded. The trial was funded by the Bill & Melinda Gates Foundation and USAID.Conclusions.Our results suggest integration of improved water quality, sanitation, and handwashing could contribute to sustainable control strategies forAscarisinfections, particularly in similar settings with recent or ongoing deworming programs. Water treatment alone was similarly effective to integrated WSH, providing new evidence that drinking water should be given increased attention as a transmission pathway forAscaris.

Summary Background Poor nutrition and infectious diseases can prevent children from reaching their developmental potential. We aimed to assess the effects of improvements in water, sanitation, handwashing, and nutrition on early child development in rural Kenya. Methods In this cluster-randomised controlled trial, we enrolled pregnant women in their second or third trimester from three counties (Kakamega, Bungoma, and Vihiga) in Kenya's western region, with an average of 12 households per cluster. Groups of nine geographically adjacent clusters were block-randomised, using a random number generator, into the six intervention groups (including monthly visits to promote target behaviours), a passive control group (no visits), or a double-sized active control group (monthly household visits to measure child mid-upper arm circumference). The six intervention groups were: chlorinated drinking water; improved sanitation; handwashing with soap; combined water, sanitation, and handwashing; improved nutrition through counselling and provision of lipid-based nutrient supplements; and combined water, sanitation, handwashing, and nutrition. Here we report on the prespecified secondary child development outcomes: gross motor milestone achievement assessed with the WHO module at year 1, and communication, gross motor, personal social, and combined scores measured by the Extended Ages and Stages Questionnaire (EASQ) at year 2. Masking of participants was not possible, but data assessors were masked. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01704105. Findings Between Nov 27, 2012, and May 21, 2014, 8246 women residing in 702 clusters were enrolled. No clusters were lost to follow-up, but 2212 households with 2279 children were lost to follow-up by year 2. 5791 (69%) children were measured at year 1 and 6107 (73%) at year 2. At year 1, compared with the active control group, the combined water, sanitation, handwashing, and nutrition group had greater rates of attaining the standing with assistance milestone (hazard ratio 1·23, 95% CI 1·09–1·40) and the walking with assistance milestone (1·32, 1·17–1·50), and the handwashing group had a greater rate of attaining the standing alone milestone (1·15, 1·01–1·31). There were no differences when comparing the other intervention groups with the active control group on any of the motor milestone measures at year 1. At year 2, there were no differences among groups for the communication, gross motor, personal social, or combined EASQ scores. Interpretation The handwashing and combined water, sanitation, handwashing, and nutrition interventions might have improved child motor development after 1 year, although after 2 years there were no other differences between groups. Future research should examine ways to make community health and nutrition programmes more effective at supporting child development. Funding Bill & Melinda Gates Foundation.

Environmental enteropathy (EE) is characterised by subclinical inflammation and hyperpermeability of the small intestine, hypothesised to be caused by recurrent ingestion of faecal bacteria. It has been suggested that EE may be a contributor to malnutrition and growth delays seen in children living in unsanitary conditions. We measured putative faecal EE markers myeloperoxidase (MPO) (ng/mL) and alpha-1-antitrypsin (AAT) (mg/g) in stool samples collected from 133 children aged 1-5 years in 16 communities enrolled in the WASH for WORMS randomised controlled trial in Timor-Leste. Samples were collected two years after a community-wide water, sanitation and hygiene (WASH) intervention that was integrated with regular deworming. Mixed effects multivariable linear regression models were used to examine the impact of the study intervention and of various WASH and infection-related factors on EE biomarkers. Children who lived in communities that received both the WASH intervention and deworming had similar AAT values as those who lived in communities that received only deworming (regression coefficient -0.14, p = 0.583), but they had a trend towards lower MPO values (coeff -0.51, p = 0.055). Younger children showed significantly higher MPO levels (coeff: -0.29, p = 0.002). No WASH variables or parasitic infections were associated with AAT levels. Household water being stored in covered containers was associated with lower MPO levels (coeff -1.75, p = 0.046). We found little evidence that a community-based WASH intervention had an impact on EE over a two-year period.

The trial found that individual handwashing and sanitation interventions significantly reduced childhood Giardia infections. Combined interventions provided no additional benefit. To reduce Giardia infection, individual interventions may be more feasible and cost-effective than combined interventions in similar rural, low-income settings., Background We evaluated effects of individual and combined water, sanitation, handwashing (WSH), and nutritional interventions on protozoan infections in children. Methods We randomized geographical clusters of pregnant women in rural Bangladesh into chlorinated drinking water, hygienic sanitation, handwashing, nutrition, combined WSH, nutrition plus WSH (N+WSH), or control arms. Participants were not masked. After approximately 2.5 years of intervention, we measured Giardia, Cryptosporidium, and Entamoeba histolytica prevalence and infection intensity by multiplex real-time polymerase chain reaction of child stool. Analysis was intention-to-treat. Results Between May 2012 and July 2013, we randomized 5551 pregnant women. At follow-up, among 4102 available women, we enrolled 6694 children into the protozoan assessment. We analyzed stool from 5933 children (aged ~31 months) for protozoan infections. Compared with 35.5% prevalence among controls, Giardia infection prevalence was lower in the sanitation (26.5%; prevalence ratio [PR], 0.75 [95% confidence interval {CI}, .64–.88]), handwashing (28.2%; PR, 0.80 [95% CI, .66–.96]), WSH (29.7%; PR, 0.83 [95% CI, .72–.96]), and N+WSH (26.7%; PR, 0.75 [95% CI, .64–.88]) arms. Water and nutrition interventions had no effect. Low prevalence of E. histolytica and Cryptosporidium (

Introduction Enteric infections are common during the first years of life in low-income countries and contribute to growth faltering with long-term impairment of health and development. Water quality, sanitation, handwashing and nutritional interventions can independently reduce enteric infections and growth faltering. There is little evidence that directly compares the effects of these individual and combined interventions on diarrhoea and growth when delivered to infants and young children. The objective of the WASH Benefits study is to help fill this knowledge gap. Methods and analysis WASH Benefits includes two cluster-randomised trials to assess improvements in water quality, sanitation, handwashing and child nutrition—alone and in combination—to rural households with pregnant women in Kenya and Bangladesh. Geographically matched clusters (groups of household compounds in Bangladesh and villages in Kenya) will be randomised to one of six intervention arms or control. Intervention arms include water quality, sanitation, handwashing, nutrition, combined water+sanitation+handwashing (WSH) and WSH+nutrition. The studies will enrol newborn children (N=5760 in Bangladesh and N=8000 in Kenya) and measure outcomes at 12 and 24 months after intervention delivery. Primary outcomes include child length-for-age Z-scores and caregiver-reported diarrhoea. Secondary outcomes include stunting prevalence, markers of environmental enteropathy and child development scores (verbal, motor and personal/social). We will estimate unadjusted and adjusted intention-to-treat effects using semiparametric estimators and permutation tests. Ethics and dissemination Study protocols have been reviewed and approved by human subjects review boards at the University of California, Berkeley, Stanford University, the International Centre for Diarrheal Disease Research, Bangladesh, the Kenya Medical Research Institute, and Innovations for Poverty Action. Independent data safety monitoring boards in each country oversee the trials. This study is funded by a grant from the Bill & Melinda Gates Foundation to the University of California, Berkeley. Registration Trial registration identifiers (http://www.clinicaltrials.gov): NCT01590095 (Bangladesh), NCT01704105 (Kenya).

We assessed the relationship of fecal environmental contamination and environmental enteropathy. We compared markers of environmental enteropathy, parasite burden, and growth in 119 Bangladeshi children (≤ 48 months of age) across rural Bangladesh living in different levels of household environmental cleanliness defined by objective indicators of water quality and sanitary and hand-washing infrastructure. Adjusted for potential confounding characteristics, children from clean households had 0.54 SDs (95% confidence interval [CI] = 0.06, 1.01) higher height-for-age z scores (HAZs), 0.32 SDs (95% CI = -0.72, 0.08) lower lactulose:mannitol (L:M) ratios in urine, and 0.24 SDs (95% CI = -0.63, 0.16) lower immunoglobulin G endotoxin core antibody (IgG EndoCAb) titers than children from contaminated households. After adjusting for age and sex, a 1-unit increase in the ln L:M was associated with a 0.33 SDs decrease in HAZ (95% CI = -0.62, -0.05). These results are consistent with the hypothesis that environmental contamination causes growth faltering mediated through environmental enteropathy.

Background Our current understanding of the composition and stability of the human distal gut microbiota is based largely on studies of infants and adults living in developed countries. In contrast, little is known about the gut microbiota and its variation over time in older children and adolescents, especially in developing countries. Methodology/Principal Findings We compared the diversity, composition, and temporal stability of the fecal microbiota of healthy children, ages 9 to 14 years, living in an urban slum in Bangladesh with that of children of the same age range in an upper-middle class suburban community in the United States. We analyzed >8,000 near full-length 16S rRNA gene sequences and over 845,000 pyrosequencing reads of the 16S rRNA V1–V3 region. The distal gut of Bangladeshi children harbored significantly greater bacterial diversity than that of U.S. children, including novel lineages from several bacterial phyla. Bangladeshi and U.S. children had distinct fecal bacterial community membership and structure; the microbiota of Bangladeshi children was enriched in Prevotella, Butyrivibrio, and Oscillospira and depleted in Bacteroides relative to U.S. children (although similar to Bangladeshi adults). Furthermore, community membership and structure in Bangladeshi children was significantly less stable month-to-month than U.S. children. Conclusions/Significance Together, these results suggest that differing environmental or genetic factors may shape the microbiota of healthy children in the two countries. Further investigation is necessary to understand the mechanisms and factors that underlie these differences, and to incorporate these findings into new strategies for the prevention and treatment of childhood and adolescent diseases.