Study design This will be a community-based intervention study. Survey, screening, enrollment
of subjects will be done in Mirpur in Dhaka city.
Subjects Participants will be recruited from two age groups: a child cohort (age 12 to 18
months) and an adult cohort (age 18 to 45 years). The child cohort will consist of two groups
of children- stunted children (LAZ score < -2) and children who are at risk of stunting (LAZ
<-1 to -2). A group of malnourished adults (BMI <18.5) will also be enrolled from the parents
of children and by a cross sectional survey from Mirpur area of Dhaka city.
Sample size:
Participants will be collected and recruited in a cross sectional manner. Based on a sample
size calculation with 10% precision and 95% confidence interval, a minimum of 35 stunted
children, 35 at risk of stunting children and 35 adults will be recruited. So, the final
sample size for endoscopy is 105 subjects (35+35+35). As endoscopy is an invasive procedure,
it was assumed that only one-third of those who would fail to respond after nutrition
intervention would give consent for endoscopy. So, basic sample size was increased three
times for a minimum of 105 (35 x 3) stunted children (LAZ <-2), 105 (35 x 3) children at risk
of stunting (LAZ <-1 to 2) and 105 (35 X 3) malnourished adults (BMI < 18.5) who would fail
to thrive. To achieve that 525 stunted children, 525 children at risk of stunting and 525
malnourished adults for nutrition interventions will be recruited as it was assumed that ~20%
of each group will fail to respond despite the nutritional intervention. For that order of
recruitment surveying at least 3977 (1312+2665) children 12-18 months old and 2100 adults
based on the current prevalence of malnutrition in the two age groups is required. For adult
control group, same number (35) of adult controls will be recruited from Dhaka Medical
College and international centre for diarrhoeal disease research,Bangladesh staff clinic by
convenient sampling method
Description of field site:
The study will be conducted among residents of Mirpur, one of the 21 administrative units of
the nation's capital, Dhaka.
Recruitment, Screening and Consenting If the participants are interested to volunteer in the
study, the designated staff will proceed to screening and consenting. Screening will consist
of a review of the inclusion and exclusion criteria listed above. If the subject is eligible
to participate, the process will proceed to consenting consisting of a thorough review of the
written consent form in a manner appropriate for the child's parents' / participants literacy
level. Prior to signing the consent form, they will have an opportunity to ask any questions
about the study. If the Field Research Assistant (FRA) determines that participants have
demonstrated adequate comprehension of the study, the consent form will be signed by the FRA
and the child's parent(s) /adult participants. If the parent(s) / adult participants are not
sufficiently literate to read and/or sign the consent form, consenting and a thumb impression
will be obtained in the presence of a witness who is not associated with the study. The
child's parent(s) / adult participants will be provided with a copy of the signed consent
form.
Detailed procedure of Directly Observed Nutritional Therapy:
After enrollment, participants will receive daily directly observed nutritional therapy.
Nutritional therapy consists egg and milk and will be provided by the study free of cost.
Duration of nutrition interventions will be 3, 2 and 2 months for stunted children, children
at risk of stunting, and malnourished adults, respectively and nutritional therapy will be
delivered between the morning and mid day meal, anytime between 10:00 am to 11:30 am.
Participants will be asked to come to the designated nutrition center (to be established at
Mirpur) daily for nutritional therapy to avoid the issue of food sharing. A staff will visit
the family's household if a participant defaults in coming to the nutrition center. In
addition to the food based nutritional therapy, participants will receive the following
nutritional and health support:
Anti-helminthic treatment (Albendazole/Pyrantel Pamoate) as per national guidelines
Parents/caregivers will be given nutritional counselling
For the child cohort, parents will be given one sachet of multiple micro-nutrient
sprinkles per day for two months.
Any inter-current illnesses detected by the study team will also be taken care of.
Food based nutritional therapy: Supplementation of the usual home diet with one egg, and 150
ml of whole milk which will provide an additional 178 kilo-calories, 11.1 g protein, and 11.5
g of fat to the daily diet of enrolled subjects. Sachets of multiple micro-nutrient powder
will be provided for use at home at dinner.
In order to clarify the role of food-based nutritional therapy, there will be comparison
subgroups of 40 stunted children and 40 children at risk of stunting who will be followed for
3 and 2 months respectively, without any nutritional therapy. At the end of the observation
period of 3 and 2 months, nutritional therapy will be provided to these children same as that
provided to the enrolled participants.
Discontinuation from the study: A child will be discontinued from the study and referred for
medical evaluation if he/she shows reluctant to feed for consecutive 7 days which includes
daily intake of 50% or less amount of food that will be offered. A new eligible child will be
recruited in his position for nutritional intervention.
Collection of biological samples:
All biological samples (blood, urine, stool, duodenal aspirates, and endoscopic biopsies)
will be collected as per the standard operative procedures (SOPs) which will be prepared for
this protocol. A total amount of 5ml blood will be collected from each participant before and
after nutritional intervention. Urine will be collected for Lactulose-Rhamnose assay, and for
metabolomic studies; breath samples will be collected for testing for small intestinal
bacterial overgrowth (SIBO). Second SIBO will be done 1-2 weeks prior to endoscopy so that
the findings from the endoscopy can be compared to the breath tests. Fecal samples will be
collected for biomarkers, Taqman array card (TAC) assays and Microbiome/Microbiota for age
Z-score (MAZ)/Bug fluorescence-activated cell sorting.
Detailed procedure of Bio-marker testing
The bio-markers going to be tested for are-
Stool bio-markers
Urine bio-markers
• Lactulose-Rhamnose test
Blood bio-markers
C- Reactive Protein
Alpha-1-acid glycoprotein
Soluble cluster of differentiation 14
Kynurenine-Tryptophan Ratio
Glucagon-like peptide-2
Insulin-like growth factor-2
Tissue Transglutaminase
Ferritin
Zinc
Pepsinogen I/II Ratio
Low density lipoprotein related receptor 1 (LRP1)
Blood plasma metabolomics
Other proposed biomarkers
• Small intestinal bacterial overgrowth
After completion of nutritional therapy, all enrolled participants will have an assessment
for response which will include:
Anthropometric measurements
Collection of one 5 ml blood sample for serum bio-markers of gut function and screening
tests for the endoscopy with biopsy procedure, if the procedure is indicated for failure
to thrive
Collection of stool specimen for bio-markers of gut function
Administration of lactulose-rhamnose solution and collection of urine for up to 2 hours
for urine bio markers of gut function
At this time, the final inclusion criterion for endoscopy with biopsy will be evaluated to
determine whether the participants failed nutritional therapy or had an adequate response, or
have an underlying disease that caused the malnutrition. Failure of nutritional therapy in
this study, or failure to respond, is defined in the absence of any other underlying diseases
such as tuberculosis:
LAZ score remains < -2 for stunted children cohort
LAZ score remains < -1 for "at risk of stunting" children cohort
BMI < 18.5 and < 10% increase in BMI for adults.
Participants who meet the study definition of "failure to respond" will be checked for
presence of any secondary cause of malnutrition like tuberculosis, carcinoma, any parasitic
infection etc. Stool microscopy for parasite identification will be done at icddr,b
parasitology lab as soon as possible after collection of stool samples. If nothing conclusive
can be found, the participants may be requested to undergo preparations for upper GI
endoscopy with biopsy, as they may benefit from further evaluation to identify the potential
cause(s) of intervention failure and to determine an appropriate therapeutic course. It will
also benefit others by validating the non-invasive bio-marker signature of Environmental
Enteric Dysfunction. Participants who will undergo endoscopy will receive necessary
treatments according to diagnosis.
Participants having celiac disease and clotting disorder (tested by prothrombin time /
International Normalized Ratio coagulopathy) will be excluded from the study. Endoscopy of
upper gastrointestinal tract will be done as per standards recommended by North American
Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN0, American College
of Gastroenterology (ACG) and American Society for Gastrointestinal Endoscopy (ASGE).
