Effects of drinking water, sanitation, handwashing, and nutritional interventions on immune status in young children: a cluster-randomized controlled trial in rural bangladesh

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<0.05), indicating the intervention promoted IL-10 driven immunoregulation. Similar reductions in ratios of pro-inflammatory cytokines to IL-10 were sustained in the intervention group after two years. After one year, IL-12/IL-4, IL-12/IL-5, IFN-γ/IL-5, and IL-12/IL-13 ratios were lower in the intervention group (−0.18 to -0.27, p<0.05), suggesting a shift towards a Th2 cytokine response. These findings suggest that the N+WSH intervention enhanced the immunoprotective and immunoregulatory responses, and suppressed/counteracted the inflammatory/immunopathological response, of the immune triad. ### Competing Interest Statement All authors received funding for either salary or consulting fees through a grant from the Bill & Melinda Gates Foundation for this study. A.L. received funding for salary through a grant from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health. ### Clinical Trial ClinicalTrials.gov ([NCT01590095][1]) ### Funding Statement This study was funded by Global Development grant OPPGD759 from the Bill & Melinda Gates Foundation to the University of California, Berkeley and by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health [grant number K01AI136885 to A.L.]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. icddr,b is grateful to the Governments of Bangladesh, Canada, Sweden, and the United Kingdom for providing core/unrestricted support. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Human subjects committees at icddr,b (PR-11063 and PR-14108), the University of California, Berkeley (2011-09-3652 and 2014-07-6561) and Stanford University (25863 and 35583) gave ethical approval for this work. A data safety monitoring committee convened by icddr,b oversaw the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Deidentified individual participant data collected for the study and a data dictionary defining each field in the set, will be made available to others. The pre-specified, registered statistical analysis plan and replication files for the study will be available with publication (https://osf.io/p9c6q/). [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01590095&atom=%2Fmedrxiv%2Fearly%2F2021%2F11%2F14%2F2021.11.10.21266206.atom