Availability of antiretroviral therapy (ART) has improved the survival and quality of life in HIV-infected individuals, including children in resource-limited settings. However, after highly active ART (HAART), viral suppression is not always accompanied by complete immune reconstitution.1 Immune recovery is frequently slow and incomplete with an initial increase in memory CD4 cells followed by an increase in naive CD4 cells.2 In a Thai study,3 which documented the safety, effectiveness, and feasibility of nonnucleoside reverse transcriptase inhibitor–based regimens as first-line HAART in treatment-naive children with advanced stage HIV infection from a resource-limited setting, it took longer for children to achieve viral suppression (of the 81 children who attained virological success at week 72 in the study, only 57 did so by week 24). In the 2NN study,4 most of the virological successes were achieved by week 24. The higher baseline virus loads in children, compared with adults, might explain this difference. Besides higher viral load in children, the relationship between viral suppression with HAART and immune recovery is dynamic and complex and involves multiple factors,5 of which nutritional status is believed to play a pivotal role.
Zinc has many effects on the immune system,6,7 but few have been observed in HIV-infected individuals. Micronutrient malnutrition has been shown to be associated with more frequent opportunistic infections, faster disease progression, and a greater incidence of HIV-related mortality8–11 and, thus, a worsening of clinical condition among HIV-infected children. Zinc may also help by reducing the occurrence of infectious/opportunistic infections12; many of these infections enhance viral replication. These effects of zinc may facilitate faster immunologic response in HIV-infected children administered HAART. Zinc supplementation has been shown in vitro to improve the lymphoproliferative responses and reduce the percentage of apoptotic cells.13
Zinc deficiency is common in Indian children.14 Various studies in HIV-infected children have documented high prevalence of zinc deficiency.15–17
The safety of zinc supplementation has been established in HIV-infected children18; however, these children were not receiving HAART. There was no increase in viral load with zinc supplementation; children receiving zinc had a reduction in morbidity caused by diarrhea.18
There have been a few studies in children evaluating the influence of zinc on outcomes in HIV-infected children.19,20 Although there are trials supporting beneficial effects of micronutrient supplementation in adults, the data in pediatric age group are scarce.21,22
Long-term zinc supplementation has been shown to delay immunologic failure and decrease diarrhea over time in HIV-infected adults supporting the use of zinc supplementation as an adjunct therapy for HIV-infected adults with poor viral control.23
Despite the potential that zinc may improve immune functioning, its impact as an adjunct to antiretroviral treatment of HIV disease has not been evaluated in children. We evaluated the effect of zinc supplementation on helper T cell (CD4) count in HIV-infected children who were started on ART.
We conducted this study to assess the immunologic effect of daily 20 mg zinc supplementation for 24 weeks in HIV-infected children older than 6 months receiving HAART.
METHODS AND SUBJECTS
This randomized, double-blind, placebo-controlled trial was conducted in the Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India, over a 3-year period from November 2009 to October 2012. The study was initiated after obtaining ethical clearance from the Institute's Ethics Committee. The children were enrolled after obtaining written informed consent from the parent/guardian. The trial was registered with Clinical Trials Registry, India (CTRI/2009/091/000971).
All HIV-1–infected children older than 6 months in whom initiation of ART was indicated as per the guidelines were eligible for the study.24 Children receiving zinc supplementation at the time of screening for enrollment, those with clinical evidence of zinc deficiency, and sick children with concurrent other infections, including pneumonia, diarrhea, or tuberculosis (TB), were excluded.
HIV-infected children are followed up in a specialty clinic at our hospital. The diagnosis of HIV-1 infection in children was confirmed as recommended in the national guidelines; antibody tests were used for children older than 18 months, whereas for younger children, HIV-DNA polymerase chain reaction was used.24 All HIV-infected children undergo a baseline evaluation that includes the following: clinical evaluation, complete blood counts, renal and liver function tests, chest radiograph, Mantoux test, CD4 count/CD4%, and other investigations as indicated.
Based on the available guidelines, children were screened for need of ART. ART was indicated if the child had a World Health Organization (WHO) stage 3 or 4 condition and/or evidence of severe immune suppression (based on CD4 count or CD4%).24 HIV-infected children in whom ART was indicated were eligible for the study.
The children underwent a clinical examination and anthropometry. These children were sampled for a CD4%, baseline HIV viral load, and serum zinc level.
These children were prescribed the first-line ART as per the guidelines issued by the National AIDS Control Organization (NACO).24 Fixed-dose combination of 3 antiretroviral drugs was used as recommended in NACO guidelines.24 The concurrent illnesses were managed as per the NACO guidelines.24
Randomization and Allocation Concealment
Children were randomized as per a computer-generated randomization list in a variable block size. The randomization was done using the statistical software STATA version 9.0 (StataCorp, College Station, TX) by an individual not involved in the study. Two strata based on the weight for height “z” score were made. Children who had a weight for height/length “z” score value greater than equal to −3 [body mass index (BMI) “z” score greater than equal to −3 in children with height greater than 110 cm; this modification was done as the weight for height/length “z” scores could not be calculated for these children] were included in stratum 1, whereas those with a value less than −3 (BMI “z” score less than −3 in children with height greater than 110 cm) were in stratum 2.
We procured identical blister packs that contained dispersible tablets with or without zinc sulfate (20 mg of elemental zinc; Bharat Immunologicals and Biologicals Corporation Limited, a Government of India Undertaking, Bulandshahr, Uttar Pradesh, India); the company had no role in the study design, conduct, or analysis of data. Each blister pack of 10 tablets had been labeled with a unique serial number according to the randomization list, which was not available to any of the investigators until all the data had been collected and entered. We maintained masking during data analysis by coding treatment allocation with 2 letters.
Children were randomized to receive either 20 mg of elemental zinc as sulfate or a similar appearing and tasting preparation of placebo, daily for 24 weeks. Both groups received 1 recommended dietary allowance (RDA) of multivitamins everyday for 24 weeks. The child's parents/guardians are instructed to administer the study drug once a day.
Children were followed up every 4 weeks for evaluation and supply of intervention. At each 4 weekly visit, the adherence to study medications and to ART was assessed by interview and examining the blister packs (pill count). Home visits by a social worker were used to ensure adherence.
Measurements at Follow-Up Visits
Children were followed up every 4 weeks for 24 weeks. At each visit, the following evaluation was conducted—clinical evaluation: detailed history regarding any concurrent illnesses, infections, physical examination, and anthropometry; laboratory evaluation: viral load, CD4%, and zinc levels were estimated again at 12 and 24 weeks. In addition, additional investigations were performed as indicated by the clinical evaluation.
The primary outcome evaluated was CD4% value at the end of 12 and 24 weeks of study intervention in the enrolled children.
The secondary outcomes that were studied included the following: change in the viral load from baseline at the end of 12 and 24 weeks of study intervention; change in anthropometric parameters: weight for age “z” score and height for age “z” score; and episodes of infectious illnesses in the study period.
Monitoring of Primary Outcome
CD4% and count were enumerated on blood collected in EDTA by whole-blood lysis method along with 2-color flow cytometry using staining with fluorescent tagged antibodies to CD3 and CD4, on a FACSCalibur flow cytometer (Becton Dickinson).25
CD4% and count were enumerated on blood collected in EDTA by whole-blood lysis method along with 2-color flow cytometry using staining with fluorescent tagged antibodies to CD3 and CD4, on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The assays were performed within a few hours of sample collection by dedicated study personnel.
HIV Viral Load
RNA was extracted from plasma, and viral load was quantified using the Amplicor HIV RNA viral load kit (Roche, Indianapolis, IN) and Cobas Taqman HIV Test.
Zinc levels in serum were estimated by atomic absorption spectrophotometry. The elemental zinc in the sample was dissociated from its chemical bonds and placed in unexcited, unionized neutral atoms' ground state. This was passed through band path width between 0.001 and 0.01 nm. The source of radiations was the hollow cathode lamp of the element. The amount of absorption of radiation is directly proportional to the amount of free radicals in the sample. Chemical standard from Merck Co. (Darmstadt, Germany) was used to plot the calibration curve, and international serum control (SERO A/S, Billingstad, Norway) was used with intermittent indoor control pool serum.26
Sample Size Calculation
With HAART alone, the CD4% values at the end of 3 months therapy have been reported to be 15% ± 5%.27 With the intervention (addition of zinc to HAART), we expected a mean increase of 5% points over the control arm (placebo + HAART). A sample size of 25 is required for each arm to detect this difference with an alpha of 0.05, power of 90%, and accounting for 10% loss to follow-up.
Data were collected on pretested structured case report forms by research staff and cross-checked. Data were managed on Microsoft Access. Double data entry was performed and the discrepancies resolved.
Statistical analysis was performed using STATA version 9.0 (StataCorp). The 2 groups (ART + placebo vs ART + zinc) were compared for changes in CD4% values over the 24-week study period. The change in viral load was evaluated. The change in anthropometry was compared; “z” scores were calculated using WHO Anthro and AnthroPlus softwares (Department of Nutrition, WHO, Geneva, Switzerland). We also compared the episodes of infectious illnesses in the 24-week study period. The change in plasma zinc levels was compared in the 2 groups. Continuous variables are presented as mean (SD) when data were normally distributed; otherwise, median (interquartile range) was used. Wilcoxon rank sum test was used to assess statistical significance of the differences in primary outcomes. For comparing normally distributed data, “t” test was used.
Eighty-three HIV-infected children were screened (Fig. 1). After 31 exclusions, 52 children were enrolled in the study. The major reasons for exclusion were TB in 12 patients, orphan children living in a care home n = 8, and refusal of consent. One child died in each group, and 1 child's parents withdrew consent (placebo arm).
The mean age of children randomized to zinc group and the placebo group was 80.4 and 86.6 months (Table 1). There was only one child younger than 18 months enrolled in the study.
The anthropometric indices were comparable in the 2 groups. Of the 52 children, 48 were randomized in stratum 1 (weight for height/length or BMI “z” score value ≥ −3). The clinical stages of the children were comparable in the 2 groups. Children who had WHO clinical stages 1 and 2 were started on ART based on their CD4% values.
The median CD4% values in the 2 groups were comparable, being 10% and 11%, respectively.
The enrolled children had high viral loads; the children in zinc group had median viral load value of 174,000 copies per milliliter as compared with 86,900 copies per milliliter in the placebo group. The baseline median serum zinc values in the zinc and placebo groups were 35.45 and 44.08 μg/dL, respectively.
Most of the children were started on a stavudine-based ART as per the available guidelines from the NACO24; 23 and 19 children received stavudine + lamivudine + nevirapine in the zinc and placebo groups, respectively, whereas the rest received zidovudine + lamivudine + nevirapine.
Children received the study medications (zinc or placebo) in addition to the ART. The adherence to the ART was 97% in both groups, whereas it was 98% for the study medications.
Serum Zinc at Follow-Up
The serum zinc values on follow-up were significantly higher than the baseline values. Although the median serum zinc values were higher in the zinc group as compared with the placebo arm at follow-up at 12 and 24 weeks, the differences were not statistically significant (Table 2).
The median CD4% value rose from 10% to 23% at 12 weeks and to 24.5% at 24 weeks in the zinc group, whereas in the placebo group, the value rose from 11% to 20% at 12 weeks and to 22% at 24 weeks. There was a trend toward a higher increase in the zinc group (Table 3).
The median viral loads at 12 and 24 weeks in the 2 groups were comparable. The baseline viral load was higher in the zinc group as compared with the placebo group (Table 4).
The log reduction in the viral load at 12 and 24 weeks was marginally higher in the zinc group as compared with the placebo group; the difference was not significant. Similarly, the numbers of children with viral load ≤47 copies per milliliter at 12 and 24 weeks in the 2 groups were similar.
Change in Nutritional Status
The anthropometric indices at 12 and 24 weeks were comparable in the 2 groups except for the weight for height “z” score at 24 weeks, which was higher in the placebo arm; this, however, has to be viewed in context of the initially higher “z” score in the children receiving placebo (Table 5).
We compared the median number of febrile episodes, acute respiratory infection episodes, diarrheal episodes, and hospitalization in the 2 groups over 12 and 24 weeks follow-up (Table 6).
Two children died, one in each group. One died within a week of enrollment because of a lower respiratory tract infection (possibly swine origin influenza); the other 4 months after the enrollment because of central nervous system TB (the child was diagnosed as having pulmonary TB in the third month after starting ART). No adverse events of zinc were observed in the study subjects.
We conducted a randomized, double-blind, placebo-controlled trial to evaluate the efficacy of zinc (20 mg) administered daily for 24 weeks on immunologic parameters in HIV-infected children newly started on ART. Children had a high viral load at initiation of HAART. There was a significant increase in serum zinc values in both the zinc and placebo groups; the rise was higher in the zinc group.
Although there was a trend toward a higher CD4% value at 12 and 24 weeks in the zinc group, the differences were not statistically significant. A sample size of 59 patients per arm would be needed to estimate a statistically significant difference in CD4% of 3%. Similarly, the changes in the viral load were comparable in the 2 groups at 12 and 24 weeks. The anthropometric indices improved marginally in the 2 groups. The morbidity pattern was also comparable in the 2 groups.
There are no similar studies reported earlier. In an observational study, a higher pre-HAART zinc level was associated with a greater increase in CD4% at 48 weeks.19 However, in a randomized controlled trial in 847 Ugandan children (HAART: n = 85) aged 1–5 years, twice the RDA of 14 micronutrients (including 10 mg of zinc) compared with a standard RDA of 6 multivitamins for 6 months did not significantly affect mortality, growth, or CD4 counts.20 In this study, only 10% of children were receiving ART.
Zinc supplementation has been shown to delay immunologic failure in HIV-infected adults.23 The investigators used 12 mg zinc in women and 15 mg in men for 18 months; 62.3% of the subjects were receiving ART.23 The mean serum zinc level in the study subjects was 60 μg/dL, which was higher than that in our study subjects. At the end of the trial, participants who received zinc supplements had significantly higher plasma zinc levels over time (P = 0.047) than those received placebo, after controlling for high sensitivity C-reactive protein (hsCRP) at baseline and over time. However, in our study, this difference in the zinc levels in the 2 groups did not reach statistical significance.
The available systematic reviews supporting beneficial effects of micronutrient supplementation in HIV-infected adults and the data in pediatric age group are scarce.21,22
The potential reasons for the absence of a statistically significant difference in CD4% in the 2 groups could be the following. The sample size was low to detect the difference; we calculated the sample size on the basis of a clinically relevant difference in CD4%. Serum zinc levels improved in the placebo arm also; this may have been because of improved nutritional status with ART and improvement in the gut absorption of zinc. Similar phenomenon of increase in the zinc levels in individuals receiving placebo has been observed in studies with long-term supplementation of zinc in conditions like TB.28 Most of the children had low serum zinc levels at baseline; therefore, the dose of zinc administered may not have been enough to have an impact on the CD4% levels even though we used a dose of 20 mg/d, which is higher than that used in prior studies in adults.
The study has some limitations. The sample size was low to detect the significance of the differences in rise of CD4%; for the expected difference of 3%, we would need a sample size of 118 (59 per arm) and for 2.5% a sample size of 170 (85 per arm); however, the difference of 5% or more is likely to be meaningful. There is no consensus on the dose of zinc supplementation; we used a uniform dose across age groups. The dose used by us is comparable with that used in studies in adults. We did not study the long-term effects of zinc supplementation; study in adults with zinc supplementation for 18 months has shown to affect immunologic failure.23 Majority of the children in the study received stavudine-based regimen, as was recommended in the national program. Stavudine is associated with lipoatrophy; however, the same typically occurs after 12 months of therapy.29,30
We conclude that supplementation of 20 mg zinc daily for 24 weeks did not have any statistically significant effect on the increase in CD4%, decrease in viral load, anthropometric indices, and morbidity profile at 12 and 24 weeks in HIV-infected children started on ART. It is likely that zinc deficiency in the study subjects affected the results. It may be worthwhile to explore the effects of different doses of zinc in children categorized on the basis of zinc status in a larger study.
The authors thank the Bharat Immunologicals and Biologicals Corporation Limited, a Government of India Undertaking, Bulandshahr, Uttar Pradesh, for supplying the zinc and placebo tablets.
1. Lederman HM, Williams PL, Wu JW, et al.; AIDS Clinical Trials Group 889 Study Team. Incomplete immune reconstitution after initiation of highly active antiretroviral therapy in human immunodeficiency virus-infected patients with severe CD4+ cell depletion. J Infect Dis. 2003;188:1794–1803.
2. Connick E, Lederman MM, Kotzin BL, et al.. Immune reconstitution in the first year of potent antiretroviral therapy and its relationship to virologic response. J Infect Dis. 2000;181:358–363.
3. Puthanakit T, Oberdorfer A, Akarathum N, et al.. Efficacy of highly active antiretroviral therapy in HIV-infected children participating in Thailand's National Access to Antiretroviral Program. Clin Infect Dis. 2005;41:100–107.
4. van Leth F, Phanuphak P, Ruxrungtham K, et al.; 2NN Study team. Comparison of first line antiretroviral therapy with regimens including nevirapine, efavirenz, or both drugs, plus stavudine and lamivudine: a randomized open-label trial, the 2NN study. Lancet. 2004;363:1253–1263.
5. Koletar SL, Williams PL, Wu J, et al.; AIDS Clinical Trials Group 362 Study Team. Long-term follow-up of HIV-infected individuals who have significant increases in CD4 cell counts during antiretroviral therapy. Clin Infect Dis. 2004;39:1500–1506.
6. Prasad AS, Meftah S, Abdallah J, et al.. Serum thymulin in human zinc deficiency. J Clin Invest. 1988;82:1202–1210.
7. Shankar AH, Prasad AS. Zinc and immune function: the biological basis of altered resistance to infection. Am J Clin Nutr. 1998;68:447S–463S.
8. Friis H, Goma E, Michaelson KF. Micronutrient interventions and the HIV pandemic. In: Friis H, ed. Micronutrients and HIV Infection. Boca Raton, FL: CRC Press; 2002:219–246.
9. Baum MK, Shor-Posner G, Lai S, et al.. High risk of HIV-related mortality is associated with selenium deficiency. J Acquir Immune Defic Syndr Hum Retrovirol. 1997;15:370–374.
10. Tang AM, Graham NM, Saah AJ. Effects of micronutrient intake on survival in human immunodeficiency virus type 1 infection. Am J Epidemiol. 1996;143:1244–1256.
11. Kupka R, Fawzi W. Zinc nutrition and HIV infection. Nutr Rev. 2002;60:69–79.
12. Mda S, van Raaij JM, de Villiers FP, et al.. Short-term micronutrient supplementation reduces the duration of pneumonia and diarrheal episodes in HIV-infected children. J Nutr. 2010;140:969–974.
13. Neves I Jr, Bertho AL, Veloso VG, et al.. Improvement of the lymphoproliferative immune response and apoptosis inhibition upon in vitro treatment with zinc of peripheral blood mononuclear cells (PBMC) from HIV+ individuals. Clin Exp Immunol. 1998;111:264–268.
14. Kapil U, Jain K. Magnitude of zinc deficiency amongst under five children in India. Indian J Pediatr. 2011;78:1069–1072.
15. Ndeezi G, Tumwine JK, Bolann BJ, et al.. Zinc status in HIV infected Ugandan children aged 1-5 years: a cross sectional baseline survey. BMC Pediatr. 2010;10:68.
16. Ndagije F, Baribwira C, Coulter JB. Micronutrients and T-cell subsets: a comparison between HIV-infected and uninfected, severely malnourished Rwandan children. Ann Trop Paediatr. 2007;27:269–275.
17. Thakur S, Gupta N, Kakkar P. Serum copper and zinc concentrations and their relation to superoxide dismutase in severe malnutrition. Eur J Pediatr. 2004;163:742–744.
18. Bobat R, Coovadia H, Stephen C, et al.. Safety and efficacy of zinc supplementation for children with HIV-1 infection in South Africa: a randomised double-blind placebo-controlled trial. Lancet. 2005;366:1862–1867.
19. Bunupuradah T, Ubolyam S, Hansudewechakul R, et al.; PREDICT study group. Correlation of selenium and zinc levels to antiretroviral treatment outcomes in Thai HIV-infected children without severe HIV symptoms. Eur J Clin Nutr. 2012;66:900–905.
20. Ndeezi G, Tylleskär T, Ndugwa CM, et al.. Effect of multiple micronutrient supplementation on survival of HIV-infected children in Uganda: a randomized, controlled trial. J Int AIDS Soc. 2010;13:18.
21. Irlam JH, Visser MM, Rollins NN, et al.. Micronutrient supplementation in children and adults with HIV infection. Cochrane Database Syst Rev. 2010;CD003650.
22. Zeng L, Zhang L. Efficacy and safety of zinc supplementation for adults, children and pregnant women with HIV infection: systematic review. Trop Med Int Health. 2011;16:1474–1482.
23. Baum MK, Lai S, Sales S, et al.. Randomized, controlled clinical trial of zinc supplementation to prevent immunological failure in HIV-infected adults. Clin Infect Dis. 2010;50:1653–1660.
24. Guidelines for HIV care and treatment in Infants and Children. 2006. Published by IAP and NACO. Available at: www.nacoonline.org
. Accessed September 19, 2009.
25. Thakar MR, Abraham PR, Arora S, et al.. Establishment of reference CD4+ T cell values for adult Indian population. AIDS Res Ther. 2011;8:35.
26. Hambidge KM, King JC, Kern DL, et al.. Pre-breakfast plasma zinc concentrations: the effect of previous meals. J Trace Elem Electrolytes Health Dis. 1990;4:229–231.
27. Wamalwa DC, Farquhar C, Obimbo EM, et al.. Early response to highly active antiretroviral therapy in HIV-1-infected Kenyan children. J Acquir Immune Defic Syndr. 2007;45:311–317.
28. Pakasi TA, Karyadi E, Suratih NM, et al.. Zinc and vitamin A supplementation fails to reduce sputum conversion time in severely malnourished pulmonary tuberculosis patients in Indonesia. Nutr J. 2010;9:41.
29. Caron-Debarle M, Lagathu C, Boccara F, et al.. HIV-associated lipodystrophy: from fat injury to premature aging. Trends Mol Med. 2010;16:218–229.
30. Palmer M, Chersich M, Moultrie H, et al.. Frequency of stavudine substitution due to toxicity in children receiving antiretroviral treatment in sub-Saharan Africa. AIDS. 2013;27:781–785.