Independent and combined effects of improved water, sanitation, and hygiene, and improved complementary feeding, on child stunting and anaemia in rural Zimbabwe: a cluster-randomised trial.

BACKGROUND: Child stunting reduces survival and impairs neurodevelopment. We tested the independent and combined effects of improved water, sanitation, and hygiene (WASH), and improved infant and young child feeding (IYCF) on stunting and anaemia in in Zimbabwe. METHODS: We did a cluster-randomised, community-based, 2 × 2 factorial trial in two rural districts in Zimbabwe. Clusters were defined as the catchment area of between one and four village health workers employed by the Zimbabwe Ministry of Health and Child Care. Women were eligible for inclusion if they permanently lived in clusters and were confirmed pregnant. Clusters were randomly assigned (1:1:1:1) to standard of care (52 clusters), IYCF (20 g of a small-quantity lipid-based nutrient supplement per day from age 6 to 18 months plus complementary feeding counselling; 53 clusters), WASH (construction of a ventilated improved pit latrine, provision of two handwashing stations, liquid soap, chlorine, and play space plus hygiene counselling; 53 clusters), or IYCF plus WASH (53 clusters). A constrained randomisation technique was used to achieve balance across the groups for 14 variables related to geography, demography, water access, and community-level sanitation coverage. Masking of participants and fieldworkers was not possible. The primary outcomes were infant length-for-age Z score and haemoglobin concentrations at 18 months of age among children born to mothers who were HIV negative during pregnancy. These outcomes were analysed in the intention-to-treat population. We estimated the effects of the interventions by comparing the two IYCF groups with the two non-IYCF groups and the two WASH groups with the two non-WASH groups, except for outcomes that had an important statistical interaction between the interventions. This trial is registered with ClinicalTrials.gov, number NCT01824940. FINDINGS: Between Nov 22, 2012, and March 27, 2015, 5280 pregnant women were enrolled from 211 clusters. 3686 children born to HIV-negative mothers were assessed at age 18 months (884 in the standard of care group from 52 clusters, 893 in the IYCF group from 53 clusters, 918 in the WASH group from 53 clusters, and 991 in the IYCF plus WASH group from 51 clusters). In the IYCF intervention groups, the mean length-for-age Z score was 0·16 (95% CI 0·08-0·23) higher and the mean haemoglobin concentration was 2·03 g/L (1·28-2·79) higher than those in the non-IYCF intervention groups. The IYCF intervention reduced the number of stunted children from 620 (35%) of 1792 to 514 (27%) of 1879, and the number of children with anaemia from 245 (13·9%) of 1759 to 193 (10·5%) of 1845. The WASH intervention had no effect on either primary outcome. Neither intervention reduced the prevalence of diarrhoea at 12 or 18 months. No trial-related serious adverse events, and only three trial-related adverse events, were reported. INTERPRETATION: Household-level elementary WASH interventions implemented in rural areas in low-income countries are unlikely to reduce stunting or anaemia and might not reduce diarrhoea. Implementation of these WASH interventions in combination with IYCF interventions is unlikely to reduce stunting or anaemia more than implementation of IYCF alone. FUNDING: Bill & Melinda Gates Foundation, UK Department for International Development, Wellcome Trust, Swiss Development Cooperation, UNICEF, and US National Institutes of Health.The SHINE trial is funded by the Bill & Melinda Gates Foundation (OPP1021542 and OPP113707); UK Department for International Development; Wellcome Trust, UK (093768/Z/10/Z, 108065/Z/15/Z and 203905/Z/16/Z); Swiss Agency for Development and Cooperation; US National Institutes of Health (2R01HD060338-06); and UNICEF (PCA-2017-0002)

Biomarker concentrations in anemic and non-anemic infants.

<p>Graphs show the plasma concentrations of hepcidin, ferritin, soluble transferrin receptor and C-reactive protein (CRP) in infants at (A) 3 months, (B) 6 months and (C) 12 months of age. The boxes indicate the 25^th and 75^th percentiles and the horizontal line indicates the median. Hepcidin, ferritin and CRP are expressed on a log scale. Full data for each biomarker are also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135227#pone.0135227.t002" target="_blank">Table 2</a>.</p

Relationships between hepcidin and other biomarkers.

<p>Correlations between (A) plasma hepcidin and ferritin and (B) plasma hepcidin and CRP. Values for non-anemic (empty circles) and anemic (solid circles) infants are combined (p<0.001 for all Spearman correlations). Plasma hepcidin, ferritin and CRP are expressed on a log scale.</p

Stunting is characterized by chronic inflammation in Zimbabwean infants.

Stunting affects one-third of children in developing countries, but the causes remain unclear. We hypothesized that enteropathy leads to low-grade inflammation, which suppresses the growth hormone-IGF axis and mediates stunting.We conducted a case-control study of 202 HIV-unexposed Zimbabwean infants who were stunted (height-for-age Z-score (HAZ) -0.5; controls) at 18 months. We measured biomarkers of intestinal damage (I-FABP), inflammation (CRP, AGP, IL-6) and growth hormone-IGF axis (IGF-1, IGFBP3) in infant plasma at 6 weeks and 3, 6, 12 and 18 months, and in paired maternal-infant plasma at birth. Adjusted mean differences between biomarkers were estimated using regression models. Multivariate odds ratios of stunting were estimated by logistic regression.At birth, cases were shorter (median (IQR) HAZ -1.00 (-1.53, -0.08) vs 0.03 (-0.57, 0.62,); P<0.001) than controls and their mothers had lower levels of IGF-1 (adjusted mean difference (95%CI) -21.4 (-39.8, -3.1) ng/mL). From 6 weeks to 12 months of age, levels of CRP and AGP were consistently higher and IGF-1 and IGFBP3 lower in cases versus controls; IGF-1 correlated inversely with inflammatory markers at all time-points. I-FABP increased between 3-12 months, indicating extensive intestinal damage during infancy, which was similar in cases and controls. In multivariate analysis, higher log10 levels of CRP (aOR 3.06 (95%CI 1.34, 6.99); P = 0.008) and AGP (aOR 7.87 (95%CI 0.74, 83.74); P = 0.087) during infancy were associated with stunting. There were no associations between levels of I-FABP, IL-6, sCD14 or EndoCAb and stunting.Stunting began in utero and was associated with low maternal IGF-1 levels at birth. Inflammatory markers were higher in cases than controls from 6 weeks of age and were associated with lower levels of IGF-1 throughout infancy. Higher levels of CRP and AGP during infancy were associated with stunting. These findings suggest that an extensive enteropathy occurs during infancy and that low-grade chronic inflammation may impair infant growth

Selection of infants into the hepcidin substudy.

<p>^¶ HIV-unexposed infants with gestational age >37 weeks, birth weight > 2500 g and available plasma samples (>120 μL). *Non anemic infants were selected based on no abnormal iron indicators (defined as hemoglobin < 105 g/L at 3 and 6 months and < 100 g/L at 12 months, serum ferritin < 12 μg/L, sTfR > 8.3 mg/L), no evidence of inflammation (defined as AGP > 1 g/L or CRP > 5 mg/L) and no acute illness (defined as diarrhea or fever in the prior week or measles in the prior 3 months). Anemic infants at each age were randomly selected from 62, 77 and 85 eligible infants at 3, 6 and 12 months based on hemoglobin (defined as <105 g/L at 3 and 6 months, and <100 g/L at 12 months of age.</p

Inflammation and cytomegalovirus viremia during pregnancy drive sex-differentiated differences in mortality and immune development in HIV-exposed infants.

Children who are HIV-exposed but uninfected have increased infectious mortality compared to HIV-unexposed children, raising the possibility of immune abnormalities following exposure to maternal viraemia, immune dysfunction, and co-infections during pregnancy. In a secondary analysis of the SHINE trial in rural Zimbabwe we explored biological pathways underlying infant mortality, and maternal factors shaping immune development in HIV-exposed uninfected infants. Maternal inflammation and cytomegalovirus viraemia were independently associated with infant deaths: mortality doubled for each log10 rise in maternal C-reactive protein (adjusted hazard ratio (aHR) 2.09; 95% CI 1.33-3.27), and increased 1.6-fold for each log10 rise in maternal cytomegalovirus viral load (aHR 1.62; 95% CI 1.11-2.36). In girls, mortality was more strongly associated with maternal C-reactive protein than cytomegalovirus; in boys, mortality was more strongly associated with cytomegalovirus than C-reactive protein. At age one month, HIV-exposed uninfected infants had a distinct immune milieu, characterised by raised soluble CD14 and an altered CD8 + T-cell compartment. Alterations in immunophenotype and systemic inflammation were generally greater in boys than girls. Collectively, these findings show how the pregnancy immune environment in women with HIV underlies mortality and immune development in their offspring in a sex-differentiated manner, and highlights potential new intervention strategies to transform outcomes of HIV-exposed children. ClinicalTrials.gov/NCT01824940

Baseline characteristics of infants and mothers.

<p>*Defined as birth weight <2500 g.</p><p>**Detailed feeding information was collected from mothers at 6 weeks, 3 months and 6 months of age, including whether any of 22 liquids (water, juice, tea, cooking oil), milks (formula, fresh, tinned), medicines (traditional, oral rehydration solution, prescribed) or solid foods (porridge, sadza, fruit, vegetables, meat, eggs) had been given to the infant. Breastfeeding was defined as exclusive, predominant or mixed at 3 months of age, according to previously published definitions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086928#pone.0086928-Iliff1" target="_blank">[19]</a>. Data were not available at 3 months of age for 6 stunted and 25 non-stunted infants.</p>†<p>In the ZVITAMBO trial, mother-infant pairs were randomized within 96 h of birth to one of 4 treatment groups (Aa, Ap, Pa, Pp), where ‘A’ was maternal vitamin A supplementation (400,000 IU), ‘P’ was maternal placebo, ‘a’ was infant vitamin A supplementation (50,000 IU) and ‘p’ was infant placebo. Full details of the trial have been published elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086928#pone.0086928-Humphrey2" target="_blank">[18]</a>.</p>‡<p>Maternal weight and BMI were measured 6 weeks postpartum.</p><p>WAZ: weight-for-age Z-score; HAZ: height-for-age Z-score; WHZ: weight-for-height Z-score; SD: standard deviation; IQR: interquartile range.</p

Biomarker levels in mothers of cases and controls.

‡<p>Biomarkers were measured on cryopreserved plasma from blood samples collected from mothers within 96 hours of delivery.</p><p>* Mothers of cases (infants who were stunted at 18 months of age) and mothers of controls (infants who were not stunted at 18 months of age).</p>†<p>Data shown are mean and standard deviation (SD) apart from IL-6, which was not normally distributed even after log transformation; data for IL-6 are therefore medians with interquartile range. For normally distributed biomarkers, unadjusted differences between means were compared using the two-sample t-test. For IL-6, unadjusted differences between medians were compared using the cendif command in Stata <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086928#pone.0086928-Newson1" target="_blank">[21]</a>.</p>#<p>Differences are biomarker values in mothers of cases minus values in mothers of controls.</p><p>** For normally distributed biomarkers, adjusted differences between means were calculated using an ordinary least-squares regression model. For IL-6, adjusted differences between medians were calculated using a median regression model. The covariates used to calculate adjusted differences in maternal biomarkers were maternal height, weight, mid-upper arm circumference and education.</p><p>IGF-1: Insulin-like growth factor-1; IGFBP3: Insulin-like growth factor binding protein 3; CRP: C-reactive protein; AGP: Alpha-1 acid glycoprotein.</p