Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
Prospective, cluster randomized, controlled evaluation
of the impact of zinc and / or micronutrient
supplementation on intestinal flora, diarrheal disease
burden, intestinal mucosal integrity and growth
among cohorts of children in Pakistan
_____________________________________________
A Global Health Program Proposal to Bill and Melinda Gates Foundation by
Prof. Zulfiqar A Bhutta
zulfiqar.bhutta@aku.edu
Tel: +92 21 4864782
Fax: +92 21 4934294
1
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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.
41
Research Assurances
* Separate attachments
2
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
I. Proposal Information
A. Organization
Organization
The Aga Khan University
Name:
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
Title
Address
First
Mohammad
name
Dean, Medical College
Aga Khan University, Stadium
Road, Karachi, Pakistan
Dr
E-mail
Web site
Khurshid
Suffix
Surname
Telephone
+92 21 4930051
Fax
+92 21 4932095
mohammad.khurshid@aku.edu
www.aku.edu
B. Project
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
Project Name:
Principal Investigator/Project Director:
Prefix
Title
Address
E-mail
Web site
First
Zulfiqar
name
Professor and Chairman, Dept of
Paediatrics
Aga Khan University, Stadium
Road, Karachi, Pakistan
Dr
Surname
Bhutta
Telephone
+92 21 4930051
Fax
+92 21 4932095
Suffix
zulfiqar.bhutta@aku.edu
www.aku.edu
Amount Requested From
Foundation ($USD):
Project Duration
(months):
Estimated Total Cost of Project
($USD):
Organization’s total revenue for
most recent audited financial year
($USD):
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
3
36
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
a) For detailed follow up of the cohort for growth, micronutrient status, morbidity and
prospective evaluation of a subset for intestinal mucosal permeability, small bowel
microflora, and breath hydrogen excretion
b) For management and treatment of diarrheal episodes, respiratory and other infections.
c) For investigations, both for routinely indicated clinical investigations and those for study
purpose only.
d) Data management and analysis of results
e) Supervisory visits of collaborators
C. Regulatory Approval Questionnaire
Please enter an “x” in the appropriate column.
Yes
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.
X
4. Project will involve research using vertebrate animals
5. Project will involve research using human subjects
X
6. Project will involve clinical trials
X
7. Project will involve use of any of the following substances:
• recombinant DNA subject to regulation
• pathogens/toxins identified as “select agents” by U.S. law:
(http://www.aphis.usda.gov/vs/ncie/pdf/agent_toxin_list.pdf)
• biohazards or genetically modified organisms
X
4
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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.
5
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 ( 40/min).
Severe illness will be defined as a temperature 38.4°C or admission to hospital or
respiratory rate 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
6
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
respiration rate ( 50/min) or chest indrawing), fever (axillary temperature 37.2°C), and
high fever (axillary temperature 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 48hour 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.
7
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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.
8
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
Studies of zinc in the
(x1000)
pathogenesis and treatment of
Etiology
episodes
Basic science
Applied clinical
Median
Range
studies
studies
Salmonella
67.2
49.6 - 126.4
3
-
Shigella sp.
97.6
57.6 – 169.6
2
2
Campylobacter
84.8
41.6 – 166.4
-
-
Vibrio cholerae
100.0
25.6 – 187.2
3
3
ETEC
152.0
107.2 – 268.8
2
-
EPEC
264.0
150.4 – 441.6
1
-
Rotavirus
406.4
262.4 – 564.8
-
-
Giardia
28.8
11.2 – 100.4
2
-
Cryptosporidium
49.6
19.2 – 131.2
-
-
Entamoeba
11.2
3.2 – 59.2
-
-
Multiple infections
200.0
136.0 – 323.2
-
-
Unknown
137.6
----
Total
1,600
----
9
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
10
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
11
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
12
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
Table II
Epidemiological information from the entire urban / rural sites (April 2006 – March
2007)
INDICATOR
BILAL
COLONY
HALA/MATIARI
TOTAL
(RURAL)
(URBAN)
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 125150 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
13
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
Pir Jhando
B.D Kaka
Bago Jamali
K.B Kaka
Matiari
S.A.S Ji Wasi
n
3
4
56
11
140
119
702
Sultan pur
Diarrhea
episodes/child
for 3 months
1.67
1.00
1.36
1.36
1.34
1.81
1.47
267
Oderolal Station
New Saeedabad
Fakir Nothiani
Hala Old
Shahmeer Rahoo
Mushtaqabad
Oderolal Village
Nobat Mari
New Hala
Total
103
63
194
2
20
258
160
147
2249
1.19
1.29
1.60
1.54
1.00
3.10
1.73
1.58
1.57
1.50
Bilal Colony
Sectors
A
B
C
D
E
F
Gulshan-eLatif
Mohd.Ali
Shah Goth
Total
n
21
26
7
9
7
22
Diarrhea
episodes/child
for 3 months
0.62
0.88
0.57
1.00
0.71
1.36
18
2.22
4
0.25
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).
14
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
15
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
16
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
17
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 indepth 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
(6-12 months
age)
Clinical
evaluation
Zinc and /or
micronutrient
supplementation
Standard
Anthropometry
Initial
exam
3
months
6
months
12
months
18
months
24
months
√
√
√
√
√
√
none
none
continued
To stop at 18
months age
none
√
√
Initiation of zinc
and/or
micronutrients
√
√
√
√
18
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Time post
recruitment
(6-12 months
age)
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)*
Sept 30, 2007
Initial
exam
3
months
6
months
12
months
18
months
24
months
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
√
* 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.
19
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 prespecified 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-toevent 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.
20
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 loglinear 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
21
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 threesix 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.
22
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 / nonsupplemented 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 weightfor 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
23
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
24
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 noninvasive 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.
25
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
26
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
27
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
28
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
5.
Sept 30, 2007
Frequency of isolation of pathogenic gram negative organisms
routine stool specimens (microflora)
from
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
29
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
30
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
1. Is the proposed research likely to lead to any patentable or
commercially exploitable results?
Narrative (Use as much space as necessary)
31
No
X
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
Yes
No
2. Will the proposed project, either at its inception or at a foreseeable
future point, depend on the 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?
X
Yes
No
3. Is the proposed project and related IP subject to any agreements
(e.g., licenses, collaborations, research or funding agreements or
any other form of agreement) with commercial, academic, or other
organizations, including other funding entities, subgrantees or
subcontractors?
X
Yes
4. Does your organization plan to assume responsibility for
maturation, production, and dissemination of the innovation itself?
No
X
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.
32
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
33
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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).
34
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
IX. Citations
1. Black RE, Morris SS, Bryce J. Where and why are 10 million children dying every
year? Lancet. 2003;361:2226-34
2. Keusch GT, Fontaine O, Bhargava A, Boschi-Pinto C, Bhutta ZA, Gotuzzo E,
Rivera JA, Chow J, Shahid-Salles SA, Laxminarayanan R. Diarrheal Diseases. In
Jamison DT, Breman JG, Measham AR, Alleyne G, Claeson M, Evans DB, Jha
P, Milla A, Musgrove P (eds). Disease Control Priorities in Developing Countries
(Second Edition), Oxford, Washington DC, 2006, pp 371-388.
3. Implementing the New Recommendations on the Clinical Management of
Diarrhea Guidelines for Policy Makers and Programme Managers, World Health
Organization 2006: http://www.who.int/child-adolescent-health /New_Publications
/CHILD_HEALTH /ISBN_92_4_159421_7.pdf accessed on 29th Jan 2007
4. Bhutta ZA, Bird SM, Black RE, Brown KH, Gardner JM, Hidayat A, Khatun F,
Martorell R, Ninh NX, Penny ME, Rosado JL, Roy SK, Ruel M, Sazawal S,
Shankar A. Therapeutic effects of oral zinc in acute and persistent diarrhea in
children in developing countries: pooled analysis of randomized controlled trials.
Am J Clin Nutr. 2000;72:1516-22.
5. Bhutta ZA, Black RE, Brown KH, Gardner JM, Gore S, Hidayat A, Khatun F,
Martorell R, Ninh NX, Penny ME, Rosado JL, Roy SK, Ruel M, Sazawal S,
Shankar A. Prevention of diarrhea and pneumonia by zinc supplementation in
children in developing countries: pooled analysis of randomized controlled trials.
Zinc Investigators' Collaborative Group. J Pediatr. 1999;135:689-97.
6. Miller M, Agqarwal R, Sentz J. Role of zinc administration in prevention of
childhood diarrhea and respiratory infections: a meta-analysis. Pediatrics (2007
in press)
7. Baqui AH, Black RE, El Arifeen S, Yunus M, Chakraborty J, Ahmed S, Vaughan
JP. Effect of zinc supplementation started during diarrhea on morbidity and
mortality in Bangladeshi children: community randomised trial. BMJ.
2002;325:1059
8. Bhutta ZA. Iron and zinc deficiency in children in developing countries. BMJ.
2007;334:104-5.
9. Bhutta ZA. The role of zinc in child health in developing countries: taking the
science where it matters. Indian Pediatr. 2004;41:429-33.
10. Jones G, Steketee RW, Black RE, Bhutta ZA, Morris SS; Bellagio Child Survival
Study Group. How many child deaths can we prevent this year? Lancet.
2003;362:65-71
11. Awasthi S; INCLEN Childnet Zinc Effectiveness for Diarrhea (IC-ZED) Group.
Zinc supplementation in acute diarrhea is acceptable, does not interfere with oral
rehydration, and reduces the use of other medications: a randomized trial in five
countries. J Pediatr Gastroenterol Nutr. 2006;42:300-5
12. Sazawal S, Dhingra U, Dhingra P, Hiremath G, Kumar J, Sarkar A, Menon VP,
Black RE. Effects of fortified milk on morbidity in young children in north India:
community based, randomised, double masked placebo controlled trial. BMJ.
2007;334:140
13. Villalpando S, Shamah T, Rivera JA, Lara Y, Monterrubio E. Fortifying milk with
ferrous gluconate and zinc oxide in a public nutrition program reduced the
prevalence of anemia in toddlers. J Nutr. 2006;136:2633-7.
14. Christofides A, Asante KP, Schauer C, Sharieff W, Owusu-Agyei S, Zlotkin S.
Multi-micronutrient Sprinkles including a low dose of iron provided as
35
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
microencapsulated ferrous fumarate improves haematologic indices in anaemic
children: a randomized clinical trial. Matern Child Nutr. 2006;2:169-80
15. Sazawal S, Black RE, Ramsan M, Chwaya HM, Stoltzfus RJ, Dutta A, Dhingra U,
Kabole I, Deb S, Othman MK, Kabole FM. Effects of routine prophylactic
supplementation with iron and folic acid on admission to hospital and mortality in
preschool children in a high malaria transmission setting: community-based,
randomised, placebo-controlled trial. Lancet. 2006;367:133-43
16. Wasantwisut E, Winichagoon P, Chitchumroonchokchai C, Yamborisut U,
Boonpraderm A, Pongcharoen T, Sranacharoenpong K, Russameesopaphorn W.
Iron and zinc supplementation improved iron and zinc status, but not physical
growth, of apparently healthy, breast-fed infants in rural communities of northeast
Thailand. J Nutr. 2006;136:2405-11.
17. Bhandari N, Taneja S, Mazumder S, Bahl R, Fontaine O, Bhan MK; Zinc Study
Group. Adding zinc to supplemental iron and folic acid does not affect mortality
and severe morbidity in young children. J Nutr. 2007;137:112-7.
18. de Benoist B, Darnton-Hill I, Lynch S, Allen L, Savioli L. Zinc and iron
supplementation trials in Nepal and Tanzania.Lancet. 2006;367:816.
19. Luabeya KK, Mpontshane N, Mackay M, Ward H, Elson I, Chhagan M, Tomkins
A, Van den Broeck J, Bennish ML. Zinc or multiple micronutrient
supplementation to reduce diarrhea and respiratory disease in South african
children: a randomized controlled trial. PLoS ONE. 2007 Jun 27;2:e541.
20. Bhandari N, Taneja S, Mazumder S, Bahl R, Fontaine O, Bhan MK; Zinc Study
Group. Adding zinc to supplemental iron and folic acid does not affect mortality
and severe morbidity in young children. J Nutr. 2007;137:112-7
21. Roy SK, Behrens RH, Haider R, Akramuzzaman SM, Mahalanabis D, Wahed
MA, Tomkins AM. Impact of zinc supplementation on intestinal permeability in
Bangladeshi children with acute diarrhea and persistent diarrhea syndrome. J
Pediatr Gastroenterol Nutr. 1992;15:289-96.
22. Salgueiro MJ, Zubillaga M, Lysionek A, Cremaschi G, Goldman CG, Caro R, De
Paoli T, Hager A, Weill R, Boccio J. Zinc status and immune system relationship:
a review. Biol Trace Elem Res. 2000;76:193-205.
23. Fraker PJ, King LE, Laakko T, Vollmer TL. The dynamic link between the
integrity of the immune system and zinc status. J Nutr. 2000;130(5S
Suppl):1399S-406S.
24. Blanchard RK, Cousins RJ. Regulation of intestinal gene expression by dietary
zinc: induction of uroguanylin mRNA by zinc deficiency. J Nutr. 2000;130(5S
Suppl):1393S-8S
25. Canani RB, Cirillo P, Buccigrossi V, Ruotolo S, Passariello A, De Luca P,
Porcaro F, De Marco G, Guarino A. Zinc inhibits cholera toxin-induced, but not
Escherichia coli heat-stable enterotoxin-induced, ion secretion in human
enterocytes. J Infect Dis. 2005;191:1072-7.
26. Lanata C, Black RE, Bische-Pinto C et al. Global burden of diarrhea and etiology.
unpublished data from CHERG. 2006.
27. Hegazy SM, Adachi Y. Comparison of the effects of dietary selenium, zinc, and
selenium and zinc supplementation on growth and immune response between
chick groups that were inoculated with Salmonella and aflatoxin or Salmonella.
Poult Sci. 2000;79:331-5.
28. Smith BL, Towers NR. Salmonellosis in sheep and zinc oxide dosing. N Z Vet J.
1986;34:37-8
36
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
29. Roy SK, Tomkins AM, Ara G, Jolly SP, Khatun W, Chowdhury R, Chakrabarty B.
Impact of zinc deficiency on vibrio cholerae enterotoxin-stimulated water and
electrolyte transport in animal model. J Health Popul Nutr. 2006;24:42-7.
30. Surjawidjaja JE, Hidayat A, Lesmana M. Growth inhibition of enteric pathogens
by zinc sulfate: an in vitro study.Med Princ Pract. 2004 Sep-Oct;13(5):286-9.
31. Raqib R, Roy SK, Rahman MJ, Azim T, Ameer SS, Chisti J, Andersson J. Effect
of zinc supplementation on immune and inflammatory responses in pediatric
patients with shigellosis. Am J Clin Nutr. 2004;79(3):444-50.
32. Rahman MJ, Sarker P, Roy SK, Ahmad SM, Chisti J, Azim T, Mathan M, Sack D,
Andersson J, Raqib R. Effects of zinc supplementation as adjunct therapy on the
systemic immune responses in shigellosis. Am J Clin Nutr. 2005;81:495-502.
33. Alam AN, Sarker SA, Wahed MA, Khatun M, Rahaman MM. Enteric protein loss
and intestinal permeability changes in children during acute shigellosis and after
recovery: effect of zinc supplementation. Gut. 1994;35:1707-11.
34. Qadri F, Ahmed T, Wahed MA, Ahmed F, Bhuiyan NA, Rahman AS, Clemens
JD, Black RE, Albert MJ. Suppressive effect of zinc on antibody response to
cholera toxin in children given the killed, B subunit-whole cell, oral cholera
vaccine. Vaccine. 2004;22:416-21.
35. Karlsen TH, Sommerfelt H, Klomstad S, Andersen PK, Strand TA, Ulvik RJ,
Ahren C, Grewal HM. Intestinal and systemic immune responses to an oral
cholera toxoid B subunit whole-cell vaccine administered during zinc
supplementation. Infect Immun. 2003;71:3909-13.
36. Albert MJ, Qadri F, Wahed MA, Ahmed T, Rahman AS, Ahmed F, Bhuiyan NA,
Zaman K, Baqui AH, Clemens JD, Black RE. Supplementation with zinc, but not
vitamin A, improves seroconversion to vibriocidal antibody in children given an
oral cholera vaccine. J Infect Dis. 2003;187:909-13.
37. Roselli M, Finamore A, Garaguso I, Britti MS, Mengheri E. Zinc oxide protects
cultured enterocytes from the damage induced by Escherichia coli. J Nutr.
2003;133:4077-82.
38. Owusu-Asiedu A, Nyachoti CM, Marquardt RR. Response of early-weaned pigs
to an enterotoxigenic Escherichia coli (K88) challenge when fed diets containing
spray-dried porcine plasma or pea protein isolate plus egg yolk antibody, zinc
oxide, fumaric acid, or antibiotic. J Anim Sci. 2003;81:1790-8.
39. Sugarman B, Epps LR. Zinc and the heat-labile enterotoxin of Escherichia coli. J
Med Microbiol. 1984;18:393-8.
40. Olivares JL, Fernandez R, Fleta J, Rodriguez G, Clavel A. Serum mineral levels
in children with intestinal parasitic infection. Dig Dis. 2003;21:258-61.
41. Ertan P, Yereli K, Kurt O, Balcioglu IC, Onag A. Serological levels of zinc, copper
and iron elements among Giardia lamblia infected children in Turkey. Pediatr Int.
2002;44:286-8.
42. Vega Robledo GB, Carrero JC, Ortiz-Ortiz L. Effect of zinc on Entamoeba
histolytica pathogenicity. Parasitol Res. 1999;85:487-92.
43. Campbell DI, Elia M, Lunn PG. Growth faltering in rural Gambian infants is
associated with impaired small intestinal barrier function, leading to endotoxemia
and systemic inflammation. J Nutr. 2003;133:1332-8.
44. Katouli M, Melin L, Jensen-Waern M, Wallgren P, Mollby R The effect of zinc
oxide supplementation on the stability of the intestinal flora with special reference
to composition of coliforms in weaned pigs. J Appl Microbiol. 1999 Oct;87(4):56473
45. Bolkent S, Yanardag R, Bolkent S, Mutlu O, Yildirim S, Kangawa K, Minegishi Y,
Suzuki H. The effect of zinc supplementation on ghrelin-immunoreactive cells
37
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
and lipid parameters in gastrointestinal tissue of streptozotocin-induced female
diabetic rats. Mol Cell Biochem. 2006;286:77-85.
46. Jamshidian M, La T, Phillips ND, Hampson DJ. Brachyspira pilosicoli colonization
in experimentally infected mice can be facilitated by dietary manipulation. J Med
Microbiol. 2004;53:313-8.
47. Kelleher SL, Casas I, Carbajal N, Lonnerdal B. Supplementation of infant formula
with the probiotic lactobacillus reuteri and zinc: impact on enteric infection and
nutrition in infant rhesus monkeys. J Pediatr Gastroenterol Nutr. 2002;35:162-8.
48. Zhang P, Carlson MP, Schneider NR, Duhamel GE. Minimal prophylactic
concentration of dietary zinc compounds in a mouse model of swine dysentery.
Anim Health Res Rev. 2001;2:67-74.
49. Di Leo V, D'Inca R, Barollo M, Tropea A, Fries W, Mazzon E, Irato P, Cecchetto
A, Sturniolo GC. Effect of zinc supplementation on trace elements and intestinal
metallothionein concentrations in experimental colitis in the rat. Dig Liver Dis.
2001;33:135-9.
50. Broom LJ, Miller HM, Kerr KG, Knapp JS. Effects of zinc oxide and Enterococcus
faecium SF68 dietary supplementation on the performance, intestinal microbiota
and immune status of weaned piglets. Res Vet Sci. 2006;80:45-54.
51. Siriken B, Bayram I, Onol AG. Effects of probiotics: alone and in a mixture of
Biosacc plus Zinc Bacitracin on the caecal microflora of Japanese quail.Res Vet
Sci. 2003;75:9-14.
52. Bhutta ZA, Nizami SQ, Isani Z, Zinc Supplementation in malnourished children
with persistent diarrhea in Pakistan. Pediatrics 1999;103:1-9
http://www.pediatrics.org/cgi/content/full/103/4/e42
53. Chang AB, Torzillo PJ, Boyce NC, White AV, Stewart PM, Wheaton GR, Purdie
DM, Wakerman J, Valery PC. Zinc and vitamin A supplementation in Indigenous
Australian children hospitalized with lower respiratory tract infection: a
randomised controlled trial. Med J Aust. 2006;184:107-12.
54. Doherty CP, Sarkar MA, Shakur MS, Ling SC, Elton RA, Cutting WA. Zinc and
rehabilitation from severe protein-energy malnutrition: higher-dose regimens are
associated with increased mortality. Am J Clin Nutr. 1998;68:742-8.
55. Sharieff W, Bhutta Z, 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
56. Mai V, Morris JG Jr. Colonic bacterial flora: changing understandings in the
molecular age. J Nutr. 2004;134:459-64.
57. Couteau D, McCartney AL, Gibson GR, Williamson G, Faulds CB. Isolation and
characterization of human colonic bacteria able to hydrolyse chlorogenic acid. J
Appl Microbiol. 2001;90:873-81
58. Sullivan PB. Studies of the small intestine in persistent diarrhea and malnutrition:
the Gambian experience. J Pediatr Gastroenterol Nutr. 2002 May-Jun;34 Suppl
1:S11-3.
59. Peret Filho LA, Brasileiro Filho G, Penna FJ. Morphological changes of the
jejunal mucosa in protracted diarrhea and their correlation with disease duration,
weight loss and serum albumin levels. Braz J Med Biol Res. 1997;30:1067-73
60. Brewster DR, Manary MJ, Menzies IS, O'Loughlin EV, Henry RL. Intestinal
permeability in kwashiorkor. Arch Dis Child. 1997;76:236-41.
61. Goto K, Chew F, Torun B, Peerson JM, Brown KH. Epidemiology of altered
intestinal permeability to lactulose and mannitol in Guatemalan infants. J Pediatr
Gastroenterol Nutr. 1999;28:282-90.
38
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
62. Haase AM, Kukuruzovic RH, Dunn K, Bright A, Brewster DR. Dual sugar
permeability testing in diarrheal disease. J Pediatr. 2000;136:232-7.
63. Albers MJ, Steyerberg EW, Hazebroek FW, Mourik M, Borsboom GJ, Rietveld T,
Huijmans JG, Tibboel D. Glutamine supplementation of parenteral nutrition does
not improve intestinal permeability, nitrogen balance, or outcome in newborns
and infants undergoing digestive-tract surgery: results from a double-blind,
randomized, controlled trial. Ann Surg. 2005;241:599-606.
64. Kalach N, Benhamou PH, Campeotto F, Dupont Ch. Anemia impairs small
intestinal absorption measured by intestinal permeability in children. Allerg
Immunol (Paris). 2007 Jan;39(1):20-2.
65. Reims A, Strandvik B, Sjovall H. Epithelial electrical resistance as a measure of
permeability changes in pediatric duodenal biopsies. J Pediatr Gastroenterol
Nutr. 2006 Nov;43(5):619-23.
66. Miele E, Pascarella F, Quaglietta L, Giannetti E, Greco L, Troncone R, Staiano
Altered intestinal permeability is predictive of early relapse in children with
steroid-responsive ulcerative colitis. Aliment Pharmacol Ther. 2007;25(8):933-9.
67. 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 Aug;100(8):791-4. Epub 2006 Mar 15.
68. Canani RB, de Horatio LT, Terrin G, Romano MT, Miele E, Staiano A,
Rapacciuolo L, Polito G, Bisesti V, Manguso F, Vallone G, Sodano A, Troncone
R. Combined use of noninvasive tests is useful in the initial diagnostic approach
to a child with suspected inflammatory bowel disease. J Pediatr Gastroenterol
Nutr. 2006 Jan;42(1):9-15.
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.
39
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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, MyoKhin, 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, PablosMendez 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
40
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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 JanFeb;(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
41
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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. IturrizaGomara, 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.
42
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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.
127.
Niesters, HGM. Molecular and diagnostic clinical virology in real time.
Clinical Microbiology and Infection 2004;10: 5–11.
43
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
44
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
45
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
-
Sept 30, 2007
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 HIVassociated lipodystrophy syndrome: An update. AIDS 2001; 15:1917-1930.
46
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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.
47
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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
48
Bhutta et al
Department of Paediatrics & Child Health
The Aga Khan University
Sept 30, 2007
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.
49