Abstract
Background: Although various micronutrient regimens have been shown to prevent and treat common infectious diseases in children, the effects of daily multivitamin (MV) and/or zinc supplementation have not been widely evaluated in young African infants.
Objective: The objective was to determine whether daily supplementation of HIV-unexposed Tanzanian infants with MVs or zinc reduces the risk of infectious morbidity compared with placebo.
Methods: In a 2 × 2 factorial, double-blind, randomized controlled trial, 2400 infants who were 6 wk of age and born to HIV-negative mothers in a low-malaria setting were randomly assigned to receive daily oral supplementation of MVs (vitamin B complex and vitamins C and E), zinc, zinc + MVs, or placebo for 18 mo. Morbidity was assessed by study nurses at monthly visits and by physicians every 3 mo and/or when the child was acutely ill.
Results: No significant differences were found in the percentage of nurse visits during which diarrhea, cough, or any other symptom were reported throughout the previous month when receiving either zinc or MVs. However, physician diagnoses of all types of diarrhea (RR = 0.88; 95% CI: 0.81, 0.96; P = 0.003), dysentery (RR = 0.84; 95% CI: 0.74, 0.95; P = 0.006), and acute upper respiratory infection (RR = 0.92; 95% CI: 0.88, 0.97; P = 0.0005) were significantly lower for infants supplemented with zinc than for those who did not receive zinc. Among the 2360 infants for whom vital status was obtained, there was a nonsignificant increase in all-cause mortality among infants who received zinc (HR = 1.80; 95% CI: 0.98, 3.31; P = 0.06) compared with those who did not receive zinc. MVs did not alter the rates of any recorded physician diagnoses or mortality. Neither zinc nor MVs reduced hospitalizations or unscheduled outpatient visits.
Conclusions: Daily zinc supplementation of Tanzanian infants beginning at the age of 6 wk may lower the burden of diarrhea and acute upper respiratory infections, but provision of MVs using the regimen in this trial did not confer additional benefit. This trial was registered at clinicaltrials.gov as NCT00421668.
Keywords: multivitamins, zinc, child morbidity, diarrhea, respiratory infection
Introduction
Diarrheal diseases and respiratory infections are among the leading causes of child mortality globally and were responsible for the deaths of 1.9 million children <5 y of age in 2011 (1). Low birth weight and early childhood undernutrition are major risk factors for morbidity and mortality from these and other infectious diseases, particularly in resource-limited areas of the world. Recent estimates indicate that 45% of all child deaths result from fetal growth restriction, anthropometric deficits, micronutrient deficiencies, or suboptimal breastfeeding (2). Therefore, interventions to improve nutritional status have great potential to reduce child morbidity and mortality.
Micronutrient supplementation is one such strategy and is particularly compelling given the important role of numerous vitamins and minerals in systemic immune function and the maintenance of local defenses, coupled with its relative cost-effectiveness (3, 4).
Several studies have evaluated the efficacy of different micronutrient regimens in preventing and/or treating common infectious diseases, including respiratory and gastrointestinal diseases. However, differences in study design and location, baseline nutritional status of the study population, composition and duration of supplementation, and disease background have led to varying results. There is clear evidence that therapeutic zinc supplementation has beneficial effects on the duration and severity of diarrhea and other morbidities (5), and the WHO has included zinc supplementation in its guidelines for clinical management of acute diarrhea for the past decade (6). However, policy recommendations do not yet exist for preventive zinc supplementation. A meta-analysis showed that preventive zinc supplementation in children reduced the incidence of diarrhea and respiratory infections by ~20% and 14%, respectively (7). A notable finding, however, was that this effect was generally not observed in infants <12 mo of age. In addition, few studies included in the analysis were conducted in sub-Saharan Africa (7). Given regional differences in maternal nutritional status, birth outcomes, and morbidity patterns, it is critical that micronutrient supplementation trials be conducted in a diversity of settings, including sub-Saharan Africa, before global recommendations can be developed.
Because zinc insufficiency may coexist with other vitamin and mineral deficiencies, simultaneous supplementation with other micronutrients may be an effective strategy. Two recent trials in Tanzania and Pakistan examined whether the provision of multiple micronutrients in addition to zinc confers additional benefits against diarrhea and other morbidities (8, 9). Both trials, however, were restricted to children ≥6 mo of age, both included iron as part of the micronutrient supplement, and both reported that multiple micronutrients either increased diarrhea morbidity or conferred no additional benefit in comparison with zinc alone. To our knowledge, there have not been any investigations of preventive zinc and multivitamin (MV) supplementation on infectious disease morbidity that have included African children <6 mo of age. Initiation of supplementation earlier in infancy may be appropriate given the fact that many lactating mothers are deficient in several micronutrients (10). To evaluate the potential role of zinc or MV supplementation in early infancy, we performed a randomized trial to determine whether daily supplementation of HIV-unexposed Tanzanian infants with MVs, zinc, or both reduced the risk of infectious morbidity compared with placebo.
Methods
Study design and participants.
The study was a randomized, double-blind, 2 × 2 factorial design trial that took place in periurban Dar es Salaam, Tanzania (clinicaltrials.gov NCT 00421668). Mothers of potentially eligible infants were recruited into the study in 1 of 2 ways: 1) pregnant women ≤34 wk gestation presenting at 1 of 3 prenatal clinics in Dar es Salaam were informed about the study and consented prenatally or 2) women were recruited from the labor ward of Muhimbili National Hospital within 12 h of delivering a healthy singleton baby. In both cases, written informed consent was obtained and mothers were asked to present at a study clinic within 1–2 wk of delivery for HIV testing. Maternal HIV status was determined using 2 sequential ELISAs that used the Murex HIV antigen/antibody (Abbott Murex) followed by the Enzygnost anti-HIV-1/2 Plus (Dade Behring) or the Enzygnost HIV Integral II Antibody/Antigen (Siemens). Any discrepancy between the first and second ELISA was resolved by a Western blot assay. Consenting mothers who were confirmed to be HIV-negative were enrolled into the study and their infants were randomly assigned to 1 of 4 regimens between 5 and 7 wk of age. Infants of multiple births and infants with congenital anomalies or other conditions that would interfere with the study procedures were excluded. Birth characteristics were obtained immediately after delivery whenever possible. We used reference data from Oken et al. (11) to calculate the percentile of birth weight for each completed week of gestation and defined small-for-gestational age as ≤10th percentile. At the time of randomization, clinical examination was performed by a study physician, history of morbidity and infant feeding practices was conducted by a study nurse, infant blood was drawn for a complete blood count, and anthropometric measurements were performed.
Institutional approval was granted by the Harvard T.H. Chan School of Public Health Human Subjects Committee, the Muhimbili University of Health and Allied Science Committee of Research and Publications, the Tanzanian National Institute of Medical Research, and the Tanzanian Food and Drugs Authority. Over the course of the study, a Data Safety and Monitoring Board met twice annually.
Randomization and masking.
Infants were randomly assigned in a factorial design to receive a daily oral dose of 1 of the following 4 regimens for 18 mo from the time of randomization: 1) zinc, 2) MVs, 3) zinc + MVs, or 4) placebo. The biostatistician in Boston prepared a randomization list from 1 to 2400 that used blocks of 20 and was stratified by study clinic. Capsules were packaged in a blister pack of 15 each and numbered boxes containing 6 blister packs were prepared containing the corresponding treatments. Each eligible infant was assigned the next numbered box of capsules at his/her respective site. The supplement used was an orange-flavored powder encapsulated in an opaque gelatinous capsule and was manufactured by Nutriset. All 4 regimens were field tested and the taste, smell, and appearance were found to be indistinguishable between groups. All study personnel and participants were blinded to treatment assignment for the duration of the study.
Procedures.
From the time of randomization to 6 mo of age, infants received 1 capsule/d, and from 7 mo of age to the end of follow-up, 2 capsules were provided daily. For infants in the zinc group, the capsule contained 5 mg of zinc. For infants in the MV group, the capsule contained 60 mg of vitamin C, 8 mg of vitamin E, 0.5 mg of thiamine, 0.6 mg of riboflavin, 4 mg of niacin, 0.6 mg of vitamin B-6, 130 mg of folate, and 1 mg of vitamin B-12. Infants in the MV + zinc group received 1 capsule containing the micronutrients listed in both the MV and the zinc groups. For children 0–6 mo of age, these doses represented between 150% and 600% of the RDA or Adequate Intake, and for children 7–12 mo of age, the doses were equivalent to 200–400% of the RDA or Adequate Intake. Mothers were shown how to push the capsule through the back of the blister pack, open the capsule, decant the powder into a small plastic cup, mix the powder with 5 mL of sterile water, and administer the solution to the child orally.
Our choice of supplement composition was based on several considerations including: 1) previous research that found deficiencies in vitamin B-12, folate, zinc, vitamin A, and vitamin E among breastfeeding women in South Africa (10) and, therefore, suggests that infant micronutrient status may be low in sub-Saharan Africa; 2) a previous clinical trial involving pregnant Tanzanian women that confirmed improved birth outcomes with supplementation of vitamin B complex, vitamin E, and vitamin C (12); and 3) findings from our previous trial of MV supplementation involving HIV-exposed children, which revealed lower rates of fever and vomiting among supplemented children (13). Dar es Salaam has been described as a malaria-endemic area, although studies have suggested that rates of malaria are declining (14). Nonetheless, owing to the potential that iron supplementation of nonanemic children may have adverse consequences in malaria-endemic regions (15), the MV supplement did not include iron.
Mothers and children were followed from the time of randomization for 18 mo, until the child’s death, or until loss to follow-up. Mothers who were enrolled during pregnancy received standard prenatal care including anthropometric assessment, intermittent prophylaxis for malaria, tetanus toxoid immunization, deworming using mebendazole, anemia assessment, and iron-folic acid supplementation. During this follow-up period, mothers and children were asked to return to the study clinic every 4 wk for data collection and standard clinical care, including growth monitoring, immunizations, routine medical treatment for illnesses, and periodic vitamin A supplementation (100,000 IU at 9 mo and 200,000 IU at 15 mo). Children who were diagnosed with anemia were treated with iron supplementation. At ~12 mo of age, CD4 and CD8 T cell counts and percentages were measured from a subset of 428 children using FACSCalibur system (Becton Dickinson) in order to determine any potential immune effect of micronutrient supplementation.
At each of the monthly follow-up visits study nurses assessed compliance by counting the number of unconsumed capsules, assessed infant feeding practices, and conducted a morbidity history with the aid of pictorial diaries that mothers were instructed to complete daily in order to document the occurrence of any of the following symptoms: diarrhea; common cold; cough; difficulty breathing; fever; refusal to eat, drink, or breastfeed; pus draining from ears; and vomiting. They also assessed symptoms that were present on the day of the visit, recorded vital signs (including measurement of the child’s temperature and respiratory rate and detection of chest indrawing), and inquired about the occurrence of any unscheduled clinic visits or hospitalizations in the past month. Diarrhea was defined as ≥3 loose or watery stools within a 24-h period. Rapid respiratory rate was defined as >50 breaths/min in infants 2–11 mo of age and >40 breaths/min among infants ≥12 mo of age.
At baseline, every 3 mo, and/or when acute illnesses were noted by the study nurse a study physician conducted a physical examination, diagnosed illnesses, and provided necessary medical treatment. Physicians underwent regular training so that they used standardized diagnostic criteria and treatment guidelines consistent with the WHO and Tanzanian Ministry of Health and Social Welfare policies. For the purposes of the analysis of physician diagnosis, “any form of diarrhea” included persistent diarrhea, acute diarrhea, dysentery, and/or intestinal parasites. Acute upper respiratory infection was defined as pharyngitis or rhinitis (both without fast breathing or chest indrawing). Acute lower respiratory infection was defined as cough or difficulty breathing, rapid respiratory rate (based on the same definition described previously), and either a fever of >38.3°C or chest retractions. “Any form of respiratory infection” included acute upper respiratory infection, acute lower respiratory infection, pulmonary tuberculosis, or other causes of pneumonia.
Children who missed their scheduled monthly follow-up appointment were visited at home and their vital status was confirmed through contact with immediate family members. In cases of child death, a verbal autopsy was performed to determine the cause of death. The cause of death forms were then coded by 2 independent pediatricians (KPM and CPD), and any differences were resolved by a third pediatrician.
Data management and analysis.
The primary outcomes of the study were the incidence of clinical symptoms of diarrhea and lower respiratory infection. Power was calculated for 1 factor in the factorial design, which represented either the zinc or MV arm. Based on findings from our previous trial, we estimated that the mean (SD) number of diarrheal and lower respiratory illnesses per child per year in the placebo group would be 3.4 (4.2) and 2.1 (1.0), respectively (16). After applying a 2-sided α-value of 0.025 to yield an overall type I error rate of 0.05 for each primary outcome and allowing for a 15% loss to follow-up and minimum power of 80%, we calculated that we would require 2400 subjects to detect a reduction of 18% in the mean number of diarrheal episodes per year. We then calculated that with this sample size we would have 90% power to detect an 8% reduction in the mean number of episodes of lower respiratory illness per year.
Data were double entered using Microsoft Access software (Microsoft Corp.) at the central study site and then converted to SAS data sets and uploaded to a secured UNIX-based server for analysis. Intent-to-treat analyses were conducted according to a pre-established data analysis plan. Descriptive statistics were used to summarize baseline characteristics of the study population. Frequencies were reported for categorical variables and the mean ± SD for continuous variables. The χ2-test and ANOVA were used to detect any differences among treatment groups. We used generalized estimating equations with the log link, binomial variance, and exchangeable correlation matrix to compare the proportion of follow-up visits in which the illness symptom had occurred in the previous 4 wk between factors. For physician diagnoses that were made during routine visits every 3 mo or during unscheduled visits during acute illness episodes, the mean number of diagnoses over the follow-up period was compared between factors using Poisson regression. In both sets of analyses we introduced interaction terms to test for joint effects between the zinc and MV factors. We also tested for effect modification by each factor and sex and low birth weight. When the P-interaction term was <0.10, stratified analyses were performed.
Although the study was not powered to detect differences in mortality, we used Cox proportional hazards modeling to explore differences in all-cause and cause-specific mortality by factor. Values in the text are means ± SDs unless otherwise indicated. All analyses were performed using SAS software (version 9.2; SAS Institute).
Results
Figure 1 shows the study profile. Between August 2007 and December 2009, 2400 infants were randomly assigned, and follow-up ended in May 2011. Median (25th and 75th percentiles) regimen compliance among children was 96 (91, 99) of the allocated regimen. With the exception of length-for-age z score, there were no significant differences in any maternal, socioeconomic, or child characteristics between the 4 groups at baseline (Table 1). Mean maternal age was ~26 y, about three-quarters of mothers had ≤7 y of education, and ~30% had not been pregnant previously. The prevalences of low birth weight and preterm birth in all groups were ~3% and 13%, respectively. One child was enrolled outside of the age criteria (at 2.4 wk) but was kept in the trial after review and approval by the Data Safety and Monitoring Board and the Institutional Review Board.
FIGURE 1.
Study profile of a randomized trial of MV and/or zinc supplementation to infants in Dar es Salaam, Tanzania. MV, multivitamin.
TABLE 1.
Baseline characteristics of enrolled Tanzanian infants and their mothers by supplementation group1
| Placebo (n = 604) | Zinc only (n = 596) | MVs only (n = 598) | Zinc + MVs (n = 602) | |
| Maternal characteristics | ||||
| Age, y | 26.5 ± 5.0 | 26.8 ± 5.1 | 26.2 ± 5.0 | 26.1 ± 5.0 |
| Formal education, n (%) | ||||
| None | 9 (1.5) | 8 (1.4) | 10 (1.7) | 9 (1.5) |
| 1–7 y | 453 (75.3) | 421 (71.1) | 416 (70.2) | 441 (73.4) |
| ≥8 y | 140 (23.3) | 163 (27.5) | 167 (28.2) | 151 (25.1) |
| Employment, n (%) | ||||
| Housewife without income | 386 (64.4) | 365 (62.3) | 337 (56.7) | 357 (59.8) |
| Housewife with income | 176 (29.4) | 175 (29.9) | 212 (35.7) | 201 (33.7) |
| Other | 37 (6.2) | 46 (7.9) | 45 (7.6) | 39 (6.5) |
| Married or cohabitating with partner, n (%) | 537 (90.0) | 534 (90.5) | 542 (91.4) | 542 (90.5) |
| Prior pregnancies, n (%) | ||||
| None | 184 (30.6) | 169 (28.6) | 205 (34.5) | 187 (31.2) |
| 1–4 | 398 (66.2) | 410 (69.3) | 375 (63.1) | 396 (66.1) |
| ≥5 | 19 (3.2) | 13 (2.2) | 14 (2.4) | 16 (2.7) |
| Midupper arm circumference, cm | 27.0 ± 3.1 | 27.1 ± 3.1 | 27.1 ± 3.1 | 26.7 ± 3.2 |
| Recruited prenatally, n (%) | 86 (14.2) | 76 (12.8) | 90 (15.1) | 92 (15.3) |
| Socioeconomic characteristics | ||||
| Daily food expenditure per person in household is <1000 TSh, n (%) | 158 (27.6) | 162 (28.6) | 164 (28.8) | 170 (29.4) |
| Household possessions,2 n (%) | ||||
| None | 171 (28.4) | 192 (32.7) | 173 (29.2) | 180 (30.0) |
| 1–3 | 358 (59.5) | 308 (52.4) | 330 (55.7) | 339 (56.5) |
| >3 | 73 (12.1) | 88 (15.0) | 90 (15.2) | 81 (13.5) |
| Child characteristics | ||||
| Age at randomization, wk | 5.9 ± 0.4 | 5.9 ± 0.4 | 5.9 ± 0.4 | 5.9 ± 0.4 |
| Male, n (%) | 293 (48.5) | 296 (49.7) | 282 (47.2) | 313 (52.0) |
| Low birth weight (<2500 g), n (%) | 18 (3.0) | 21 (3.6) | 21 (3.6) | 22 (3.7) |
| Born <37 wk gestational age, n (%) | 77 (14.0) | 80 (14.6) | 66 (12.0) | 67 (12.1) |
| Born <34 wk gestational age, n (%) | 15 (2.7) | 14 (2.6) | 12 (2.2) | 22 (4.0) |
| Born small for gestational age (<10th percentile), n (%) | 45 (8.4) | 47 (8.7) | 52 (9.7) | 47 (8.7) |
| Apgar score ≤7 at 5 min after birth, n (%) | 9 (1.6) | 17 (3.1) | 5 (0.9) | 10 (1.8) |
| Hemoglobin, g/dL | 10.7 ± 1.6 | 10.6 ± 1.6 | 10.7 ± 1.4 | 10.6 ± 1.4 |
| Length-for-age z score3 | −0.17 ± 1.28a | −0.33 ± 1.92a,b | −0.26 ± 1.20a,b | −0.37 ± 1.23b |
| Weight-for-length z score | 0.05 ± 1.32 | 0.16 ± 1.33 | 0.14 ± 1.29 | 0.15 ± 1.31 |
| Weight-for-age z score | −0.16 ± 1.05 | −0.23 ± 0.97 | −0.17 ± 0.99 | −0.26 ± 1.03 |
According to the χ2-test or ANOVA, there were no significant differences in baseline characteristics among treatment groups (P > 0.05) with the exception of length-for-age z score (P = 0.03). Values are means ± SDs or percentages. MV, multivitamin; TSh, Tanzanian shilling.
From a list that includes sofa, television, radio, refrigerator, and fan.
Labeled means in a row without a common letter differ, P < 0.05.
Table 2 shows the effect of MV and zinc supplementation on the occurrence of common infectious morbidities, hospitalizations, and unscheduled outpatient visits, as recorded by the study nurses at the monthly clinic visits. When children who received zinc were compared with children who did not receive zinc, there were no significant differences in the percentage of visits during which diarrhea (RR: 0.93; 95% CI: 0.82, 1.05; P = 0.26), cough (RR: 0.95; 95% CI: 0.90, 1.01; P = 0.10), or any other symptom had been experienced during the previous month. In addition, the occurrence of hospitalizations (RR: 1.50; 95% CI: 0.83, 2.70; P = 0.18) and unscheduled outpatient visits (RR: 1.18; 95% CI: 0.98, 1.43; P = 0.08) did not vary significantly in children receiving zinc. With the exception of pus draining from the ears, MVs did not affect the occurrence of any of the morbidity symptoms, hospitalizations (RR: 1.18; 95% CI: 0.67, 2.11; P = 0.56), or unscheduled outpatient visits (RR: 1.11; 95% CI: 0.92, 1.34; P = 0.28). There was, however, a nonsignificant interaction between treatment groups in the case of diarrhea (P-interaction = 0.06). Subsequent stratified analysis revealed that in comparison with the placebo group, children in the zinc group had significantly fewer reported cases of diarrhea (RR: 0.83; 95% CI: 0.69, 0.99; P = 0.04), whereas children in the MV (RR: 0.91; 95% CI: 0.77, 1.08; P = 0.28) and zinc + MV (RR: 0.95; 95% CI: 0.80, 1.13; P = 0.59) groups did not.
TABLE 2.
Effect of daily MVs and zinc on the occurrence of common infectious morbidities in Tanzanian infants by nurse evaluation1
| Received zinc2 |
Received MV3 |
||||||||
| Yes (n = 1198) |
No (n = 1202) |
Yes (n = 1200) |
No (n = 1200) |
||||||
| Morbidity reported in the past 4 wk | % (n event/visit)4 | % (n event/visit)4 | RR (95% CI)5 | P5 | % (n event/visit)4 | % (n event/visit)4 | RR (95% CI)5 | P5 | Pint6 |
| Diarrhea | 3.7 (547/14,703) | 4.0 (584/14,725) | 0.93 (0.82, 1.05) | 0.26 | 3.9 (566/14,611) | 3.8 (565/14,817) | 1.02 (0.90, 1.16) | 0.74 | 0.06 |
| Cough | 22.1 (3266/14,782) | 23.4 (3474/14,823) | 0.95 (0.90, 1.01) | 0.10 | 22.8 (3,350/14,697) | 22.7 (3,390/14,908) | 1.01 (0.95, 1.07) | 0.73 | 0.78 |
| Difficulty breathing | 1.1 (159/14,777) | 1.0 (141/14,818) | 1.15 (0.89, 1.47) | 0.29 | 1.1 (161/14,692) | 0.9 (139/14,903) | 1.16 (0.90, 1.49) | 0.25 | 0.51 |
| Cough + fever | 4.9 (723/14,782) | 5.3 (789/14,823) | 0.90 (0.81, 1.01) | 0.08 | 5.2 (760/14,697) | 5.0 (752/14,908) | 1.02 (0.91, 1.13) | 0.78 | 0.53 |
| Cough plus7 | 1.7 (247/14,782) | 1.8 (261/14,823) | 0.95 (0.78, 1.15) | 0.57 | 1.8 (268/14,697) | 1.6 (240/14,908) | 1.14 (0.94, 1.39) | 0.17 | 0.80 |
| Cough with rapid respiratory rate8 | 0.1 (16/14,782) | 0.1 (14/14,823) | 0.98 (0.40, 2.43) | 0.97 | 0.1 (15/14,697) | 0.1 (15/14,908) | 0.85 (0.34, 2.10) | 0.72 | 0.99 |
| Fever | 10.0 (1471/14,780) | 10.4 (1539/14,821) | 0.95 (0.88, 1.03) | 0.18 | 10.3 (1,514/14,694) | 10.0 (1,496/14,907) | 1.01 (0.94, 1.09) | 0.78 | 0.80 |
| Cold | 21.3 (3153/14,782) | 22.5 (3329/14,822) | 0.95 (0.90, 1.01) | 0.09 | 22.0 (3,226/14,697) | 21.8 (3,256/14,907) | 1.01 (0.95, 1.07) | 0.82 | 0.82 |
| Vomiting | 1.8 (261/14,779) | 1.7 (249/14,819) | 1.05 (0.87, 1.26) | 0.60 | 1.9 (272/14,691) | 1.6 (238/14,907) | 1.16 (0.97, 1.39) | 0.11 | 0.79 |
| Refusal to eat, drink, or breastfeed | 2.3 (336/14,780) | 2.5 (368/14,817) | 0.90 (0.77, 1.06) | 0.22 | 2.5 (363/14,691) | 2.3 (341/14,906) | 1.07 (0.91, 1.26) | 0.38 | 0.75 |
| Pus draining from ears | 0.5 (74/14,778) | 0.5 (73/14,817) | 1.06 (0.74, 1.52) | 0.75 | 0.4 (59/14,691) | 0.6 (88/14,904) | 0.65 (0.45, 0.93) | 0.02 | 0.91 |
| Hospitalizations | 0.2 (34/14,620) | 0.1 (21/14,669) | 1.50 (0.83, 2.70) | 0.18 | 0.2 (29/14,528) | 0.1 (26/14,761) | 1.18 (0.67, 2.11) | 0.56 | 0.63 |
| Unscheduled outpatient visits | 2.0 (282/14,286) | 1.7 (237/14,358) | 1.18 (0.98, 1.43) | 0.08 | 1.90 (270/14,194) | 1.72 (249/14,450) | 1.11 (0.92, 1.34) | 0.28 | 0.46 |
MV, multivitamin.
Received zinc “yes” refers to children who received zinc alone as well as those who received zinc and MVs. Received zinc “no” refers to all children who received MVs alone and those who received the placebo.
Received MVs “yes” refers to children who received MVs alone as well as those who received zinc and MVs. Received MVs “no” refers to all children who received zinc alone and those who received the placebo.
Total number of events occurring during follow-up, defined as being reported in the 28 d (4 wk) before visit or being present on the day of the evaluation.
RR, 95% CI, and corresponding P values were obtained from generalized estimating equations with the binomial variance, log link, and exchangeable working covariance structure.
P-interaction effect.
Cough plus defined as cough with one or more of the following events: difficult breathing, chest retractions, and refusal to eat, drink, or breastfeed.
Cough with rapid breathing on the day of the evaluation (respiratory rate: >60/min in infants <2 mo old, >50/min in infants 2–11 mo old, >40/min in infants 12–59 mo old).
Examination of effect modification showed that zinc lowered the burden of diarrhea in boys (RR: 0.84; 95% CI: 0.71, 0.99; P = 0.04) but not in girls (RR = 1.07; 95% CI: 0.89, 1.29; P = 0.49; P-interaction = 0.05). Conversely, zinc increased the occurrence of fever among low birth weight infants (RR: 1.66; 95% CI: 1.09, 2.53; P = 0.02) but had no effect among infants born with a birth weight of ≥2.5 kg (RR: 0.95; 95% CI: 0.87, 1.02; P = 0.17; P-interaction = 0.02). The occurrence of unscheduled outpatient visits was also higher among low birth weight infants who received zinc (RR: 5.00; 95% CI: 1.15, 21.69; P = 0.03); however, no differences were observed among infants with birth weights of ≥2.5 kg (RR: 1.17; 95% CI: 0.96, 1.41; P = 0.11; P-interaction = 0.009). Similarly, low birth weight infants who received MVs had a cough more often than those who did not receive MVs (RR: 1.48; 95% CI: 1.08, 2.02; P = 0.02), whereas there was no difference among infants with a birth weights of ≥2.5 kg (RR: 1.00; 95% CI: 0.94, 1.06; P = 0.94; P-interaction = 0.02).
The effect of MVs and zinc on infectious morbidities diagnosed by the study physician is shown in Table 3. The low burden of malaria in the study setting is worth noting: the average number of physician diagnoses over the course of follow-up was <1/child across all treatment groups. Rates of any type of respiratory infection (RR: 0.93; 95% CI: 0.89, 0.97; P = 0.0006), acute upper respiratory infection (RR: 0.92; 95% CI: 0.88, 0.97; P = 0.0005), any form of diarrhea (RR: 0.88; 95% CI: 0.81, 0.96; P = 0.004), and dysentery (RR: 0.84; 95% CI: 0.74, 0.95; P = 0.006) were significantly lower among children who received zinc compared with children who did not receive zinc. MVs did not significantly change the rates of physician diagnosis of acute upper respiratory infection, acute lower respiratory infection, pulmonary tuberculosis or other causes of pneumonia, any form of respiratory infection, acute diarrhea, dysentery, persistent diarrhea, intestinal parasites, any form of diarrhea, uncomplicated malaria, severe malaria, or pallor/anemia. There were no significant interactions between treatment arms for any of the morbidities.
TABLE 3.
Effect of daily MVs and zinc on the incidence of common infectious morbidities in Tanzanian infants by physician diagnosis1
| Received zinc2 |
Received MV3 |
||||||||||||
| Yes |
No |
Yes |
No |
||||||||||
| Physician diagnosis | n | Diagnosis, n4 | n | Diagnosis, n4 | RR (95% CI)5 | P5 | n | Diagnosis, n4 | n | Diagnosis, n4 | RR (95% CI)5 | P5 | Pint6 |
| Acute upper respiratory infection | 1094 | 3.39 ± 2.60 | 1072 | 3.70 ± 2.80 | 0.92 (0.88, 0.97) | 0.0005 | 1082 | 3.52 ± 2.68 | 1084 | 3.57 ± 2.73 | 0.98 (0.94, 1.03) | 0.43 | 0.35 |
| Acute lower respiratory infection | 1040 | 0.63 ± 0.95 | 1034 | 0.63 ± 0.95 | 1.01 (0.91, 1.13) | 0.86 | 1039 | 0.64 ± 0.96 | 1035 | 0.62 ± 0.94 | 1.02 (0.92, 1.14) | 0.69 | 0.56 |
| Pulmonary tuberculosis or other causes of pneumonia | 1027 | 0.003 ± 0.07 | 1022 | 0.001 ± 0.03 | 3.01 (0.31, 28.86) | 0.34 | 1027 | 0.002 ± 0.06 | 1023 | 0.002 ± 0.04 | 0.99 (0.14, 7.05) | 0.99 | — |
| Diagnosis of any form of respiratory infection | 1096 | 3.94 ± 2.91 | 1074 | 4.26 ± 3.06 | 0.93 (0.89, 0.97) | 0.0006 | 1083 | 4.09 ± 2.97 | 1087 | 4.11 ± 3.01 | 0.99 (0.95, 1.03) | 0.62 | 0.26 |
| Acute diarrhea | 1033 | 0.46 ± 0.77 | 1028 | 0.50 ± 0.85 | 0.91 (0.81, 1.03) | 0.15 | 1032 | 0.48 ± 0.79 | 1029 | 0.47 ± 0.83 | 1.02 (0.90, 1.16) | 0.75 | 0.29 |
| Dysentery | 1048 | 0.43 ± 0.79 | 1040 | 0.52 ± 0.87 | 0.84 (0.74, 0.95) | 0.006 | 1045 | 0.49 ± 0.85 | 1043 | 0.46 ± 0.80 | 1.05 (0.93, 1.19) | 0.42 | 0.25 |
| Persistent diarrhea | 1028 | 0.003 ± 0.05 | 1022 | 0.009 ± 0.10 | 0.33 (0.09, 1.23) | 0.10 | 1027 | 0.009 ± 0.10 | 1023 | 0.003 ± 0.05 | 1.99 (0.81,11.01) | 0.10 | 0.71 |
| Intestinal parasites | 1030 | 0.14 ± 0.38 | 1023 | 0.15 ± 0.40 | 0.94 (0.74, 1.17) | 0.56 | 1029 | 0.14 ± 0.39 | 1024 | 0.14 ± 0.39 | 0.97 (0.77, 1.21) | 0.76 | 0.33 |
| Diagnosis of any form of diarrhea | 1054 | 1.00 ± 1.18 | 1043 | 1.14 ± 1.33 | 0.88 (0.81, 0.96) | 0.003 | 1049 | 1.09 ± 1.29 | 1048 | 1.05 ± 1.23 | 1.03 (0.95, 1.12) | 0.43 | 0.24 |
| Uncomplicated malaria | 1052 | 0.87 ± 1.08 | 1037 | 0.91 ± 1.08 | 0.96 (0.87, 1.05) | 0.33 | 1048 | 0.88 ± 1.07 | 1041 | 0.90 ± 1.09 | 0.98 (0.89, 1.07) | 0.63 | 0.92 |
| Severe malaria | 1032 | 0.06 ± 0.24 | 1023 | 0.06 ± 0.24 | 1.05 (0.73, 1.52) | 0.79 | 1028 | 0.05 ± 0.22 | 1027 | 0.06 ± 0.25 | 0.79 (0.55, 1.15) | 0.22 | 0.34 |
| Pallor/anemia | 1029 | 0.16 ± 0.45 | 1026 | 0.15 ± 0.42 | 1.10 (0.88, 1.37) | 0.41 | 1030 | 0.15 ± 0.42 | 1025 | 0.16 ± 0.45 | 0.88 (0.70, 1.09) | 0.25 | 0.42 |
MV, multivitamin.
Received zinc “yes” refers to children who received zinc alone as well as those who received zinc and MVs. Received zinc “no” refers to all children who received MVs alone and those who received the placebo.
Received MVs “yes” refers to children who received MVs alone as well as those who received zinc and MVs. Received MVs “no” refers to all children who received zinc alone and those who received the placebo.
Mean ± SD diagnoses over the course of follow-up.
RRs, 95% CIs, and corresponding P values were obtained from generalized estimating equations with the Poisson distribution and log link and by using the log of the follow-up time as the offset variable.
P-interaction term.
Stratified analyses illustrated that zinc reduced the rate of dysentery in boys (RR: 0.73; 95% CI: 0.61, 0.84; P = 0.004) but not girls (RR: 0.98; 95% CI: 0.81, 1.17; P = 0.79; P-interaction = 0.02). The sex of the child also modified the effect of zinc on uncomplicated malaria. Girls who received zinc had a reduced rate of uncomplicated malaria compared with girls who did not receive zinc (RR: 0.86; 95% CI: 0.75, 0.98; P = 0.02); however, no difference was observed among boys (RR: 1.06; 95% CI: 0.93, 1.20; P = 0.39; P-interaction = 0.02). Birth weight did not modify the effect of zinc on any physician diagnoses. Likewise, the sex of the child or the birth weight did not influence the effect of MVs on any physician diagnoses.
Among the subgroup of study participants with immunologic measures available at 51.3 ± 2.2 wk, CD4 T cell percent was 35.0 ± 7.4 in infants who received zinc compared with 34.5 ± 8.0 in those not receiving zinc (P = 0.51). However, infants who received zinc and MVs had a CD4 T cell percentage of 36.7 ± 7.4 compared with 34.3 ± 8.0 among children who received placebo (P = 0.03). There were no group differences in mean CD8 T cell percentages or CD4:CD8 ratios noted (all, P > 0.20).
After a median follow-up of 17.8 mo, there were 45 deaths among the 2360 study participants with a known vital status. Of the 40 children who were lost to follow-up, 8 received the placebo, 11 received zinc only, 15 received MVs only, and 6 received zinc and MVs. We observed a nonsignificant increase in all-cause mortality among children who received zinc compared with children who did not receive zinc (HR: 1.80; 95% CI: 0.98, 3.31; P = 0.06). MVs did not significantly alter the risk of all-cause mortality (HR: 0.73; 95% CI: 0.40, 1.32; P = 0.30). Information on the cause of death was available for 39 study participants. Respiratory illness (n = 13), diarrheal disease (n = 5), malaria (n = 7), other infectious diseases (n = 8), and neonatal conditions (n = 2) were the primary causes of deaths. There were no significant differences in cause-specific mortality between treatment groups (all, P > 0.05).
Discussion
In this randomized, double-blind trial in 2400 HIV-unexposed Tanzanian infants, we found that zinc supplementation significantly lowered rates of physician diagnoses of diarrhea and acute upper respiratory infections. Zinc specifically reduced the rates of dysentery, particularly among boys. The occurrence of diarrhea, as reported at monthly clinic visits with study nurses, was also significantly reduced among children who received only zinc in comparison with placebo. MVs did not alter the occurrence of any recorded morbidities, and neither zinc nor MVs reduced hospitalizations or unscheduled outpatient visits. Although our study was not powered to detect differences in mortality, we observed a nonsignificant increase in mortality among infants who received zinc. Furthermore, in a subset of children with immunologic measures available at 12 mo of age, mean CD4 T cell percent was slightly but significantly higher among children who received zinc and MVs in comparison with placebo. To our knowledge, this is the first study of preventive zinc and MV supplementation to be conducted among young infants in sub-Saharan Africa. Given the large global burden of diarrhea and acute respiratory infections in young children, we believe that the 12% reduction in diarrhea, 16% reduction in dysentery, and 8% reduction in acute upper respiratory infections among children who received zinc are clinically significant and potentially of major public health importance.
Although, to our knowledge, no other studies have assessed the effects of daily zinc supplementation in tandem with MVs from such a young age, our findings can be compared with trials of zinc vs. placebo on infant morbidity. Osendarp et al. (17) reported that daily zinc supplementation from 4 to 24 wk of age did not reduce morbidity from diarrhea or respiratory infections in Bangladeshi infants. Likewise, a large trial of preventive zinc and/or folic acid supplementation involving Nepalese children 1–35 mo of age saw no differences in the frequency or duration of diarrhea or acute lower respiratory infection between treatment groups (18). However, weekly zinc supplementation for 12 mo was found effective in reducing the incidence of diarrhea and pneumonia among urban Bangladeshi infants aged 2–12 mo (19). Similarly, a study in Delhi, India, involving infants 6–11 mo of age, reported that a short course of daily zinc supplementation for 2 wk effectively reduced the subsequent number and duration of diarrhea episodes (20). In contrast, a multicenter study involving infants 1–5 mo of age with acute diarrhea found that infants who received therapeutic zinc supplementation for 14 d had more days of diarrhea and similar prevalence of pneumonia and respiratory infection compared with the placebo group (21). Differences in the age of study participants, dose and duration of supplementation, and length of follow-up make it difficult to identify the reasons behind these contrasting results. However, it is important to note that, to our knowledge, our study provided supplementation for the longest period of time and followed infants well into their second year of life, capturing the period during which the incidence of infectious morbidities typically rises.
Our findings build on previous investigations into the possible effects associated with the addition of multiple micronutrients to zinc supplementation regimens among older infants and children. We previously reported that the same MV regimen did not affect the risk of mortality among HIV-exposed Tanzanian infants, but it did reduce episodes of vomiting and fever (13). A different study of daily zinc and/or multiple micronutrient supplementation involving rural Tanzanian children 6–60 mo of age showed that zinc supplementation significantly decreased the rate of diarrhea and protected against fever without localizing signs; however, multiple micronutrient supplementation actually increased the rate of diarrhea by approximately one-quarter (9). Similarly, in a trial of micronutrient powders with or without zinc among Pakistani children 6–18 mo of age, Soofi et al. (8) observed an increase in the proportion of days with diarrhea as well as higher rates of bloody diarrhea and chest indrawing among children in the 2 micronutrient groups compared with a nonsupplemented control group. An earlier, relatively small study that provided zinc with or without multiple micronutrients to Peruvian children 6–35 mo of age with persistent diarrhea showed similar results: zinc supplementation tended to reduce morbidity, whereas the addition of MVs increased morbidity (22). However, it is worth noting that iron was included in the multiple micronutrient supplement that was evaluated in these 3 trials (8, 9, 22), whereas our MV regimen did not include iron. When taken as a whole, these findings do not support routine MV supplementation as an effective means of reducing infant and child morbidity, even in resource-limited settings where diets are likely poor.
Our findings of increased CD4 T cell percentage with zinc and MV supplementation suggest one possible immune effect of supplementation. Although evidence on the topic is particularly limited in HIV-negative children, we previously reported an increase in CD4 counts among HIV-infected pregnant women in Tanzania who received a similar MV regimen (23). Zinc and multiple micronutrients have also been shown to increase CD4 counts in HIV-infected adults receiving highly active antiretroviral therapy (24, 25). However, in a safety and efficacy study involving 96 HIV-infected children in South Africa, daily supplementation with 10 mg of elemental zinc for 6 mo did not significantly change the percentage of CD4+ T lymphocytes (26). In an HIV-negative elderly population, Fortes et al. (27) reported that zinc increased the number of CD4 cells. Although the compilation of these findings is encouraging, more research is needed to better understand the potential mechanisms by which certain nutrients could be affecting this marker of immunologic function.
Although not statistically significant, our finding of a possible increase in the risk of all-cause mortality among children who received zinc is generally inconsistent with the bulk of the published evidence on this topic. Our results were unexpected, especially considering the fact that zinc reduced rates of diarrhea and acute upper respiratory infections, and we saw no differences in rates of hospitalizations or unscheduled clinic visits between treatment groups. Our trial did not have sufficient power to assess cause-specific mortality, which could have helped to elucidate possible causal pathways driving this apparent effect. A meta-analysis concluded that therapeutic zinc supplementation for the treatment of diarrhea significantly reduces child mortality (5). Although the effect of preventive zinc supplementation on mortality appears to be weaker, Brooks et al. (19) reported a 17% reduction in all-cause mortality among children who received weekly zinc supplementation for 12 mo, and most other studies, including a large study from Pemba Island, Tanzania, have reported a nonsignificant reduction in mortality (18, 28, 29). Clearly, more research is needed to better understand the mechanisms through which zinc may affect certain causes of mortality and to resolve differences that could be attributed to variations in study design and subject characteristics.
Several strengths and limitations of our study deserve comment. As previously noted, to our knowledge, our study is the first to examine the effects of preventive zinc and MV supplementation in African infants 6 wk of age. We enrolled a large number of participants and conducted intensive, monthly follow-up activities over an extended period of time. This allowed us to evaluate the effects of supplementation during the period when rates of morbidity typically increase. We also assessed morbidity in a comprehensive manner, using symptom diaries, monthly nurse evaluations, and regular physician examinations. The discordance in results between the nurse evaluations and the physician diagnoses is notable. One possible explanation is that mild illnesses were only detected by study nurses at the monthly follow-up visits, whereas more severe symptoms prompted a visit with study physicians (30). Other possible limitations include lack of vitamin D in the supplement regimen and what might be considered a low dose of zinc given the possibility of impaired zinc absorption in African children (31).
In summary, daily zinc supplementation of HIV-unexposed Tanzanian infants at 6 wk of age significantly reduced physician-diagnosed cases of diarrhea and acute upper respiratory tract infections. Simultaneous provision of MVs did not appear to confer additional benefit. Further investigation into the optimal timing, frequency, and duration of supplementation is needed before policy recommendations can be formulated.
Acknowledgments
We are grateful to the field and study staff for their tireless efforts: Esther Kibona (deceased), Frank Killa, Michel Alexander, Phares Zawadi, Susie Welty, Rachel Steinfeld, Anne Marie Darling, James Okuma, Angela Jardin, Elizabeth Long, Jenna Golan, and Emily Dantzer. We thank Roland Kupka for expert advice. We thank the members of the Data Safety and Monitoring Board: Paul Jacques, Davidson Hamer, Roger Mbise, and Zul Premji.
CMM analyzed the data and wrote the manuscript; KPM designed the study, supervised data collection, and provided input to the manuscript; RK provided input to the study design, oversaw data collection, and reviewed the manuscript; SA oversaw all laboratory aspects of the study and reviewed the manuscript; DS supervised the statistical analysis and reviewed the manuscript; WWF designed the study, provided input to the statistical analysis, and reviewed the manuscript; CPD designed the study, oversaw study implementation, contributed to the statistical analysis, and provided input to the manuscript. All authors read and approved the final manuscript.
References
- 1.WHO [Internet]. Geneva (Switzerland): WHO; [cited 2013 Sep 10]. Available from: http://www.who.int/gho/child_health/mortality/mortality_causes_region_text/en/index.html.
- 2.Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, de Onis M, Ezzati M, Grantham-McGregor S, Katz J, Martorell R, et al. Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet 2013;382:427–51. [DOI] [PubMed] [Google Scholar]
- 3.Bhutta ZA, Das JK, Rizvi A, Gaffey MF, Walker N, Horton S, Webb P, Lartey A, Black RE, Lancet Nutrition Interventions Review Group; Maternal and Child Nutrition Study Group. Evidence-based interventions for improvement of maternal and child nutrition: what can be done and at what cost? Lancet 2013;382:452–77. [DOI] [PubMed] [Google Scholar]
- 4.Brown KH, Hess SY, Vosti SA, Baker SK. Comparison of the estimated cost-effectiveness of preventive and therapeutic zinc supplementation strategies for reducing child morbidity and mortality in sub-Saharan Africa. Food Nutr Bull 2013;34:199–214. [DOI] [PubMed] [Google Scholar]
- 5.Walker CL, Black RE. Zinc for the treatment of diarrhoea: effect on diarrhoea morbidity, mortality and incidence of future episodes. Int J Epidemiol 2010;39 Suppl 1:i63–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.WHO/UNICEF. Clinical management of acute diarrhoea. Geneva (Switzerland): WHO; 2004. [Google Scholar]
- 7.Brown KH, Peerson JM, Baker SK, Hess SY. Preventive zinc supplementation among infants, preschoolers, and older prepubertal children. Food Nutr Bull 2009; 30 1 Suppl:S12–40. [DOI] [PubMed] [Google Scholar]
- 8.Soofi S, Cousens S, Iqbal SP, Akhund T, Khan J, Ahmed I, Zaidi AK, Bhutta ZA. Effect of provision of daily zinc and iron with several micronutrients on growth and morbidity among young children in Pakistan: a cluster-randomised trial. Lancet 2013;382:29–40. [DOI] [PubMed] [Google Scholar]
- 9.Veenemans J, Schouten LR, Ottenhof MJ, Mank TG, Uges DR, Mbugi EV, Demir AY, Kraaijenhagen RJ, Savelkoul HF, Verhoef H. Effect of preventive supplementation with zinc and other micronutrients on non-malarial morbidity in Tanzanian pre-school children: a randomized trial. PLoS One 2012;7:e41630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Papathakis PC, Rollins NC, Chantry CJ, Bennish ML, Brown KH. Micronutrient status during lactation in HIV-infected and HIV-uninfected South African women during the first 6 mo after delivery. Am J Clin Nutr 2007;85:182–92. [DOI] [PubMed] [Google Scholar]
- 11.Oken E, Kleinman KP, Rich-Edwards J, Gillman MW. A nearly continuous measure of birth weight for gestational age using a United States national reference. BMC Pediatr 2003;3:6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Fawzi WW, Msamanga GI, Urassa W, Hertzmark E, Petraro P, Willett WC, Spiegelman D. Vitamins and perinatal outcomes among HIV-negative women in Tanzania. N Engl J Med 2007;356:1423–31. [DOI] [PubMed] [Google Scholar]
- 13.Duggan C, Manji KP, Kupka R, Bosch RJ, Aboud S, Kisenge R, Okuma J, Fawzi WW. Multiple micronutrient supplementation in Tanzanian infants born to HIV-infected mothers: a randomized, double-blind, placebo-controlled clinical trial. Am J Clin Nutr 2012;96:1437–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Wang SJ, Lengeler C, Mtasiwa D, Mshana T, Manane L, Maro G, Tanner M. Rapid Urban Malaria Appraisal (RUMA) II: epidemiology of urban malaria in Dar es Salaam (Tanzania). Malar J 2006;5:28. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ, Dutta A, Dhingra U, Kabole I, Deb S, Othman MK, et al. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 2006;367:133–43. [DOI] [PubMed] [Google Scholar]
- 16.Fawzi WW, Mbise R, Spiegelman D, Fataki M, Hertzmark E, Ndossi G. Vitamin A supplements and diarrheal and respiratory infections among children in Dar es Salaam, Tanzania. J Pediatr 2000;137:660–7. [DOI] [PubMed] [Google Scholar]
- 17.Osendarp SJ, Santosham M, Black RE, Wahed M, van Raaij JM, Fuchs GJ. Effect of zinc supplementation between 1 and 6 mo of life on growth and morbidity of Bangladeshi infants in urban slums. Am J Clin Nutr 2002;76:1401–8. [DOI] [PubMed] [Google Scholar]
- 18.Tielsch JM, Khatry SK, Stoltzfus RJ, Katz J, LeClerq SC, Adhikari R, Mullany LC, Black R, Shresta S. Effect of daily zinc supplementation on child mortality in southern Nepal: a community-based, cluster randomised, placebo-controlled trial. Lancet 2007;370:1230–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Brooks WA, Santosham M, Naheed A, Goswami D, Wahed MA, Diener-West M, Faruque AS, Black RE. Effect of weekly zinc supplements on incidence of pneumonia and diarrhoea in children younger than 2 years in an urban, low-income population in Bangladesh: randomised controlled trial. Lancet 2005;366:999–1004. [DOI] [PubMed] [Google Scholar]
- 20.Malik A, Taneja DK, Devasenapathy N, Rajeshwari K. Short-course prophylactic zinc supplementation for diarrhea morbidity in infants of 6 to 11 months. Pediatrics 2013;132:e46–52. [DOI] [PubMed] [Google Scholar]
- 21.Walker CLF, Bhutta ZA, Bhandari N, Teka T, Shahid F, Taneja S, Black RE. Zinc during and in convalescence from diarrhea has no demonstrable effect on subsequent morbidity and anthropometric status among infants <6 mo of age. Am J Clin Nutr 2007;85:887–94. [DOI] [PubMed] [Google Scholar]
- 22.Penny ME, Marin RM, Duran A, Peerson JM, Lanata CF, Lonnerdal B, Black RE, Brown KH. Randomized controlled trial of the effect of daily supplementation with zinc or multiple micronutrients on the morbidity, growth, and micronutrient status of young Peruvian children. Am J Clin Nutr 2004;79:457–65. [DOI] [PubMed] [Google Scholar]
- 23.Fawzi WW, Msamanga GI, Spiegelman D, Urassa EJ, McGrath N, Mwakagile D, Antelman G, Mbise R, Herrera G, Kapiga S, et al. Randomised trial of effects of vitamin supplements on pregnancy outcomes and T cell counts in HIV-1-infected women in Tanzania. Lancet 1998;351:1477–82. [DOI] [PubMed] [Google Scholar]
- 24.Kaiser JD, Campa AM, Ondercin JP, Leoung GS, Pless RF, Baum MK. Micronutrient supplementation increases CD4 count in HIV-infected individuals on highly active antiretroviral therapy: a prospective, double-blinded, placebo-controlled trial. J Acquir Immune Defic Syndr 2006;42:523–8. [DOI] [PubMed] [Google Scholar]
- 25.Baum MK, Lai S, Sales S, Page JB, Campa A. Randomized, controlled clinical trial of zinc supplementation to prevent immunological failure in HIV-infected adults. Clinical Infectious Diseases 2010;50 (12):1653–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Bobat R, Coovadia H, Stephen C, Naidoo KL, McKerrow N, Black RE, Moss WJ. Safety and efficacy of zinc supplementation for children with HIV-1 infection in South Africa: a randomised double-blind placebo-controlled trial. Lancet 2005;366:1862–7. [DOI] [PubMed] [Google Scholar]
- 27.Fortes C, Forastiere F, Agabiti N, Fano V, Pacifici R, Virgili F, Piras G, Guidi L, Bartoloni C, Tricerri A, et al. The effect of zinc and vitamin A supplementation on immune response in an older population. J Am Geriatr Soc 1998;46:19–26. [DOI] [PubMed] [Google Scholar]
- 28.Sazawal S, Black RE, Ramsan M, Chwaya HM, Dutta A, Dhingra U, Stoltzfus RJ, Othman MK, Kabole FM. Effect of zinc supplementation on mortality in children aged 1–48 months: a community-based randomised placebo-controlled trial. Lancet 2007;369:927–34. [DOI] [PubMed] [Google Scholar]
- 29.Yakoob MY, Theodoratou E, Jabeen A, Imdad A, Eisele TP, Ferguson J, Jhass A, Rudan I, Campbell H, Black RE, et al. Preventive zinc supplementation in developing countries: impact on mortality and morbidity due to diarrhea, pneumonia and malaria. BMC Public Health 2011;11 Suppl 3:S23. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Schmidt WP, Arnold BF, Boisson S, Genser B, Luby SP, Barreto ML, Clasen T, Cairncross S. Epidemiological methods in diarrhoea studies–an update. Int J Epidemiol 2011;40:1678–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Manary MJ, Abrams SA, Griffin IJ, Quimper MM, Shulman RJ, Hamzo MG, Chen Z, Maleta K, Manary MJ. Perturbed zinc homeostasis in rural 3–5-y-old Malawian children is associated with abnormalities in intestinal permeability attributed to tropical enteropathy. Pediatr Res 2010;67:671–5. [DOI] [PubMed] [Google Scholar]