Effects of lipid-based nutrient supplements and infant and young child feeding counseling with or without improved water, sanitation, and hygiene (WASH) on anemia and micronutrient status: Results from 2 cluster-randomized trials in Kenya and Bangladesh

Background Anemia in young children is a global health problem. Risk factors include poor nutrient intake and poor water quality, sanitation, or hygiene. Objective We evaluated the effects of water quality, sanitation, handwashing, and nutrition interventions on micronutrient status and anemia among children in rural Kenya and Bangladesh. Design We nested substudies within 2 cluster-randomized controlled trials enrolling pregnant women and following their children for 2 y. These substudies included 4 groups: water, sanitation, and handwashing (WSH); nutrition (N), including lipid-based nutrient supplements (LNSs; ages 6-24 mo) and infant and young child feeding (IYCF) counseling; WSH+N; and control. Hemoglobin and micronutrient biomarkers were measured after 2 y of intervention and compared between groups using generalized linear models with robust SEs. Results In Kenya, 699 children were assessed at a mean ± SD age of 22.1 ± 1.8 mo, and in Bangladesh 1470 participants were measured at a mean ± SD age of 28.0 ± 1.9 mo. The control group anemia prevalences were 48.8% in Kenya and 17.4% in Bangladesh. There was a lower prevalence of anemia in the 2 N intervention groups in both Kenya [N: 36.2%; prevalence ratio (PR): 0.74; 95% CI: 0.58, 0.94; WSH+N: 27.3%; PR: 0.56; 95% CI: 0.42, 0.75] and Bangladesh (N: 8.7%; PR: 0.50; 95% CI: 0.32, 0.78; WSH+N: 7.9%, PR: 0.46; 95% CI: 0.29, 0.73). In both trials, the 2 N groups also had significantly lower prevalences of iron deficiency, iron deficiency anemia, and low vitamin B-12 and, in Kenya, a lower prevalence of folate and vitamin A deficiencies. In Bangladesh, the WSH group had a lower prevalence of anemia (12.8%; PR: 0.74; 95% CI: 0.54, 1.00) than the control group, whereas in Kenya, the WSH+N group had a lower prevalence of anemia than did the N group (PR: 0.75; 95% CI: 0.53, 1.07), but this was not significant (P = 0.102). Conclusions IYCF counseling with LNSs reduced the risks of anemia, iron deficiency, and low vitamin B-12. Effects on folate and vitamin A varied between studies. Improvements in WSH also reduced the risk of anemia in Bangladesh but did not provide added benefit over the nutrition-specific intervention. These trials were registered at clinicaltrials.gov as NCT01590095 (Bangladesh) and NCT01704105 (Kenya). Detailed descriptions of the 2 trials have been previously published (9–13). The trial was implemented in 3 counties in western Kenya—Kakamega, Bungoma, and Vihiga. Geographically matched village clusters were block-randomized into 1 of 8 study arms: chlorine treatment of drinking water (W); improved sanitation limiting exposure to feces (S); handwashing with soap (H); combined WSH; IYCF counseling plus small-quantity LNSs (N); combined WSH+N; active control in which children were visited by a health promoter monthly; and passive control with no intervention household visits. Villages were eligible for inclusion into the trial if they were rural, with most of the population reliant on unimproved sanitation facilities, without widespread access to chlorinated water, and without any active water, sanitation, handwashing, or nutrition programs. Households were eligible if there was a woman in her second or third trimester of pregnancy who planned to live in the community for ≥2 y and who could speak Kiswahili, Luhya, or English. Intervention clusters were formed by including a minimum of 6 eligible pregnant women residing in ≤3 neighboring villages. There were no buffer zones between clusters due to geographic constraints. Community health promoters (CHPs) were nominated by women in the community and trained to provide the intervention-specific behavior change activities as well as instructions for provision of supplements or hardware use. In both the active control and intervention arms, they were trained to measure midupper arm circumference to identify and provide referrals for potential cases of severe acute malnutrition. Each intervention package consisted of a comprehensive behavior change promotion program, including key messages; visual aids in the form of flip charts, posters, and reminder cue cards; interactive activities with songs, games, and pledges; and the distribution of arm-specific hardware, products, or supplements (10). CHPs were instructed to visit homes once per month throughout the trial, although visit frequency declined during the second year of the trial (10). In the active control arm, CHPs visited households at the same frequency as in the intervention arms. In the N intervention arm, households received monthly rations of micronutrient-fortified, small-quantity LNSs (Nutriset) when children were between the ages of 6 and 24 mo. The composition of the LNSs in comparison to the Recommended Nutrient Intakes for children is shown in Supplemental Table 1. At the start of the trial, supplements were distributed by CHPs, whereas in the second year of the study, project staff provided the monthly rations. Supplements were provided for the index child plus any other age-eligible siblings in the household. Caregivers were instructed to mix one 10-g sachet into the child’s complementary foods twice per day. Key messages focused on standard IYCF recommendations, including maternal dietary diversity during pregnancy and lactation, early initiation of breastfeeding, exclusive breastfeeding from age 0 to 6 mo and continued breastfeeding through 24 mo, timely introduction of complementary foods at 6 mo, dietary diversity, feeding frequency, and feeding during illness. In the WSH intervention arm, CHPs advocated for a variety of behaviors to improve water quality, sanitation, and handwashing practices within the home. Specifically, they promoted treatment of drinking water with sodium hypochlorite using either chlorine dispensers installed at the point-of-collection in study villages or bottled chlorine provided directly to households. They also used chlorine test strips to spot-check household chlorine concentrations during monthly visits; results were used to improve counseling. Existing latrines were upgraded and improved by installing a plastic slab with a tight-fitting lid. Households with no latrine were provided with a new one. CHPs provided a “sani-scoop” with a paddle to remove animal and human fecal material from the yard surrounding the home and child potties for each child aged <3 y in the study compound. Households were also provided with 2 dual tippy-tap handwashing stations, one near the latrine and a second near the cooking area. Stations were designed to have 2 foot-pedal–operated jerry cans that could be tipped to dispense a small stream of either soapy water or rinse water (14). CHPs filled the soapy water dispenser with soap every 3 mo. In the WSH+N arm, all of the aforementioned activities, hardware, and supplements were provided. Adherence to LNS recommendations was high (>90%) in the N arms, but there were no differences in complementary feeding practices between groups (10, 15). Adherence to the WSH interventions varied, but was generally lower than the LNS adherence (10). The WASH Benefits Bangladesh Trial was implemented in subdistricts of the Gazipur, Kishoreganj, Mymensingh, and Tangail districts. The study area was selected because it had low groundwater iron and arsenic according to data obtained from the Department of Public Health Engineering/British Geological Survey/Department for International Development National Hydrochemical Survey and a survey conducted before study initiation (16, 17) (Supplemental Figure 1); and it had no major water, sanitation, or focused nutrition programs underway or planned. Clusters were randomized by geographic blocks into 1 of 7 study arms: chlorine treatment of drinking water (W); improved sanitation limiting exposure to feces (S); handwashing with soap (H); combined WSH; IYCF counseling plus small-quantity LNSs (N); combined WSH+N; or a control group that received no intervention. In contrast to Kenya, there was no active control arm. Households with a pregnant woman were invited to participate in the trial if they planned to reside in the study communities for ≥2 y. The subsequent child or children, in the case of multiple births, born to those women were considered the study index children. Intervention clusters were formed with 8 eligible pregnant women in nearby proximity with a minimum buffer zone of 1 km between clusters to minimize intervention spillover effects. Local community members were recruited to serve as CHPs for the trial. Individuals who lived within walking distance of an intervention cluster, who had completed ≥8 y of formal education, and who passed a written and oral exam were considered eligible. CHPs attended an arm-specific training session at the start of the trial plus quarterly refresher trainings. Training sessions focused on the behavioral recommendations and intervention hardware usage, as well as communication and active listening techniques, and approaches for collaborative problem solving with the enrolled mothers. CHPs were instructed to visit study households at least once per week in the first 6 mo and fortnightly thereafter, although in practice, visits averaged 6 times/mo throughout the trial (9). Each intervention package consisted of arm-specific behavioral recommendations, hardware, or supplements delivered by the health promoter. There were no promoter visits or other intervention activities in the control-group households. The N intervention was similar to that described for Kenya. LNSs were delivered monthly by CHPs and provided for the index child only. IYCF recommendations were adapted from those developed by the Alive and Thrive program in Bangladesh (18). In the WSH intervention arm, households were provided a 10-L vessel with a lid, a tap, and a regular supply of 33-mg sodium dichloroisocyanurate (NaDCC) tablets (Medentech) to treat and safely store their drinking water. Households were encouraged to fill the vessel, add 1 tablet, and wait 30 min before drinking the water. Households that did not have access to an improved latrine with a slab and functional water seal in their compound were provided either a new latrine or improvements to an existing latrine. Households were also provided a scoop that could be used to clean the home environment and a child potty for all children aged <3 y for the safe disposal of child feces. Handwashing practices were promoted and supported by providing 2 handwashing stations, one placed near the latrine and the second placed near the kitchen. Each station contained a water reservoir with a tap, a basin to collect the rinse water, and a soapy water bottle. CHPs provided a regular supply of detergent for making the soapy water. The WSH+N intervention group received all of the aforementioned behavioral interventions, hardware, and products. The adherence to LNS supplementation and all behavioral recommendations was high across all groups throughout the trial (9) and dietary diversity was significantly greater in the 2 N arms (19). Interventions were randomly assigned at the cluster level with the use of codes generated by an off-site investigator independent of the data collection team via a random-number generator. Groups of adjacent clusters were block-randomized into the 6 intervention arms or a double-sized passive control arm (in Bangladesh) or double-sized active control arm and single-sized passive control arm (in Kenya). Participants were informed of their group assignment after the baseline survey. Masking participants was not possible due to the nature of the interventions. Although data collectors were not directly informed of the random assignment, they may have inferred the intervention group through observation of materials within the home during subsequent household visits. Masked technicians completed the laboratory analyses. In both trials, substudy clusters were selected from the N, WSH, WSH+N, and control arms (active in Kenya and passive in Bangladesh). Clusters were selected based on the logistical feasibility of the preservation and collection of biological specimens, as well as their transport to the central laboratory. The primary objective of the substudies was to assess biomarkers of environmental enteric dysfunction, which will be reported elsewhere. Index households with live-born infants residing in selected clusters were invited to participate in the substudy activities. Exclusion criteria included symptoms of moderate to severe dehydration on the day of the survey, as follows: 1) restless, irritable; 2) sunken eyes; 3) drinks eagerly, thirsty; and 4) pinched skin returns to normal position slowly or the child being listless or unable to perform their normal activities. In both trials, after consent and enrollment, enumerators administered a baseline questionnaire asking respondents to report on their household characteristics, animal and other asset ownership, food insecurity status using the Household Food Insecurity Access Scale (20) in Bangladesh and the Household Hunger Scale (21) in Kenya, and WASH conditions within the home. In addition to the 2 follow-up visits that all study children received, children in the substudies were visited at 3 additional times for data and biological sample collection. Anemia and micronutrient status were measured at the third of these follow-up visits. In Kenya, this occurred ∼2 y after the start of intervention activities. In Bangladesh, fieldwork was delayed by ∼4 mo at the third follow-up point due to civil unrest, which also caused inconsistency in the timing of follow-up visits in some clusters. Substudy visits were conducted within the home in Bangladesh and at a central site in the community in Kenya. For each child, a maximum of 7.7 mL blood was collected by a trained phlebotomist via venipuncture into a Sarstedt monovette serum collection tube and a Sarstedt monovette lithium heparin trace element–free plasma collection tube. Hemoglobin concentrations were measured in venous whole blood at the point of collection using a portable spectrophotometer (Hemocue 301) that was regularly checked using standardized quality-control procedures. In Kenya, malaria was tested using rapid diagnostic test kits (Alere; SD Bioline Malaria Ag P.f/P.f/P.v) at the time of collection. In Kenya, blood samples were placed on ice packs, centrifuged at 3500 rpm for 15 min at ambient temperature within a mean of 1 h of collection, and then plasma and serum aliquots were placed on ice packs in a cooler where they remained for ∼2.5 h until they could be transferred to a −20°C freezer. Within ∼2 wk of collection, samples were subsequently transferred to −80°C freezers at the Kenya Medical Research Institute (KEMRI) in Nairobi for long-term storage. In Bangladesh, blood samples were placed on ice, transported to the project laboratory, centrifuged at 3500 rpm for 15 min at ambient temperature, and stored in a −80°C freezer. The mean time from blood collection to centrifugation was 8.5 h. Specimens were shipped on dry ice to collaborating laboratories for analysis. Serum retinol-binding protein (RBP), ferritin, soluble transferrin receptor (sTfR), α-1 acid glycoprotein (AGP), and C-reactive protein (CRP) were measured using ELISA methods (22) (VitMin Lab). Quality-control material from the CDC and Biorad Liquicheck Controls were used to ensure the accuracy and precision of the analysis. A manufacturer lot problem with the AGP assay affected approximately half the Bangladesh samples and there was insufficient sample volume available to rerun the samples, so those results are not presented. Hepcidin was measured using a commercially available ELISA kit and controls (Peninsula Laboratories) following the manufacturer's protocol at the International Center for Diarrheal Disease Research, Bangladesh, or KEMRI laboratories. Serum vitamin B-12 and folate concentrations were measured using chemiluminescence on a Roche e411 with Roche B-12 and folate III reagents, standards, and controls to ensure accuracy and precision of the assay at the USDA Western Human Nutrition Research Center in Davis, California. External quality control was monitored through the CDC Vital-External Quality Assurance Program. Intra-assay CVs from replicate measures for all micronutrient biomarkers, CRP, and AGP were <10% in Bangladesh and <12% in Kenya. The highest CV was found in the hepcidin assay in Kenya (11.5%), which was driven by 7 samples with a large variation. Removing those subjects from the analysis did not substantially change the interpretation of our results and so we have elected to keep them in the analysis. In Bangladesh, whole-blood samples were tested for thalassemia and hemoglobin E (HbE) traits using electrophoresis at Dhaka Shishu Hospital. In Kenya, DNA was extracted from packed blood cell samples and tested for sickle cell trait and α-thalassemia by polymerase chain reaction using proprietary kits (Qiagen) (23, 24) at KEMRI/Wellcome Trust laboratories in Kilifi, Kenya. The sample size for the substudy was based on detecting a difference of ≥0.26 SDs in standardized log environmental enteric dysfunction biomarkers (reported separately) between any intervention arm and the control arm assuming 80% power, a 2-sided type 1 error of 5%, and an intracluster correlation coefficient of 0.15, resulting in a sample size of n = 375 children/group, assuming 54 clusters/arm and 7 children/cluster (https://osf.io/qa43y/). Using this number, we estimated that we would be able to detect a mean difference in hemoglobin between groups of 3.4–3.9 g/L or a difference in the prevalence of anemia of 11.3–11.9 percentage points based on an assumed mean hemoglobin of 103 g/L and prevalence of anemia of 47–85%, based on previously published data from children in nearby study areas (25, 26). The intracluster correlation was estimated to be between 0.6 and 0.14 for hemoglobin and 0.07 and 0.10 for anemia, based on data from 3 large studies in India, Indonesia, and Vietnam (27–29). Mothers provided written informed consent for themselves and their infants. The study protocol was reviewed and approved by the Committee for the Protection of Human Subjects at the University of California, Berkeley, the Institutional Review Board at Stanford University, the Ethical Review Committee at the International Center for Diarrheal Disease Research, Bangladesh, and the Scientific and Ethics Review Committee at the KEMRI. Children with hemoglobin <70 g/L or who tested positive for malaria with fever or sickle cell disease (HbSS) were referred for treatment. Detailed analysis plans were prespecified and publicly posted (https://osf.io/dsrv2). Analyses were independently replicated by 2 investigators (CDA and KAB) using R version 3.5.0 (R Foundation for Statistical Computing) and Stata version 14 (StataCorp LLC). All investigators were blinded to group assignment until primary analyses were completed. Hemoglobin concentrations were adjusted for altitude, and a hemoglobin cutoff of 110 g/L was used to define anemia (30). We further calculated the prevalence of mild (hemoglobin of 100–109 g/L), moderate (hemoglobin of 70–99 g/L), and severe (hemoglobin <70 g/L) anemia. Micronutrient deficiencies were defined in the following ways: iron deficiency as either ferritin <12 µg/L or sTfR >8.3 mg/L (22); vitamin A deficiency as RBP <0.83 µmol/L (31); folate deficiency as <10 nmol/L (32, 33); and vitamin B-12 deficiency as <150 pmol/L and depletion as 150–221 pmol/L (34). We also examined low hepcidin concentrations using a cutoff of 5.5 ng/mL (35). Further, high folate was categorized as >45.3 nmol/L (36). Elevated CRP (>5 mg/L) or AGP (>1 g/L; in Kenya only) was used to define inflammation (37). We estimated the mean difference in each of the biomarkers between each intervention group and the control group using generalized linear models with robust SEs controlling for clustering at the study block level. Skewed outcomes were log-transformed for analysis. We also calculated the prevalence difference and prevalence ratio between groups using a linear probability model for the prevalence differences and a binomial distribution with a log link for the prevalence ratios, each with robust SEs at the study block level. Our primary inference was the unadjusted between-group differences in these parameters. The primary models were not adjusted for inflammation because this might be on the causal pathway between the interventions and the outcomes. In secondary analyses, we additionally adjusted for prespecified baseline covariates that may be potentially associated with the outcomes, including child age, sex, birth order, maternal age, height, educational level, household food insecurity category, number of children aged <18 y in the household, number of individuals living in the compound, distance to the primary water source, housing materials, household assets, animal ownership, malaria infection, and thalassemia, sickle cell, or HbE traits. Further, we also considered the potential effects of inflammation on the interpretation of the deficiency prevalence estimates. Ferritin, sTfR, and RBP have been found to be sensitive to the acute-phase response (37, 38). We used the regression method proposed by the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia research group (39–41). Finally, we considered the potential for interaction between the intervention group and child age, hemoglobinopathy trait, sex, and household food insecurity status. In additional analyses, and to examine the potential for bias due to losses to follow-up, we examined whether there were differences in the characteristics of those with missing data compared with those who were included in this analysis. We conducted an inverse probability of censoring–weighted (IPCW) analysis that reweighted the analysis population to reflect the original enrolled population (42, 43). The reference population consisted of all children selected for the substudy, excluding known pregnancy losses. Baseline measurements including maternal parity, age, education, household hunger score, number of children aged <18 y in the household, number of people living in the compound, distance to primary water source, improved primary water source, roofing quality, asset index score, animal ownership, and timing of measurement in the cluster were used to predict missingness. Missingness mechanism parameters were estimated using logistic regression, and targeted maximum likelihood was used for treatment effect inference. In post hoc analysis, we additionally examined the micronutrient concentrations by study month in Bangladesh to examine seasonal patterns.