Our primary outcomes include: any bacteria or protozoa infection at age 12 months after birth; individual pathogens or pathogen groups; child gut microbiome composition; and household source water quality. We include viral, protozoal and bacterial pathogens responsible for the vast majority of enteric pathogen infections and global disease burden.96 97 (link) While we measure viral pathogens using the TAC assay, they will be excluded from the combined enteropathogen prevalence primary outcome measure, because waterborne transmission is unlikely to dominate for these viral pathogens.98–101 (link) In addition to the aforementioned reasons related to child development and infection risk, measuring pathogens at 12 months will give us the greatest power to detect a difference, given higher levels of infection at that age than in younger children. We will measure gut microbiome using 16S rRNA gene amplicon sequencing in the full sample at 12 months and in a random subset of 200 children with complete data at 3, 6 and 9 months, evenly distributed between intervention and control groups; dyads eligible for subset sampling will include those with complete stool sample collection and unchanged intervention exposure conditions. The 12-month samples will allow us to compare all study children at a common time, when all children are consuming drinking water and once the gut microbiome has become relatively established102 (link); the longitudinal samples will allow for comparison of development of the microbiome over time between the two groups. Microbiome outcomes include alpha and beta diversity metrics, and identification of enriched taxonomic groups. We also include household source water quality as a primary exposure outcome, as understanding whether exposure to microbial contaminants is altered is considered a critical aspect of evaluation of WASH projects.71 103 (link)
Additional non-primary outcomes include pathogen count, pathogen community similarity (measured using Jaccard Similarity Index), diarrhoea, child growth and prior enteropathogen infection (measured using serology on dried blood spot samples). We will measure additional water quality exposure measures, as well as measures of exposure to the improved water system, such as fidelity of the intervention (eg, improvements to water quantity and coverage of household taps) and receipt of the intervention by community members (eg, reductions in water insecurity, increased water use). These fidelity and uptake measures will be collected at all time points through direct observation and respondent report. Available minimal detectable effect sizes are summarised in table 1 and calculations are further detailed in the online supplemental material.