Zinc, Vitamin A, and Micronutrient Supplementation in Children withDiarrhea: A Randomized Controlled Clinical Trial of Combination Therapy

versus Monotherapy

ath Naik, PhD, Krishnan Rajendran, PhD,

diarrhea and moderate dehy itamin A (group 1), zinc plusother micronutrients plus vita o (group 4) as an adjunct toUNICEF United Nations Childrens Fundoral rehydration solution. Duration, volume of diarrhea, and consumption of oral rehydration solution were com-pared as outcome variables within the supplemented groups and with the placebo group.Results The 167 study subjects included 41 in group 1, 39 in group 2, 44 in group 3, and 43 in group 4. All 3 sup-plemented groups demonstrated a significant reduction in outcome variables (P < .0001) compared with the pla-cebo group. Group 3 had the lowest reduction of outcome variables and group 2 had a speedy recovery, butdifferences among the supplemented groups were not statistically significant.Conclusions Supplementation with a combination of micronutrients and vitamins was not superior to zinc alone,confirming the clinical benefit of zinc in children with diarrhea. (J Pediatr 2011;159:633-7).

Childhood malnutrition and diarrhea are common in developing countries and responsible for a high proportion ofdeaths in children.1 Substantial progress has been made in the treatment of diarrhea in children, with the introductionof reduced-osmolarity oral rehydration solution (ORS) and zinc supplementation supported by strong scientific evi-

dence.2-8 At present, the World Health Organization (WHO) and United Nations Childrens Fund (UNICEF) jointly recom-mended zinc supplementation for children with diarrhea.9 Despite strong supportive evidence, zinc has not yet been globallyaccepted as a therapeutic agent.10,11

Vitamin A deficiency is a major public health problem in developing countries.12 Vitamin A supplementation trials havedocumented reduced severity, duration, and even mortality due to diarrhea.13,14 Deficiencies of copper, iron, folate, vitaminB12, and selenium are also common in children of most developing countries and are responsible for increased severity ofinfection, inflammatory lesions, and reduced antibody response.15-18 The therapeutic effect of combined supplementationof these micronutrients and vitamins has not yet been studied, however.We evaluated the therapeutic impact of supplementation with zinc, zinc plus vitamin A, and a combination of micronu-

trients and vitamins (ie, zinc, iron, copper, selenium, vitamin B12, folate and vitamin A) on diarrhea in children. Our primaryhypothesis was that combined supplementation with micronutrients and vitamins might have a better therapeutic effect com-pared with supplementation with zinc alone. All micronutrient deficiencies could then be corrected simultaneously.

Methods

We conducted a hospital-based, double-blind, randomized, placebo-controlled clinical trial at the Dr B. C. RoyMemorial Hos-pital for Children, Kolkata, India betweenMarch 1999 andMay 2001. The study was not registered in the clinical trial registry asthe study was intra-mural in nature and at the time of initiation of the study registry as not mandatory. Male children were cho-sen for ease of separate collection of stool and urine samples. The children ranged in age from 6 to 24months and had a history ofacute watery diarrhea (more than 3 episodes within the last 24 hours) of less than72 hours duration. All of the children had moderate dehydration, manifested by

From the National Institute of Cholera and EntericDiseases, Kolkata, India (P.D., U.M., S.D., T.N., K.R.);and Department of Pediatric Medicine, Dr. B.C. RoyMemorial Hospital for Children, Kolkata, India (M.C.)

Supported by the Indian Council of Medical Research, anintramural project of the National Institute of Cholera andEnteric Diseases, Kolkata, India. The authors declare noconflicts of interest.

HIV Human immunodeficiency virus

ORS Oral rehydration salt solution

RDA Recommended daily allowanceWHO World Health Organizatiodration were randomized to receive zinc plus placebo vmin A (group 2), zinc plus vitamin A (group 3), or placeband Mrinal Kanti Chatterjee, MD

Objective Tocompare the clinical efficacyof supplementationof zinc, zinc plus vitaminA, and zinc plus combinationofmicronutrients and vitamins (iron, copper, selenium, vitamin B12, folate, and vitamin A) on acute diarrhea in children.Study design This was a double-blind, randomized, placebo-controlled trial. Children aged 6 to 24 months withPhalguni Dutta, PhD, Utpala Mitra, MBBS, Shanta Dutta, MD, PhD, Trailokya N0022-3476/$ - see front matter. Copyright 2011 Mosby Inc.All rights reserved. 10.1016/j.jpeds.2011.03.028

n

633

THE JOURNAL OF PEDIATRICS www.jpeds.com Vol. 159, No. 4clinical signs and symptoms of thirst: irritability; sunken eyes;dry mouth, lips, and tongue; and loss of skin elasticity.The sample size was calculated under the assumption that

the average duration of diarrhea in the supplemented groupswill be significantly lower (25%) compared with a meanduration of 66.4 32.3 hours after initiation of standardtreatment.19 Considering a 5% level of significance, 80%power, and 10% dropout, the minimum sample size was cal-culated as 42 children in each of the 4 groups. However, forfear of more dropouts, we randomized 44 children in eachgroup from a total of 176 children.Children clinically diagnosed with severe undernutrition

(wasting) or another systemic illness (eg, septicemia, pneu-monia, urinary tract infection, otitis media) or chronic un-derlying disease (eg, tuberculosis, liver diseases) or needingintensive care (eg, life support system, blood transfusion, totalparental nutrition) were excluded from the study. Childrenwho were exclusively breast-fed also were excluded. Childrenwho had received antibiotics before enrollment or received vi-tamin A supplementation within the previous 6 months wereexcluded. The childrens human immunodeficiency virus(HIV) status was not assessed, because HIV screening is notroutinely done in children with acute diarrhea. Moreover,theHIVprevalence in the catchment population of this regiondid not warrant routine HIV testing of the study population.Children who fulfilled the inclusion and exclusion criteria

were randomized to 4 treatment groups according to a ran-dom number table. Randomization was done blindly and in-dependently to allocate a patient to specific numbered bottleof supplementation or placebo. The serial code numbers werekept in a sealed envelope, and the groups were identified onlyafter study completion. The children in group 1 received 20mg elemental zinc (twice the Recommended Daily Allowance[RDA]) daily and a single oral dose of placebo of vitamin A onadmission. Group 2 children received micronutrient combi-nation (twice the RDA of all micronutrients and vitamins:zinc, 20 mg; iron, 10 mg; copper, 2 mg; selenium, 40 mg;vitamin B12, 1.4 mg; folate, 100 mg) daily and a single oraldose of vitamin A on admission following national guidelines(age 1 year, 200 000 IU). Group 3 chil-dren received 20 mg of elemental zinc (twice the RDA) dailyand a single oral dose of vitamin A on admission accordingto national guidelines. Group 4 children received placebo ofmicronutrients and vitamins and a single oral dose of placeboof vitamin A on admission. Micronutrients, vitamins, or pla-cebo were given in 2 daily doses for 14 days even after recov-ery. All of the supplemental micronutrients and placebo werein syrup form with a similar taste and appearance. They wereprepared by Greenco Biologicals (Kolkata, India) accordingto our specifications and packaged in identical-looking bot-tles. VitaminA and its placebowere prepared in samemanner.This study was approved by the Scientific Advisory Com-

mittee and Institutional Ethics Committee of the National In-stitute of Cholera and Enteric Diseases in Kolkata, India.Before enrollment, informed written consent was obtainedfrom the parents of each child after the study procedurewas described in detail. A complete clinical history was634obtained from the parents, and a thorough physical examina-tion was done. Children were weighed unclothed on a scalewith a sensitivity of 1 g. Length and mid-arm circumferencealso were measured. Stool samples were collected on admis-sion in sterile McCartney bottles, using sterile rectal cathetersand were processed for detection of established enteropatho-gens, including bacteria, viruses, and parasites, usingstandard procedures. Children received the WHO-recommended reduced-osmolarity ORS for correction ofinitial dehydration and as maintenance therapy, matchingthe stool volume and loss in vomitus until cessation of di-arrhea.20 Intravenous fluid (ie, Ringers lactate solution)was available for a child who developed severe dehydrationor intractable vomiting during the hospital stay, in accor-dance with WHO guidelines.20 No children received anydrug therapy during the study period.Immediately after correction of initial dehydration, feed-

ing was resumed in all children. Breast-fed children wereallowed to continue breast-feeding; others were allowed totake formula milk or animal milk. Older children continuedon their normal diet before the onset of illness. Plain waterwas offered. Children were followed up in the hospital untilrecovery or for 5 days if recovery did not occur within thattime period. Intake and output data were measured every 8hours and recorded.The duration of diarrhea was calculated as hours from the

passage of the last liquid stool. Recovery was defined as pas-sage of soft stool, formed stool, or no stool for 18 hours. Stoolloss was measured using preweighed disposable diapers ona scale with a sensitivity of 1 g. Urine was separated from stoolusing a urine collection bag. Vomitus was weighed using pre-weighed gauze pads. Body weight was recorded after correc-tion of initial dehydration and then every morning between10 and 10:30 a.m. until recovery. The following measure-ments were recorded daily: number of stools, number ofvomiting episodes, stool output (g), intake of ORS (mL),intake of plain water (mL), intake of liquid food (mL), intakeof intravenous fluid (mL) if required for correction of dehy-dration, and body weight (g). Children were discharged fromthe study after the passage of formed stool. Mothers were ad-vised at the time of discharge to bring their children to thehospital if they developed complications.The checklist of treatment assignment was decoded for the

experimental groups. The 4 study groups were comparedbased on clinical characteristics on admission and isolationof enteropathogens using the c2 test. The Mantel-Haenszelc2 test was used for testing recovery rates. One-way analysisof variance post hoc testing was carried out to compare thequantitative measures of duration of diarrhea, volume ofstool output, and intake of ORS in the 4 treatment groups.Survival function analysis was used to assess the recoverystatus of the 4 groups.

Results

A total of 176 male children aged 6 to 24 months were ini-tially recruited into the study; however, 9 children droppedDutta et al

on numerous randomized controlled trials conducted by

Table I. Characteristics of children in the 4 study groups at t

ParametersGroup 1(n = 41)

Age, months 12.3 (4.4)Body weight, kg 6.9 (0.8)Length, cm 69.3 (5.1)MAC, cm 12.3 (0.9)Nutritional status (weight for length z score; WHO standard), n (%)

Normal ($1) 11 (26.8)Mild undernutrition (

zinc levels may not truly reflect zinc status, because levels mayfluctuate due to changes in diet, degree of hypoproteinaemia,acute infection, or inflammatory conditions.Some earlier studies failed to detect a beneficial effect of zinc

supplementation in patients with rotavirus and E coliassociated diarrhea.10,11,24,25 However, the present studydemonstrates the potential impact of zinc on diarrheawhen ro-

in patients receiving zinc plus vitamin A supplementationcompared with those receiving zinc supplementation alone.Children in developing countries are often malnourished

and have deficiencies of multiple micronutrients and vita-mins that necessitate supplementation.29 We expected tofind a better therapeutic effect with combined therapy, butfailed to document this effect. However, the trend toward

Table III. Outcome variables of the 4 study groups

ParameterGroup 1(n = 41)

Group 2(n = 39)

Group 3(n = 44)

Group 4(n = 43) P value

Patients recovered within 5 days, n (%) 38 (93) 39 (100) 43 (98) 29 (68)*

therapy with micronutrients and vitamins in children withdiarrhea. n

We thank Mr. P. K. Sinha, Managing Director, Greenco Biological,Kolkata, India, for preparing the study syrups and placebo used inthis trial.

Nutr Rev 2007;65:218-32.

13. Barreto ML, Santos LMP, Assis AMO, Araujo MP, Farenzena GH,

Santos PAB. Effect of vitamin A supplementation on diarrhoea and acute

lower respiratory tract infections in young children in Brazil. Lancet

1994;344:228-31.

14. Fawzi WW, Mbise R, Fataki M, Hertzmark E, Ndossi G, Spiegelman D,

et al. Vitamin A supplements and diarrheal and respiratory tract infections

among children in Dar es Salaam, Tanzania. J Pediatr 2000;137:660-7.

15. Beisel WR. Single nutrients and immunity. Am J Clin Nutr 1982;35:417-

October 2011 ORIGINAL ARTICLES12. United Nations Administrative Committee on Coordination/ Sub-

Committee on Nutrition in collaboration with International Food Pol-

icy Research Institute (IFPRI). 4th Report on the World Nutrition

Situation - Nutrition throughout the Life Cycle. Geneva: ACC/SCN

publications Jan 2000.Submitted for publication Aug 19, 2010; last revision received Jan 19, 2011;

accepted Mar 18, 2011.

Reprint requests: Phalguni Dutta, PhD, National Institute of Cholera and

Enteric Diseases, P33 CIT Road, Scheme XM, Beliaghata, Kolkata-700010,

India. E-mail: drpdutta@yahoo.com

References

1. World Health Organization. The global burden of disease: 2004 update.

Geneva, Switzerland: World Health Organization; 2008.

2. World Health Organization, United Nations Childrens Fund, US Agency

for International Development, JohnsHopkins Bloomberg School of Pub-

lic Health. Implementing the new recommendations on the clinical man-

agement of diarrhoea: guidelines for policy makers and programme

managers. Geneva, Switzerland: World Health Organization; 2006.

3. Roy SK, Tomkin AM, Akramuzzaman SK, Behrens MH, Haider RH,

Mahalanabis D, et al. Randomized controlled trial of zinc supplementa-

tion in malnourished Bangladeshi children with acute diarrhea. Arch Dis

Child 1997;77:196-200.

4. Sachdev HPS,Mittal NK,Mittal SK, Yadav HS. A controlled trial on util-

ity of oral zinc supplementation in acute dehydrating diarrhoea in in-

fants. J Pediatr Gastroenterol Nutr 1988;7:877-81.

5. Dutta P, Mitra U, Datta A, Niyogi SK, Dutta S, Manna B, et al. Impact of

zinc supplementation in malnourished children with acute watery diar-

rhoea. J Trop Pediatr 2000;46:259-63.

6. Bahl R, Baqui A, BhanMK, Bhatnagar S, Black RE, Brooks A, et al. Effect

of zinc supplementation on clinical course of acute diarrhea. J Health

Popul Nutr 2001;19:338-46.

7. Bhatnagar S, Bahl R, Sharma PK, Kumar GT, Saxena SK, BhanMK. Zinc

with oral rehydration therapy reduces stool output and duration of di-

arrhea in hospitalized children: a randomized controlled trial. J Pediatr

Gastroenterol Nutr 2004;38:34-40.

8. Bhutta ZA, Bird SM, Black RE, BrownKH, Gardner JM, Hidayat A, et al.,

Zinc Investigators Collaborative Group. Therapeutic effects of oral zinc

in acute and persistent diarrhea in children in developing countries:

pooled analysis of randomized controlled trials. Am J Clin Nutr 2000;

72:1516-22.

9. WHO/UNICEF Joint statement. Clinical management of acute diarrhea.

2004; WHO/FCH/CAH/04.7. World Health Organization, Geneva.

10. Patel A, Mamtani M, Dibley MJ, Badhoniya N, Kulkarni H. Therapeutic

value of zinc supplementation in acute and persistent diarrhea: a system-

atic review. PLoS ONE 2010;5:e10386.

11. Long KZ, Rosado JL, Fawzi W. The comparative impact of iron, the B

complex vitamins, vitamin C, and E, and selenium on diarrheal patho-

gen outcomes relative to the impact produced by vitamin A and zinc.Zinc, Vitamin A, and Micronutrient Supplementation in Children wTrial of Combination Therapy versus Monotherapy68.

16. Thurnham DI. Micronutrients and immune function: some recent de-

velopments. J Clin Pathol 1997;50:887-91.

17. Goodman KI, Salt WB, 2nd Vitamin B12 deficiency: important new

concepts in recognition. Postgrad Med 1990;88:147-50. 153-8.

18. Beckett GJ, Arthur JR, Miller S, McKenzie RC. Selenium. In: Hughes A,

Bendich A, Darlington G, eds. Dietary enhancement of human immune

function. Totowa, NJ: Humana Press; 2004. p. 217-40.

19. Dutta P, Mitra U, Manna B, Niyogi SK, Roy K, Mondal C, et al.

Double-blind, randomised controlled clinical trial of hypo-osmolar

oral rehydration salts solution in dehydrating acute diarrhoea in se-

verely malnourished (marasmic) children. Arch Dis Child 2001;84:

237-40.

20. World Health Organization. The treatment of diarrhea: A manual for

physicians and other senior health workers4th rev of doc. WHO/

CDD/SER/80.2. WHO 2005 ISBN 92 4 159318 0 World Health Organi-

zation, Geneva.

21. Bhandari N, Mazumder S, Taneja S, Dube B, Black RE, Fontaine O, et al.

A pilot test of the addition of zinc to the current case management pack-

age of diarrhea in a primary health care setting. J Pediatr Gastroenterol

Nutr 2006;42:253-5.

22. Bhandari N, Mazumder S, Taneja S, Dube B, Agarwal RC,

Mahalanabis D, et al. Effectiveness of zinc supplementation plus oral re-

hydration salts compared with oral rehydration salts alone as a treatment

for acute diarrhea in a primary care setting: a cluster randomized trial.

Pediatrics 2008;121:e1279-85.

23. Awasthi S, INCLEN Childnet Zinc Effectiveness for Diarrhea (IC-ZED)

Group. Zinc supplementation in acute diarrhea is acceptable, does not

interfere with oral rehydration and reduces the use of other medications:

a randomized trial in five countries. J Pediatr Gastroenterol Nutr 2006;

42:300-5.

24. Patel A, Dibley MJ, Mamtani M, Badhoniya N, Kulkarni H. Zinc and

copper supplementation in acute diarrhea in children: a double-blind

randomized controlled trial. BMC Med 2009;7: doi: 10.1186/1741-

7015-7-22.

25. Patel A, Dibley MJ, Mamtani M, Badhoniya N, Kulkarni H. Influence of

zinc supplementation in acute diarrhea differs by the isolated organism.

Int J Pediatr 2010; 2010: doi:10.1155/2010/671587.

26. Gopalan C, Sastri BVR, Balasubramanian SC. Nutritive Value of Indian

Foods (1985), Revised and Updated (1989), by Narasinga Rao BS, Pant

KC, Deosthale YG (Reprinted 2000). ICMR publication, Ansarinagar,

New Delhi, India.

27. Varlery PC, Torzillo PJ, Boyce NC,White AV, Stewart PA, Wheaton GR,

et al. Zinc and vitamin A supplementation in Australian indigenous chil-

dren with acute diarrhea: a randomized controlled trial. Med J Aust

2005;182:530-5.

28. Rahman MM, Vermund SH, Wahed MA, Fuchs GJ, Baqui AH,

Alvarez JO. Simultaneous zinc and vitamin A supplementation in Ban-

gladeshi children: randomized double-blind controlled trial. BMJ

2001;323:314-8.

29. Fuchs GJ. Possibilities for zinc in the treatment of acute diarrhea. Am J

Clin Nutr 1998;68(Suppl):S480-3.ith Diarrhea: A Randomized Controlled Clinical 637

Zinc, Vitamin A, and Micronutrient Supplementation in Children with Diarrhea: A Randomized Controlled Clinical Trial of Com ... Methods Results Discussion References