Effects of water quality, sanitation, handwashing and nutritional interventions on enteric pathogen seroconversion in rural Bangladesh: a cluster randomised trial

Background: Improvements to water quality, sanitation, handwashing (WASH) and nutrition may prevent enteric pathogen infection during the first two years of life. Few randomised controlled trials have measured pathogen-specific effects of WASH and nutritional interventions. Methods: Within a 2x2 factorial cluster-randomized trial of WASH and nutritional interventions in Bangladesh, we tested dried blood spot specimens with multiplex assay for IgG responses to 12 enteric pathogens: Norovirus, Campylobacter, LT-producing enterotoxigenic E. coli (LT-ETEC), Shigella, non-typhoidal and typhoidal Salmonella enterica, Vibrio cholerae, Cryptosporidium, Giardia, Entamoeba histolytica, Taenia solium, and Strongyloides stercoralis. Primary analysis included children with IgG measurements at ~24 months, and secondary analysis assessed early effects in a sub-study at ~14 months. We estimated age-specific seroprevalence and marginal seroprevalence differences for WASH and nutrition effects. Trial is registered with ClinicalTrials.gov ( NCT01590095 ). Findings: Across the four arms (450 clusters), 4817 samples were tested for IgG responses, 2453 samples at the primary endpoint. Seroprevalence increased with age, consistent with intense early-life transmission. For highest transmission pathogens, >94% of children had seroconverted to norovirus, LT-ETEC and Campylobacter by 12-18 months. WASH effects were modest and pathogen-specific but generally protective. At 24 months, children that received WASH interventions had less antibody against Shigella (-4.5%, 95% CI -7.6% to -1.5%), non-typhoidal S. enterica (-4.0%, 95% CI -6.5% to -1.5%), and Giardia (-3.4%, 95% CI -6.7% to -0.2%), although not after multiple-testing correction. Nutrition had limited effects on most enteric pathogens, but reduced non-typhoidal S. enterica at 14 months (-6.4%, 95% CI: -9.8% to -3.0%). Interpretation: Multiplex serology provided evidence that WASH may modestly reduce cumulative exposure to selected enteric pathogens, but household-level interventions were insufficient to substantially disrupt transmission in this high-exposure setting. Funding: National Institutes of Health (R01AI166671, R01AI106878, R01AI134814). The original WASH Benefits trial was funded by Gates Foundation (OPPGD759).