December 2007 Vol 8 After Meeting.indd Effect of Zinc Supplementation on Morbidity due to Acute Diarrhoea in Infants and Children in Sanaa, Yemen A randomized controlled double blind clinical trial *Muna A M Elnemr1 and Ahmed K Abdullah2 عند احلاد االسهال املراضة املصاحبة حلاالت على الزنك امداد تأثير (اليمن) صنعاء في �األطفال التعمية مزدوجة العشوائية مضبطة سريرية دراسة اهللا عبد قائد أحمد ، النمر محمد عبده منى دراسة الطريقة: هذه ــة. اخلامس ــن س األطفال دون لدى احلاد ــهال لإلس املصاحبة والوفيات املراضة على الزنك إعطاء تأثير ــة امللخص: الهدف: دراس لألمومة والطفولة السابني ــفى مستش 2006 في 2005 وأكتوبر ــبتمبر س الفترة بني أجريت في ، التعمية ــوائية مزدوجة العش مضبطة ــريرية س إلى ــوائيا عش احلاالت ــيم مت تقس ــهرا. 6-48ش بني أعمارهم تراوحت احلاد، ــهال باإلس أصيبوا ــة) اخلامس ــن س (دون 180طفال باليمن ل في صنعاء ملدة احلاالت متابعة ومتت كاذبا، عقارا الثانية مت إعطاء اموعة بينما ــبوعني أس ملدة بالفم الزنك ــيتيت اس محلول األولى اموعة أعطيت مجموعتني العينة 2:59 في مقابل الزنك أخذوا الذين األطفال 1.39 عند اإلسهال عدد نوبات متوسط من قلل الزنك إعطاء النتائج أن النتائج: أظهرت شهرين. كما هو الضابطة) العينة ــي 5.47 ف مقابل الزنك أخذت التي العينة (3.57 في ــهال كل نوبة إس في باليوم ــل مرات التغوط ــك قل كذل ــة. الضابط نوبات من حدوث قلل اإلسهال بنوبات اإلصابة ــبوعني أثناء أس ملدة الزنك اخلالصة: إعطاء املتابعة. مدة في ــهال نوبة إس في كل البراز حجم في احلال لسهولة وذلك كبيرة فائدة ذو يعتبر الزنك باستعمال احلاد ــهال لإلس املراضة املصاحبة معدالت تقليل إن هذه النوبات. ــدة وكذلك قلل ش ــهال اإلس األطفال. إسهال على للسيطرة يستعمل أن ميكن ولهذا ، ثمنه ولرخص استعماله ، اليمن. ، إسهال الكلمات: زنك مفتاح SULTAN QABOOS UNIVERSITY MEDICAL JOURNAL DECEMBER 2007 VOL 7, NO. 3, P. 219-225 SULTAN QABOOS UNIVERSITY© SUBMITTED - 1ST APRIL 2007 ACCEPTED - 29TH JULY 2007 1Department of Pediatrics, Faculty of Medicine, University of Science and Technology, Sana’a, Yemen; 2Department of Physiology, Faculty of Medi- cine, Thamar University, Sana’a, Yemen *To whom correspondence should be addressed. Email: munaabdo@hotmail.com ABSTRACT Objectives: To study the impact of zinc administration on the morbidity and mortality attributed to diarrhoea among children less than 5 years old. Methods: The study design was a randomized double blinded controlled clinical trial, held at Elsabeen Hospital for Maternity and Childhood, Sana’a, Yemen. The study was conducted during the period September 2005 to October 2006 on 80 children less than 5 years old with acute diarrhoeal episodes. They were randomly allocated to two groups; one of them received a placebo and the other received zinc acetate syrup for 4 days. Both groups were followed up for 2 months. Results: Zinc was able to decrease the mean number of diarrhoeal episodes: .39 in the intervention group versus 2.59 in the control group. It also reduced the mean frequency of stools per day in each attack (3.57 in the intervention group versus 5.47 in the control group) and the volume of stool in each attack during the follow-up period. Moreover, zinc was significantly more palatable. Conclusion: We can conclude from the study that administration of zinc for two weeks during acute diarrhoeal episodes could decrease the incidence of further diar- rhoeal episodes as well as the severity of these episodes. The lower rates of child morbidity with zinc treatment represent substantial benefits from a simple and inexpensive intervention that can be incorporated in existing efforts to control diarrhoeal disease. Key Words: Zinc; Diarrhoea; Children; Yemen. C L I N I C A L A N D B A S I C R E S E A R C H M U N A A B D O M U H A M M E D E L N E M R A N D A H M E D K A I D A B D U L L A H 220 Diarrhoea represents a leading cause of under 5 mortality in developing countries, includ-ing Yemen and many other countries of the Eastern Mediterranean.1 Overall under 5 mortality was estimated by World Health report to be 113 per 100,000 live births of which diarrhoea accounted for 17%, while that of measles represented only 4% and that of malaria 3%. Hence, reducing the burden of diarrhoea in these countries will significantly reduce overall mortality in such countries. Zinc deficiency is highly prevalent among children in developing countries.2 Diarrhoea causes loss of zinc in the stools, which exacerbates the zinc deficiency in children with acute diarrhoea. Provi- sion of zinc during diarrhoea is thus rationalized.3 Therapeutic studies of giving zinc during diarrhoea proved that zinc administration during and for 14 days from the onset of acute attack will reduce the sever- ity of diarrhoea.4 Moreover, zinc therapy for diarrhoea has been shown to be beneficial in controlled trails, in reducing the need for antibiotic therapy and increasing the use of ORT (oral rehydration therapy).5 In anoth- er study in 2004, Baqui et al. found that zinc sulphate has an antimicrobial effect on the enteric pathogens in vitro.6 Unlike other essential micronutrients such as iron and vitamin A, there are no conventional tissue reserves of zinc that can be released or sequestered quickly in response to variations in dietary supply. It is recognized that the equivalent of approximately one third (~450 mg) of total body zinc exchanges between the blood stream and other tissues.7 The major source of zinc in- take is through diet, with the transcellular uptake oc- curring in the distal duodenum and proximal jejunum, potentially facilitated by specific transporters, such as zinc transporter protein-1 (ZnTP-1).8 The intestine also serves as the major conduit for zinc elimination from the body with almost 50% of the daily zinc losses oc- curring in the gut. However, much of the zinc that is secreted into the intestine is subsequently reabsorbed and this process serves as an important point of regu- lation of zinc balance. Other routes of zinc excretion include the urine, which accounts for approximately 15% of total zinc losses, and epithelial cell desquama- tion, sweat, semen, hair, and menstrual blood, which together account for approximately 17% of total zinc losses.9 The mechanism by which zinc (Zn) improves diarrhoea is not known but could result from the fact that Zn inhibits cAMP-induced Cl secretion by block- ing basolateral membrane K channels. Given the high prevalence of micronutrient deficiencies and infectious diseases in children of developing coun- tries, interventions to reduce infant and pre-school morbidity are a public health priority.10 M E T H O D S This study was conducted in the Oral Rehydration Therapy Centre at Elsabeen Teaching Hospital in Sana’a,Yemen. The following criteria were used to include subjects in the study: infants and children with acute diarrhoeal episodes aged 3 months to 2 years of both sexes either admitted to the medical ward or not. The following subjects were excluded from this study: infants with malnutrition (body weight <60% of the median for age and sex and/or stunting (low height for age ) or wasting (low weight for height) accord- ing to the 1995 WHO Standard Deviation Classifica- tion (The Use and Interpretation of Anthropometry , Geneva,1995). Infants with co-infections were also ex- cluded as well as those with bloody diarrhoea. The study was a prospective randomized double blinded controlled clinical trial. Sample size was calcu- lated according to the suitable formula: employing the EpiInfo package using an expected reduction of mortal- ity and morbidity of 20%. The confidence interval was 95%; power of the test 90%; unexposed: exposed ratio: Advances in Knowledge In contrast to previous studies, this is the first study in Yemen to utilise a special liquid preparation containing only zinc (dose = 20mg/ 5ml) with an acceptable taste. This study supports the efficacy of zinc in acute diarrhoea among children under five years. Applications to Patient Care Efficacy of zinc as an agent that could reduce the recurrence and duration of episodes of diarrhoea in children less than five years which, in turn, implies that zinc supplementation could be used routinely for every child with acute diarrhoea as a prophylactic measure. E F F E C T O F Z I N C S U P P L E M E N TAT I O N O N M O R B I D I T Y D U E T O A C U T E D I A R R H O E A I N I N FA N T S A N D C H I L D R E N I N S A N A A , YE M E N 221 1:1. Disease in unexposed: 30% (in Yemen); Risk Ratio: .33. Using the sample size collection of EpiInfo 2000, the number of children was 100 per group. The total number of children was 200. The following sampling technique was used: the children were randomly allocated into two groups as soon as the enrolment was completed. The randomiza- tion was undertaken by blinded trained co-investiga- tors in the study location and by the principal investi- gators. The control group received ORT and advice to the mothers on feeding during diarrhoeal episodes. The intervention group received zinc acetate 20mg daily in the form of Diazar syrup from Indimedica Pharmaceu- ticals, India, which was undistinguishable from the pla- cebo bottles. Treatment began as soon as the episode of diarrhoea started and continued for 14 days, in addi- tion to ORT and the advice to the parents. The data collectors taught the mothers how to use the zinc syrup, and visited or called on the mother or the care taker at day 7 and day 14 and then every 2 weeks for 2 months. Children and infants eligible for the study were enrolled during the acute attack of di- arrhoea. Each infant and child in the first episode was submitted to the following: 1. A detailed and complete clinical history including previous diarrhoeal episodes and their duration; 2. An evaluation using a questionnaire designed to collect: personal data; regular follow up of the acute episodes and the subsequent attacks; adher- ence to the zinc therapy and occurrence of death and 3. Complete clinical examination with special atten- tion to nutritional status, dehydration status and chest examination. Both groups were followed by observing and reporting the different outcomes mentioned in the record data form: name, age, sex, residency, weight, height, date of the first episode and its duration and frequency of stools, acceptability of the zinc medication, adherence to the treatment, as well as the number of episodes, amount and frequency in each episode, the duration of hospital stay, if any, and death and its cause, if any. It was very difficult to measure the amount of each mo- tion so that we trained the data collectors and the par- ents to approximate the amount. Training was given to the care givers and data collectors to approximate the amount of stools per motion according to accommoda- tion of the diaper to each motion. ST UDY D E FI N I TI O N S An acute diarrhoeal episode was defined as three or more loose, liquid stools or at least one loose stool containing blood in a 24 hour period. An acute diar- rhoeal episode, which led to inclusion of the patient, was defined as one that started during the 48 hours before the visit to the centre. Resolution is defined as three consecutive days free from disease. The diagnosis of under nutrition was based on the WHO’s The Use and Interpretation of Anthropometry, which uses us- ing weight for age, weight for height and height for age. Stunting was defined as height for age <2 standard de- viations, while wasting was defined as weight for height < 2 standard deviations and under weight as weight for age < 2 standard deviations. Figure 1: Mean number of episodes, mean fre- quency of stool in these attacks, and mean dura- tion of hospital stay during follow up. Figure 2: Average amount of stools per motion during follow up period M U N A A B D O M U H A M M E D E L N E M R A N D A H M E D K A I D A B D U L L A H 222 STATI STI C A L A N A LY SI S Data were collected and analysed using the SSPS pro- gramme for data analysis, and appropriate tests were used to test significant differences; p value of less than 0.05 was considered significant. E TH I C S The ethical review committees of the hospital approved the study procedures. Because this was a community based treatment trial, we obtained consent from par- ents that explained the purpose of the study and the potential risks and benefits of the new treatment. We obtained verbal or written informed consent for data collection (depending on education of the parent) from parents of both the intervention and comparison group children. Verbal consent was taken from the few who were illiterate or where the data collectors did not take the written consent in the first visit. The majority pro- vided written consent. R E SULTS In this double blind controlled randomized study, 180 children were enrolled with 92 in the control group and 88 in the intervention group. Ten children were excluded from the study because of the discovery of underlying chronic diseases after enrolment and other ten children dropped out during the period of follow up due to difficult communication, 6 from the inter- vention group and the rest from the control group. The mean age of the children was14.10 months in the inter- vention group and 14.38 months in the control group. In the intervention group, 56 (63.6%) were males; 32 (36.4%) were females. In the control group, 51 (55.4%) were males and 41 (44.6%) were females. The two group were not statistical significant in term of gender. Mean weight and height among the enrolled chil- dren are illustrated in Table 1. It shows that there were no significant differences in the age, weight and height between the intervention and control groups. There were no significant differences in the mean duration of the initial attacks of diarrhoea (before start- ing intervention) between the two groups [Table 2]. The mean durations of the initial attack (in days) in both groups were not significant; for the interven- tion group = 6.62 ±1.369; for the control group = 6.43 ±1.181. The mean number of diarrhoeal episodes during the follow up period was significantly lower in the inter- vention group compared to the control group (Table 2 and Figure 1]. The mean frequency of motions in each Table 1: Distribution of enrolled children, both groups, according to age, sex, and height Table 2: Mean number of episodes, mean frequency of stool in these attacks and mean duration of hos- pital stay during follow up P valueStd. error Mean Std. deviationMeanTreatment group <0.051.0429.778months 14.10InterventionAge 1.187 11.383months 14.38control <0.050.24472.2953KG 8.167Interventionweight 0.6162 5.9102KG 8.776control <0.051.06399.9799cm 71.856InterventionHeight 1.1639 11.1639cm 71.217control Treatment group Mean Std. Deviation Std. Error mean p value Mean No. of episodes during follow up Intervention 1.39 1.077 0.115 <0.05 control 2.59 1.224 0.128 Mean frequency of motions in episodes during follow up intervention 3.57 2.116 0.226 <0.05 control 5.47 1.708 0.179 Mean Duration of hospital stay(day) Intervention 2.00 0.000 0.000 <0.05 Control 3.17 0.983 0.401 E F F E C T O F Z I N C S U P P L E M E N TAT I O N O N M O R B I D I T Y D U E T O A C U T E D I A R R H O E A I N I N FA N T S A N D C H I L D R E N I N S A N A A , YE M E N 223 episode during the period of follow up was significantly lower in the intervention group. Zinc was acceptable regarding palatability more than the control syrup, as shown in Table 3. The data collectors had trained the mothers to assess approximately the amount of stools; the results showed that Zinc was able to reduce the average amount of stools per motion during the follow up period as shown in Table 4 and Figure 2. On the whole, the present study suggests that zinc did not decrease the duration or the frequency of diar- rhoea in the initial attack. No mortality was reported during the study or subsequent the follow up period in either group. D I S C U S S I O N In our study population, 88 children received zinc sup- plementation during and after diarrhoea, with ages ranging from 3 months to four years. There was a downward trend in the incidence of further episodes of diarrhoea and in the mean frequency of motions in each episode during the follow up period in the zinc treatment group, but not in the comparison group, sug- gested a benefit of zinc. The reductions in duration of diarrhoea, although not significant, also suggest a ben- efit of zinc. In a randomized controlled study in India in 2002, 11zinc supplementation substantially reduced the in- cidence and severity of diarrhoea, the two important determinants of diarrhoea-related mortality and mal- nutrition. This intervention also substantially reduced the proportion of children who experienced recurrent diarrhoea. The reduction in the duration of diarrhoeal epi- sodes is consistent with earlier studies.12 A meta-analy- sis of five studies of zinc treatment for acute diarrhoea found a summary estimate for reduction in duration of 16%. Possible mechanisms for the effect of zinc treat- ment on the duration of diarrhoea include improved absorption of water and electrolytes by the intestine, 13 faster regeneration of gut epithelium, increased levels of enterocyte brush border enzymes, 14 and enhanced immune response, leading to early clearance of diar- rhoeal pathogens from the intestine. Several controlled trials have shown a preventive ef- fect of routine zinc supplementation on the incidence of diarrhoea15 and acute lower respiratory infection. However, these studies provided daily zinc supplemen- tation for a period of 6-12 months, which is often not feasible in large scale programmes. Three studies, in which zinc was given for two weeks during and after diarrhoea, found reductions in episodes of diarrhoea in the subsequent two to three months period without ad- ditional zinc.4, 11, 16 The same result were found in a pre- vious study, in which the zinc-supplementation group showed a decrease of 39 percent (95 percent confidence interval, 6 percent to 70 percent) in the mean number of watery stools per day (p = 0.02).17 Table 3: Acceptability of Zinc syrup and placebo to children in both groups Table 4: Average amount of stools per motion during follow up period Treatment group Yes No p value n % within group n % within group Total Acceptability Intervention 80 90.9% 8 9.1% 88 <0.05 Control 70 76.1% 22 23.9% 92 <0.05 Average Amount of stool per motion in attacks during follow up period Mild Moderate Large p value No. % within group No. % within group No % within group Intervention 64 73.6% 4 4.6% 0 O% < 0.05 Control 56 61.5% 22 24.2% 9 9.9% Total 120 67.4% 26 14.6% 9 5.1% M U N A A B D O M U H A M M E D E L N E M R A N D A H M E D K A I D A B D U L L A H 224 The lower rates of additional episodes of diarrhoea in this and other studies indicate that the reduction in incidence could be due to a systemic effect of zinc, probably through enhanced immune function. The re- duction in hospital admissions for diarrhoea could have been due to effects of zinc on episode duration, or re- duced incidence. An important aspect of our study is that it shows a reduction in the volume of each bowel motion in the subsequent episodes from the use of zinc as a treatment for diarrhoea, although in this study we depended on the care taker evaluation in determining the stool volume. Three acute-diarrhoea trials with ap- propriate outcome measures all found reductions in di- arrhoeal severity in zinc-supplemented children com- pared with control children. Of the two trials conducted in India, one found 18% fewer diarrhoeal stools per day (p < 0.1) 11 and the other found 39% fewer watery stools per day (p < 0.02). 15 In the only hospital-based trial of acute diarrhoea, zinc-supplemented children had a 28% lower measured diarrhoeal stool output per day (p = 0.06) 18. The trial conducted in India reported a 21% lower diarrhoeal stool frequency (p = 0.08).11 Two hos- pital-based trials in Bangladesh and India found a 37% lower measured stool output (p < 0.02) in zinc-supple- mented children.17, 19 In a study that investigated the effect of zinc on the duration of illness and the stool quantity in acute watery diarrhoea of infants aged less than 6 months in 2002, Zinc supplementation did not affect diarrhoea duration or stool volume in young infants, although young infants tolerated zinc doses.20 A recently published randomised trial from India found a large reduction in overall mortality in infants who were small for gestational age and supplemented daily with zinc from 1 to 9 months of age.11 In our study, there were no differences in mortality between the two groups and this may be due to a relatively small sam- ple. C O N C L U S I O N Zinc supplementation in this study substantially re- duced the subsequent incidence of severe diarrhoea and frequency of motions, the two important determinants of diarrhoea-related mortality and malnutrition. This intervention also substantially reduced the proportion of children who experienced recurrent diarrhoea. The effects are large enough to merit routine use of zinc during acute diarrhoea in developing countries. Prompt measures to improve the zinc status of de- ficient populations are warranted. The potential ap- proaches to achieve this goal include: food fortification; dietary diversification; cultivation of plants that are zinc dense or have a decreased concentration of zinc absorption inhibitors; zinc supplementation for select- ed groups of children. Future studies should assess the impact of increased zinc intakes on childhood mortality in developing coun- tries. For facilitating intervention, there is a need to ob- tain reliable estimates of zinc deficiency, particularly in developing countries. Moreover, longer duration of zinc administration after the initial attack should be studied, for example a duration of 3-4 months of daily zinc supplementation. The intervention we evaluated is simple and inexpensive and can be incorporated into existing diarrhoeal disease control efforts. A C K N OW L E D GE ME N TS This investigation received technical and financial sup- port jointly from WHO Eastern Meditranean Region (EMRO), Division of communicable Diseases (DCD), and the Special Programme for Research and Training in Tropical Diseases (TDR)). We also grateful to Professor Yeha Raja, Sanaa Uni- versity, Department of Community Medicine, for his help and support during statistical analysis. We would like also to thank with appreciation Dr Omar Ali Baash- our, paediatric specialist, Elsabeen Paediatric Teaching Hospital, Sanaa, and the rest of the paediatricians, gen- eral practitioners and nursing stuff in that hospital for their cooperation and support during the perod of data collection and follow up. R E F E R E N C E S 1. Sazawal S, Black RE, Menon VP, Dinghra P, Caulfield LE, Dinghra U, et al. Zinc supplementation in infants born small for gestational age reduces mortality: a prospective, randomized, controlled trial. Pediatrics 2001; 108:1280- 1286. 2. Sandest HH. Zinc deficiency : a public health problem. Am J Child 1991; 145:853-859. 3. Zinc investigators collaborative group.Therapeutic effect of oral zinc in acute diarrhoea. Am J Clin Nutr 2000; 72:1516-1522. 4. Ruel MT, Rivera JA, Santizo MC, Lonnerdal B, Brown KH. Impact of zinc supplementation on morbidity from diarrhoea and respiratory infections among rural Guate- malan children. Pediatrics 1997; 99:808-813. 5. Baqui AH, Black RE, El-Arifeen S, Yunus M, Zaman K, Begum N, et al. Zinc therapy for diarrhoea increased the use of oral rehydration therapy and reduced the use of E F F E C T O F Z I N C S U P P L E M E N TAT I O N O N M O R B I D I T Y D U E T O A C U T E D I A R R H O E A I N I N FA N T S A N D C H I L D R E N I N S A N A A , YE M E N 225 antibiotics in Bangladeshi children. J Health Popul Nutr 2004; 22:440-442. 6. Surjawidjaja, Hidajat A, Lesmana M. Growth inhibition of enteric pathogens by zinc sulphate: an in vitro study. Med Princ Pract 2004; 13:286-289. 7. Juyal R, Osmamy M, Black RE, Dhingra U, Sarkar A, Dh- ingra P, et al. Efficacy of micronutrient fortification of milk on morbidity in preschool children. Am J Clin Nutr 2005; 82:1032-1039. 8. Walker CF, Black RE. Zinc and the risk for infectios dis- ease. Ann Rev Nutr 2004; 24:255-275. 9. Fraker PJ, King LE, Laakko T, Vollmer TL. The dynamic link between the integrity of immune system and zinc status. Nutr 2000; 130:1399-1406. 10. Bhutta ZA. The Role of Zinc in health in developing countries. Indian Pediatr 2004; 41:429-433. 11. Roy SK, Tomkins AM, Akramuzzaman SM, Behrens RH, Mahalanabis D, Fuchs G. Randomised controlled trial of zinc supplementation in malnourished Bangladeshi chil- dren with acute diarrhoea. Arch Dis Child 1997; 77:196- 200. 12. Golden BE, Golden, MHN. Zinc, sodium and potas- sium losses in the diarrhoeas of malnutrition and zinc deficiency. In: Mills CF, Bremner I, Chesters JK, eds. Trace elements in man and animals TEMA 5. Aberdeen: Rowett Research Institute, 1985. p. 228-232. 13. Bettger WJ, O’Dell BL. A critical physiological role of zinc in the structure and function of biomembranes. Life Sci 1981; 28:1425–1438. 14. Ruel MT, Rivera JA, Santizo MC, Lonnerdal B, Brown KH. Impact of zinc supplementation on morbidity from diarrhoea and respiratory infections among rural Guate- malan children. Pediatrics. 1997; 99:808-813. 15. Sachdev HPS, Mittal NK, Mittal SK, Yadav HS. A con- trolled trial on utility of oral zinc supplementation in acute dehydrating diarrhea in infants. J Pediatr Gastro- enterol Nutr 1988; 7:877-781. 16. Sazawal S, Black RE, Bhan MK, Jalla S, Bhandari N, Sinha A, et al. Zinc supplementation in young children with acute diarrhea in India. N Engl J Med 1995; 333:839- 844. 17. Sachdev HPS, Mittal NK, Yadav HS. Oral zinc supple- mentation in persistent diarrhoea in infants. Ann Trop Paediatr 1990;10:63-69. 18. Roy SK, Tomkins AM, Akramuzzaman SM, Behrens RH, Haider R, Mahalanabis D, et al. Randomised controlled trial of zinc supplementation in malnourished Bang- ladeshi children with acute diarrhoea. Arch Dis Child 1997; 77:196-200. 19. Bhandari N, Bahl R, Taneja S, Strand T, Molbak K, Ulvik RJ et al. Substantial reduction in severe diarrheal mor- bidity by daily zinc supplementation in young north In- dian children. Pediatrics 2002; 109:e86.