Environmental and behavioural exposure pathways associated with diarrhoea and enteric pathogen detection in 5-month-old, periurban Kenyan infants: a cross-sectional study

Objectives The aim of this study was to test whether household environmental hygiene and behavioural conditions moderated associations between diarrhoea and enteric pathogen detection in infants 5 months of age in Kenya and pathogen sources, including latrine access, domestic animal co-habitation and public food sources. Design Cross-sectional study utilising enrolment survey data of households participating in the Safe Start cluster-randomised controlled trial. Setting Kisumu, Kenya. Participants A total of 898 caregivers with 5-month (22 week ± 1 week) aged infants were enrolled in the study and completed the enrolment survey. Primary and secondary outcome measures Outcomes were (1) caregiver-reported 7-day diarrhoea prevalence and (2) count of types of enteric viruses, bacteria and parasites in infant stool. Exposures and effect modifiers included water access and treatment, cohabitation with domestic animals, sanitation access, handwashing practices, supplemental feeding, access to refrigeration and flooring. Results Reported handwashing after handling animals (adjusted odds ratio (aOR)=0.20; 95% CI=0.06 to 0.50) and before eating (aOR=0.44; 95% CI=0.26 to 0.73) were strongly associated with lower risk of caregiver-reported diarrhoea, while cohabitation with animals (aOR=1.54; 95% CI=1.01 to 2.34) living in a household with vinyl-covered dirt floors (aOR=0.60; 95% CI=0.45 to 0.87) were strongly associated with pathogen codetection in infants. Caregiver handwashing after child (p=0.02) or self-defecation (p=0.03) moderated the relationship between shared sanitation access and infant exposure to pathogens, specifically private latrine access was protective against pathogen exposure of infants in households, where caregivers washed hands after defecation. In the absence of handwashing, access to private sanitation posed no benefits over shared latrines for protecting infants from exposure. Conclusion Our evidence highlights eliminating animal cohabitation and improving flooring, postdefecation and food-related handwashing, and safety and use of cow milk sources as interventions to prevent enteric pathogen exposure of young infants in Kenya. Trial registration number NCT03468114 This cross-sectional study uses baseline data collected from 5-month-old (22 weeks ± 1 week) infants and their caregivers at the point of enrolment into Safe Start cluster-randomised controlled trial of an infant food hygiene behaviour change intervention in Kisumu, Kenya (Clinical Trials identifier: {“type”:”clinical-trial”,”attrs”:{“text”:”NCT03468114″,”term_id”:”NCT03468114″}}NCT03468114, Pre-results stage as of 18 Oct. 2022). The formative work and trial protocol, including the estimation of sample size for evaluating trial impact, are described elsewhere.21–25 Kisumu is a city of approximately 490 000 people (Kisumu county integrated development plan 2013–2017) in the western region of Kenya. The study site includes communities in two low-income periurban neighbourhoods in Kisumu. The study was approved by the scientific and ethical review committees at Great Lakes University of Kisumu (Ref. No. GREC/010/248/2016), London School of Hygiene and Tropical Medicine (Ref. No. 14695) and University of Iowa (IRB ID 201804204). Caregivers with 5-month-old infants living in the catchment area of participating Community Health Volunteers (CHVs) participated in a survey and provided a sample of infant stool for microbial analysis at enrolment into the trial. We defined eligibility of an infant as being 22 weeks (±1 week) of age, as verified by birth registration card, who resided in one of the two study neighbourhoods. We enrolled caregivers who were responsible for care of the infant during the day and were at least 18 years of age. Study participants and CHVs provided input on the goals, design and implementation of the Safe Start Study through prestudy knowledge dissemination meetings and formative research.21 25 The outcomes for this study are: (1) 7-day caregiver-reported diarrhoea prevalence prior to enrolment, where diarrhoea is defined as three or more loose, watery stools in the previous 24 hours; and (2) the sum count of enteric pathogens detected in infant stool. The study was described in the caregiver’s natural language, and a signed copy of the consent form was left for their records. On verification of eligibility and consent, caregivers were interviewed to collect data about household socioeconomic conditions, access to water and sanitation infrastructure, animal ownership and hygiene practices. In anticipation that infant breast feeding and feeding practices may vary day to day and may be subject to response bias or recall bias, we asked caregivers about overall dietary history and foods given to infants in the last day. Caregivers were then provided several sterile commercially produced diapers and a sterile Ziploc bag, and asked to use these until the infant defecated. Diapers prevented cross-contamination via, for example, caregivers collecting infant faeces with dirt attached from the ground. Caregivers were instructed to fold the diaper, place it into the storage bag and store it in a cool dark place out of the reach of children and animals. The research team returned within 24 hours to collect the diaper, placed it in a cooler on ice packs and transported it to the laboratory within 5 hours of collection from the household. If the infant did not defecate in the first 24 hours, the team returned each day up to 5 days after enrolment to assess whether the infant had defecated. If no faeces could be collected, the infant was de-enrolled from the study. Lab technicians unwrapped diapers in biosafety cabinets and used sterile stool collection scoops to transfer 200 mg of stool into Zymobiomics Shield Collection tubes, which were vortexed on a bead beater for 20 min and then processed according to the manufacturer’s instructions for the ZymoBIOMICS DNA/RNA extraction mini-kit (Zymo Research, Irvine, California, USA). One molecular-grade water only sample was prepared each day of sample processing as a process contamination control. Approximately half (n=383) of samples were spiked with 3 µL of 1.8 × 106 CFU/µL of live bacteriophage MS2 to serve as a process control to assess for inhibition and efficiency in DNA and RNA recovery. Samples were transported on dry ice to the University of Iowa and stored at −80℃ until analysis. A total of 23 gene targets of pathogen of interest in the TaqMan assays were used to assess pathogen presence in infants. Pathogen gene targets were Adenovirus 40–41 Fibre, Adenovirus broad species Hexon, Rotavirus NSP3, Norovirus GI ORF 1–2, Norovirus GII ORF 1–2, Aeromonas aerolysin toxin aerA, Campylobacter jejuni/C. coli (cadF), Enterohemorrhagic Escherichiap coli (EHEC) 0157 rdb, Enteroaggregative E. coli (EAEC) aatA and aaiC, Enteropathogenic E. coli (EPEC) bfpA and eae, Enterotoxigenic E. coli (ETEC) elt and est, Clostridioides difficile tcdB, Salmonella enterica ttr, Shigella spp virG, Vibrio cholerae hlyA, Giardia duodenalis Assemblage A triosephosphate isomerase (TPI), Giardia duodenalis Assemblage A triosephosphate isomerase (TPI), Cryptosporidium spp 18S, C. hominus LIB13 and C. parvum LIB13.26 27 For each sample, 40 µL of extract was mixed with 5 µL of nucleic acid-free water, 50 µL of 2X RT-buffer, 0.6 µL of 50 mg/mL bovine serum albumin (to reduce inhibition) and 4 µL of 25X AgPath enzyme from the AgPath-ID One-Step Reverse Transcription-Polymerase Chain Reaction (RT-PCR) kit (Thermo Fisher, Waltham, Massachusetts, USA) and pipetted into a well on a compartmentalised TaqMan card that included primer and probe assays in duplicate for each gene. TaqMan assays were completed in either a ViiA7 or QuantStudio8 instrument (Thermo Fisher, Waltham, Massachusetts, USA) for cycling conditions: 45°C for 20 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. A subset of samples that included both low and high Cq results were analysed on both machines and compared toconfirm that results did not vary between machines, before proceeding with further use of both machines. We defined a sample as positive if a gene target amplified within a 35 Cycle threshold (Cq35). If multiple gene targets were used to detect one type of pathogen, amplification of either gene (EAEC aaiC/aatA, EPEC bfpA/eae, ETEC elt/est) was considered positive for the general type of pathogen. The pathogen-specific detection patterns used to define the pathogen count variable for this analysis are described elsewhere.28 Independent variables representing point sources of faeces that could contaminate the environment with enteric pathogens included household latrine design and location of the latrine, sharing of a latrine, ownership of domestic animals, whether animals are typically kept inside the household and observation of rodents or their faeces in the household. Improved sanitation was defined as a flush, pour flush, ventilated pit latrine or pit latrine with an impermeable slab, according to WHO/UNICEF JMP criteria.29 Although they do not represent point sources of faeces, food ingredients and especially animal-based ones, as well as community drinking water sources could be sources of pathogens that originate outside the household. Thus, primary and secondary water sources and whether the primary water source is intermittent, and feeding the infant solid or liquid (other than breastmilk) foods were also defined as pathogen sources. A basic water source was defined as a piped tap to household or compound, a public tap, tube well, borehole, protected spring, protected hand dug well or rainwater, according to WHO/UNICEF Joint Monitoring Programme criteria, available within 30 min round trip.29 Independent variables representing intermediate environmental conditions or behaviours that could modify pathogen transmission pathways and prevent exposure included household flooring (soil/surfaces), treating drinking water after collection, prior and recent (in the last day) breastfeeding status, presence of a handwashing station with soap and water and self-reported handwashing at critical times. Three of these critical handwashing times are behaviours that could modify hand cleanliness after touching human faeces or animals, while three were focused specifically on modification of food sources being prepared within the household. Potential confounders included in this analysis are marital status of caregiver, maternal education, household wealth, presence of multiple children under 5 years of age, infant preterm birth status, rotavirus vaccination status and prior or current breastfeeding practices. Principal components analysis with Promax rotation of 15 household assets (bicycle, motorbike, car, refrigerator, mobile phone, wrist or pocket watch, wall clock, radio, cassette or CD player, television, DVD player, microwave oven, presence of grates on the windows and doors, use of electricity for lighting and use of propane or electricity for cooking) resulted in a household wealth variable which was stratified into five quintiles. All analysis were conducted with R software V.4.0.3 (R Foundation for Statistical Computing, Vienna, Austria). Associations between exposures and the binary indicator for 7-day caregiver-reported diarrhoea were evaluated using logistic regression. Bivariate associations were evaluated between each exposure and self-reported diarrhoea, and fully adjusted relationships between the exposures and the outcome was evaluated by running a single model with the confounder variables listed above and all exposure variables included. Associations between exposures and the ordinal categorical pathogen count variable were estimated using ordinal logistic regression. As with caregiver-reported diarrhoea, we evaluated bivariate and fully adjusted relationships. In both fully adjusted models, multicollinearity among the exposure variables resulted in non-identifiable or nearly-non-identifiable model, resulting in the exclusion of some redundant exposure variables. In addition, some confounder variables had insufficient variability to have estimable effects and hence were removed. Finally, caregivers reporting a lack of breast feeding were so rare that this variable was excluded from the data. Logistic regression results are reported as ORs, with 95% CIs, for having diarrhoea, and ordinal logistic regression results are reported as ORs for having a higher count of pathogen types in stool versus fewer pathogen types. A random effect was initially included in models to adjust for spatial clustering, but was removed due to lack of variation in outcomes between villages. Our moderation analyses tested whether hygiene of intermediate exposure pathways modified the relationships between point sources of pathogen contamination and infant health outcomes. Lack of access to a latrine with a barrier between users and excreta, and/or sharing unclean latrines with others can result in infant exposure to pathogens in human faeces15 20 30 through faeces being tracked by feet onto household floors, where infants play and place objects or hands that have been on the floor in their mouth. We hypothesise that floor type moderates the effect of latrine access on pathogens in infants such that permeable dirt floors that absorb liquids and sustain microbial growth, and that are harder to clean and disinfect, increase the association between human sanitation and pathogens in children compared with impermeable floors, like vinyl, concrete, or tile. Lack of or sharing latrine access can result in infant exposure to pathogens when caregivers do not wash hands after self-defecation or cleaning a child and then place hands in the infant’s mouth. We hypothesise that handwashing after defecation or child defecation moderates the effect of latrine access on infant health, such that human sanitation will be associated with pathogens in children among caregivers not washing hands after self or child defecation but not among caregivers who wash hands after self/child defecation. Like with latrines, an association between domestic animals or rodents and enteric infections in children could be caused by exposure of infants to floors contaminated with animal faeces.12 We hypothesise that floor type can moderate this risk such that a dirt floor increases the association between domestic animals kept in or near the household or the presence of rodents and pathogens in children compared with households with impermeable floors. Zoonotic transmission of pathogens to infants could also occur through hands of caregivers who touch domestic animals or their faeces, and then place hands in the infant mouth. We hypothesise that washing hands after handling animals moderates the effect of animal or rodent presence in the household on pathogens in children such that caregivers not washing hands after handling animals increases the association between animals or rodents and pathogens in infants compared with caregivers who wash hands after touching animals. Animal or human faeces contamination on hands can be introduced into infant food during preparation or feeding and ingested by the infant while handwashing after defecation and animal handling could prevent transmission. We hypothesise that food-related handwashing also moderates the association between faeces sources (latrine access and the presence of animals or rodents in the household) and pathogens in infants such that caregivers (1) washing hands before preparing food, (2) washing hands before eating or (3) washing hands before feeding the infant decreases the association between latrines, domestic animals and rodents with pathogens in infants, compared with caregivers who do not wash hands at these times. Infant supplemental foods include street foods prepared by vendors, raw fruits and prepackaged commercial products (eg, pasteurised milk) that could contain contamination. Cooking (eg, porridge), washing (eg, fruit) or storing these foods can mitigate or enhance these external food system-based pathogen transmission risks.31 We hypothesise that access to a refrigerator for food storage moderates the pathway between supplemental foods and pathogens in infants such that a lack of refrigeration increases the association between supplemental food and pathogens in infants compared with households with refrigeration. Similarly, reliance on unsafe water sources can increase the chances of pathogen infection through water, but water treatment can reduce or eliminate this contamination. We hypothesise that filtering, boiling or chlorinating water after collection moderates the association between type of water source and pathogens in infants such that not treating drinking water increases the association between household drinking water source and pathogens in infants compared with households who treat their drinking water. These hypotheses were tested one at a time by adding an interaction term to the fully adjusted pathogen count model; the large number of potential confounders and exposure variables prohibited the simultaneous inclusion of all interaction terms being estimable. For each hypothesis listed above we ran an ordinal logistic regression model of the form Moderation effects were then tested using a likelihood ratio test comparing the additive model excluding the interaction term to the full model including the interaction term.