Evaluation of the impact of zinc and / or micronutrient ...
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
[pic]
Prof. Zulfiqar A Bhutta
zulfiqar.bhutta@aku.edu
Tel: +92 21 4864782
Fax: +92 21 4934294
Table of Contents
I. Proposal Information 3
II. Executive Summary 5
III. Goals and Objectives 6
IV. Project Design & Implementation 8
V. Date Management Monitoring, Evaluation, & Dissemination 22
VI. Optimizing Public Health Outcomes and Intellectual Property Plans to Achieve Global Access 24
VII. Organizational Capacity and Management Plan 26
VIII. Budget Narrative 28
IX. Citations 29
X. Appendices 36
A. Milestone Summary Table and Timeline *
B. Budget Spreadsheet *
C. Financial and Tax Information *
D. Biographical Information 36
XI. Additional Guidelines 41
A. Research Assurances 41
* Separate attachments
I. Proposal Information
A. Organization
|Organization Name: |The Aga Khan University |
|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 |Suffix | |
|Title |Dean, Medical College |Telephone |+92 21 4930051 |
| |Aga Khan University, Stadium Road, Karachi, Pakistan| Fax |+92 21 4932095 |
|Address | | | |
| | | |
|E-mail |mohammad.khurshid@aku.edu |
|Web site |aku.edu |
B. Project
|Project Name: |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 |
Principal Investigator/Project Director:
|Prefix |Dr |First name |Zulfiqar |Surname |Bhutta |Suffix | |
|Title |Professor and Chairman, Dept of Paediatrics |Telephone |+92 21 4930051 |
| |Aga Khan University, Stadium Road, Karachi, Pakistan| Fax |+92 21 4932095 |
|Address | | | |
| | | |
|E-mail |zulfiqar.bhutta@aku.edu |
|Web site |aku.edu |
|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 for |
|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 |
|For management and treatment of diarrheal episodes, respiratory and other infections. |
|For investigations, both for routinely indicated clinical investigations and those for study purpose only. |
|Data management and analysis of results |
|Supervisory visits of collaborators |
C. Regulatory Approval Questionnaire
Please enter an “x” in the appropriate column.
| |Yes |No |
|1. 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: | |x |
|• recombinant DNA subject to regulation | | |
|• pathogens/toxins identified as “select agents” by U.S. law: | | |
|() | | |
|• biohazards or genetically modified organisms | | |
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 province of 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 monitoring for 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 morbidity patterns 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:
1. 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.
2. 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:
1. To assess the impact of preventive zinc supplementation on the incidence of viral or bacterial diarrhea among children in the above cohort.
2. To 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.
3. To 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 diarrhea and additional morbidity such as acute lower respiratory tract infections, pneumonia and days with severe illness. Diarrhea will be defined as three or more loose or watery stools in 24 hours, and children will be considered to have recovered after three days without diarrhea. Acute lower respiratory tract infection will be diagnosed if the child has reported difficulty in breathing and rapid breathing ([pic]40/min). Severe illness will be defined as a temperature [pic]38.4°C or admission to hospital or respiratory rate [pic]50/min or chest indrawing. Dysentery will be defined as diarrhea with visible blood or mucus in stools, severe acute lower respiratory tract infections (worsening of existing infection or new onset of cough or difficulty in breathing with high respiration rate ([pic]50/min) or chest indrawing), fever (axillary temperature [pic]37.2°C), and high fever (axillary temperature [pic]38.4°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 hours with 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
• IMCI Integrated 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
| |Number of diarrheal deaths (x1000) |Studies of zinc in the pathogenesis and treatment of |
|Etiology | |episodes |
| | | |Basic science studies |Applied clinical studies |
| |Median |Range | | |
| | | | | |
|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, interactions and 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
Our understanding of the role of intestinal microflora has been hampered by the technical limitations in assessing small bowel flora/overgrowth in a quantitative and rigorous fashion. Attempts in the past to assess small bowel flora used sterile, plugged tubes that could be introduced into the small bowel and aspirated; the aspirate was then quantitatively cultured and flora examined. There are significant limitations inherent in this technique including imprecision in placing the sterile tube, differences in amount of small bowel fluid leading to differential results from the attempts to aspirate; the regional nature of the sampling, among others. This is also a difficult technique to justify in a study population that includes infants and young children. Similarly studies of intestinal morphology have required invasive biopsy techniques which are invasive and thus not possible in most ambulatory and community settings (58, 59). There is thus an urgent need to develop surrogate or indirect measures to assess intestinal mucosal integrity and function. The development of methods such as intestinal permeability testing through dual sugar absorption tests thus represents a major advance and allows the indirect assessment of small bowel intestinal function (60-63). This technique has been used in a variety of settings to evaluate health outcomes (64-74). Intestinal permeability testing has thus made it possible to evaluate intestinal mucosal regeneration and function in a variety of states including invasive diarrhea (75), in response to nutritional therapy (76-78) and chemotherapy (79). Recently the potential adverse effects of iron supplementation on intestinal mucosa have also been shown using this technique (80).
Similarly breath hydrogen testing (BHT) has been used as a non-invasive measure of small bowel bacterial overgrowth (52, 81-83). In this proposal, we propose to use a series of non-invasive techniques that will assist in allowing us to assess the status of intestinal flora and intestinal mucosal function in a cohort of infants in community settings.
Additional consideration with studies of intestinal microflora and zinc:
While it is entirely feasible to study the alterations in intestinal microflora and gut ecology in children on small number of children, it must be recognized that these biological changes and the information derived thereof is critically dependent upon ecological pressure and environmental factors. As in antimicrobial resistance patterns of flora, the pressure on organisms are not merely related to changes within the host but the entire background of community and population exposures (84). Studies of microflora in infancy also indicate significant impact of environmental organisms and exposure rates (85-87). This is readily recognizable by the rapid intestinal colonization with pathogenic enterobacteriacae observed among Pakistani newborn infants in both hospital and community settings (88, 89).
In the case of long term supplementation with zinc and its impact on intestinal flora the effects on children will greatly depend upon environmental exposure and population density and levels of exposure. From a programmatic perspective these effects would differ greatly were a population exposed to this nutrition intervention effecting gut flora, as opposed to exposure in a small group of children. While both kinds of studies are feasible, given the call for large scale zinc supplementation programs (90, 91), in our proposal we are nesting the detailed study of intestinal function and microflora within a larger cohort for the specific purpose of simulating programmatic circumstances and obtaining critically relevant information.
The potential of addressing the impact of zinc and / or micronutrient supplementation on intestinal flora and diarrheal disease outcomes in population settings in Pakistan:
The aforementioned information gaps with regards to preventive and therapeutic zinc studies merit evaluation in appropriate and representative population settings. The community research program of the department of Paediatrics & Child Health at the Aga Khan University in Karachi (Pakistan) has been closely involved in nutrition and diarrheal diseases research with several urban and rural sites, stable representative populations, basic surveillance systems and data management systems. These sites have yielded important information on diarrhea epidemiology (92-94) and the impact of micronutrient interventions on diarrhea and health outcomes (55, 95). The general incidence of diarrhea in these settings has ranged from 2.3 – 6.4 episodes per child per year with some 10% of cases representing bloody diarrhea and rotavirus episodes accounting for almost 40% of acute diarrhea episodes. These settings, especially sites which have not had recent nutrition and diarrhea related interventions, offer a unique opportunity for evaluating the potential impact of zinc supplementation on gut ecology, intestinal mucosal function, diarrheal pathogens and outcome of therapy of specific types of diarrhea.
Study Design
In this study of 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, we plan to study a birth cohort of children, who will be enrolled for supplementation between 6 and18 months of age. The children will be randomly allocated in a cluster randomized design to micronutrient supplementation (with and without zinc) or a control group receiving no supplements. This entire cohort will be followed up for a number of functional outcomes and morbidity patterns (especially diarrhea and respiratory infections). A subset from this larger cohort will be studied in depth to additionally evaluate the impact of supplementation strategies on intestinal microbial flora, intestinal permeability and patterns of breath hydrogen excretion. We will use a number of serial laboratory measures and the following strategies for achieving our objectives.
Selection of study sites:
The two urban and rural population sites chosen for this study, Bilal Colony (urban Karachi) and Matiari (rural Sindh), have well established community and health system liaison, basic demographic surveillance and field centers for research. These specific sites have also been selected because they have been the location of several large cluster randomized trials, are well mapped with household level GIS but have not had recent micronutrient and diarrhea interventions. These sites are representative of typical conditions in urban and rural settings in Pakistan, and there is a close working relationship with community, civic society leaders and public health department facilities
1. Matiari: Matiari is a rural site for study, situated in North Eastern part of Sindh, about 200 km away from Karachi. Matiari has 721 villages with population of about 250,000 including 45,000 children < 5 years and several functional health centers
2. Bilal & Awami Colony: Bilal Colony is a urban squatter settlement in the industrial area of Karachi. The population consists of mixed ethnic groups of Punjabi, Baluchi and Pathan migrants from northern areas of Pakistan. Bilal Colony has a population about 65,000 with 11,000 children < 5 years.
Table II below highlights some of the features from the study areas
Table II
Epidemiological information from the entire urban / rural sites (April 2006 – March 2007)
|Indicator |Bilal Colony |Hala/Matiari |Total |
| |(urban) |(rural) | |
|Population size |65,502 |318,226 |383,728 |
|Villages |-- |954 |954 |
|Households |8858 |47121 |55979 |
|Number of children under 2 years |4030 |24595 |28625 |
|Number of children 2- 5 years |5809 |39255 | |
|Total births ( in last one year) |1027 |11417 |12444 |
| | | |(3111 per quarter) |
|Diarrhea surveillance data (from April – August 2006) |
|Diarrhea prevalence (last 24 hours) |6% |8% | |
|ORS use rates (%) |45% |34% | |
|Zinc use rates (%) |0.4% |Nil | |
Sample size estimation:
We are detailing the sample size estimation for the main cohort and the subset separately below
Sample size estimation and cluster allocation for the main study
Give the population size at these two sites, we plan a cluster-based design with allocation of the rural population into village/locality based clusters/blocks of about 125-150 households each and the urban population clusters/blocks of 50 households and an estimated 25 eligible children each. We estimate that the overall population in both areas can be divided into 600 clusters of varying sizes consisting of at least 25-40 births annually in each cluster. This age group represents the highest risk for diarrheal diseases and growth faltering.
Table below indicates the data obtained in the time period indicated above for diarrhea rates in both Matiari and Bilal colony. The two highlighted locations in Bilal are a bit detached from the main population and have varied rates for diarrhea. These areas will not be included in the main study in order to reduce inter- cluster variability. The data indicate that the average diarrhea burden in the rural population (with the exception of one area) areas ranges from 4.0 – 6.4 episodes per child per year where as in the urban area (barring the two outlying areas) it ranges from 2.3 – 5.4 episodes per child per year.
Table
|Matiari Cluster | |Diarrhea episodes/child|Bilal Colony |n |Diarrhea |
| |n |for 3 months |Sectors | |episodes/child for 3|
| | | | | |months |
|Pir Jhando |3 |1.67 |A |21 |0.62 |
|B.D Kaka |4 |1.00 |B |26 |0.88 |
|Bago Jamali |56 |1.36 |C |7 |0.57 |
|K.B Kaka |11 |1.36 |D |9 |1.00 |
|Matiari |140 |1.34 |E |7 |0.71 |
|S.A.S Ji Wasi |119 |1.81 |F |22 |1.36 |
|Sultan pur |702 |1.47 |Gulshan-e-Latif |18 |2.22 |
|Oderolal Station |267 |1.19 |Mohd.Ali Shah Goth|4 |0.25 |
|New Saeedabad |103 |1.29 | | | |
|Fakir Nothiani |63 |1.60 | | | |
|Hala Old |194 |1.54 | | | |
|Shahmeer Rahoo |2 |1.00 | | | |
|Mushtaqabad |20 |3.10 | | | |
|Oderolal Village |258 |1.73 | | | |
|Nobat Mari |160 |1.58 | | | |
|New Hala |147 |1.57 | | | |
|Total |2249 |1.50 |Total |114 |1.10 |
We have estimated sample size for cluster randomized trials based on the formula for unmatched studies by Hayes and Bennett, (96).
For the rural clusters we have made the following estimates:
• clusters will each contribute 50 child years of observation to the study
• average diarrhea incidence in the "comparison" arm is 3.4 episodes per child year
• 95% of clusters will have diarrhea incidence rates which lie between 1 and 6 episodes per child year (CV = 0.37)
• the intervention will reduce diarrhea incidence by 20% (i.e. to an average of 2.72 episodes/child/year)
• level of statistical significance = 5%
• power required = 90%
Given the potential attrition expected, the possible geographic clusters in both areas, potential problems that may arise at cluster level in health system setting and the need to have a minimum rural sample of 50 clusters per arm, we have planned for allocation of the population to 56 clusters each in the rural area (168 rural clusters).
For the urban clusters in order to achieve 80% power to detect a 20% reduction in diarrhea from 4 episodes/child/year to 3.2, we estimate that 36 clusters per arm will be needed (with an estimated 25 child years per cluster). Give the population available in Bilal Colony, lower attrition rates and the planned recruitment over 18 months, we shall plan to divide the urban area into 108 clusters to be equally allocated to the three intervention groups. Thus we are confident of achieving independent sample size for significance in the urban cohort as well. Thus altogether 276 clusters will be allocated to the three intervention groups.
Of a potential 34,000-50,000 diarrhea episodes are expected annually over the course of the study, a large proportion will be recognized and treated clinically by CHWs, other health care staff and further investigated within the public health system as required. While we will target collecting stool specimens from the maximum number of diarrhea episodes, this will only be possible during morning working hours to allow for processing and storage. We estimate that adequate samples from even 30% of the acute diarrhea episodes (~ 15000 episodes) will give us the requisite sample size needed for laboratory investigations within the clinical diarrhea episodes
Sample size estimation for the subset studied for intestinal function and microflora
It is extremely difficult to calculate sample size for this type of study as the technology is so novel that the differences that may be found are not known and there are very few data from studies looking microflora changes following zinc supplementation. That is one of the main considerations for nesting this study within a larger study adequately powered for clinical benefits.
In addition, we have relied upon our own findings of altered breath hydrogen (BHT) excretion patterns following zinc supplementation as the basis for some estimates, which suggested that there was an 2-3 folds increase in BHT among children receiving zinc for 14 days (52). If we assume that about a third of infants will have gram negative organisms in their intestinal microflora by 9 months of age (range 20-40%), which increase to 45% after zinc supplements we will need about 165 children per arm. Given the potential drop outs expected in this sub-group because of the sequential investigations, we are assuming a 20% drop out rate and will plan to recruit 200 children per intervention group for the in-depth sub-study with proportional allocation to urban and rural clusters (an average 2 children for each of the 168 rural clusters and 1 child per year per urban cluster). This random cluster based allocation will be determined by a computer generated list with replacement in the event of drop outs.
Many of the published studies on intestinal microflora report data from much smaller samples than we propose: To illustrate, several previous studies of microflora in infants have reported sample sizes ranging from 12-70 (97-100) The largest sample used was in a study comparing flora between age groups in European countries in 230 individuals (101). Given our proposed sample size (n= 600) we are confident that this study will provide unique longitudinal data on the relationship of long term zinc and micronutrient supplementation, intestinal mucosal function and enteric flora.
Randomization and intervention allocation
The clusters will be randomly allocated to the following cluster-specific interventions by an independent statistician using a set of indicators (households, population size of children under 2 years of age, geospatial location in relation to health system facilities and a set of socio-economic indicators)
Group A (controls) will receive no specific nutrition intervention other than the standard of care for preventive health education by the Lady Health Workers (CHWs), which currently does not include any nutrition commodities. In addition there will be health worker training in integrated management of childhood illness (IMCI) and facility strengthening for child survival interventions in all clusters. Specifically these children will however receive oral rehydration and 10 days course of treatment with 20 mg zinc daily for treatment of acute diarrhea episodes. These refresher CHW trainings will include a specific focus on family education and mobilization for exclusive breastfeeding (for about 6 months) and appropriate complementary feeding using home available foods.
Group B (additional daily multiple micronutrients, excluding zinc, as Sprinkles). In addition to the health systems interventions listed above, this group of children will receive a daily sachet of these micronutrients (Sprinkles) for 12 months from recruitment (i.e. between 6-18 months age). These Sprinkles will be added to commonly used and home available weaning foods. The Sprinkles will be prepared by Genera Pakistan Pvt. Ltd (Rawalpindi) to required specifications as single serving sachets each containing microencapsulated iron (12.5 mg), vitamin C (50 mg), vitamin A (300 µg), vitamin D (5 µg) and folic acid (150 µg).
Group C (additional daily multiple micronutrients, including zinc, as Sprinkles). In addition to the health systems and nutrition interventions listed above, this group of children will receive a daily sachet of micronutrients for 12 months from recruitment (i.e. between 6-18 months age) to be added to commonly used and home available weaning foods. In addition to the micronutrients specified above, these Sprinkles will also contain 10 mg elemental zinc sulfate.
The two types of Sprinkles will have identical packing and each sachet batch will be labeled with a pre-specified code which will be held by the manufacturer (Genera Pharma) and the Chairman of the external study oversight group. The Sprinkles will be provided every month by the concerned Community Health Workers (CHWs) as part of the government distribution system for Groups B and C. All field and study staff will remain blinded as to the content of the Sprinkles and the code. This approach has been successfully pilot tested in several districts of Pakistan. The CHWs and government health system staff will provide these commodities to families and children and regular intake will be supervised by additional logistics supervisory team staff who will also undertake monthly visits in target villages.
Community assent, Informed consent & Participation
The investigators in the department of Paediatrics and AKU have well established protocols for informed consent. We intend to share the information about the project with local government leaders, community leaders and members from all participating villages and Bilal Colony. In addition parents of all eligible households, especially mothers, will be provided information about the study both verbally and through a written illustrated information sheet (102). Following an objective assessment of understanding of the project by a standard interview, the families will be invited to join the follow up protocol and provide a written informed consent. The same procedure will be applied to the families identified for the in-depth study. In all instances parents will be informed of the right to withdraw from the study without prejudice.
Community cohort establishment, follow up and data collection
An independent data collection system will be established through trained research data collectors (DCs) in addition to the routine monitoring and evaluation by the village based CHWs. The latter are residential local health workers with the primary responsibility for maternal and child care activities. Our program will work in very close liaison with these staff who will also collect sentinel information and provide information on diarrhea episodes. All children 0-3 months of age and those born in the clusters in the first 6 months of the trial will be potentially eligible for inclusion in the study and will be identified through the regular demographic surveillance and baseline survey. These children will be enrolled for inclusion at birth or identification at the baseline survey (for those between 0-3 months of age) and allocated study numbers. The mothers of all consented infants within specific clusters will be encouraged to exclusively breastfeed and receive specific interventions as specified above once they reach 6 months of age.
The following system for data collection will be established in the study sites:
1. The existing CHWs will continue to perform their routine work (including breastfeeding and complementary feeding promotion) and will be informed of the study and interventions. A pilot agreement to administer Sprinkles through the CHW program in the target areas has been reached with the national CHW program of the government of Pakistan. The CHW program maintains its own data system for vital events which will be available to the research group and will be used for corroboration of primary sentinel events (births, referrals, hospitalizations, deaths). Once a child is identified with acute diarrhea, the CHWs will provide the standard treatment until the diarrheal episode is resolved. These children will be treated as per the IMCI protocol and receive the standard course of treatment with low osmolality ORS and oral zinc sulfate (daily dose of 20 mg for 10 days) as per standard WHO/UNICEF guidelines (103). Referral for intravenous rehydration or hospitalization will also be made as per IMCI guidelines and data recorded. For children with suspected dysentery antibiotic treatment will be provided as per standard guidelines of the government of Pakistan (oral nalidixic acid 55 mg/kg/day and adjustment thereafter as per clinical response or culture results).
2. The CHWs will be given mobile phone cards to notify the mobile research team of physicians & laboratory technicians who are in the field site during working hours (0900-1700 hours) and will obtain the requisite laboratory investigations.
3. Each mobile research team (two rural and one urban team) will consist of a medical officer and a technician. For every such child with diarrhea so identified, the mobile research team with collect a stool specimen for routine examination, virology, molecular biology studies for common pathogens and bacterial cultures. If the diarrheal episode persists for longer than 1 week, another stool sample (or rectal swab if stool sample is unavailable) will be obtained and processed for microscopy, culture, viral and molecular studies. After initial processing in the field microbiology office and microscopy, the stool samples will be transported in PGB transport media and frozen to the main research laboratories at the Aga Khan University for further processing (in case of Bilal Colony) or Hyderabad Microbiology Research Laboratories (from rural Matiari site)
4. The entire cohort will be evaluated at 3 monthly intervals by trained community health workers (CHWs) who are provided transportation to respective sites. The CHWs will also identify all children between 0-43 months if age for inclusion in the first survey round and all new births for possible enrolment to the trial within the first six months. All recruited children in study groups will be will be monitored for growth status, morbidity and hospitalizations. Standard anthropometric assessments will be undertaken by the data collection teams using electronic weighing scales (sensitivity 10 g, Seca, Japan) and Infant Stadiometers (104). The CHWs will obtain information about any morbidity, admissions to public hospital and overnight stays in private clinics, and also record deaths if any. If a child has died, two supervisors trained to do verbal autopsies will visit the family within 2 weeks of the child's death to try to identify the cause, using a standard verbal autopsy method that has been adapted and extensively pre-tested for local use. We shall also analyze reports of overnight admission to private clinics on the basis of the history given by the family.
5. A subset of these children will be randomly identified using a computer allocation for in-depth evaluation. This subset will be followed by a separate group of CHWs and medical officers who will visit them on a weekly basis. As indicated in the section detailing the sample size estimation, we shall target recruiting 200 children from each allocation group (600 children in all) who in addition to regular clinical examination and anthropometry, will undergo the following sequential in-depth serial evaluations until 24 months of age. These children will undergo serial interventions and evaluations as per the following schedule (Table III)
Table III
Protocol for sequential assessment of the subset
|Time post recruitment |Initial exam |3 |6 |12 months |18 |24 months |
|(6-12 months age) | |months |months | |months | |
|Zinc and /or micronutrient |none |none |Initiation of zinc and/or |continued |To stop at 18 months |none |
|supplementation | | |micronutrients | |age | |
|Standard Anthropometry |√ |√ |√ |√ |√ |√ |
|Stool specimen for |√ |√ |√ |√ |√ |√ |
|microscopy, culture and | | | | | | |
|Intestinal microflora | | | | | | |
|estimation (fluorescent in | | | | | | |
|situ hybridization and flow| | | | | | |
|cytometry) | | | | | | |
|Intestinal permeability |√ |√ |√ |√ |√ |√ |
|test | | | | | | |
|Breath Hydrogen Test |√ |√ |√ |√ |√ |√ |
|Complete blood count & |√ | |√ | |√ | |
|micronutrient status | | | | | | |
|(ferritin, zinc, retinol) | | | | | | |
|Zinc pool estimate (using | | |√ | |√ | |
|stable isotopes)* | | | | | | |
* this will be done in 30 children (10 from each group) in collaboration with Drs Nancy Krebs (University of Colorado, Denver)
We shall give every participating child's parents/guardians a laminated card with the child's and their household's identification information and a control number printed on it for verification. We shall ask the family to maintain the card, show it to the community health worker or data collector when she visits, and also to present it in the hospital or to the family physician if the child is admitted. The children will be visited fortnightly at home by the CHW who, at every visit, shall deliver a new set of 14 Sprinkles sachets labeled with the child's identification number, collect the previous fortnight's left over Sprinkles (either empty or used sachets), and record level of adherence to the supplement. This will also be independently overseen by a logistics team at monthly intervals.
Given the need to undertake 12 months supplementation (18 months in the urban cohort) and follow up, each infant initiated on supplementation at 6 months of age will receive it until 18 months of age, with continued follow up for growth and morbidity for an additional 6 months thereafter. The estimated beneficiaries for the duration of the project in the 210 clusters are an approximate 29,000 households with estimated 6000 births in the first 6 months (the recruitment phase of the study) who will be prospectively followed up between 6-24 months of age. An additional 3000 infants 0-3 months of age who are identified in the first survey round will also be eligible for inclusion in the study. These numbers and the inclusion of all children in the respective clusters are necessary to simulate programmatic conditions and assess the full ecological extent of the impact of the interventions on the subset as well.
All acute diarrhea episodes in the participating cohort or study groups will be identified by CHWs, private practitioners and public health sector facilities. These children will be treated as per the IMCI protocol and receive the standard course of treatment (WHO/UNICEF) with low osmolality ORS. It must be noted that although zinc has been recommended by WHO/UNICEFfor the treatment of diarrhea, the government of Pakistan has as yet not ratified the use of zinc for the management of diarrhea in the public sector (103). We anticipate therefore that the background rates for zinc use these urban and rural settings will be negligible and a recent pilot cross-sectional survey corroborates this (Table II).
Adjustment for loss to follow up
Given that the cluster numbers have been adjusted for potential loss to follow up, we will not attempt any replacement for the main study clusters. We have similarly adjusted the in-depth study sample for a possible 20% loss to follow up. This will be done by a pre-specified computerized allocation per cluster to the in-depth study at the outset. Thus for each rural cluster with a targeted 2 children for the in-depth study, a random set of 5 numbers will be generated for sequential allocation in the event of drop out or loss to follow up. The corresponding numbers for the urban cohort are 3 children per cluster. These numbers will be generated and provided as required by the study oversight team and independent statistician.
Data management, analysis plan and statistical methods
We plan to use Visual Foxpro® to manage our data, with stringent range, consistency, and logical checks. Real time data entry will be used to ensure data quality and accuracy. We shall use double data entry and manual checking of frequencies during data cleaning. We shall perform intent to treat analysis (all children will be included in analyses irrespective of their adherence to the supplement) and we shall include all data gathered during the intervention period of 18 months per child. For children leaving the study area or withdrawing from the study, we shall include the data until the date of censorship.
Person-time analysis will be done with actual follow-up as denominator. For the effect on incidence of diarrhea episodes we shall estimate relative risk using Poisson regression; and for prevalence, we shall estimate odds ratio using GLM for binomial outcomes (maximum likelihood logit estimation for grouped data). In both estimations, we shall use robust clustered standard error estimation, the clustering variable being the child. This modified sandwich estimator is unbiased for cluster correlated data regardless of the settings. For the effect on total mortality and cause-specific mortality, we shall use Cox regression with exact handling for ties (STATA version 9.2, StataCorp, College Station, TX). For analysis of adverse events and admissions, we shall use Anderson Gill time-to-event survival methods in Cox regression (105) with robust estimation of standard errors to account for multiple events per child or within household. In these analyses, a relative rate (RR) of greater than 1 indicates a higher event rate in the intervention groups than in the control group (increased risk with intervention) and a value of less than 1 is consistent with the intervention being protective. In Cox regression models, we shall model an interaction term of time and treatment as a continuous variable. Effect of age will also be assessed by modeling an interaction term of age and treatment. To assess the effect of duration of supplementation on intervention effects, we will calculate Nelson-Aalen cumulative hazard estimates. Based on Nelson-Aalen estimates, we shall stratify time since start of supplementation as less than 90 days and more than 90 days. We shall assess trends across three age groups (6–11 months, 12-17 months and 18-24 months) and the two time strata with the Mantel-Cox method (Strate Mantel-Cox procedure in STATA 9.2, which estimates a one step Newton approximation to the log-linear Poisson regression coefficient). The last time period i.e. 18-24 months will estimate the residual impacts of the supplementation and differences if any between the two intervention groups and controls over the 6 months following the end of supplementation.
B. Major Activities and Milestones (Implementation Plan)
Objective 1 – Recruitment and Training of Staff
Activity 1 – To recruit, select, orient and train staff for this study
Staff comprising of Research Medical Officers, Field Supervisors, Social Scientists, Community Mobilizers, Data Collectors, Technicians, Community Health Workers and Admin and Finance Officers will be recruited, selected, oriented with the University’s policies and field research procedures, and trained as per needs of the study to enable them to carry out their responsibilities efficiently and effectively. Specialized training will be provided to Field Staff for study procedures, community mobilization, follow up and data collection through written manuals, presentations, role playing and videos. These are established protocols for staff training in field activities, data collection and verification etc that will be used for these activities.
The milestone 1 will be achieved once the entire team is hired, and trained. Timeline for this activity is first quarter of the study (3 months) from the starting date.
Objective 2 – Census of Field Sites and cluster allocation
Activity 2 – To conduct a Household Survey for collection of family data at study sites
A team of Data Collectors, supervised by Field Supervisors will conduct house to house baseline census to establish the baseline community denominators. The objective of this activity is to collect complete household census and socio-economic indicators. The data will be double entered in real time and cleaned. A GIS data base established at a household level.
The milestone 2 will be achieved once the complete census data has been collected and compiled on computers. This activity will start from the last month of first quarter and will go till end of 2nd quarter of the study.
Activity 3 – Cluster finalization and randomization
Although we have information from the sites form the census conducted in 2006, in light of the freshly conducted baseline household census in Activity 1, a de-novo cluster allocation of the population to the three intervention groups will be done blindly by the study oversight group.
The milestone 3 will be achieved once the complete demarcation of clusters has been undertaken and allocation of the clusters finalized. This activity will be completed in the last month of second quarter of the study and upon receiving complete census data conducted in Activity 2.
Objective 3 – Consent and Recruitment of Study Subjects
Activity 4 – To conduct Household survey by trained Community Health Workers (CHWs) to identify the cohort of infants eligible for the study
A system of data collection will be established through resident CHWs and CHWs with an objective to obtain information from each clusters to identify full term pregnant women and infants between 0-3 months of age. The teams of CHWs will be actively supervised by Field Supervisors and RMOs will also provide necessary assistance as and when required. We shall recruit the cohort of eligible infants and obtain the requisite consent after due discussion and information sharing.
The milestone 4 will be achieved once we have the information on identified infants and the requisite consent for participation by the data collection team. The timeline for this activity is third quarter of the study.
Objective 4 –Supplementation of Micronutrient and Zinc and regular follow up of the Study Population
Activity 5 – Initiation of supplementation with Micronutrient and / or Zinc at 6 months of age in intervention clusters
During this intervention initiation phase (quarters 4-5) The micronutrient and / or zinc intervention will initiated in the intervention clusters (and to the 400 infants who will be part of the in-depth study in Groups B and C). While the administration will be done by CHWs, the supervision will be undertaken by research supervisory teams.
This milestone 5 will be achieved at the end of quarter 9 as all identified and eligible children would have received daily micronutrients and / or Zinc for 12 months each. This activity will be conducted from the end of the third quarter and will go until quarter 9 with each eligible infant receiving supplement from 6-18 months of age.
Activity 6 – Regular follow up of study population by trained data collection teams
As indicated earlier two types of data collection and survey regimen are planned.
1. A quarterly general data collection system will target obtaining information from all participating infants and children in the clusters and principally collect data on pre-specified proforma. This data collection team will consist of trained CHWs who will visit each participating village/area in the clusters every three months. The general data collection teams (10 in all) will consist of CHWs supervised by a field supervisor who will be mobile.
2. A second group of in-depth data collection teams (detailed below) will obtain regular information from participating in-depth households (600 infants) weekly as per protocol. Because of the limited number of children per cluster undergoing in-depth study, these teams will cover multiple clusters and will be fully blinded to cluster allocations.
Each in-depth data collection team shall consist of a pediatric medical officer, laboratory technician, CHW and will be mobile. The families undergoing in-depth evaluation will require additional mobilization and support as these children will undergo sequential clinical evaluation, growth monitoring and relevant laboratory investigations every three-six months and it is critical to maintain close rapport with them. The CHWs, technicians and medical officers from this in-depth data collection team will work in close liaison with the local CHWs and family physicians specifically focusing on recruiting, ensuring compliance and follow up. These in-depth data collection teams will visit each of the participating households at fortnightly intervals and additionally these families will also be provided a contact telephone number to inform the team leader in the event of problems or illnesses requiring attention.
The milestone 6 will be achieved as the primary data is collected. Since this data collection will be at regular (bi-weekly or quarterly) intervals, the timeline will span from the last month of 3rd quarter to second month of 11th quarter.
Objective 5 – Clinical and Laboratory evaluation of supplemented / non-supplemented children
Activity 7 – Clinical and Anthropometric assessment
A clinical examination will be undertaken every fortnight and duly recorded. Anthropometric assessment will include weight, length, head circumference and arm circumference following standard protocols similar to those used in the construction of the 2004 World Health Organization (WHO) international growth reference standards (103). WHO Anthro 2005 software will be used to calculate height, weight and weight-for height percentiles, Z-scores, and malnutrition category relative to the international growth reference. In addition to the regular follow up plan, a computerized system of record keeping for serial investigations (as per Table III) will be maintained and the field work plan drawn up in advance by the medical officer and technologists of each in-depth study team.
The milestone 7 for this activity will be achieved by estimating growth rate of assessed children. Timeline for this activity will also span from 4th quarter to 11th quarter.
Laboratory investigations, Sample collection, processing, transportation and storage
With the exception of the small number to total body zinc exchangeable pool estimations, all other investigations will be undertaken in domiciliary settings using [protocols that have been previously used in the field by our research group (55, 94, 95, 106). All collection and transport of samples will be done as early as possible during the day with a defined work plan for the in-depth assessment team. While blood samples, urine and breath specimens will be obtained during the team visit, the collection of stool specimens will require coordination with the CHW and local CHW who will work with the families to ensure timely availability of fresh specimens. The protocols for collection of fecal samples will be brought to the study centre and blood samples will be collected at the centre and refrigerated on delivery or collection. The department of Paediatrics research program has considerable field experience with sample preparation and processing in field settings as a similar exercise was undertaken for the National Nutrition Survey (2002) (107) and the Sindh urinary iodine survey (2007). Each mobile field team will be equipped with a battery operated freezer, a portable centrifuge for sample preparation in the field, a liquid nitrogen container for freezing specimens and storage until transportation to the main laboratory (in either Hyderabad or Karachi). These laboratories have storage facilities with ultra-low deep freezers, which be enhanced in capacity as part of this project.
Entire emphasis is on non-invasive tests which can be accepted by families, implemented in the field and repeated at regular intervals.
Activity 8 – To conduct Breath Hydrogen Test (BHT)
The flora of children is expected to change as a child develops. We shall undertake breath hydrogen determination in the in-depth study group after a lactose feed to determine whether alterations in breath hydrogen excretion patterns occur which may be suggestive of small bowel bacterial overgrowth (i.e. with a delayed peak of breath hydrogen excretion). Breath hydrogen testing in a study with this sample size should be able to definitively determine whether the use of zinc leads to an alteration in breath hydrogen which presumes small bowel overgrowth. The breath hydrogen determinations will be made over time in each child as indicated in Table III. Comparisons will be made at the time of study entry in the three groups and sequentially at 3, 6, and 9 months after the initiation of the study, allowing for cross comparisons in the 3 groups at these time points.
The milestone 8 will be achieved by assessing breath hydrogen tests amongst assessed children. Timeline for this activity will also span from 4th quarter to 11th quarter.
Activity 9 – To conduct Intestinal Permeability Testing:
Intestinal permeability will be assessed using Lactulose /Rhamnose method (52, 79). Children will be given 400 mg/kg of Lactulose mixed with 100 mg/kg of rhamanose orally and urine will be collected over the next 6 hours and stored at -20oC till analysis. We have successfully adopted this method for ambulatory use and have undertaken the requisite analysis on relatively small urinary samples using HPLC .
The milestone 9 for this activity will be the assessment of sequential intestinal permeability tests in the cohort of 600 infants and children selected for in-depth study. The timeline will span from 4th quarter to 11th quarter.
Activity 10 – To conduct stool analysis among children with diarrheal episodes
To conduct stool examination & microbiological studies in acute diarrhea episodes, diarrheal stool samples will be collected from the earliest time possible from the start of a diarrheal episode from those cases identified during follow up of the in-depth study subset. Stool samples, (10-50 ml) will be collected by field workers in designated labeled containers during home visits or at health center presentation and transported as described previously. Depending upon clinical condition and state of dehydration, blood cultures, electrolytes, SGPT and creatinine will be obtained.
For Stool examination, Stool microscopy will be done within 6 hours of collection for the following pathogens:
• Parasites: Stool samples will be examined by microscopy using saline and iodine preparations, formol-ether concentration, and by modified acid fast and modified trichrome staining to look for helminth ova and protozoa (e.g., Isospora, Entamoeba histolytica, Cyclospora, Microsporidia). The ProSpecT Giardia Microplate ELISA Assay (Remel), will be used to detect Giardia lamblia (108). The presence and species of Cryptosporidium in the stool will be determined by nested PCR RFLP on stool DNA using primers specific for the small subunit ribosomal RNA as described previously (109). DNA will be extracted using a using a QIAamp Stool Mini kit (Qiagen) according to the manufacturer’s instructions.
• Bacteria: Stool cultures will be performed using the Bactec ® system to identify diarrhea causing pathogens (e.g., Shigella spp., Aeromonas spp., Vibrio spp., Salmonella spp., Campylobacter spp.). A nested PCR will be used to detect B. fragilis toxin in stool (110). Diarrheagenic Escherichia coli will be identified from colonies by a multiplex PCR based on the amplification of virulence genes from Shiga toxin-producing (stx1, stx2, and eae), enteropathogenic (eae and bfp), enterotoxigenic (stII and lt), enteroinvasive (virF and ipaH), enteroaggregative (aafII), and diffuse adherent (daaE) E. coli as described previously (111).
• Viruses: Enteric viruses including rotavirus, noroviruses, adenoviruses and astroviruses, will be identified by IDEIA™ and RIDAscreen® or ProSpecT® ELISAs performed according to the manufacturers’ instructions (112).
Activity 11: Characterization of intestinal microflora:
Fresh fecal samples will be collected every 3 months as described previously from children who do not have acute diarrhea and stored at -70° C until transport and analysis. Intestinal microflora will be characterized using the recently developed non-invasive 16s ribosomal RNA analysis on stool specimens (113-117) (see specific methods below). Older techniques such as small bowel intubation and quantitative cultures of small bowel flora have been found to provide a less accurate assessment of intestinal flora; this technique is also subject to more bias related to position of the sampling tube. In addition 16 s ribosomal RNA analysis, PCR and DNA microarrays have the capability to identify and characterize gut flora that may not be amenable to routine culture techniques (118). Samples will be available for assessment of intestinal flora on a quarterly basis and characterization of flora will be performed at baseline, 3 months, 6 months, 9 months and 12 months post-recruitment
We plan to use fluorescent in-situ hybridization and flow cytometry to assess differences in the fecal microbiota as a consequence of zinc or micronutrient supplementation. This technique has been widely used to assess differences in the intestinal microbiota under various conditions (119-121). The protocol will be adapted from a previously published report in which fecal microbiota was analyzed in a cohort of healthy humans from Europe (119). Briefly, a portion of the stool sample will be suspended in phosphate buffered saline (PBS), homogenized with glass beads and subsequently fixed with paraformaldehyde. The fixed fecal sample will then be stored at –70°C until later analysis at the Intestinal Microbiology laboratory of the GRASP Digestive Diseases Center at Tufts University in Boston. At the time of analysis the samples will be briefly centrifuged to remove debris. The samples will then be hybridized with a series of species-specific and generic bacterial fluorochrome-labeled oligonucleotide probes along with unlabeled competitor probes overnight. The samples will then be washed and resuspended in PBS with fetal bovine serum and analyzed on a FACScalibur flow cytometer (BD Biosciences) in the Tufts University Flow Cytometry core facility. The fluorescent emissions will be acquired using Cellquest (BD Biosciences) and analyzed with Summit software (Dakocytomation). The relative amounts of each bacterial species will be expressed relative to the amount of total bacteria detected by the generic eubacterial probe. Previously, others have demonstrated that the predominant phylogenetic groups in Europeans are Eubacterium rectale-Clostridium coccoides, Bacteroides-Prevotella and Faecalibacterium prausnitzii (120). In comparison, we anticipate that there will be significant differences in the intestinal microbiota of infants and children in these communities in Pakistan. Specifically, we expect to find relatively more gram negative phyla, such as Enterobacteria within the fecal microbiota of these children as has been previously demonstrated (121). We expect that we will find more gram negative phyla in the children who receive zinc by sprinkles than in those who receive the zinc in food; this will be more than seen in the flora of children who received microflora but no zinc.
The milestones 10-11 for all requisite stool testing, microflora determination will be based on obtaining sequential results of stool microscopy and culture from children with acute diarrhea and microflora studies from the in-depth study subgroup of children. The timeline for both activities 10 and 11 will span from 4th quarter to 11th quarter.
Activity 12: Biochemical assessment for hematology and micronutrient assessment
CBC, plasma zinc, ferritin, CRP
These planned investigations will be undertaken on the in-depth cohort as indicated in Table II. We shall obtain a 3 ml sample of blood from a venepuncture using standardized methods, before feeds and as much as possible between 0900-1100 in the morning. The sample of blood will be spun in a portable field centrifuge and aliquoted for analysis using micromethods:
1. The 1 ml EDTA blood will be analysed with a KX-21 automated hematology analyser for a detailed haemogram, including hemoglobin, total leucocyte count, and three-part differential (122).
2. Serum ferritin will be estimated on 150μl of serum using a microparticle enzyme immunoassay (MEIA) using an IMX® Analyzer (Abbott Lab).
3. Serum C reactive protein will be estimated using fluorescence polarization immunoassay technology using a TDxFLx® Analyzer (Abbott)
4. Plasma zinc will be estimated on 500 μl plasma using standard Atomic Absorption Spectrophotometry on a Thermo Elemental SOLLAR M Series atomic absorption spectrometer (123, 124)
These analyses will be conducted at the main Nutrition Research laboratory at the Aga Khan University on a daily basis.
The milestone 12 for hematological and micronutrient investigations will be based on obtaining sequential results of stool microscopy and culture from children with acute diarrhea and microflora studies from the in-depth study subgroup of children. The timeline will span from 4th quarter to 11th quarter.
Objective 6 – Estimation of Zinc Pool
Activity 13 – Administration of Zinc Isotopes and collection for Urine samples for Identification of exchangeable Zinc pool
The exchangeable zinc pool (EZP) is defined as the estimate of the total size of the combined pools of zinc that exchange with zinc in plasma within approximately 2-3 days. The EZP will be calculated by dividing the mass of intravenous isotope dose by the enrichment value at the y-intercept of the linear regression of a semi-log plot of urine enrichment data from days 4-8 after isotope administration (125).This may be an improved measure of total body zinc status in contrast to static plasma zinc estimation which may not reflect total body zinc status. In addition to serum biochemistry and micronutrient measurements, we plan to obtain EZP estimates in a subset of 30 children at two different time points (6 and 12 months respectively) as an improved estimate of body zinc status. The children earmarked for these estimations will be randomly identified from the in-depth group and these families will be specifically consented separately for this investigation.
EZP size will be determined by intravenous administration of 70Zn enriched stable isotope (approximately 45 μg/kg) at study time points, followed by spot urine collections from days 3-7 after intravenous tracer administration. The dose is infused over approximately 1 minute into a peripheral vein; the syringe is rinsed 2-3 times with 0.5 normal saline in the presence of research personnel.
Isotope preparation and administration: Zinc oxide powder enriched with 70Zn will be obtained from Trace Science International (Ontario, Canada) and prepared (126). An accurately weighed dose of enriched isotope solution will be administered intravenously to each subject via an infusion catheter in a peripheral vein. A 3-way stop-cock will be used to allow a rinse of the dose vial with an equal volume of normal saline.
Urine collections: Amount of isotope excreted in the urine will be accomplished through spot urine sample collections over five days: 1-2 samples /day x 5 days (total ( 6 samples/infant); volume of each urine collection is ~ 20 ml. If possible, an initial, baseline sample will be collected before isotope administration at birth; 2 baseline samples will be required at 6 mo to allow for correction for any residual isotope excretion from dose at birth. Prior to urine sample collections, the infant’s perineum will be cleaned with de-ionized water and filter paper. A zinc-free urine bag will be applied and urine collected. Once collected, the urine will be stored at -20(C until analysis. Research personnel responsible for the metabolic studies will be trained by Dr. Krebs, and supervised by Dr. Bhutta. Dr Krebs is a recognized global authority in this field and has ongoing collaborations with Dr Bhutta at the Aga Khan University with community based studies in EZP measurements among low birth weight infants in community settings.
Sample preparation and analyses for Zn enrichment: Preparation of individual urine samples includes wet digestion with concentrated nitric acid and H2O2. Dried samples are ashed on a hot plate and chelated to remove major minerals. Zinc concentrations will be measured using a Thermo Elemental SOLLAR M Series atomic absorption spectrometer. Zinc isotope enrichment in urine will be determined from isotope ratio measurements by VG Plasma Quad 3 (VG Elemental, Cheshire, UK) ICP-mass spectrometer.
The milestone 13 for this activity will be achieved in terms of estimation of exchangeable Zinc pool for the selected subset of 30 infants and children at the two time points specified. Given the analysis period required, the estimated timeline spans from 4th quarter to 11th quarter.
Objective 7 – Data Management
Activity 14 – Real-time Data Entry, cleaning and data management
We shall enhance and improve the existing systems for data entry and management in Matiari and Bilal Colony. All collected data will be cross-checked by the field supervisors at field offices on a daily basis and transferred on weekly basis to the Data Management Center at Aga Khan University from the field stations. Prior to data entry, all forms will be checked for completeness and consistency as well as coding of open- ended responses and area codes, etc. In case of inconsistency or missing responses, the editors will flag the errors/omissions and consult the interviewers for possible explanations.
For real-time data entry, databases and entry screens will be developed using Microsoft FoxPro. The entry screens will employ range and consistency checks and skips to minimize entry of erroneous data. Special arrangements will be made to enforce referential integrity of the collected database so that all data tables are related to each other without problem. A sub sample of the data (5%) will be manually checked to examine data entry errors and to monitor error rates of data entry operators. The data will be double entered.
The milestone 14 will be achieved by reviewing regular, bi-weekly reports of the data cleaning and error checking and the development of a clean locked data set for analysis by the middle of quarter 11. The timeline will span from 2nd quarter to 11th quarter.
Activity 15 – Data Safety and Monitoring activities by an External Study Oversight group:
We aim to establish an external independent study oversight group which will function as the data safety and monitoring board (DSMB). The main functions of the study oversight group will be to
1. Evaluate the study design & procedures for scientific validity
2. Evaluate the cluster data and develop a randomization protocol
3. Receive data on adverse events and fatalities during the course of the study
4. Receive unblinded data reports at three time points (quarter 6, 9 and 12) with the option of unblinding themselves if needed
5. Approving the release of blinding codes once the final data set is cleaned and locked.
While it is anticipated that the study oversight group will largely work by email link, we would propose at least one face to face meeting of the group at the mid point of the study. We have budgeted for a meeting of the DSMB at the mid point of the study in Pakistan but do recognize that for a variety of reasons including costs, this meeting could be held in the USA with the investigators traveling there if needed. We would propose four names for this external oversight group with the requisite broad expertise in this area. These include Prof Robert E. Black (Johns Hopkins University), Prof Larry Moulton (Johns Hopkins University), Prof Kenneth H Brown (UC Davis & Helen Keller International) and Prof Stanley Zlotkin (Hospital for Sick Children, Toronto).
The milestone 15 will be achieved by successful conclusion of the study oversight group deliberations at various time points specified and the time line for this is the entire duration of the study.
Activity 16 – Data Analysis of computerized data
The detailed analytical plan has been specified previously. This exercise will be undertaken at various stages of the project in a blinded fashion for reporting to the study oversight group which will also serve as the data safety and monitoring board. The analysis will be undertaken blindly once the data set is cleaned and locked (quarter 12) and the codes will be broken once cleared by the study oversight committee.
The Primary and Secondary outcome variables that will be analyzed for the three groups of infants include
Primary
1. Weight for age, height for age, weight for height z scores (% below -2 Z scores).
2. Intestinal permeability at 3, 6, 9 and 12 months post-supplementation
3. Ratio of gram negative to gram positive species of in the fecal microflora
4. Relative amount of bacterial species to total bacteria by eubacterial probe
5. Frequency of isolation of pathogenic gram negative organisms from routine stool specimens (microflora)
Secondary variables assessed will include:
1. Episodes of acute watery diarrhea
2. Episodes of bloody diarrhea and dysentery
3. Episodes of lactose intolerance
4. Episodes of prolonged diarrhea
5. Frequency of isolation of pathogens from diarrheal episodes
6. Frequency of co-morbid conditions such as febrile episodes, acute respiratory infections etc.
7. Micronutrient status at 6 & 12 months post-supplementation
External input and expertise will also be sought from Dr Simon Cousens (London School of Hygiene & Tropical Medicine) on the data for analysis techniques and advices as collaborator and therefore some international travel has also been budgeted in this activity
This milestone 16 will be achieved upon successful data compilation and analysis as per plan. The timeline for this activity will span from the end of the 3rd quarter to the 12th quarter.
Objective 8 – Report Writing
Activity 17 – Development of Final Report
We anticipate generating 6 monthly technical and financial reports for the project with the final report of the study shared with Gates Foundation at the end of the 12th quarter.
The milestone 17 will be achieved upon successful submission of final report. The timeline for the development and submission of this report will be the 12th quarter of the project.
C. Challenges
There are several major challenges in this project, ranging from communal agreement for participation in this intervention (since this a cluster design), to ensuring that the family members understand the project and interventions and thus comply with this. In particular, the in-depth study group of 600 infants will require close support and follow up to ensure compliance. Since we will be replenishing the micronutrient supply every fortnight, we will need to ensure that these are consumed as per instructions and that default rates are low.
The collection of fecal specimens and other laboratory investigations in field settings is a challenge in terms of logistics and quality assurance. Additionally, the timely processing of biological specimens and transportation to the field laboratories and reference laboratory at AKU while maintaining the cold chain is critical. The administration of intestinal permeability dual-sugar absorption tests and breath hydrogen testing will require trained technicians capable of undertaking testing over several hours in field circumstances. This will require adequate training in field procedures and senior level quality control measures.
Fortunately the principal investigator and his research team have been extensively involved in similar projects for many years and have the requisite expertise to undertake this. An extensive community-based support system exists and we have conducted several cluster randomized studies in health system settings. We are therefore confident that we can get the requisite buy-in from the community members and health department for these interventions.
There has been extensive recent formative work on complementary feeding initiatives in this area and a previous study of Sprinkles in Bilal Colony was well accepted. We have undertaken studies of diarrhea epidemiology in both rural and urban settings and as indicated earlier, have extensive experience with field stool, urine and blood sample preparation and processing. As part of this project we will have the mobile teams for field work and timely sample transportation to the main laboratories at AKU Karachi and Hyderabad. A system of freezing specimens with liquid Nitrogen will be put in place. We are also making adequate provision for ultra-low deep freezers for specimen storage until processing in both Karachi and Boston.
V. Monitoring, Evaluation and Dissemination
Quality Assurance
To ensure proper implementation of the intervention, the field supervisors will make spot checks and will arrange monthly refresher group sessions of the first-line health workers in which the problems encountered will be discussed and resolved. In addition, the data collection activity will be carried out by teams consisting of CHWs and DCs and; will be further monitored by field supervisors who will perform a check on a subset (5%) of households. Similarly all laboratory procedures have a quality check by periodic rechecking and standardization of the procedures. The Micronutrient Research Laboratories at AKU has a quality assurance system in place in collaboration with the biochemistry laboratories of ICDDRB and with the Q-Rad system in UK.
Dissemination:
The principal products of this study will be high quality scientifically valid publications in reputed international journals. We would consider other methods of sharing results in discussion with the Gates Foundation which may include a seminar or workshop discussing the implications and relevance of these findings. Clearly this will depend upon the nature of the findings but we anticipate that the results of the functional implications of large scale zinc and or micronutrient supplementation will be of great interest to the nutrition and child health community.
Ethical consideration:
Confidentiality:
Confidentiality of all the data collected from the population will be guaranteed through existing systems of data storage and anonymization of forms. Participant privacy and confidentiality in electronic and printed data, publications, and reports during and following completion of the study will be fully ensured. The field site offices and central storage facilities at AKU are fully secure with electronic access and monitoring around the clock.
Informed consent:
Informed consent will be obtained from the parent/guardian of the children at the time of recruitment in the study. An illustrated, simple to understand, information sheet will be prepared which will be shared with the families a day prior to the consent. The latter will only take place after a standardized interview with the team supervisor/medical officer ensuring their understanding of the project and its implications. Written informed consent will then be obtained and participants will be informed that they have the right to withdraw from the study at any time without any penalty in terms of care. Withdrawal from the study or refusal to participate will not in any way affect their ability to receive any services offered to the community by the study.
A similar but separate written consent process will be undertaken after due explanation for the families randomly identified for the in-depth study. All signed informed consent forms will be retained in the study files and a copy supplied to the Ethics Review Committee of AKU.
Ethical approval:
The study Proposal has been reviewed by the Research Ethics Review Committee (ERC) of the Aga Khan University, Karachi, Pakistan and a provisional clearance has been granted (attached). The committee has asked for some clarifications in its July meeting and final approval is expected in the main meeting of the ERC in late August or September 2007.
VI. Optimizing Public Health Outcomes and Intellectual Property Plans to Achieve Global Access
A. Overview
Although, a considerable amount of work has been done on zinc supplementation during illnesses and diarrhea in particular, programs for large scale supplementation with zinc for the prevention of diarrhea and stunting have as yet not begun. It is also notable that as yet there have been few studies of the impact of such supplementation strategies on gut ecology and gut function. Critically, the information on differential mechanisms of action and benefits of zinc in various types of diarrhea is lacking. This proposed body of work aims to evaluate the impact of zinc in a representative cohort of infants in urban and rural Pakistan employing some of the best available tools and non-invasive methods available. The findings of this study will help us fill some of these gaps and provide much needed information of relevance to future programs.
B. Optimizing Public Health Outcomes
If the findings of this study indicate no adverse effects and negative impacts on gut flora, this will add great support to the planned programs for scaling up such micronutrient interventions. Given the unexpected negative results from the recent iron and zinc supplementation trial in Pemba (90) World Health Organization in general and the governments of developing countries in particular will have a role in disseminating the knowledge obtained from this study and utilize its recommendation in their health policies in order to improve health of their children.
C. Intellectual Property (IP) Plan.
| |Yes |No |
|1. Is the proposed research likely to lead to any patentable or commercially exploitable results? | |X |
|Narrative (Use as much space as necessary) |
| |Yes |No |
|2. Will the proposed project, either at its inception or at a foreseeable future point, depend on the | |X |
|use of technologies, materials, or other inventions that may conflict with goals of global access in | | |
|terms of either cost or availability in the developing world? | | |
| |Yes |No |
|3. Is the proposed project and related IP subject to any agreements (e.g., licenses, collaborations, | |X |
|research or funding agreements or any other form of agreement) with commercial, academic, or other | | |
|organizations, including other funding entities, subgrantees or subcontractors? | | |
| |
| |Yes |No |
|4. Does your organization plan to assume responsibility for maturation, production, and dissemination of|X | |
|the innovation itself? | | |
| |
C. Commitment to Sharing Data and Materials
The Aga Khan University & Tufts University fully intends to share the findings of this project with the global science and public health community. The modality for this will be through peer reviewed scientific publications and a possible dissemination meeting.
VII. Organizational Capacity and Management Plan
A. Organizational Capacity and Facilities
Aga Khan University
Aga Khan University was established as a fully autonomous University in Pakistan under an Order by the President of Pakistan on March 16, 1983. The general supervision and control of the affairs of the University and the power to lay down the policies of the University vests with the Board of Trustees, which consists of a Chairman (nominated by the Chancellor).
Over recent years, AKU researchers have helped expand the boundaries of knowledge in a variety of disciplines. Our faculty in the past few years has achieved greater success in competing for external grants, both at the national and international levels. Research capacity and performance are now key factors in appointment and promotion of faculty. Research is also an active part of the curricula of the undergraduate, graduate and postgraduate programmes.
The strong commitment of the University to research is reflected in its increased investment in this area e.g. construction of a dedicated multi-million dollar Juma Research Building with state of the art facilities including a Bio Safety Level 3 laboratory, and well equipped multi-disciplinary and specialised laboratories for bench research scattered across the University.
Collaborative Research
The University is cognizant of its obligations to society and its innumerable outreach programmes, driven by societal needs and problems in both the bio-medical and social sciences in diversified areas such as infectious diseases, cancer, reproductive health and women issues, cardiovascular diseases, genetics, nutrition, human development, mental health and education, are testimony to this commitment. In addition, AKU also collaborates with organisations of national and international repute such as the Pakistan Medical and Research Council and the Karolinska Institute, as part of its engagement with the wider research community. The University places major emphasis on multidisciplinary and collaborative research.
Infrastructure for Research
1. URC and Research Ethics Committees
The University has a well developed infrastructure to support research activities and maintain the research ethics at international norms. The URC is the main monitoring and implementing body supported by Ethical Review Committee and Ethical Committee for Research on Animals. In addition to this University has developed Authorship Guidelines and a draft document on Intellectual Property Rights is currently under discussion in URC. AKU has received major grants for research ethics trainings and capacity development from the NIH (USA), the Wellcome Trust and is also a member of the steering committee of the Global Forum for Health Research.
2. The Research Office
University also has a Research Office which helps researchers on various research related matters including preparation of proposal, and annual reports of research projects. A research data base is also maintained by the Research Office which has the details of all funded projects conducted at AKU to date. Dissemination of information on funding opportunities and faculty research work and publications is also being looked after by this office.
3. The Grants & Contracts Office
University’s Grants and Contracts Office has considerable level of expertise and help faculty in preparing the budget, reviewing the financial aspects and assist grant recipients in managing the grants. This office works in close coordination with the Research Office and other university support offices.
4. Laboratories
To support researchers, AKU has recently developed new research facilities. These include a Juma Research Building which started functioning in November 1999. There is a capacity of 32 workstations for scientists who intend to carry out laboratory investigations in infectious diseases, and other research involving molecular biology and immunology. It has biosafety level 2 and biosafety level 3 facilities with an area to work on experimental animals. Inaugurated in 2005, the Nutrition Research Lab is a latest addition to research laboratories. It is well equipped to support the research activities focusing on women and child health. This facility could be used in all research that approach to utilize modern molecular biology and molecular genetics and tissue culture techniques.
To further support the cancer research, a university wide cancer tissue and data bank has been
established very recently.
5. Library
At the Aga Khan University, there is the Health Sciences Library with one of the best collections of medical literature in Pakistan. The library subscribes to more than 500 journals that are available on paper based as well as electronic format. In addition to this there are more than 22,000 books as well as other material on the electronic format and in the audiovisual department of the library. The library also has established linkages with other regional libraries to provide rapid access to users, for literature and other scientific material.
B. Management and Staffing Plan
For this study, one project supervisor, two senior field supervisors and four field supervisors for each site will be needed to oversee the progress of the project. In addition, two community mobilizers per site will be needed for overseeing census and cluster integrity in the study area, besides various other technical and junior staff mentioned in the budget. The Principal Investigator will be actively overseeing the progress at various stages along with other co-investigators to ensure timely completion of scheduled tasks in line of the study protocols.
For each data collection team, one medical officer and two community health workers will be needed, who will visit allocated clusters on regular fortnightly basis. Five such teams will be constituted for Bilal Colony and five teams for Matiari. Additional data collectors will be needed for revisits of household of sick children and their follow up. Additional workers will be needed to perform breath hydrogen test and intestinal permeability under supervision of medical officers.
Data management will need at least one data manager, two data supervisors per site and fifteen data entry personnel for real time data entry. An analyst will be needed on full time basis to check and analyze the data from time to time.
The investigators percent time allocation has been described under the heading Biographical Information Section X (Appendices).
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69. Duran B. The effects of long-term total parenteral nutrition on gut mucosal immunity in children with short bowel syndrome: a systematic review. BMC Nurs. 2005 Feb 1;4(1):2.
70. Andreassen BU, Paerregaard A, Schmiegelow K, Rechnitzer C, Heilman C, Hartmann B, Holst JJ, Michaelsen KF. Glucagon-like peptide-2 (GLP-2) response to enteral intake in children during anti-cancer treatment. J Pediatr Gastroenterol Nutr. 2005 Jan;40(1):48-53.
71. Rosenfeldt V, Benfeldt E, Valerius NH, Paerregaard A, Michaelsen KF. Effect of probiotics on gastrointestinal symptoms and small intestinal permeability in children with atopic dermatitis. J Pediatr. 2004 Nov;145(5):612-6.
72. Campbell DI, McPhail G, Lunn PG, Elia M, Jeffries DJ. Intestinal inflammation measured by fecal neopterin in Gambian children with enteropathy: association with growth failure, Giardia lamblia, and intestinal permeability. J Pediatr Gastroenterol Nutr. 2004 Aug;39(2):153-7.
73. Kukuruzovic R, Brewster DR, Gray E, Anstey NM. Increased nitric oxide production in acute diarrhoea is associated with abnormal gut permeability, hypokalaemia and malnutrition in tropical Australian aboriginal children. Trans R Soc Trop Med Hyg. 2003 Jan-Feb;97(1):115-20.
74. Campbell DI, Lunn PG, Elia M. Age-related association of small intestinal mucosal enteropathy with nutritional status in rural Gambian children. Br J Nutr. 2002 Nov;88(5):499-505.
75. Kukuruzovic R, Robins-Browne RM, Anstey NM, Brewster DR. Enteric pathogens, intestinal permeability and nitric oxide production in acute gastroenteritis. Pediatr Infect Dis J. 2002;21:730-9.
76. Lima NL, Soares AM, Mota RM, Monteiro HS, Guerrant RL, Lima AA. Wasting and intestinal barrier function in children taking alanyl-glutamine-supplemented enteral formula. J Pediatr Gastroenterol Nutr. 2007;44:365-74.
77. Rabbani GH, Teka T, Saha SK, Zaman B, Majid N, Khatun M, Wahed MA, Fuchs GJ. Green banana and pectin improve small intestinal permeability and reduce fluid loss in Bangladeshi children with persistent diarrhea. Dig Dis Sci. 2004 Mar;49(3):475-84.
78. Lima AA, Brito LF, Ribeiro HB, Martins MC, Lustosa AP, Rocha EM, Lima NL, Monte CM, Guerrant RL. Intestinal barrier function and weight gain in malnourished children taking glutamine supplemented enteral formula. J Pediatr Gastroenterol Nutr.2005 Jan;40(1):28-35.
79. Tooley KL, Saxon BR, Webster J, Zacharakis B, McNeil Y, Davidson GP, Butler RN. A novel non-invasive biomarker for assessment of small intestinal mucositis in children with cancer undergoing chemotherapy. Cancer Biol Ther. 2006;5:1275-81.
80. Nchito M, Friis H, Michaelsen KF, Mubila L, Olsen A. Iron supplementation increases small intestine permeability in primary schoolchildren in Lusaka, Zambia. Trans R Soc Trop Med Hyg. 2006;100:791-4
81. Pereira SP, Khin-Maung-U, Bolin TD, Duncombe VM, Nyunt-Nyunt-Wai, Myo-Khin, Linklater JM. A pattern of breath hydrogen excretion suggesting small bowel bacterial overgrowth in Burmese village children. J Pediatr Gastroenterol Nutr. 1991;13:32-8
82. de Boissieu D, Chaussain M, Badoual J, Raymond J, Dupont C. Small-bowel bacterial overgrowth in children with chronic diarrhea, abdominal pain, or both. J Pediatr. 1996;128:203-7
83. Dos Reis JC, de Morais MB, Oliva CA, Fagundes-Neto U. Breath hydrogen test in the diagnosis of environmental enteropathy in children living in an urban slum. Dig Dis Sci. 2007;52:1253-8.
84. Okeke IN, Laxminarayan R, Bhutta ZA, Duse AG, Jenkins P, O'Brien TF, Pablos-Mendez A, Klugman KP. Antimicrobial resistance in developing countries. Part I: recent trends and current status. Lancet Infect Dis. 2005 Aug;5(8):481-93
85. Lund B, Edlund C, Rynnel-Dagoo B, Lundgren Y, Sterner J, Nord CE. Ecological effects on the oro- and nasopharyngeal microflora in children after treatment of acute otitis media with cefuroxime axetil or amoxycillin-clavulanate as suspensions. Clin Microbiol Infect. 2001;7:230-7.
86. Lund B, Edlund C, Barkholt L, Nord CE, Tvede M, Poulsen RL. Impact on human intestinal microflora of an Enterococcus faecium probiotic and vancomycin. Scand J Infect Dis. 2000;32:627-32.
87. Fanaro S, Chierici R, Guerrini P, Vigi V. Intestinal microflora in early infancy: composition and development. Acta Paediatr Suppl. 2003;91:48-55.
88. Adlerberth I, Jalil F, Carlsson B, Mellander L, Hanson LA, Larsson P, Khalil K, Wold AE. High turnover rate of Escherichia coli strains in the intestinal flora of infants in Pakistan. Epidemiol Infect. 1998 Dec;121(3):587-98.
89. Adlerberth I, Carlsson B, de Man P, Jalil F, Khan SR, Larsson P, Mellander L, Svanborg C, Wold AE, Hanson LA. Intestinal colonization with enterobacteriaceae in Pakistani and Swedish hospital-delivered infants. Acta Paediatr Scand. 1991 Jun-Jul;80(6-7):602-10
90. Bhutta ZA. Lancet nutrition series
91. Bryce J Lancet Nutrition series
92. Ali NK, Bhutta ZA. A review of rotavirus diarrhea in Pakistan: how much do we know? J Coll Physicians Surg Pak. 2003;13:297-301
93. von Seidlein L, Kim DR, Ali M, Lee H, Wang X, Thiem VD, Canh do G, Chaicumpa W, Agtini MD, Hossain A, Bhutta ZA, Mason C, Sethabutr O, Talukder K, Nair GB, Deen JL, Kotloff K, Clemens J. A multicentre study of Shigella diarrhea in six Asian countries: disease burden, clinical manifestations, and microbiology. PLoS Med. 2006 Sep;3:e353.
94. Zafar A, Sabir N, Bhutta ZA. Frequency of isolation of shigella serogroups/serotypes and their antimicrobial susceptibility pattern in children from slum areas in Karachi. J Pak Med Assoc. 2005;55:184-8
95. Fischer Walker CL, Bhutta ZA, Bhandari N, Teka T, Shahid F, Taneja S, Black RE; Zinc Study Group. Zinc supplementation for the treatment of diarrhea in infants in Pakistan, India and Ethiopia. J Pediatr Gastroenterol Nutr. 2006;43:357-63
96. Hayes RJ, Alexander ND, Bennett S, Cousens SN. Design and analysis issues in cluster-randomized trials of interventions against infectious diseases. Stat Methods Med Res. 2000;9:95-116
97. Levanova LA, Aleshkin VA, Vorob'ev AA, Afanas'ev SS, Surikova EV, Aleshkin AV. [State of normal intestinal microflora in preschool children living in the ecologically adverse region] Zh Mikrobiol Epidemiol Immunobiol. 2002 Jan-Feb;(1):64-7
98. Rochet V, Rigottier-Gois L, Sutren M, Krementscki MN, Andrieux C, Furet JP, Tailliez P, Levenez F, Mogenet A, Bresson JL, Meance S, Cayuela C, Leplingard A, Dore J. Effects of orally administered Lactobacillus casei DN-114 001 on the composition for activities of the dominant faecal microbiota in healthy humans. Br J Nutr. 2006 Feb;95(2):421-9
99. Sokol H, Seksik P, Rigottier-Gois L, Lay C, Lepage P, Podglajen I, Marteau P, Dore J. Specificities of the fecal microbiota in inflammatory bowel disease. Inflamm Bowel Dis. 2006;12:106-11.
100. Korshunov VM, Potashnik LV, Efimov BA, Volodin NN, Korshunova OV, Gyr K, Frei R, Reber H, Ierendorzh D. [Intestinal microflora in children from Mongolia, Russia, and Switzerland]Zh Mikrobiol Epidemiol Immunobiol. 2001;(2):61-4
101. Mueller S, Saunier K, Hanisch C, Norin E, Alm L, Midtvedt T, Cresci A, Silvi S, Orpianesi C, Verdenelli MC, Clavel T, Koebnick C, Zunft HJ, Dore J, Blaut M. Differences in fecal microbiota in different European study populations in relation to age, gender, and country: a cross-sectional study. Appl Environ Microbiol. 2006;72:1027-33
102. Bhutta ZA. Beyond Informed Consent. Bull WHO 2004; 82:771-7
103. WHO/UNICEF Improved management of diarrhea: guidelines. 2003
104. de Onis M, Onyango AW, Van den Broeck J, Chumlea WC, Martorell R. Measurement and standardization protocols for anthropometry used in the construction of a new international growth reference. Food Nutr Bull. 2004;25:S27-36.
105. Therneau TM, Hamilton SA. RhDNase as an example of recurrent event analysis. Stats Med 1998; 16: 2029-2047.
106. Nizami SQ, Bhutta ZA, Weaver L, Preston T. Helicobacter Pylori colonization in infants in a peri-urban community in Karachi, Pakistan. J Paediatr Gastroenterol Nutr. 2005; 41: 191-4.
107. Government of Pakistan & UNICEF. National Nutrition Survey (2001-2). Islamabad. 2002
108. Aldeen, W. E., K. Carroll, A. Robison, M. Morrison, and D. Hale. 1998. Comparison of nine commercially available enzyme-linked immunosorbent assays for detection of Giardia lamblia in fecal specimens. J Clin Microbiol 36:1338-40
109. Xiao, L., L. Escalante, C. Yang, I. Sulaiman, A. A. Escalante, R. J. Montali, R. Fayer, and A. A. Lal. 1999. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol 65:1578-83
110. Sharma, N., and R. Chaudhry. 2006. Rapid detection of enterotoxigenic Bacteroides fragilis in diarrheal faecal samples. Indian J Med Res 124:575-82
111. Vidal, M., E. Kruger, C. Duran, R. Lagos, M. Levine, V. Prado, C. Toro, and R. Vidal. 2005. Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections. J Clin Microbiol 43:5362-5
112. Banerjee, I., B. P. Gladstone, A. M. Le Fevre, S. Ramani, M. Iturriza-Gomara, J. J. Gray, D. W. Brown, M. K. Estes, J. P. Muliyil, S. Jaffar, and G. Kang. 2007. Neonatal Infection with G10P[11] Rotavirus Did Not Confer Protection against Subsequent Rotavirus Infection in a Community Cohort in Vellore, South India. J Infect Dis 195:625-632
113. Hart AL, Staaq AJ, Frame M. Role of Gut Flora in Health and Disease. Alimentari Pharmcol ther 2002; 16: 1383-93.
114. Stroup SE, Roy S, Mchele J, Maro V, Ntabaguzi S, Siddiquie A, Kang G, Guerrant RL, Kirkpatrick BD, fayer T, Herbein J, Ward H. Haq R, Houpt ER, Real Time PCR detection and speciation of cryptosporidial infection using Scorpion Probes. J Med Micro 55; 1217-22.
115. Ward H, Leav B. Cryptosporidia: New Insights . CID 2003 36: 903-908.
116. Durmaz B, Dalqalar M, Durmaz R. Prevalence of ETBF in patients with diarrhea. Anaerobe 2005 11 318-321.
117. Guarner F. Enteric Flora in Health and Disease. 2006, Digestion, 73 S1: 5-12.
118. Pathela P, Hasan KZ, Roy E et al ETBF diarrhea in children 0-2 years of age in rural Bangladesh. Journal Infectious Disease 2005; 191: 1245-1252.
119. Lay C, Sutren M, Rochet V, Saunier K, Dore J, Rigottier-Gois L.Design and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota. Environ Microbiol. 2005;7:933-46.
120. Fallani M, Rigottier-Gois L, Aguilera M, Bridonneau C, Collignon A, Edwards CA, Corthier G, Dore J. Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. J Microbiol Methods. 2006;67:150-61.
121. Bardhan PK, Albert MJ, Alam NH, Faruque SM, Neogi PK, Mahalanabis D. Small bowel and fecal microbiology in children suffering from persistent diarrhea in Bangladesh. J Pediatr Gastroenterol Nutr. 1998;26:9-15.
122. NCCLS document H26-A. Performance goals for the internal quality control of multichannel hematology analyzers; Approved Standard. NCCLS, Wayne, PA. 1996.
123. Smith JC, Holbrook JT, Danford DE. Analysis and evaluation of zinc and copper in human plasma and serum. Am J Clin Nutr 1985. 4: 627-38
124. Paracha PI, Jamil A. Assessment of micronutrient (iron, vitamin A and zinc) status in pre-school children of North West Frontier Province, Pakistan. 1998
125. Miller LV, Hambidge KM, Naake VL, Hong Z, Westcott JL, Fennessey PV. Size of the pools that exchange rapidly with plasma zinc in humans: alternative techniques for measuring and relation to dietary zinc intake. J Nutr 1994; 124:268-76.
126. Krebs NF, Westcott JE, Arnold TD, Kluger BM, Accurso FJ, Miller LV, Hambidge KM. Abnormalities in zinc homeostasis in young infants with cystic fibrosis. Pediatr Res 2000;48:256-261.
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Clinical Microbiology and Infection 2004;10: 5–11.
X. Appendices
Standard Operating Procedures (SOP) Data Management Unit Hala project
A. Table and Timeline
The Milestone Table and Timeline are attached.
B. Budget Spreadsheet
Attached
C. Financial and Tax Information
AKU is a non-profit institution and a copy of the Institution’s Charter is attached with this proposal. AKU is also tax exempt and a copy of the certificate is also attached.
D. Biographical Information
1.
Bhutta, Zulfiqar, A
Principal Investigator
Professor and Chairman, Department of Paediatrics and Child Health, Aga Khan University,
date of employment 1986
Responsibilities include: Principal Investigator and Overall Supervision of the project
% Time to be allocated: 25%
Education:
- Degree(s): M.B.,B.S, DCH, M.R.C.P., F.C.P.S, Ph.D., FRCPCH,
- Year of Highest Degree: 1996
- Discipline: Sciences (Doctoral)
- Institution: Karolinska Institute, Stockholm, Sweden
Representative Publication
1. Bhutta ZA, Black RE, Brown KH, Gardner JM, Gore S, Hidayat A, Khatun F, Martorell R, “Prevention of diarrhea and pneumonia by zinc supplementation in children in developing countries: pooled analysis of randomized controlled trials”. Ninh NX, Penny ME, Rosado JL, Roy SK, Ruel M, Sazawal S, Shankar A. Journal of Pediatrics 1999;135:689-697.
2. Fischer Walker CL, Bhutta ZA, Bhandari N, Teka T, Shahid F, Taneja S, Black RE; Zinc Study Group. Zinc supplementation for the treatment of diarrhea in infants in Pakistan, India and Ethiopia. J Pediatr Gastroenterol Nutr. 2006;43:357-63.
3. Mahomed K, Bhutta Z, Middleton P. Zinc supplementation for improving pregnancy and infant outcome.Cochrane Database Syst Rev. 2007 Apr 18;(2):CD000230
4. Haider B, Bhutta Z. Multiple-micronutrient supplementation for women during pregnancy. Cochrane Database Syst Rev. 2006;(4):CD004905
5. Sharieff W, Bhutta ZA, Schauer C, Tomlinson G, Zlotkin S. Micronutrients (including zinc) reduce diarrhoea in children: The Pakistan sprinkles diarrhoea study. Arch Dis Child. 2006; 91:573-9
2.
Nizami, Shaikh, Q
Co-investigator
Professor, Department of Paediatrics and Child Health, Aga Khan University,
Date of employment: August 1989
Responsibilities include: Overall supervision of the project
% Time to be allocated: 20%
Education:
- Degree(s): MBBS, MCPS (Pak), FCPS (Pak)
- Year of Highest Degree: 1981
- Discipline Pediatrics
- Institution The Aga Khan University
Representative Publication
1. Nizami SQ, Bhutta ZA, Siddiqui A A, Lubbad L. Enhanced detection rate of typhoid fever in children in a periurban slum in Karachi, Pakistan using polymerase chain reaction technology. Scand J Clin Lab Invest 2006;66:429-436
2. Nizami SQ, Bhutta ZA, Hasan R Incidence of acute respiratory infections in children 2 months to 5 years of age in periurban communities in Karachi Pakistan. J Pak Med Assoc 2006;56(4):163-66
3. Nizami SQ Bhutta ZA, Weaver L, Preston Helicobacter pylori colonization in infants in periurban community in Karachi Pakistan. J Pediatr Gastroenterol Nutr. 2005 Aug;41(2):191-194
4. Nizami SQ, Bhutta ZA, Hasan R, Husen Y Role Of Chest X-Ray In Diagnosis Of Lower Respiratory Tract Infections In Children Less Than Five Years Of Age in Community. J Pak Med Sci, 2005; 21(4):417-21
5. Nizami S Q, Bhutta ZA, Molla AM, Efficacy of traditional Rice Lentil yogurt diet, lactose free milk protein based and soy protein formulae in management of secondary lactose intolerance with acute childhood diarrhoea, J Trop. Pediatr, 1996; 42:133-37
3.
Zaidi, Anita, K.M.
Co-investigator
Associate Professor, Department of Paediatrics and Child Health, Aga Khan University,
date of employment 2003
Responsibilities include: Supervision of Lab investigations and clinical assessment monitoring
% Time to be allocated: 20%
Education:
- Degree(s): M.B.,B.S., M.S
- Year of Highest Degree: 1999
- Discipline: Science (MS in Epidemiology)
- Institution: Harvard School of Public Health
Representative Publication
1. Zaidi AKM, Awasthi S, DeSilva HJ. Burden of Infectious Diseases in South Asia. BMJ 2004; 328:811-5.
2. Zaidi AKM, Khan E. Pediatric tuberculosis – A developing country perspective Ceylon Medical Journal 2004. In press
.
3. Khan TA, Zaidi AKM. Acute Respiratory Infections in Pakistan – Have we made any progress? J Coll Physicians and Surgeons Pakistan. 2004. In press.
4. Thaver D, Critchley J, Zaidi AKM, Bhutta ZA. Fluoroquinolones in the treatment of Enteric Fever. Cochrane Review and Meta-analysis. 2004. In press.
5. Zaidi AK, Hasan R, Bhutta ZA. Typhoid Fever. N Engl J Med. 2003; 348 (12):1182-4. Letter
4.
Wanke, Christine, A
Co-investigator
Professor & Division Chief, Department of Public Health and Family Medicine, Division of Nutrition and Infection, Tufts University Medical School, Boston, MA, USA,
date of employment 2006
Responsibilities include: Clinical Microbiologist
% Time to be allocated 15%
Education:
- Degree(s): B.A. ; M.D.
- Year of Highest Degree: 1980
- Discipline: Medicine
- Institution: University of Wisconsin, Madison, WI
Representative Publication
1. Wanke CA, Gerrior J, Kantaros J, Coakley E, Albrecht M. Recombinant human growth hormone improves the fat redistribution syndrome in patients with HIV. AIDS 1999; 13:2099-2104.
2. Phanuphak P, Grayson M, Sirivicahyakul S, Suwanagool S, Ruxrungthan K, Hanvanich M, Ratanasuwn W, Ubolyam S, Hughes M, Wanke CA, Hammer SM. A comparison of two dosing regimens of zidovudine in Thai adults with early asymptomatic HIV infection: conducting clinical trials in South-East Asia. Australia-New Zealand Journal of Medicine 2000; 30 (1):11-20.
3. Wanke CA, Silva M, et al. Weight loss and wasting remain common complications in individ-uals infected with HIV in the era of highly active antiretroviral therapy. CID 2000; 31: 803-805.
4. Wanke CA. Nutritional/Metabolic Status- Time to Change our Assumptions. Editorial Comment. The AIDS Reader 2000; 10: 544.
5. Shevitz AH, Wanke CA, Falutz J, Kotler DP. Clinical Perspectives on HIV-associated lipodystrophy syndrome: An update. AIDS 2001; 15:1917-1930.
5.
Hendricks, Kristy, M
Co-investigator
Associate Professor, Tufts University Medical School, Boston, MA, USA,
date of employment 2000
Responsibilities include: Nutrition Advisor
% Time to be allocated: 15%
Education:
- Degree(s): B.S.; M.S.; Sc.D.
- Year of Highest Degree: 1987
- Discipline: Nutritional Biochemistry
- Institution: Boston University, Boston, Massachusetts, USA
Representative Publication
1. Cohen S, Hendricks K, et al. Chronic nonspecific diarrhea: A complication of fat restriction. Amn J
Dis of Childhood 113: 490-492, 1979.
2. Cohen S, Hendricks K, et al. Chronic nonspecific diarrhea: Dietary relationships. Pediatrics 64:402-7, 1979.
3. Shaikh S, Molla AM, Islam A, Billoo AG, Hendricks KM, Snyder JD. A traditional diet as part of oral
therapy in severe acute diarrhea in young children. Journal of Diarrheal Disease Research 9:258-263, 1991.
4. Bhutta ZA, Molla AM, Issani Z, Badruddin S, Hendricks KM, Snyder JD. Dietary management of persistent
diarrhea: Comparison of a traditional rice-lentil based diet with soy formula. Pediatrics 88: 1010-1018, 1991.
5. Badruddin SH, Islam A, Hendricks KM, Bhutta ZA, Shaikh S, Snyder JD, Molla AM. Dietary risk factors for acute and chronic diarrhea in Karachi, Pakistan. American Journal of Clinical Nutrition 54:745-749, 1991.
6.
Ward, Honorine, D
Co-investigator
Associate Professor of Medicine, Division of Geographic Medicine and Infectious Diseases, Tufts-New England Center, Boston, MA, USA,
date of employment 2004
Responsibilities include: Molecular Biologist
% Time to be allocated 15%
Education:
- Degree(s): M.B.;B.S (US, equivalent, M.D.); Post-doc
- Year of Highest Degree: 1988
- Discipline: Parasitology
- Institution: Boston University, Boston, Massachusetts, USA
Representative Publication
1. Verdon, R., Keusch, G.T., Tzipori, S., Grubman, S.M., Jefferson, D.M. and Ward, H.D. An in vitro model of infection of human biliary epithelial cells by Cryptosporidium parvum. J Infect Dis 175:1268-72. 1997.
2. Ward, H.D. and Cevallos, A.M. Cryptosporidium: Molecular basis of Host-Parasite Interaction. Adv Parasitol 40:151-85. 1998.
3. Joe, A., Verdon, R. Keusch, G.T., Tzipori, S., and Ward, H.D. Attachment of Cryptosporidium parvum sporozoites to human intestinal epithelial cells. Infect Immun. 66:3429-32. 1998.
4. Barnes, D.A., Bonnin, A., Huang, J-X., Gousset, L., Wu, J., Gut, J., Doyle, P., Dubremetz, J-F., Ward, H., and Petersen, C. A novel multi-domain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion. Mol Biochem Parasitol. 96:93-110. 1998.
5. Cevallos A. M, Zhang X, Waldor M, Jaison S, Tzipori S, Zhou X, Neutra M, Ward H. D. Molecular cloning and expression of a gene encoding Cryptosporidium parvum glycoproteins gp40 and gp15. Infect Immun 68:4108-4116. 2000
XI. Additional Required Narrative for Compliance with Regulatory Guidelines
A. Research Assurances
1. Animal Research
There will not be any Animal Research subjects in this study.
2. Research on Human Subjects
• Human Subjects Consent Form:
A consent form and translation has been submitted to the ERC for approval and is being shared with the foundation as desired
• IRB/IEC (Institutional Review Board/Independent Ethics Committee) approval:
The main ethics review committee is the south of Pakistan is at the Aga Khan University. We have submitted the proposal to the committee and received provisional approval (submitted earlier). The final approval may be granted at the committee meeting of Aug 2007.
As per usual research policies of Aga Khan University, funding bodies are not liable to claims for adverse events, as the University when accepting and executing projects takes this responsibility upon itself.
-----------------------
[1] If you fall within one of the first five categories please include your IRS tax determination letter in Appendix A. If you are a non-U.S. charitable organization, please see fiscal status link
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