Bhutta et al Sept 30, 2007
Department of Paediatrics & Child Health
The Aga KhanUniversity
Prospective, cluster randomized, controlled evaluation of the impact of zinc and / or micronutrient supplementation on intestinal flora, diarrheal disease burden, intestinal mucosal integrity and growth
among cohorts of children in Pakistan
______
A Global Health Program Proposal to Bill and Melinda Gates Foundation by
Prof. Zulfiqar A Bhutta
Tel: +92 21 4864782
Fax: +92 21 4934294
Table of Contents
I. Proposal Information3
II.Executive Summary5
III.Goals and Objectives6
IV.Project Design & Implementation8
V.Date Management Monitoring, Evaluation, & Dissemination22
VI.Optimizing Public Health Outcomes and Intellectual Property Plans to Achieve Global Access 24
VII.Organizational Capacity and Management Plan26
VIII.Budget Narrative28
IX.Citations 29
X.Appendices36
A.Milestone Summary Table and Timeline*
B.Budget Spreadsheet*
C.Financial and Tax Information*
D.Biographical Information36
XI.Additional Guidelines41
A.Research Assurances41
* SeparateattachmentsI. Proposal Information
A. Organization
U.S. Tax Status (Refer to Tax Status Definitions)[1]: / Non-profit
Institutional Official authorized to submit and accept grants on behalf of organization:
Prefix / Dr / First name / Mohammad / Surname / Khurshid / SuffixTitle / Dean,MedicalCollege / Telephone / +92 21 4930051
Address / Aga KhanUniversity, Stadium Road, Karachi, Pakistan / Fax / +92 21 4932095
E-mail /
Web site /
B. Project
Principal Investigator/Project Director:
Prefix / Dr / First name / Zulfiqar / Surname / Bhutta / SuffixTitle / Professor and Chairman, Dept of Paediatrics / Telephone / +92 21 4930051
Address / Aga KhanUniversity, Stadium Road, Karachi, Pakistan / Fax / +92 21 4932095
E-mail /
Web site /
Amount Requested From Foundation ($USD): / Project Duration (months): / 36
Estimated Total Cost of Project ($USD):
Organization’s total revenue for most recent audited financial year ($USD):
Charitable Purpose:
To evaluate the role of zinc supplementation in children in general health improvement and diseases such as diarrhea of varied etiology and its impact on intestinal microbial flora. Zinc supplementation is being promoted on wide scale as a preventive and therapeutic agent for diarrhea and other illnesses in children, though effect of its routine supplementation has not yet been fully evaluated. Hence it is of vital importance to look at the impact of routine zinc supplementation in children.Project Description:
This is a prospective, cluster randomized, controlled evaluation of a cohort of young infants in both urban and rural settings to receive multiple micronutrient supplementation with and without zinc and, evaluation of these children for episodes of diarrhea and respiratory infections, intestinal microbial flora and intestinal permeability. The funds will be used fora)For detailed follow up of the cohort for growth, micronutrient status, morbidity and prospective evaluation of a subset for intestinal mucosal permeability, small bowel microflora, and breath hydrogen excretion
b)For management and treatment of diarrheal episodes, respiratory and other infections.
c)For investigations, both for routinely indicated clinical investigations and those for study purpose only.
d)Data management and analysis of results
e)Supervisory visits of collaborators
C. Regulatory Approval Questionnaire
Please enter an “x” in the appropriate column.
Yes / No1.Project will involve collaboration with for-profit companies / x
2.Project will involve use or creation of intellectual property / x
3.Proposal contains proprietary information / X
If you marked “yes” for any of the above statements, please complete Section VI.B.
4.Project will involve research using vertebrate animals / x
5.Project will involve research using human subjects / x
6.Project will involve clinical trials / x
7.Project will involve use of any of the following substances:
•recombinant DNA subject to regulation
•pathogens/toxins identified as “select agents” by U.S. law:
(
•biohazards or genetically modified organisms / x
II. Executive Summary
Zinc has recently been recommended by WHO for the treatment of acute diarrhea in children.There is a large body of literature available about its effectiveness in the prevention and enhancing recovery form several childhood illnesses such as diarrhea and respiratory infection. The forthcoming Lancet series on undernutrition recommends scaling-up zinc supplementation programs globally in order to address stunting in countries with high burden of undernutrition. However, despite the evidence of efficacy, the potential mechanisms of action of zinc have not been fully elucidated and importantly, the ecological and biological implications of long term supplementation at population level, not assessed. Given the current thinking on modes of administration, it is likely that zinc supplementation programs will consist of co-administration of zinc with other micronutrients as either supplements or in fortified food products. There is a thus a particular need to assess the impact of routine supplementation of zinc in young growing children, and look at its impact on intestinal microbial flora, and relationship with gut mucosa integrity and co-morbidities. We propose to evaluate the relationship if any, of intestinal microbial flora, intestinal permeability, morbidity patterns and response to various enteric pathogens in a representative birth cohort randomly allocated to receive daily zinc and micronutrients from 6-18 months age and a control population.
Infants will be recruited at birth in two study sites i.e. an urban settlement (Bilal Colony) in Karachi and a rural population of Matiari district in interior Sindh provinceof Pakistan. After a baseline census and informed consent, all births in the first six months of the study will be eligible for inclusion in the study. Exclusive breastfeeding for the first six months will be actively promoted and infants allocated to supplementation at six months of age. We expect a cohort of 2000 infants to be thus followed from birth to 24 months of age. The study areas will be randomly allocated to three cluster groups. One group of clusters (A) will be used as controls; infants in the second cluster group (B) will receive daily micronutrient supplementation in the form of Sprinkles and the third group (C) will receive daily zinc (10 mg) in addition to micronutrient supplementation. Regular follow up and monitoringfor morbidity and growth will be established and a subset from each cluster group, following detailed explanation and a special consent, will undergo a regular panel of non-invasive investigations for assessment of stool pathogens and microflora, intestinal permeability and breath hydrogen excretion (at 2 weeks, 3, 6, 12, 18 and 24 months age). All children with diarrhea and illnesses will receive standard case management as per IMCI protocol.
An independent data safety and monitoring board (DSMB) will be constituted to oversee the safety aspects of the study and conduct at least one interim analysis a mid point of the study. A DSMB meeting is planned at that time in Karachi and budgeted for. All the data collected from censuses, fortnightly follow up, morbidity episodes and laboratory investigations will be processed in real time using Visual Fox Pro. Association of growth, intestinal mucosal integrity, associated diarrhea and co-morbidities and microflora will be assessed by repeated measured ANOVA, GLM, logistic regression survival analysis and STATA 9.2.
III. Goals and Objectives
A. Goals and Attributable Benefit
Goals: To study the effect of programmatically relevant zinc and/or multiple micronutrient supplementation between 6-18 months of age in representative urban and rural cohorts on (a) growth and morbiditypatterns b)patterns of intestinal microflora in health and carriage of potential enteric pathogens causing diarrheal diseases and (c) intestinal mucosal integrity as measured by intestinal permeability measurements and (d) recovery patterns from common causes of childhood diarrhea
Attributable benefit:
This study will provide important information relevant to the safety and benefits of large scale preventive micronutrient and zinc supplementation programs. Given the push to move towards introduction of micronutrient fortification and supplementation strategies, this information will be relevant to scaling up of such programs in diverse settings.
B. Objectives
Primary objectives:
- To evaluate the impact of zinc and/or micronutrient supplementation on growth, morbidity patterns and outcomes among children between 6-18 months of age in population settings.
- To evaluate the patterns of intestinal microbial flora colonization and intestinal permeability in a subset of children within the cohort receiving regular zinc and/or micronutrient supplementation in comparison with a control group.
Secondary objectives:
1To assess the impact of preventive zinc supplementation on the incidence of viral or bacterial diarrhea among children in the above cohort.
2To evaluate overall impact on micronutrient status (hemoglobin, serum ferritin and plasma zinc, adjusted for CRP,) among children supplemented with zinc and/or micronutrients as compared to those not supplemented.
3To assess the impact, if any, of zinc and / or micronutrients supplementation in a subset of infants on bioavailability of zinc and total body zinc pool using stable isotopes.
Morbidity assessment and definitions
Our primary outcomes for assessing the additive impact of zinc supplementation will be episodes of diarrheaand additional morbidity such as acute lower respiratory tract infections, pneumonia anddays with severe illness. Diarrhea will be defined as three ormore loose or watery stools in 24 hours, and children will be consideredto have recovered after three days without diarrhea. Acutelower respiratory tract infection will be diagnosed if the childhas reported difficulty in breathing and rapid breathing (40/min).Severe illness will be defined as a temperature 38.4°C or admissionto hospital or respiratory rate 50/min or chest indrawing. Dysenterywill be defined as diarrhea with visible blood or mucus in stools, severeacute lower respiratory tract infections (worsening of existinginfection or new onset of cough or difficulty in breathing withhigh respiration rate (50/min) or chest indrawing), fever (axillarytemperature 37.2°C), and high fever (axillary temperature38.4°C).
- Diarrhea: Passage of three or more semi liquid or watery stools or one large watery stool in last 24 hours. The diarrhea classification system is based predominantly on the appearance of the stool. This will include all type of diarrhea in local terminologies
- Diarrheal episode: Presence of diarrhea lasting at least 48 hourswith less than 48-hour diarrhea free interval if any. If there is more than 48 hours interval between diarrheal days; recurrence of diarrhea after 48 hours interval will be counted as second episode.
- Duration of diarrhea: number of days since beginning of diarrhea until the last diarrheic stool before two normal stools or a 24 hour period without stools".
- Dysentery: Passage of semi soft or liquid stools with visible blood and/or mucus.
- Mortality: Number of death in children during last one year due to any cause.
- Diarrhea related mortality: No. of deaths in children due to severe dehydration or complications following diarrhea
- IMCIIntegrated Management of childhood illnesses: A WHO protocol for integrated management of common childhood illnesses (IMCI) at first level of health care facility.
IV. Project Design and Implementation
A. Project Design
Background and Rationale
Current status of zinc in childhood diarrhea:
Despite major advances in our understanding of its pathogenesis and epidemiology, childhood diarrhea, it remains a major cause of morbidity and mortality among children under five globally with an estimated 1.6 million deaths annually (1, 2). In recent years the management of diarrhea has advanced with the introduction of low osmolality ORS and oral zinc supplementation (10-20 mg/day for 10-14 days) (3-6). In addition, regular supplementation with zinc has also been shown to reduce inappropriate antimicrobial prescribing (7) thus increasing the impetus for implementing this intervention at scale in health systems (8). The use of zinc for the treatment of diarrheal episodes has been suggested as a major strategy for reducing diarrhea associated child mortality in developing countries (9). In addition, widespread zinc deficiency has been implicated with growth failure and preventive zinc supplementation has been suggested as a strategy for reducing diarrhea burden and stunting in developing countries (Lancet series on Maternal and Child Undernutrition 2007, forthcoming).
A number of strategies have been suggested for increasing the coverage of preventive and therapeutic zinc therapy including fortification and widespread use for all episodes of diarrhea (10, 11). These strategies and the impetus for increasing the use of zinc in health systems suggest that we will soon see large scale population exposure to this intervention for extended periods of time. While the use of zinc for diarrhea therapy would mean intermittent courses of treatment for 10-14 days at a time (perhaps 2-3 such courses per child per year), preventive strategies would necessitate alternative strategies such as fortification of commonly used commodities such as milk (12, 13) or through Sprinkles for home fortification of complementary foods (14). Given the recent issues arising from the adverse effects observed after large scale use of iron supplements in malaria endemic areas (15) and the failure of zinc supplementation to impact health outcomes when given in some settings in south Asia (16-18), it is imperative that our understanding of the implications for the long term use of zinc in diverse settings is improved. Populations with widespread zinc deficiency also have concomitant multiple micronutrient deficiencies such as iron, vitamin A, iodine and folic acid. In view of the relationship of several micronutrient deficiencies and health outcomes in children (19, 20), it is unlikely that single nutrient interventions will be instituted at scale. Thus while short of zinc for the treatment of diarrhea are feasible, preventive zinc supplementation of zinc is likely to happen in combination with other micronutrients through fortification or supplements such as Sprinkles.
Efficacy of zinc in various types of diarrhea and prevention of enteropathy:
The mechanisms of action of zinc in the treatment and prevention of diarrhea are probably multi-factorial and several mechanisms have been proposed. These include promotion of intestinal mucosal repair and improvement of intestinal permeability (21, 22), improvement of non-specific immune status (23) as well as impact on intestinal mucosal secretory response due to production of uroguanylin (24). More strikingly, while experimental differences have been found in the response to Escherichia coli enterotoxin or cholera toxin (25), indicating that not all types of diarrhea may respond equally to zinc therapy.
Despite the plethora of evidence of the role of zinc in the treatment of diarrhea, the evidence base of studies evaluating the efficacy of zinc in the pathogenesis, prevention and treatment of various diarrhea etiologies is very limited (26-43) (Table 1). Although there is some information from animal studies indicating that dietary zinc may affect intestinal colonization, microflora and mucosal repair (44-51). there is surprisingly little information about the role of zinc and intestinal colonization and microflora in human beings. In a previous study evaluating the impact of zinc supplementation among malnourished children with persistent diarrhea in Pakistan, we observed a pattern of breath hydrogen excretion by day 14 suggestive of a significant increase in breath hydrogen excretion by day 14 suggestive of small bowel bacterial overgrowth (52).
Table I
Global burden of diarrhea due to specific pathogens and corresponding studies of zinc
Etiology / Number of diarrheal deaths (x1000) / Studies of zinc in the pathogenesis and treatment of episodesMedian / Range / Basic science studies / Applied clinical studies
Salmonella / 67.2 / 49.6 - 126.4 / 3 / -
Shigella sp. / 97.6 / 57.6 – 169.6 / 2 / 2
Campylobacter / 84.8 / 41.6 – 166.4 / - / -
Vibrio cholerae / 100.0 / 25.6 – 187.2 / 3 / 3
ETEC / 152.0 / 107.2 – 268.8 / 2 / -
EPEC / 264.0 / 150.4 – 441.6 / 1 / -
Rotavirus / 406.4 / 262.4 – 564.8 / - / -
Giardia / 28.8 / 11.2 – 100.4 / 2 / -
Cryptosporidium / 49.6 / 19.2 – 131.2 / - / -
Entamoeba / 11.2 / 3.2 – 59.2 / - / -
Multiple infections / 200.0 / 136.0 – 323.2 / - / -
Unknown / 137.6 / ----
Total / 1,600 / ----
Unanswered questions with regards to the use of zinc in childhood:
Given the possibility that zinc supplements will be used in large scale population settings in both preventive and therapeutic programs, it is important that we have adequate information as to the implications of such programs (using zinc alone or in combination with other micronutrients) on short and medium term outcomes. These include information on its safety and ecological impact, interactionsand effect on intestinal mucosa when used with other micronutrients. The variable outcomes (19), including adverse effects (53, 54), reported from zinc supplementation in children from various settings also suggest that further information must be obtained on the factors associated with such diversity of responses.
The amount of information available on the benefit of zinc in various types of diarrhea is relatively limited. The most frequent use of zinc is in acute watery diarrhea and while current recommendations do not differentiate between various types of diarrhea; it is still not clear if zinc supplementation is equally effective in diarrhea due to all causes. While the public health relevance of this information may not be readily evident, this is of key importance in the post-rotavirus vaccination scenario.
One key issue relating to the use of zinc as a nutrient supplement for children is also the concern that luminal zinc may alter intestinal flora with effects have either beneficial or detrimental effects on the host/ child. It is possible that the presence of zinc in the intestine may promote proliferation of beneficial organisms or assist in maintaining homeostasis of the normal intestinal microflora. Data from a previous small study by our group in malnourished children receiving zinc supplements suggested that the use of zinc in children was associated with abnormal breath hydrogen excretion patterns (52) suggesting the possibility of small bowel overgrowth. In another recent study evaluating the impact of supplementation of children with micronutrients on diarrhea in Karachi, the group receiving micronutrients (including zinc) and probiotics had the worse outcome (55). The question of whether zinc has the potential to alter large bowel flora remains unanswered. While small bowel overgrowth can contribute to malabsorption and diarrhea, alterations in colonic flora may contribute to general health as flora in the large bowel contribute metabolites which impact health (56, 57). There is good evidence that colonic microflora content or balance can contribute to health by the maintenance of B vitamin levels, the promotion of normal development of the immune system and assist in maintaining the normal intestinal ecosystem that protects against harmful pathogens and also contribute to the generation of carcinogens and tumor promoters.
Strategies and limitations for assessing small bowel function, intestinal permeability and intestinal flora in children