Introduction. The Journal of Nutrition Community and International Nutrition

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1 The Journal of Nutrition Community and International Nutrition Lipid-Based Nutrient Supplements Plus Malaria and Diarrhea Treatment Increase Infant Development Scores in a Cluster-Randomized Trial in Burkina Faso 1 3 Elizabeth L Prado, 4 * Souheila Abbeddou, 4 Elizabeth Yakes Jimenez, 6,7 Jérôme W Somé, 4,8 Zinewendé POuédraogo, 8 Steve A Vosti, 5 Kathryn G Dewey, 4 Kenneth H Brown, 4,9 Sonja Y Hess, 4 and Jean-Bosco Ouédraogo 8 Departments of 4 Nutrition and 5 Agricultural and Resource Economics, University of California Davis, Davis, CA; Departments of 6 Individual, Family, and Community Education and 7 Family and Community Medicine, University of New Mexico, Albuquerque, NM; 8 Institut de Recherche en Sciences de la Santé/Direction Régionale de l Ouest (Health Sciences Research Institute/Western Regional Directorate), Bobo-Dioulasso, Burkina Faso; and 9 Bill & Melinda Gates Foundation, Seattle, WA Abstract Background: Adequate nutrition is necessary for the rapid brain development that occurs during infancy. Objectives: We tested the hypothesis that the provision of small-quantity, lipid-based nutrient supplements (SQ-LNSs) plus malaria and diarrhea treatment positively affects infant development. We also tested the effect of various doses of zinc provided in SQ-LNSs or in a tablet. Methods: In a partially masked, cluster-randomized controlled trial, communities in rural Burkina Faso were stratified by selected characteristics and then randomly assigned within strata to the intervention (IC; 25 communities, 2435 children) or the nonintervention (NIC; 9 communities, 785 children) cohorts. IC children were randomly assigned to 4 groups. As secondary outcomes, a subsample of 3 of these 4 groups (n = 747) and of the NIC (n = 376) were assessed for motor, language, and personal-social development at age 18 mo by using the Developmental Milestones Checklist II. The 3 IC groups received 20 g SQ-LNSs/d containing 0 or 10 mg added zinc with a placebo tablet or 20 g SQ-LNSs/d containing 0 mg added zinc with a tablet containing 5 mg Zn. All IC groups received treatment of malaria and diarrhea from age 9 to 18 mo. Data collectors and participants were aware of allocation to the IC or NIC but did not know the particular IC subgroup. Results: Children in the IC scored 0.34 (95% CI: 0.21, 0.46), 0.30 (95% CI: 0.15, 0.44), and 0.32 (95% CI: 0.16, 0.48) SDs higher in motor, language, and personal-social development, respectively, than did children in the NIC (All P < 0.001). Children who received different amounts of zinc did not differ significantly in any of the scores. No effect on caregiver-child interaction was found. Conclusion: In rural Burkina Faso, the provision of SQ-LNSs to infants from age 9 to 18 mo, regardless of added zinc content, plus malaria and diarrhea treatment positively affected motor, language, and personal-social development at age 18 mo. This trial was registered at clinicaltrials.gov as NCT J Nutr 2016;146: Keywords: infant nutrition, infant development, diarrhea, malaria, language development, motor development, personal-social development, lipid-based nutrient supplements, zinc Introduction Brain development occurs rapidly during early life through a dynamic process of interaction between biology and experience. The plasticity of the developing brain makes it both vulnerable to insult from negative influences and amenable to recovery through positive influences during this period. Millions of children in low- and middle-income countries (LMICs) 10 faceahostofnegative influences, such as poor nutrition, a high burden of infection, and lack of stimulation from the environment (1). Interventions in early life to protect children from these influences have high potential to positively affect development and lifelong outcomes (2). Further research is needed to determine whether interventions to protect children from negative influences can, in fact, change their developmental trajectories. The provision of smallquantity, lipid-based nutrient supplements (SQ-LNSs) is a new strategy to reduce infant undernutrition. SQ-LNSs (20 g or ; kcal/d) were designed as a type of home fortification of complementary foods to provide key nutrients from age 6 to 24 mo, as children transition from exclusive breastfeeding to sharing the meals of the household. Four randomized controlled trials investigated the developmental effects of provision of small- or medium-quantity ã 2016 American Society for Nutrition. 814 Manuscript received October 9, Initial review completed December 11, Revision accepted February 10, First published online March 9, 2016; doi: /jn

2 lipid-based nutrient supplements (LNSs) during infancy, starting at age 6 11 mo and ending at age mo. Three of these studies did not find any effect of doses ranging from 20 to 54 g (; kcal)/d on the age of attainment of developmental milestones (3, 4) or on GriffithÕs Mental Development Scale scores (5). One trial in Ghana showed that a higher percentage of children who received 20 g SQ-LNSs/d from age 6 to 12 mo walked independently at age 12 mo compared with a nonsupplemented group (6). Additional data are needed to clarify the effects of SQ-LNSs on infant development and to determine the optimal amount of zinc to include in SQ-LNSs. Although the WHO recommends 5 mg Zn per daily dose of some homefortification products, such as micronutrient powders (7), the optimal amount of zinc to include in SQ-LNSs to support healthy brain development is not yet known. The objectives of the current study were to evaluate whether SQ-LNSs and malaria and diarrhea treatment from age 9 to 18 mo positively affect motor, language, and personal-social development scores among children in rural Burkina Faso and to test the effect of various doses of zinc included in SQ-LNSs. We also examined the effects of the intervention on caregiver-child interactions, which may mediate any effects of the intervention on developmental scores. Caregivers may treat children who are small for their age as younger than they actually are and thus may not provide age-appropriate stimulation to an undernourished child. A reduced prevalence of diarrhea, malaria, and other infections may also mediate any effects of the intervention on development, although few studies, to our knowledge, have reported the association between illness prevalence and developmental outcomes in children under age 5 y (8). To add to this evidence base, we also report the associations of diarrhea prevalence, fever prevalence, and malaria incidence from age 9 to 18 mo with 18-mo motor, language, and personal-social scores. Methods Study participants and design This study was a partially masked, cluster-randomized controlled trial conducted from April 2010 to July 2012 in the Dandé Health District in rural Burkina Faso, in communities of ,000 inhabitants with minimal access to formal education and health services. Young children in the area are affected by holoendemic malaria transmission and a high 1 This publication is based on research funded by a grant to the University of California, Davis, from the Bill & Melinda Gates Foundation. 2 Author disclosures: EL Prado, S Abbeddou, E Yakes Jimenez, JW Somé, ZP Ouédraogo, SA Vosti, KG Dewey, SY Hess, and J-B Ouédraogo, no conflicts of interest. KH Brown has worked as a consultant and later as an employee for the Bill & Melinda Gates Foundation. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation. 3 Supplemental Methods, Supplemental Results, Supplemental Tables 1 and 2, and Supplemental Figure 1 are available from the Online Supporting Material link in the online posting of the article and from the same link in the online table of contents at *To whom correspondence should be addressed. elprado@ucdavis.edu. 10 Abbreviations used: DMC, Developmental Milestones Checklist; DS, developmental subsample; FCI, Family Care Indicators; HFIAS, household food insecurity access scale; HQ, housing quality; IC, intervention cohort; LAZ, length-for-age z score; LMIC, low- and middle-income country; LNS, lipid-based nutrient supplement; LNS-TabZn5, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus 5-mg Zn tablet; LNS-Zn0, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus placebo tablet; LNS-Zn5, 20 g lipid-based nutrient supplements/d containing 5 mg added zinc plus placebo tablet; LNS-Zn10, 20 g lipid-based nutrient supplements/d containing 10 mg added zinc plus placebo tablet; NIC, nonintervention cohort; RDT, rapid diagnostic test; SQ-LNS, small-quantity lipid-based nutrient supplement; WLZ, weight-for-length z score; ZPP, zinc protoporphyrin. prevalence of stunting and underweight (9). The study reported here was embedded in a larger trial, the objective of which was to compare zincrelated functional responses, such as growth and morbidity, among children who received varying doses of zinc in SQ-LNSs or as a waterdispersible tablet. The methods of the larger trial and primary outcomes are reported by Hess et al. (10). Here, we report the secondary developmental outcomes in a subset of children who participated in the larger trial. For the subset reported here, the trial included 2 levels of randomization: the community and the child. First, 34 communities were stratified by selected indicators (population size, proximity to road and the city of Bobo-Dioulasso, and health clinic affiliation) and then were assigned by computer-generated random assignment within strata to participate in the intervention cohort (IC; 25 communities) or in the nonintervention cohort (NIC; 9 communities). The IC and NIC clusters were balanced with regard to the stratification factors. Second, 2435 eligible children in the IC communities were assigned by a random allocation sequence to 1 of the following supplement groups: 1) 20 g SQ- LNSs/d containing 0 mg added zinc and a placebo tablet (LNS-Zn0), 2) 20 g SQ-LNSs/d containing 5 mg added zinc and a placebo tablet (LNS- Zn5), 3) 20 g SQ-LNSs/d containing 10 mg added zinc and a placebo tablet (LNS-Zn10), or 4) 20 g SQ-LNSs/d containing 0 mg added zinc and a tablet containing 5 mg Zn (LNS-TabZn5). In the larger trial, this second level of randomization was by household cluster. However, only 1 child/household was included in the developmental subsample; therefore, in this study, this second level of randomization was by individual child. Children were considered eligible if they were 8.8 to 9.9 mo of age, resided permanently in the Dandé Health District, planned to be available during the study period, and if written parental consent was obtained. Children were excluded on the basis of the following criteria: hemoglobin <50 g/l (11), weight-for-length <70% of the median of the National Center for Health Statistics/WHO growth reference (12), presence of bipedal edema, severe illness warranting hospital referral, congenital abnormalities potentially interfering with growth, chronic medical condition (e.g., malignancy) requiring frequent medical attention, known HIV infection of the infant or mother, history of allergy to peanuts, history of anaphylaxis or serious allergic reaction to any substance, requiring emergency medical care, and concurrent participation in any other clinical trial. Once enrolled, children were excluded if absent from the study for >3 wk. The SQ-LNS products were developed and produced by Nutriset SAS. Twenty grams of SQ-LNSs contained 118 kcal, 2.6 g protein, 9.6 g fat, 4.46 g linoleic acid (18:2n26), 0.58 g a-linoleic acid (18:3n23), 400 mg vitamin A (retinyl acetate), 0.3 mg thiamine, 0.4 mg riboflavin, 4 mg niacin, 1.8 mg pantothenic acid, 0.3 mg vitamin B-6, 0.5 mg vitamin B-12, 80 mg folic acid (pteroyl monoglutamic acid), 30 mg vitamin C (L-ascorbic acid), 5 mg vitamin D (cholecalciferol), 6 mg vitamin E (d,la-tocopherol acetate), 30 mg vitamin K (phylloquinone 5%), 280 mg calcium (tricalcium phosphate), 0.34 mg copper, 90 mg iodine, 6 mg iron, 40 mg magnesium, 1.2 mg manganese, 190 mg phosphorus, 200 mg potassium, and 20 mg selenium. Data collectors and participants were aware of the allocation of IC or NIC but were blind to the IC groups. The LNS-Zn0 product contained 0.29 mg Zn/20-g sachet from the raw ingredients. The LNS-Zn5 and LNS-Zn10 products contained an additional 5 mg and 10 mg Zn, respectively, in the form of zinc sulfate with a margin of 610%. Because zinc absorption may be inhibited when zinc is given with SQ-LNSs and/or mixed with food, participants were instructed to feed the tablet to the child at least half an hour before or after any meal or snack, including SQ-LNSs. Thus, the LNS-TabZn5 group was considered a positive control for possible effects of zinc on study outcomes. At the time of enrollment, maternal and child anthropometric measurements and family socioeconomic characteristics were obtained for all children. Further details are provided in Supplemental Methods and in Hess et al. (10). At enrollment, project staff provided all children free medical treatment in accordance with national policy: children with diarrhea received oral rehydration salts, children with fever received paracetamol, and children with a positive rapid diagnostic test (RDT) received antimalarial treatment, which consisted of artemisinin-based combination therapy and paracetamol. Children with hemoglobin <80 g/l Lipid-based nutrient supplements and development 815

3 received anthelmintic treatment and iron supplements (2 6 mg iron/kg body weight for 30 d). Children with any other illness requiring medical follow-up were referred to the health center. Children in the IC were visited weekly during the 9-mo intervention period for the delivery of intervention products and for the collection of morbidity and adherence data, by maternal report and by collecting empty supplement packages (13). The fieldworkers also evaluated the child for diarrhea, fever, and malaria. In case of reported fever, the fieldworker measured the childõs body temperature and performed an RDT. Body temperature was also measured routinely once a month. In case of uncomplicated diarrhea, fever, and RDT-confirmed malaria, the fieldworker provided the same treatment as described above. Diarrhea, fever, and malaria with complications and any other cases of severe illness were referred to the health center for evaluation and treatment. In the NIC, 785 eligible children were enrolled in the trial. At enrollment, project staff evaluated and treated their health conditions in the same way as for children in the IC. They were subsequently visited after a period of 9 mo, during which they did not receive any supplements, visits, morbidity monitoring, or treatment from project personnel. These children received 20 g SQ-LNSs/d containing 10 mg Zn during the subsequent 9 mo (age mo). At 18 mo of age, participants were asked to visit their local community health clinic, at which time project staff conducted the developmental assessments. Ethical approval for the study procedures was obtained from the Institutional Review Board of the University of California, Davis, and the Comité dõethique Institutionnel du Centre Muraz, Bobo Dioulasso. The study was registered with the US NIH as a clinical trial ( NCT ). Participants in 18-mo developmental assessment Figure 1 shows the trial profile. A subsample of 446 children from the NIC and 980 children from 3 of the 4 IC groups (LNS-Zn0, LNS-Zn10, and LNS-TabZn5) were randomly selected for motor, language, and personal-social assessment at 18 mo of age. This selection was accomplished through a specialized SAS program (SAS Institute), which randomly assigned children to be assessed or not assessed within treatment group and time block. Children from 3 of 4 IC groups were chosen because constraints on personnel resources precluded the assessment of all children. Thus, we targeted the minimum dose of zinc (LNS-Zn0), the maximum dose of zinc (LNS-Zn10), the positive control group (LNS-TabZn5), and the NIC. The middle dose of zinc (LNS-Zn5) was not included in the developmental assessment subsample (DS). The data collectors were unaware which groups had been selected for developmental assessment, thus maintaining blinding. With this design, a sample size of 244 children/group provided 80% power to detect an effect size of 0.3 SDs in a 2-sided test with an a of We increased the target by one-third in the NIC to account for cluster effects of IC compared with NIC, assigned by community, bringing the sample size to ;325, and by an additional one-third in all groups to account for potential attrition, bringing the sample to ;445 in the NIC and ;325 in each IC group. Developmental assessment measures We assessed motor, language, and personal-social development with the use of the Developmental Milestones Checklist (DMC) II (14). This tool evaluates motor, language, and personal-social development through both interviewing the caregiver and observing the child. We extended the first version of the DMC (15) for this study, creating the DMC-II. The internal reliability (CronbachÕs a) and interinterviewer and test-retest reliability (PearsonÕs r) of the DMC-II scores were >0.7. For a detailed description of the adaptation of the checklist and its psychometric properties, see Prado et al. (14). The score for each DMC-II subscale was calculated as the sum of the item scores in that subscale. Caregiver-child interaction We assessed caregiver-child interactions with the use of the Family Care Indicators (FCI) interview (16), which was developed by UNICEF and validated in Bangladesh (17). For each of 6 activities, mothers reported whether they, the childõs father, or any other adult had engaged in that activity with the child in the past 3 d (e.g., told stories, sang songs). 816 Prado et al. Before using this tool, we confirmed that these activities were appropriate for the study context by interviewing 14 community mothers regarding the activities they did with their children. We calculated the caregiver-child interaction score as the sum of these 18 item scores (6 activities for each of the 3 categories of potential caregivers). FCI score We also evaluated overall developmental stimulation in the home by using the FCI items, including the items indicating whether any caregiver had engaged the child in the 6 activities described above and 12 additional items regarding toys and books in the home. We also confirmed that these items were appropriate through the same interviews with community mothers, described above. We calculated the overall FCI score as the sum of these 18 items. Although the caregiver-child interaction score was examined as a trial outcome, the overall FCI score was used as a covariate and effect modifier in analyses on developmental scores. Malaria, fever, and diarrhea Diarrhea was defined as the presence of $3 loose or liquid stools/24 h. Fever was defined as any reported fever by the caregiver or any elevated measured auricular temperature (>37.5 C). Diarrhea and fever prevalence was calculated as the percentage of total days of observation during which the child experienced the condition. An episode of fever was defined as the period starting the day that the child first had fever after a fever-free period of 2 d and ending on the last day that the child had fever that was followed by at least 2 d without fever. Any episode of reported or confirmed fever associated with a positive RDT was defined as malaria. A child was considered at risk of malaria if he or she did not receive any antimalarial treatment within the previous 21 d. Malaria incidence was defined as the number of new episodes of malaria per 100 child-days at risk. Child malaria incidence, child fever prevalence, and child diarrhea prevalence were square-root transformed to eliminate skewness. This information was available for IC children only. Statistical analyses A statistical analysis plan for the analyses presented here was posted to the project website ( before the code was broken to reveal to the investigators which children had been assigned to each IC supplement group. This plan included prespecified covariates and effect modifiers, as described below. All analyses were conducted by using SAS version 9.3. Analysis was by intention to treat. Group characteristic comparisons. We examined whether children in the 4 trial groups were matched on selected baseline characteristics. For the continuous variables, P values were estimated by using linear mixed models with a random effect of community on the intercept to account for cluster randomization by community. For the categorical variables, P values were estimated by using generalized linear mixed models for the binary variables and Wald chi-square test for the multinomial variables, also with community as a random effect. To examine potential bias in the selection of the subsample, we compared the DS (n = 1122) with the children who were enrolled in the trial but not included in the subsample (non-ds; n = 2098) on the same baseline characteristics with the same types of models. Calculation of developmental scores and categorical outcomes. We computed z scores on the basis of the distribution of our sample by standardizing to a mean of zero and an SD of 1. For each DMC-II score, we examined the lowest decile (10%) and the lowest quartile (25%) of the total sample as a proxy for children likely to be experiencing a severe (lowest 10%) and moderate-to severe (lowest 25%) developmental delay. Effect of intervention. We estimated the differences between the 4 trial groups with the use of linear mixed models for continuous outcomes and generalized linear mixed models for categorical outcomes, with a random effect of community and fixed effects of cohort (IC

4 FIGURE 1 Trial profile. *This group was not included in the developmental sample. LNS, lipid-based nutrient supplement; NCHS, National Center for Health Statistics; NIC, nonintervention cohort. compared with NIC) and IC group (LNS-Zn0, LNS-Zn10, and LNS- TabZn5) nested within cohort. This model provided estimates of the difference between the 3 IC groups as well as the difference between the IC and the NIC. If the effect of IC group was significant at P<0.05, we used Tukey-KramerÕs test for post hoc pairwise comparisons between groups. We estimated each model by first adjusting for the childõs age at developmental assessment only and then by adjusting for child age plus any of the following variables that were independently associated with each outcome score at P< 0.1: all baseline variables in Table 1; baseline child capillary zinc protoporphyrin (ZPP) adjusted for malaria, maternal height, number of children under age 5 y in the household; housing quality (HQ) index; household food insecurity access scale (HFIAS) score; FCI score; developmental assessment data collector; and 4 baseline dietary indicators (dietary diversity, meal frequency, breastfeeding, and animal source food; for a description of these variables, see Supplemental Methods). The DMC-II scores were negatively skewed, due to a small number of children with very low scores. Thus, we also performed a sensitivity analysis excluding outliers >3 SDs from each score mean to determine whether these outliers had undue influence on the estimates of the differences between groups. Effect modifiers. We examined the following 11 effect modifiers: child sex; baseline child length-for-age z score (LAZ), weight-for-length z score (WLZ), and hemoglobin concentration; baseline maternal height, BMI, education, and marital status; HQ index; HFIAS score adjusted for season and year; and FCI score. In each model, we added the interaction between the effect modifier and cohort (IC compared with NIC) as well as the interaction between the effect modifier and IC group (LNS-Zn0, LNS-Zn10, and LNS-TabZn5) nested within cohort. All models included a random effect of community and fixed effect of child age at developmental assessment. If any interaction was significant at the P < 0.1 level, we further explored the nature of the interaction. For the continuous effect modifiers, we estimated the effect (e.g., IC compared with NIC) at the 10th and 90th percentiles of the continuous variable (e.g., baseline child LAZ). For the categorical effect modifiers, we examined the effect (e.g., IC compared with NIC) in each category (e.g., each quartile of HFIAS score). Results Group characteristic comparisons. Table 1 presents the characteristics of the trial groups at the individual level. The characteristics of the IC compared with the NIC at the cluster level are presented in Supplemental Table 1. Of the variables presented in Table 1, children in the 4 trial groups differed significantly only in baseline WLZ. Children in the LNS-TabZn5 group had higher mean WLZ scores at baseline than the children in the LNS-Zn10 group and the NIC. Children in the DS had a significantly lower WLZ at baseline than did children not included in the DS. They also differed significantly in maternal marital status. IC children in the DS (n = 746) differed significantly from IC children who were not included in the DS (n = 1689) in that they had lower diarrhea prevalence during the study period (Table 1). Lipid-based nutrient supplements and development 817

5 TABLE 1 Characteristics of the DS by trial group and of children who were enrolled in the trial but not included in the DS (non-ds) 1 DS Difference 4 DS groups between combined 4 DS groups, P (n = 1122) Difference LNS-Zn0 (n = 255) LNS-Zn10 (n = 248) LNS-TabZn5 (n = 243) NIC (n = 376) Non-DS group (n = 2098) between DS and non-ds, P Baseline child characteristics Male sex, n (%) 124 (49) 128 (52) 120 (49) 194 (52) (50) 1055 (50) 0.93 LAZ WLZ a,b b a b, Malaria-positive RDT, n (%) 157 (62) 156 (63) 138 (57) 254 (68) (63) 1273 (61) 0.40 Hemoglobin, g/dl Baseline maternal characteristics Age, y BMI (kg/m 2 ), n (%) , (15) 34 (14) 35 (14) 58 (15) 164 (15) 290 (14) (80) 192 (78) 186 (77) 302 (80) 884 (79) 1691 (81) (5) 20 (8) 22 (9) 16 (4) 72 (6) 116 (6) Maternal education, n (%) No formal or informal education 144 (56) 125 (50) 141 (58) 257 (68) 667 (59) 1253 (61) Any informal education and/or 84 (33) 89 (36) 74 (30) 81 (22) 328 (29) 599 (29),1 y of formal education At least 1 y of formal education 27 (11) 34 (14) 28 (12) 38 (10) 127 (11) 216 (10) Maternal marital status, n (%) Sole wife 152 (60) 142 (57) 149 (61) 204 (54) 647 (58) 1067 (52) First wife in a polygamous household 32 (13) 45 (18) 29 (12) 60 (16) 166 (15) 352 (17) Second wife or higher in a 65 (25) 57 (23) 63 (26) 101 (27) 286 (26) 592 (29) polygamous household Single, divorced, or widow 6 (2) 3 (1) 2 (1) 11 (3) 22 (2) 54 (3) Child morbidity during the study period Malaria incidence, episodes/ child-days at risk Fever prevalence, % of days in the study Diarrhea prevalence, % of days in the study Data collected at 18-mo developmental assessment Child age at developmental assessment, mo FCI z score Values are means 6 SDs unless otherwise indicated. For continuous variables, P values were estimated by using linear mixed models with a random effect of community on intercept to account for cluster-randomization by community. For categorical variables, P values were estimated by using generalized linear mixed models for the binary variables and Wald chi-square test for the multinomial variables, also with community as a random effect. Labeled means in a row without acommon superscript letter differ, P, DS, developmental subsample; FCI, Family Care Indicators; LAZ, length-for-age z score; LNS-TabZn5, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus 5-mg Zn tablet; LNS-Zn0, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus placebo tablet;lns- Zn10, 20 g lipid-based nutrient supplements/d containing 10 mg added zinc plus placebo tablet; NIC, nonintervention cohort; RDT, rapid diagnostic test; WLZ, weight-forlength z score. Effect of intervention on motor, language, and personalsocial scores. Table 2 shows the mean motor, language, and personal-social development z scores in the 3 IC groups separately, the 3 IC groups together, and the NIC at age 18 mo. Adjusting for cluster randomization and child age at developmental assessment, children in the IC scored significantly higher than those in the NIC in motor, language, and personalsocial development. Scores in the 3 IC groups were not significantly different from each other. Adjusting for covariates that were independently associated with each score at P<0.1 (see Table 2 footnotes) resulted in similar estimates of the difference between the IC and NIC and no significant differences between the 3 IC groups. Excluding outliers >3 SDs from each score mean did not change the pattern of effects (for details, see Supplemental Results). Effect of intervention on categorical outcomes. Table 3 presents the effect of the intervention on the risk of being in the lowest decile of scores. The effect on risk of being in the lowest quartile is shown in Supplemental Table 2. Similar to the findings for the mean scores, no significant differences were found between the 3 IC groups; however, large differences were found between the IC and NIC. Children in the NIC were significantly more likely to be in the lowest quartile and decile of motor, language, and personal-social scores. These results were consistent when controlling for covariates that were independently associated with each score. Modifiers of the effect of intervention. Four of 11 factors modified the effect of the intervention (IC compared with NIC) on motor scores: baseline child LAZ (P=0.005), FCI score (P=0.080), 818 Prado et al.

6 TABLE 2 Motor, language, and personal-social development and caregiver-child interaction scores at age 18 mo by intervention group 1 Difference between IC and NIC groups IC group Difference between IC groups 3 IC groups Covariate-adjusted z Score LNS-Zn0 (n = 255) LNS-Zn10 (n = 248) LNS-TabZn5 (n = 243) P 2 Covariate-adjusted P combined (n = 746) NIC (n =376) Effect size 2 (95% CI) effect size (95% CI) Motor *** (0.22, 0.57) *** (0.21, 0.46) Language *** (0.21, 0.52) *** (0.15, 0.44) Personal-social *** (0.19, 0.55) *** (0.16, 0.48) Caregiver-child interaction (20.20, 0.05) (20.21, 0.05) 1 Values are unadjusted means 6 SDs unless otherwise indicated. P values were estimated by using linear mixed models. ***P, FCI, Family Care Indicators; HFIAS, household food insecurity access scale; HQ, housing quality; IC, intervention cohort; LAZ, length-for-age z score; LNS-TabZn5, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus 5-mg Zn tablet; LNS-Zn0, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus placebo tablet; LNS-Zn10, 20 g lipid-based nutrient supplements/d containing 10 mg added zinc plus placebo tablet; NIC, nonintervention cohort; WLZ, weight-for-length z score; ZPP, zinc protoporphyrin. 2 Adjusted for community to account for cluster randomization and for child age at developmental assessment. 3 Adjusted for community and child age; baseline child LAZ, WLZ, hemoglobin concentration, malaria (positive rapid diagnostic test), and log ZPP adjusted for malaria; baseline breastfeeding, dietary diversity, and animal source food; baseline maternal BMI; number of children under age 5 y in the household, HQ index, data collector, and FCI score. 4 Adjusted for community and child age; child sex; baseline child LAZ, WLZ, hemoglobin concentration, malaria (positive rapid diagnostic test), and log ZPP adjusted for malaria; baseline dietary diversity, meal frequency, and animal source food; baseline maternal education; HQ index; data collector; and FCI score. 5 Adjusted for community and child age; child sex; baseline child LAZ, WLZ, hemoglobin concentration, and malaria (positive rapid diagnostic test); baseline meal frequency and animal source food; number of children under age 5 y in the household, HFIAS, data collector, and FCI score. 6 Adjusted for community and child age, baseline child LAZ and hemoglobin concentration, dietary diversity, baseline maternal education, HFIAS, data collector, and HQ index. baseline maternal education (P=0.082), and HFIAS (P=0.098). Only the FCI score modified the effect of intervention on language scores (P =0.099). Only the HQ index modified the effect of intervention on personal-social scores (P =0.095). Larger positive effects of the intervention on motor scores were found in children with lower baseline LAZs. For children in the 10th percentile of baseline LAZ (22.5 SDs), the difference between the IC and NIC was 0.60 SDs (95% CI: 0.39, 0.81; P< 0.001), whereas for children in the 90th percentile (LAZ = 0.2 SDs), the difference was 0.19 (20.03, 0.40; P=0.08). Similarly, there was a trend for larger positive effects on motor scores in households with a lower FCI score (10th percentile FCI score = 6 B: 0.49; 95% CI: 0.28, 0.70; P<0.001; 90th percentile FCI score = 11 B: 0.23; 95% CI: 0.01, 0.44; P=0.038). There was also a trend for larger positive effects of the intervention on motor scores in children of mothers with no education (B: 0.48; 95% CI: 0.28, 0.68; P<0.001) than in those with nonformal education (B: 0.15; 95% CI: 20.13, 0.42; P=0.91) or formal education (B: 0.43; 95% CI: 0.03, 0.82; P = 0.28), and in children in the second quartile of HFIAS score (B: 0.76; 95% CI: 0.42, 1.10; P<0.001) compared with the other 3 quartiles (all P > 0.1). For language scores, a trend toward greater effects was also found in households with a low FCI score. For children in the 10th percentile of baseline FCI, the difference between the IC and NIC was 0.45 SDs (95% CI: 0.24, 0.66 SDs; P<0.001), whereas for children in the 90th percentile, the difference was 0.21 SDs (20.01, 0.43 SDs; P = 0.07). For personal-social scores, a greater effect of the intervention was found in children in the second HQ quartile (B: 0.52; 95% CI: 0.27, 0.77; P= 0.001) than in the other 3 quartiles (all P > 0.09). Supplemental Figure 1 shows the mean motor and language z scores in each tertile of baseline LAZ and in each tertile of FCI. For details, see Supplemental Results. Effect of intervention on caregiver-child interaction. Although the caregiver-child interaction score was strongly associated with the developmental scores (all P < ), it did not differ between the IC and NIC or between the 3 IC groups (Table 2), showing that this did not mediate the effect of the intervention on developmental scores. These results were consistent when controlling for covariates and when excluding outliers. Association between child illness and developmental scores. The association between child malaria incidence from age 9 to 18 mo and 18-mo scores, adjusting only for community and child age, was significant for motor scores (B: 20.18; 95% CI: 20.33, 20.03; P=0.02), approached significance for language scores (B: 20.15; 95% CI: 20.32, 0.01; P=0.07), and was nonsignificant for personal-social scores (B: 20.06; 95% CI: 20.22, 0.10; P=0.49). However, none of these associations was significant when controlling for additional covariates that were independently associated with the developmental scores (motor B: 20.11; 95% CI: 20.24, 0.03; P=0.14; language B: 20.14; 95% CI: 20.30, 0.01; P=0.07; personal-social B: 20.03; 95% CI: 20.18, 0.13; P = 0.74). These additional covariates are listed in the footnotes to Table 2. Child fever prevalence was not associated with motor or language scores, with or without controlling for covariates (all P > 0.16). Fever prevalence showed a marginally significant positive association with personal-social scores (B: 0.09; 95% CI: 0.00, 0.19; P= 0.05), which was not significant when controlling for covariates (B: 0.07; 95% CI: 20.02, 0.15; P = 0.13). Child diarrhea Lipid-based nutrient supplements and development 819

7 TABLE 3 Percentage of children in the lowest decile of developmental scores by intervention group 1 Difference between IC and NIC groups IC group, n (%) Difference between IC groups Covariate-adjusted OR (95% CI) LNS-TabZn5 3 IC groups combined (n = 243) P 2 Covariate-adjusted P (n = 746), n (%) NIC (n = 376), n (%) OR 2 (95% CI) LNS-Zn10 (n = 248) LNS-Zn0 (n = 255) Children in the lowest decile of motor scores 16 (6) 22 (9) 16 (7) (7) 57 (15) 2.35*** (1.57, 3.50) *** (1.36, 3.23) Children in the lowest decile of language scores 18 (7) 19 (8) 11 (5) (6) 44 (12) 2.03** (1.31, 3.15) * (1.13, 3.64) Children in the lowest decile of personal-social scores 14 (5) 12 (5) 16 (7) (6) 53 (14) 2.77*** (1.66, 4.61) *** (1.63, 6.18) 1 Values are unadjusted n (%) unless otherwise indicated. P values and ORs were estimated by using generalized linear mixed models. *P, 0.05, **P, 0.01, ***P, FCI, Family Care Indicators; HQ, housing quality; IC, intervention cohort; LAZ, length-for-age z score; LNS-TabZn5, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus 5-mg Zn tablet; LNS-Zn0, 20 g lipid-based nutrient supplements/d containing 0 mg added zinc plus placebo tablet; LNS-Zn10, 20 g lipid-based nutrient supplements/d containing 10 mg added zinc plus placebo tablet; NIC, nonintervention cohort; WLZ, weight-for-length z score; ZPP, zinc protoporphyrin. 2 Adjusted for community to account for cluster randomization and for child age at developmental assessment. 3 Adjusted for community and child age, baseline child LAZ and WLZ, data collector, and FCI score. The model with the full set of covariates did not converge; therefore, covariates were removed beginning with the weakest predictor of motor z score, until the model converged. 4 Adjusted for community and child age; child sex; baseline child LAZ, WLZ, hemoglobin concentration, malaria (positive rapid diagnostic test), and log ZPP adjusted for malaria; baseline dietary diversity, meal frequency, and animal source food; baseline maternal education; HQ index; data collector; and FCI score. 5 Adjusted for community and child age; child sex; baseline child LAZ, WLZ, hemoglobin concentration, and malaria (positive rapid diagnostic test); baseline meal frequency and animal source food; number of children under age 5 y in the household; household food insecurity access; data collector; and FCI score. prevalence was not associated with motor, language, or personal-social scores, with or without controlling for covariates (all P > 0.33). Discussion In this cluster-randomized controlled trial, we found significant positive effects of the provision of 20 g SQ-LNSs/d, along with monitoring and treatment of malaria and diarrhea, from age 9 to 18 mo on motor, language, and personal-social development scores. The intervention also significantly reduced the proportion of children in the lowest decile and quartile of all 3 developmental scores at age 18 mo, which was a proxy for children likely to be experiencing severe and moderate-to-severe developmental delay. Additional zinc provided in SQ-LNSs (10 mg compared with 0 mg/20-g sachet) or provided separately in a daily tablet (5 mg) did not significantly affect developmental scores. The intervention did not affect caregiver-child interactions, suggesting that this did not mediate the effect on developmental scores. The intervention increased developmental scores by approximately 0.3 SDs, which is equivalent to an advance of ;1 1.5 mo of age in this sample. We derived this estimate from a previously reported cross-sectional analysis in 214 children who participated in this trial and who were evaluated with the DMC- II at age mo (14). In this analysis, motor scores increased by ;1.8 points with each month of age, which is equivalent to 0.3 SDs on the basis of the distribution of the 18-mo sample reported here. Language scores increased by ;0.9 points (0.3 SDs) with each month of age, and personal-social scores increased by ;1 point (0.2 SDs) with each month of age. Given that the duration of the intervention was 9 mo, an advance of mo of age is a substantial effect. The magnitude of the effect of this intervention was comparable to or larger than the magnitude of effects that have been found in other early childhood interventions. Nutrition interventions in children under age 2 y in LMICs have generally found smaller effects on child development, with a recent metaanalysis of 18 studies reporting a pooled effect size of 0.09 SDs (18). The pooled studies were designed to test the effect of iron, zinc, gangliosides, multiple micronutrients, or protein-energy supplements. The effect that we found was closer to the effect of interventions that enhanced psychosocial stimulation in children under age 2 y in LMICs, which was 0.42 SDs in a meta-analysis of 21 studies (18). Preschool programs for children ages 3 5 y have also shown effects of smaller or similar magnitude to this intervention, with a meta-analysis of studies in the United States reporting a pooled effect of 0.23 SDs on cognitive development (19) and a meta-analysis of studies in other countries reporting a pooled effect of 0.31 SDs (20). The finding that IC children were mo ahead of NIC children in motor, language, and personal-social development is consistent with 1 previous trial in Ghana, which found a positive effect of SQ-LNSs on walking at age 12 mo (6), but is inconsistent with 3 previous trials in Malawi and Haiti that did not find effects of LNSs on milestone achievement (3, 4) or on 18-mo GriffithÕs Mental Development Scale scores (5). One possible explanation is that the increase in scores was due to the provision of malaria and diarrhea treatment for IC but not NIC children from age 9 to 18 mo or to the combination of treatment with SQ-LNSs. However, the incidence of malaria was relatively low (mean of 0.56 episodes/100 child-days at risk in the developmental sample) and likewise the prevalence of diarrhea was 820 Prado et al.

8 relatively low (mean of 2.9% of days in the study in the DS); therefore, it is questionable whether such infrequent treatment would result in such large effects. In addition, in the IC groups, diarrhea prevalence, fever prevalence, and malaria incidence were not strongly related to any developmental scores. However, it cannot be ruled out that weekly surveillance and early detection and treatment of malaria and diarrhea accounted for at least part of the observed effects. It is also possible that weekly visits from fieldworkers increased the familyõs focus on the child and changed the care that IC children received. However, we did not detect any difference in the care that IC compared with NIC children received on the basis of the FCI score or the caregiver-child interaction score. An increased focus on the child might also increase the motherõs attentiveness to the child and the accuracy of her report of her childõs development. However, when we examined the motor z score based on 10 DMC-II items that were observed by the data collector, we found a significant difference between the IC and NIC, just as on the parent-report score, suggesting that such a bias in the maternal report cannot fully account for the results. Another possible explanation for the variability in results across studies is that SQ-LNSs are effective for promoting development in certain populations and contexts but not others, depending on factors such as the initial nutrient deficiency and developmental status of the children. Indeed, even within this study population, we found evidence that the magnitude of the effect of the intervention seemed to be greater in certain subgroups. The effect on motor development was greater in children with lower initial nutritional status (P =0.005), as indicated by lower LAZs. This is consistent with studies in Chile (21), Bangladesh (22), and Indonesia (23), which found larger effects of nutrition interventions on child development in undernourished participants. There was also a trend for greater effects of the intervention on motor (P =0.080) and language (P=0.099) development in children from households with lower developmental stimulation. These findings are consistent with both the vulnerability and plasticity of brain development during infancy. Although children in households with little developmental stimulation were behind their peers in motor and language development, they also showed a response to the intervention that was larger than children in high-stimulation households (see Supplemental Figure 1). With regard to the effect of 10 mg Zn included in SQ-LNSs or 5 mg Zn provided as a dispersible zinc tablet between meals, no significant effects were found. The lack of effects from added zinc was not due to an absence of zinc deficiency in this population, because 35% of the children had low plasma zinc concentrations at baseline (10). This had increased to 54% by age 18 mo, with no difference between the IC and NIC. No differences were found between any of the intervention groups in mean plasma zinc concentration at age 18 mo, despite high reported adherence to LNS (97% 6 6%) and tablet (97% 6 7%) consumption, suggesting that zinc intake, absorption, or uptake may not have been as high as expected (10, 13). The lack of effects of zinc is consistent with previous studies of the impact of zinc on child development. Of 9 previous randomized infant zinc supplementation trials, only 1 showed a positive effect of zinc on mental development and this benefit was found only in children who also received psychosocial stimulation. Four of these 9 trials showed positive effects on at least 1 measure of motor development (24). Our study did not show any adverse effect of supplementary zinc, which was found in 1 previous trial (25). One limitation of the study is that trial personnel were aware of the allocation of community to the IC and NIC groups. We were able to avoid this problem with regard to the IC group by coding all SQ-LNS and tablet packets, such that no personnel were aware of group allocation; however, it was not possible to hide which communities received weekly visits for morbidity surveillance, which distinguished IC groups from the NIC group. Therefore, we cannot rule out that this knowledge may have led to a systematic bias in the way the developmental interviews and observations were conducted or interpreted by the interviewers. However, the interviewers received regular training and were closely supervised to minimize this bias. Future studies can avoid this potential bias by providing morbidity surveillance and treatment to the control group. Another potential limitation with regard to external validity is that there were significant differences between DS and non-ds children with regard to a number of baseline variables. A sample size of 3220, which was determined by a power analysis to detect differences between the 5 trial groups, provides statistical power to detect very small differences between 2 groups (DS and non-ds). Although we found significant differences between DS and non-ds groups in baseline WLZ, maternal marital status, and diarrhea prevalence, the absolute differences were small (0.09 SDs, 2 6 percentage points). Thus, they are likely of little practical significance, and it is unlikely that the developmental sample is not representative of the full sample. In addition, in adjusted analyses, we controlled for any variables that were associated with the developmental scores. Since 2013, the government of Burkina Faso has adopted a policy to scale up optimal infant and young child feeding practices, which includes promotion of optimal breastfeeding, adequate complementary feeding, hygienic conditions for feeding, and appropriate home stimulation for infants and young children. Our study supports the evidence base for this policy and suggests that SQ-LNSs are a promising product as part of a program package, at least for children age 9 18 mo. This is further supported by the positive effects of the intervention on linear growth and other outcomes (10) and the acceptability of SQ-LNSs. In a subset of 349 children in the study, 92% of mothers reported that the child consumed SQ-LNSs easily. One limitation is that weekly surveillance for malaria and diarrhea may not be sustainable on a large scale. However, as discussed earlier, it is unlikely that the developmental effects were entirely due to this aspect of the intervention. A full discussion of the findings and policy implications of the ilins (International Lipid-Based Nutrient Supplements) Project is available in the Summary of Key Findings document at the ilins Project website ( In conclusion, our findings suggest that, in a population such as this one in Burkina Faso, SQ-LNSs from age 9 to 18 mo together with the provision of treatment for malaria and diarrhea can improve motor, language, and personal-social development. The loss of developmental potential in early childhood is a critical problem in LMICs. The findings show that interventions such as this one, perhaps in particular for those who are vulnerable due to factors such as low LAZ and low household stimulation, can be effective to address this loss and to promote healthy brain development in children. Acknowledgments We thank Rosemonde Guissou who contributed to the coordination of the ilins-zinc project. Vincent Gnimassou, Déborah Siry, Mariâtre Rayaisse, and Elise Kéré collected the DMC-II and anthropometric data. Bernadin Sanou, Aminata Lipid-based nutrient supplements and development 821