This is a community-based intervention study which will be undertaken at Mirpur, Dhaka, Bangladesh. 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 stunted children (length for age Z score, LAZ < -2), children who are at risk of stunting (length for age Z score <-1 to -2) and child controls. The adult cohort will consist of malnourished adult cases (Body Mass Index <18.5) and adult controls. After screening the participants for any organic diseases and application of inclusion/exclusion criteria, they will receive nutrition interventions. Participants eligible for study will be tested for potential bio markers of environmental enteropathy (stool, urine and serum) once before and once after the nutritional intervention. Participants who will fail to respond to nutritional therapy (measured by anthropometric assessment) will become candidates for upper gastrointestinal endoscopy with biopsy. The study will include duodenal biopsies from a control group of children from University of Virginia Hospital in Charlottesville, Virginia who will undergo upper GI endoscopy as part of their clinical care as per the standard clinical protocol followed at the hospital. Adult controls (BMI > 18.5) for endoscopy will be collected from international centre for diarrhoeal disease research,Bangladesh staff clinic, Dhaka Medical College and Hospital (DMCH), Dhaka and Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.

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

• Regeneration gene 1Beta
• Neopterin
• Myeloperoxidase
• Alpha-1-antitrypsin
• Calprotectin
• The TaqMan Array Card for enteropathogen
• Gut Microbiome analysis
• Bug Fluorescence-Activated Cell Sorting.

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).

Inclusion Criteria:

Inclusion criteria for stunted children: All of the following criteria must be met for a subject to be eligible to participate in the study-

• Parent(s) willing to sign consent form
• Child age 12-18 months
• LAZ < -2
• Parent(s) willing to bring child to the study site every day for 3 months for nutritional therapy
• Parents willing to have child undergo laboratory investigations and upper GI endoscopy and biopsy if the child fails to respond to nutritional therapy

Inclusion criteria for children who are at risk of stunting: All of the following criteria must be met for a subject to be eligible to participate in the study -

• Parent(s) willing to sign consent form
• Child age 12-18 months
• LAZ between <-1 to -2
• Parent(s) willing to bring child to the study site every day for 2 months for nutritional therapy
• Parents willing to have child undergo endoscopy and biopsy if the child fails to respond to nutritional therapy

Inclusion criteria for malnourished adults: All of the following criteria must be met for a subject to be eligible to participate in the study -

• Willing to sign consent form
• Age 18-45 years
• BMI < 18.5
• Willing to visit the study site every day for 2 months for nutritional therapy
• Willing to undergo endoscopy and biopsy if he/she fails to respond to nutritional therapy

Selection criteria for adult controls:

An equal number of age and sex matched adult controls will be enrolled form the subjects with functional dyspepsia (FD) with apparently normal health (normal BMI) and absence of organic diseases. FD subjects will be identified from the Gastroenterology outpatient department of Dhaka Medical College Hospital and international centre for diarrhoeal disease research, Bangladesh staff clinic who will undergo upper GI endoscopy for evaluation of dyspepsia and have normal endoscopic findings. Dyspepsia will be defined by pre-defined criteria (Rome III criteria) by a structured questionnaire which has been translated and validated in Bengali.

Exclusion Criteria:

Exclusion criteria for stunted children: Meeting any of the following criteria will exclude a subject from study participation -

• Severe acute malnutrition (SAM), severe anemia (<8 g/dl), tuberculosis, other chronic diseases or any congenital disorder or deformity
• Diarrhoea: Ongoing episode of diarrhoea, history of persistent diarrhoea in the past month or history of acute diarrhoea in the past 7 days
• Known allergy to eggs or milk or milk intolerance

Exclusion criteria for children who are at risk of stunting: Meeting any of the following criteria will exclude a subject from study participation -

• Severe acute malnutrition (SAM), severe anemia (<8 g/dl), tuberculosis, other chronic diseases or any congenital disorder or deformity
• Diarrhoea: Ongoing episode of diarrhoea, history of persistent diarrhoea in the past month or history of acute diarrhoea in the past 7 days
• Known allergy to eggs or milk or milk intolerance

Exclusion criteria for malnourished adults: Meeting any of the following criterions will exclude a subject from study participation-

• Severe anemia (<8 g/dl), tuberculosis and other chronic diseases including diabetes mellitus or any congenital disorder or deformity
• Pregnant women, lactating women, drug abusers, known psychiatric disorders
• High clinical suspicion of cancer or other chronic or acute diseases that may cause malnutrition. Adult participants who fulfilled the inclusion criteria and not excluded through history and clinical examination based exclusion criteria will undergo screening tests for confirmation.