Effect of zinc added to a daily small-quantity lipid-based nutrient supplement on diarrhoea, malaria, fever and respiratory infections in young children in rural Burkina Faso: A cluster-randomised trial

Objective: Preventive zinc supplementation in the form of tablets or syrup reduces the incidence of diarrhoea and acute lower respiratory tract infections (RTI), but its effect on malaria is inconsistent. When zinc is administered with other micronutrients or foods, its effect is also uncertain. We assessed the effects of different amounts and sources of zinc on the frequency of diarrhoea, malaria, fever and RTI in young children. Design, setting and populations: This communitybased, double-blind, placebo-controlled, clusterrandomised trial of 2435 children 9 months of age was carried out between April 2010 and July 2012 in rural southwestern Burkina Faso. Interventions: Participants were randomly assigned at the concession level to receive daily 1 of 4 interventions for 9 months: (1) 20 g small-quantity lipid-based nutrient supplement (SQ-LNS) without zinc and placebo tablet, (2) 20 g SQ-LNS with 5 mg zinc and placebo tablet, (3) 20 g SQ-LNS with 10 mg zinc and placebo tablet or (4) 20 g SQ-LNS without zinc and 5 mg zinc tablet. Participants were visited weekly in their homes for morbidity surveillance for 9 months, and those with uncomplicated diarrhoea and malaria received treatment from the study field workers in the community. Main outcomes: Incidence and longitudinal prevalence of diarrhoea, malaria, fever, and lower and upper RTI by intervention group. Results: The incidence of diarrhoea, malaria and fever was 1.10 (±1.03 SD), 0.61 (±0.66 SD) and 1.49 (±1.12 SD) episodes per 100 child-days at risk, respectively, and did not differ by intervention group (p=0.589, p=0.856 and p=0.830, respectively). The longitudinal prevalence of acute lower RTI (0.1%; 95% IC 0.10.2%) and of upper RTI (7.8%; 95% IC 7.1-8.4%) did not differ among groups (p=0.234 and p=0.501, respectively). Conclusions: Inclusion of 5 or 10 mg zinc in SQ-LNS and provision of 5 mg zinc dispersible tablet along with SQ-LNS had no impact on the incidence of diarrhoea, malaria and fever or the longitudinal prevalence of RTI compared with SQ-LNS without zinc in this population. The morbidity results reported in the current paper are from the iLiNS-Zinc study, a community-based, double-blind, placebo-controlled, cluster-randomised trial that took place between April 2010 and July 2012 in the Dandé health district in southwestern Burkina Faso. This region has a high prevalence of stunting (31.8%) and underweight (19.3%) among young children,20 poor food security and holoendemic malaria transmission.21 The study design has been described in detail by Hess et al.21a The study was registered with the US National Institutes of Health as a clinical trial (http://www.ClinicalTrials.gov; {“type”:”clinical-trial”,”attrs”:{“text”:”NCT00944281″,”term_id”:”NCT00944281″}}NCT00944281). A total of 34 villages were selected for inclusion in the study based on their accessibility during the rainy season. Target villages were stratified by health clinic affiliation, average population size, and distance from the paved road and Bobo-Dioulasso, and assigned to the intervention cohort or the non-intervention cohort. Because morbidity was not assessed in the non-intervention cohort, this paper will focus just on the intervention cohort, which consisted of 25 villages. A statistician from the University of California Davis, who was blinded to the intervention groups, generated a random allocation sequence at the level of the concession (extended family compound) using SAS V.9.3 (SAS Institute Inc, Cary, North Carolina, USA) to assign eligible children in the intervention cohort to one of four intervention groups. Every concession had a 1/8 chance of receiving one of the eight colour codes (two colours for each group to reinforce the blinding). During the trial, all participants, field staff, study statistician and investigators were blinded to the intervention groups. Potentially eligible children were identified by two censuses at a 1-year interval (November–December 2009 and 2010) in participating communities. Children were eligible if they were 8.80–9.99 months old, a permanent resident of Dandé health district, and their caregivers planned to be available during the study period and accepted home visits for data collection. Children were excluded when they had haemoglobin (Hb) concentration <50 g/L, weight-for-length <70% of the National Center of Health Statistics (NCHS) reference median,22 bipedal oedema, other severe illness requiring hospital referral, a congenital abnormality or chronic medical condition, allergy towards peanuts or history of anaphylaxis or serious allergic reactions to any substance requiring emergency medical care, or were concurrently participating in any other clinical trial. Written informed consent was obtained from one of the child's primary caregivers. If the caregiver was illiterate, an impartial witness who was present during the consent process confirmed by co-signing that the information in the consent document was accurately explained to the participant, and that consent was freely given. During the enrolment visit, length and weight were measured, as described below. All children were screened for malaria parasites using a rapid diagnostic test (RDT, histidine-rich protein II; SD BIOLINE Malaria Ag P.F/Pan, Standard Diagnostics, INC, Kyonggi-do, Korea). If the RDT was positive, the child received antimalarial treatment (amodiaquine-artesunate, 1 tablet/day for 3 days) and an antipyretic (paracetamol, 1/2 tablet × 3/day for 3 days). In case of fever with negative RDT, an antipyretic was provided for 3 days. Children with reported diarrhoea at the time of enrolment were treated with oral rehydration salts (ORS: 1 sachet/day for 4 days). Hb concentration was measured by Hemocue (Hemocue 201+, HemoCue AB, Ängelholm, Sweden). Children with Hb <80 g/L received iron supplements (ferrous sulfate, 2–6 mg iron/kg body weight/day, depending on anaemia severity) for 30 days and an anthelminthic (200 mg mebendazole/day) for 3 days. After being enrolled, participants in the intervention cohort were assigned to receive one of four interventions from 9 to 18 months of age: (1) SQ-LNS without zinc and placebo tablet (LNS-Zn0), (2) SQ-LNS with 5 mg zinc and placebo tablet (LNS-Zn5), (3) SQ-LNS with 10 mg zinc and placebo tablet (LNS-Zn10) or (4) SQ-LNS without zinc and 5 mg zinc tablet (LNS-TabZn5). Eligible twins were both enrolled in the study and received the same intervention and follow-up; however, only one randomly selected twin was included in the data analysis. SQ-LNS, zinc and placebo tablets were produced by Nutriset SAS (Malaunay, France). All SQ-LNS products had the same appearance, aroma and flavour, and the zinc and placebo dispersible tablets were identical in appearance and flavour. The composition of SQ-LNS was the same for the four intervention formulations except for their zinc content. One sachet of 20 g SQ-LNS provided 118 kcal, 6 mg of iron, 5 or 10 mg added zinc for LNS-Zn5 and LNS-Zn10, respectively, and 19 other micronutrients.17 The zinc tablet provided during this study is the same dispersible tablet provided by UNICEF and used in programmes for diarrhoea treatment in many countries, except the zinc content was 5 mg per tablet, provided as zinc sulfate. Caregivers were instructed on how to administer the study supplements and were advised to continue breast feeding and to feed diverse local foods. Caregivers were also instructed to administer 20 g SQ-LNS per day in two separate servings, preferably mixed in a small portion of the child's meal, and to give the dispersible tablet once a day at least 30 min away from meals and SQ-LNS. The latter instruction was provided to optimise zinc absorption. From 9 to 18 months, children were visited weekly by trained field data collectors. During the first part of the study, the data collectors provided one plastic cup of 140 g of SQ-LNS and a blister package containing eight dispersible tablets (zinc or placebo) each week. Later in the study, the cups were replaced by seven 20 g sachets of SQ-LNS. Adherence was assessed by obtaining information on SQ-LNS and tablet consumption, and collecting any remaining SQ-LNS and tablets and empty packages.23 Field data collectors used a structured questionnaire to collect a weekly morbidity history, including the child's general state, appetite, number of semiliquid/liquid stools, presence of blood or mucus in stools, vomiting, fever, signs of respiratory tract infections, and any treatment received by the child either from study staff members or outside the study. If the child had a reported fever during the previous 24 h, auricular temperature was measured, and an RDT and blood smear slide were performed. As a quality control measure, auricular temperature was also measured once per month for all children independent of the caregiver's report. In the case of reported diarrhoea, fever with a negative RDT and fever with a positive RDT, the child was treated according to the Burkina Faso national guideline, as described above. Children were referred to the nearest health clinic for any danger signs (convulsions, lethargy or coma, persistent vomiting or inability to eat or drink), diarrhoea and malaria with complications, suspicion of lower respiratory tract infection, and any other symptoms requiring medical attention. Field data collectors resided in their assigned village, so that caregivers could seek treatment for the child outside of the regularly scheduled visit day. Field data collectors who were approached for unscheduled evaluations followed the same procedures outlined above. On average, each field data collector was assigned to monitor 86±41 children during the entire study period. The 25 data collectors were supervised on a weekly basis by field supervisors. The work of the morbidity team was also continuously supervised by a trained nurse and two study physicians. Field data collectors and supervisors were retrained every 4–5 months to avoid any violation of the study protocol. Anthropometric measurements were performed at baseline when children were 9 months old. All measurements were taken in duplicate. Weight was measured to the nearest 0.05 kg (Seca 383, Hamburg, Germany) and length to the nearest 0.1 cm (portable length board Seca 417, Hamburg, Germany). In case of disagreement between the first two measurements (greater than 0.1 kg for weight and 0.5 cm for length), a third measurement was performed. The average of the two closest values was used in the statistical analysis. Weight-for-length z-score (WLZ), weight-for-age z-score (WAZ) and length-for-age z-score (LAZ) were calculated using the SAS macros for the WHO Child Growth Standards.24 Data on feeding practices and child dietary intake were collected at baseline using a food frequency questionnaire that elicited information on consumption of predefined semisolid or solid food groups during the previous 24 h. Variables assessing breast feeding, meal frequency, dietary diversity and animal source food consumption were constructed based on the WHO indicators for assessing infant and young child feeding practices.25 Demographic and socioeconomic data were collected within 2 weeks after enrolment. Data were obtained on maternal education and marital status, Household Food Insecurity Access Scale (HFIAS),26 number of children under 5 years in the household, and livestock possession and housing quality, as described in more detail by Hess et al.27 During monthly interviews, the caregiver was asked whether the participating child slept under a mosquito net the night preceding the visit, and whether the child received a high-dose vitamin A capsule during the preceding month. Photos of vitamin A supplements were shown to the caregiver to help differentiate between high-dose vitamin A supplements and the poliovirus oral vaccine. The longitudinal prevalences of diarrhoea and malaria were the primary outcomes. A total sample size of 2332 participants (583 per group) was needed to detect (with a significance of p<0.05 and power >0.80) ≥20% reduction in diarrhoea prevalence and malaria prevalence among the four groups, assuming an attrition rate of 15%. The expected effect sizes (0.22 SDs) were based on effects observed in previous zinc supplementation trials.4 6 7 21 28 A total of 2435 children were enrolled in the study and 2364 of them were included in the final analyses after excluding participants with less than 30 days of morbidity observations. Diarrhoea was defined as caregiver report of three or more liquid or semiliquid stools during a 24 h period. An episode of diarrhoea was defined as the period starting the day the child first had diarrhoea following a diarrhoea-free period of 2 days, and ending on the last day the child had diarrhoea that was followed by ≥2 days without diarrhoea. The episode was considered to be severe when associated with observed signs of dehydration, reported presence of faecal blood, reported presence of six or more liquid or semi-liquid stools in 24 h, or when the episode lasted ≥14 days. Fever was defined as: (1) any reported fever by the caregiver, whether or not the fever was confirmed by measured temperature during the last 24 h; or (2) any elevated measured auricular temperature (≥37.5°C). An episode of fever was defined as the period starting the day the child first had fever following a fever-free period of 2 days, and ending on the last day the child had fever that was followed by ≥2 days without fever. The episode of fever was considered to be unrelated to malaria (and classified as non-malaria fever) when there was no positive RDT for any day of the episode and within 2 days of the episode. After enrolment, malaria was defined as the presence of reported or confirmed fever during the 24 h preceding the morbidity visit, associated with a positive RDT. A malaria episode was defined as the presence of a new episode of fever and positive malaria RDT obtained 21 days after a previously treated malaria episode. An episode of malaria was considered severe when it was accompanied by seizures, unconsciousness or respiratory distress (presence of wheezing/stridor or chest in-drawing). Acute lower respiratory illness (ALRI) was defined as any episode in which the caregiver reported cough with respiratory difficulties (wheezing/stridor or chest in-drawing). An episode of ALRI ended on the last day the child had ALRI that was followed by at least 3 days free of respiratory distress. Acute upper respiratory illness (AURI) was defined as any episode in which the caregiver reported cough and a purulent nasal discharge. An episode of AURI ended on the last day the child had AURI that was followed by at least 7 days free of purulent nasal discharge. Incidence was defined as the number of new episodes of a disease per 100 days at risk and longitudinal prevalence as the per cent of total days of observation (or ‘recalled’ days) on which the disease was present (ie, the numerator is the total number of days with a disease and the denominator is the total number of days of observation). In the case of malaria, for each episode, the 21 days following the diagnosis of malaria were removed from the total days at risk. All data collection forms were checked for data quality (completeness, consistency, etc) by field supervisors and quality control agents. Data were double entered using EpiData V.3.1 (EpiData Association, Odense, Denmark) and the data sets were reviewed and validated on a weekly basis before appending to the previous cumulated database. Project coordinators routinely reviewed the data sets for errors (eg, study identification number, date of visit, group assignment, inconsistent variables, biologically implausible values and missing values) using Stata V.11.2 (StataCorp, Texas, USA) syntaxes. Original data collection forms were used for correction in cases of data entry errors identified by the coordinators. A statistical analysis plan was written by the study investigators and published to the project website (http://www.ilins.org) prior to the start of data analysis. The investigators remained blinded until consensus on primary conclusions was reached. Data analyses were completed using SAS V.9.3 (SAS Institute Inc, Cary, North Carolina, USA). Descriptive analysis was performed for baseline characteristics. Weighted means were calculated by weighting the mean number of illness days and prevalence by the total number of observation days, and by weighting the mean number of episodes and incidence rates by the total number of days at risk of a specific illness. The incidence or prevalence in the four intervention groups was compared by using binomial logistic regression and the events/trials syntax for the response variable (SAS GLIMMIX procedure), controlling for baseline characteristics and allowing for overdispersion. The following covariates were included in the different models: participant’s sex, baseline LAZ and WLZ (continuous), baseline Hb (continuous, only for malaria outcomes), iron supplementation at baseline (provision or not), and feeding practice indicators (child meets minimum requirement or not, only for diarrhoea outcomes); maternal education (no education, no formal education or <1 year formal education, ≥1 year formal education) and marital status/rank (sole wife in household, first wife in a polygamous household, and second wife or higher in a polygamous household), number of children under 5 years in the household (≤1 child, 2 children and ≥3 children); household food insecurity access score adjusted for season (season-adjusted HFIAS, quartiles), proxy of hygiene and water quality (quartiles), household livestock possession index (quartiles) and month/year of enrolment. Continuous variables were used when possible (normally distributed), and were categorised according to international standards if available (eg, WHO on infant and child feeding practices25), were constructed based on the study setting context (eg, maternal education), or were categorised into quartiles when other transformations were not useful. Because randomisation was carried out at the concession level, all analyses included random effects of concession. Pairwise comparisons between groups were carried out with a Tukey-Kramer adjustment to control for overall type I error. A set of effect modifiers (sex, baseline continuous WLZ, baseline continuous and categorical (<1.5 or ≥1.5 SD29) LAZ, days at risk of illness by type of illness, maternal education and marital status (categorised as above), month/year of enrolment, seasonal adjusted HFIAS score (quartiles), housing quality (quartiles, for diarrhoea only), and iron supplementation at enrolment) were selected a priori and were assessed individually in the models with all the covariates, by including the potential effect modifier as a main effect and in an interaction with the intervention group variable. Stratified analyses were performed to assess the nature of the interaction when the interaction term was significant at the 5% level. Final morbidity analyses included only children with at least 30 days of morbidity observations. However, in a sensitivity analysis, the inclusion of all available data did not affect the results (see online supplemental table S1). Data are presented as means±SD, unless otherwise noted. p Values <0.05 were considered statistically significant.