Community-based Clinical Trial With Microbiota-directed Complementary Foods (MDCFs) Made of Locally Available Food Ingredients for the Management of Children With Primary Moderate Acute Malnutrition
Community-based Clinical Trial With Microbiota-directed Complementary Foods (MDCFs) Made of Locally Available Food Ingredients for the Management of Children With Primary Moderate Acute Malnutrition
Background (brief):

 Burden: A total of 52 million children under 5 are suffering from acute malnutrition
 globally, of whom 33 million have moderate acute malnutrition (MAM). In Bangladesh, more than
 2 million children suffer from MAM. According to Bangladesh Demographic Health Survey 2014
 26%, 25% and 17% of children aged less than two years are stunted, underweight and wasted
 respectively.

 Knowledge gap: It has been already demonstrated that children with SAM have immature gut
 microbiota that is partially corrected with treatment. Children with MAM have an increased
 risk of mortality, infections and impaired physical and cognitive development compared to
 well-nourished children. Although the global caseload of MAM is much greater than that of
 SAM, the condition has not received the same level of attention or priority. Through our
 previous and ongoing research we now know about the members of the gut microbiota that can
 promote growth in children and also about certain food ingredients that promote the
 proliferation of such beneficial microbiota. However, this knowledge needs to be applied on a
 sufficiently powered community-based clinical trial.

 Relevance: The rationale for this study is to assess whether long-term administration of
 complementary food made of locally available food ingredients can stimulate the proliferation
 of growth promoting members of the gut microbiota and have a positive impact on child growth.
 Such a food (the microbiota directed complementary food; MDCF-2) has been identified through
 our recently concluded Pre-proof of concept trial done on children with primary MAM. We would
 now like to do a clinical community-based trial of this potential MDCF-2 in the management of
 children with primary MAM.

 Hypothesis: Complementary foods made of locally available food ingredients that stimulate the
 proliferation of growth promoting gut microbiota (MDCF-2) will improve clinical outcomes.

 Methods: We will conduct a proof of concept (POC) clinical trial in 12-18 months old children
 with primary MAM (Weight-for-Length Z-score, WLZ between -2 and -3). This study will be
 conducted at Bauniabadh, Radda MCH-FP (Maternal and Child Health- Family Planning) clinic,
 Gabtoli of Mirpur area and possibly at the Special Nutrition Unit run by Terre des Hommes in
 Kurigram. We will produce MDCF-2 at the icddr,b Food Processing Laboratory or nutrition
 centre established at the site in sufficient quantities for clinical study. This formulation
 will be matched in energy density and micronutrient content of ready-to-use supplementary
 foods (RUSFs) used for MAM in Bangladesh and other countries, and will meet all other
 requirements for a complementary/supplementary food for 12-18 months old children with MAM.
 We will test MDCF-2 and the current RUSF standard of care for primary MAM to see the effect
 on growth, proteomics and metabolomics of an intervention for 12 weeks, with a 4-week
 post-intervention phase.

 Hypothesis to be tested:

 In a hypothesis testing research proposal, briefly mention the hypothesis to be tested and
 provide the scientific basis of the hypothesis, critically examining the observations leading
 to the formulation of the hypothesis.

 Complementary foods made of locally available food ingredients that stimulate the
 proliferation of growth promoting gut microbiota (MDCF) will provide a new way to improve
 clinical outcomes, for example by improving growth of children with MAM.

 Specific Objectives:

 To investigate the efficacy of complementary food made of locally available food ingredients
 that can stimulate the proliferation of growth promoting gut microbiota (Microbiota-Directed
 Complementary Food; MDCF-2) in (i) promoting repair of microbiota immaturity (ii) promoting
 proliferation of beneficial bacteria (iii) improving both ponderal and linear growth in
 children (iv) improving the metabolomic profile with MAM
Background:

 Moderate acute malnutrition (MAM), a major global health problem, is defined as wasting (i.e.
 weight-for-height between < -2 and -3 Z-scores of the WHO Child Growth Standards) and/or
 mid-upper-arm circumference (MUAC) greater or equal to 115 mm and less than 125 mm. According
 to the Global Nutrition Report 2017, 8% or 52 million under-five children were acutely
 malnourished globally in 2016. Stunting affected 23% or 155 million children. Approximately
 one in 6 children under 5 years in South Asia suffered from MAM in 2013 (i.e. 17%). These
 children are at increased risk of severe acute malnutrition (SAM), and have a three times
 higher risk of mortality from common communicable diseases than the well-nourished peers.
 Bangladesh has one of the highest childhood malnutrition burdens in the world. According to
 Bangladesh Demographic Health Survey (BDHS) 2014, the prevalence of stunting among under-five
 children is 36%, among them 12% suffer from severe stunting (Length-for-Age-Z score, LAZ
 <-3). Around 15% of children are wasted (Weight-for-Length Z score, WLZ <-2); more than 2
 million children suffer from MAM, while 3% or 450,000 children suffer from the deadly form of
 SAM. Malnutrition costs Bangladesh an estimated US $1 billion a year. According to WHO
 recommendations, infants and children aged 6-59 months with MAM need to consume
 nutrient-dense foods to meet their extra needs for weight and height gain and functional
 recovery. Currently there are no evidence-informed recommendations on the composition of
 supplementary foods used to treat children with MAM. In situations of food shortage,
 supplementary foods have been used to treat children with moderate acute malnutrition.
 Interventions to address under nutrition should therefore include a strong component of MAM
 management. MAM prevention should be taken into consideration in food security and other
 development strategies as the situation becomes critical in populations where food insecurity
 is rampant. Food insecurity has become a worldwide concern due to the increasing number of
 people who remain undernourished amounting to 842 million, approximately 12% of the total
 world's population. From the National Micro nutrient Status Survey in Bangladesh which we
 conducted in 2011-12, severe insecurity of food was found most commonly in slum settlements
 (17.2 %), compared to 12.3% at the national level, 12% in rural areas and 12.4% in urban
 areas of the country. Since food insecurity cannot be overcome quite readily, it is important
 therefore to develop interventions that depend upon locally available food ingredients and
 are able to harness the beneficial power of the gut microbiota on infant and child growth.

 One of the major factors limiting the impact of nutrition intervention is the inability of
 the malnourished children to increase their intake to meet increased metabolic demands. In
 collaborative studies between icddr,b and the Gordon Lab at Washington University in St.
 Louis during the Jump start Phase of the Breast Milk, Microbiota and Immunity (BMMI) Project,
 we applied Random Forests, a machine-learning-based approach, to bacterial 16S rRNA
 (ribosomal ribonucleic acid) datasets generated from monthly fecal samples obtained from a
 birth-cohort of children living in an urban slum of Dhaka, Bangladesh. These children
 exhibited consistently healthy growth (WLZ -0.32+0.98). Bacterial strains were identified
 whose proportional representation defines a healthy gut microbiota as it assembles during the
 first 2-3 postnatal years. In a randomized clinical trial at icddr,b of two therapeutic foods
 (imported ready-to-use therapeutic food [RUTF (Ready to Use Therapeutic Food), Plumpy'Nut]
 versus locally prepared rice/lentil-based Khichuri and Halwa) in Bangladeshi children with
 severe acute malnutrition (SAM), it was observed that the microbiota immaturity is
 incompletely and only transiently improved, with children remaining markedly stunted and
 underweight throughout the follow-up period. Bangladeshi children with MAM also exhibited
 significant microbiota immaturity, although less severe than children with SAM. Microbiota
 immaturity thus serves as a potential biomarker to identify infants at risk for
 undernutrition and to monitor treatment and prevention strategies.

 Investigators recently developed ready-to-use therapeutic foods using locally available food
 ingredients-rice, lentil, and chickpeas that are culturally relevant and acceptable. They
 found through a double-blind RCT (Randomized Controlled Trial) that chickpea-based and
 rice-lentil-based RUTF were as effective as the commercial peanut based-RUTF and well
 accepted by children with SAM. Through a combination of the above mentioned RCT and clinical
 translational studies, they have identified growth promoting age-discriminatory beneficial
 microbiota and locally available food ingredients that support proliferation of these
 beneficial microbiota. Besides, results of earlier studies done on gnotobiotic animals in
 Washington University Centre for Genome Science has led us to suggest that a combination of
 food ingredients (chickpea, soy flour, peanut and green banana) will be worth studying with
 respect to the diet's impact on stimulating proliferation of growth-discriminatory microbiota
 as well as cost and sustainability. To assess the degree to which the results obtained from
 the gnotobiotic mouse and piglet models translate to humans, investigators recently performed
 a RRC (Research Review Committee) and ERC (Ethecial Review Committee)approved study, icddr,b
 protocol (PR-16099) 'Pre-Proof of Concept clinical trials to optimize lead
 microbiota-directed complementary food (MDCF) prototypes for their ability to repair
 microbiota immaturity and establish their organoleptic acceptability 'and successfully
 completed the study. This study was designed to test the effects of three locally produced
 MDCF prototypes (MDCF-1, MDCF-2 and MDCF-3) and a locally produced rice-lentil-based RUSF.
 The objective of this pilot study was to demonstrate a Pre-proof of Concept that certain
 complementary foods would have a beneficial effect on young children suffering from moderate
 acute malnutrition by stimulating the proliferation of particular members of the gut
 microbiota that are known for their growth promoting effect. In that pilot study they
 investigated microbiota-for-age Z score as well as the impact of the proliferation of good
 members of the gut microbiota on certain body systems. The results of the Pre-POC pilot trial
 conclusively showed that one of the three microbiota directed complementary foods namely
 (MDCF-2; composed of a combination of chickpea, soy flour, peanut, green banana, oil, sugar
 and micronutrients) was associated with increased levels of certain amino acids, that have a
 key role in development of the long bones, development of the brain and increased production
 of IGF-1. This was done using the state-of-the-art DNA aptamer based SomaLogic scan. And the
 results suggest that this candidate MDCF-2 is effective in stimulating the growth of growth
 promoting members of the gut microbiota, for example Faecalibacterium prausnitzii. Thus,
 MDCF-2 promotes gut microbiota that induces the hormone insulin-like growth factor 1 (IGF-1),
 which promotes bone growth and remodelling.

 Based on this evidence investigators would now like to do a much larger clinical trial using
 the most promising MDCF which is MDCF-2 with the primary end point being linear growth. This
 trial would be on children with primary MAM.

 Research Design and Methods Investigators will conduct a clinical trial among 12-18 months
 old children with primary MAM (WLZ <-2 to -3).

 Study design: Randomized controlled intervention trial. Study site: This study will be
 conducted at Bauniabadh, RADDA MCH-FP (Maternal and Child Health - Family Planning) clinic in
 Mirpur area and Gabtoli of Dhaka city, and possibly at the Special Nutrition Unit run by
 Terre des Hommes in Kurigram.

 Study participants: The study participants will be 12-18 months old children of either sex
 with MAM (WLZ <-2 to -3).

 Initial screening and enrolment: Children will be screened and enrolled through household
 surveys by Field Research Assistants (FRAs) following pre-specified inclusion criteria.
 Fulfilling the enrollment criteria and upon receiving the consent for study participation
 from the parents or legal guardians, the children with the respective mother/caregiver will
 be enrolled and randomly assigned to one of the two arms according to computer-generated
 random numbers. The code of assigned type of diet will be kept in closed opaque envelopes for
 each individual, and will be opened only when the caregiver signs the consent form.

 Study design:

 Arm 1 - Rice-lentil based RUSF (rationale: reference standard of care for MAM; based on
 knowledge of its effects on the gut microbiota or microbiota immaturity) Arm 2 - MDCF2
 (Microbiota Directed Complementary Food 2) with four complementary food ingredients
 (rationale: lead with evidence from Pre-POC clinical trials to optimize lead
 microbiota-directed complementary food prototypes for their ability to repair microbiota
 immaturity and positive effects on growth) Fecal sample collection Weekly fecal samples
 (1-2g) will be collected within 30 minutes of excretion (at home and transported back to the
 icddr,b) using the liquid nitrogen containing dry shipper. Specimens will be stored at -80°C
 before being sent to the Gordon Lab analysis of microbiota maturity (measured before, during
 and after cessation of treatment with MDCF and RUSF) and PCR (Polymerase Chain
 Reaction)-based assessment of enteropathogen burden in fecal samples (measured before and
 after MDCF treatment).

 1-2 gm of fecal samples will be collected from each child at enrollment, weekly during the
 1st month of intervention, and 4 weekly during the 2nd and 3rd month of intervention and at
 the end of post-intervention phase. Assuming 62 study participants /arm, a total of 992 fecal
 specimens will be collected in this study. 2 gm of faecal samples will be collected from
 mothers at the time of enrollment.

 Urine sample collection 2 mL of urine samples will be collected from each child at
 enrollment, one week after enrollment and monthly once in the intervention and
 post-intervention phases. Assuming 62 study participants /arm, a total of 744 urine samples
 will be collected in this study. 5 mL of urine samples will be collected from mothers at the
 time of enrollment.

 Blood sample collection 2 mL of blood samples each will be collected from each child prior to
 intervention, end of first month of intervention and just after the intervention is complete.
 A total of 372 plasma samples will be collected in this study. 5 mL of blood samples will be
 collected from mothers at the time of enrollment.

 Maternal nutritional status is associated with child nutritional status, as shown through the
 results of our eight country MAL-ED (Malnutrition and Enteric Diseases) study. In addition,
 neonatal and other maternal factors were early determinants of lower length-for-age, and
 their contribution remained important throughout the first 24 months of life.9 The other
 maternal factors, investigators believe, would include maternal gut microbiota as well as
 maternal blood amino acid profile. Initiatives to address childhood stunting should also
 consider improvements to the composition of complementary foods (i.e., higher protein) and
 strategies to reduce gut pathogen exposure. As such, they will record maternal height and
 weight. In order to understand the biological state of nutrition of the mother, enrolment
 samples of stool, blood and urine will be asked of the mother. These samples will be analyzed
 for gut microbiota, and proteomics, and the results correlated with those of the enrolled
 children.

 Feeding sessions The children and mothers/caregivers will be requested to come to the
 nutrition centers established at the sites preferably between 9-11 am and 3-5 pm on day 1.
 The mothers will be requested not to give any food and breast milk in the 2 hours preceding
 the observed meal time. The child will be offered 25 grams of MDCF or RUSF as decided by
 random allocation in each of the two meals between 9-11 am and 3-5 pm. The mothers will be
 asked to spoon feed the pre-weighed diets to their children until s/he refuses to eat, as
 described below. After a two-minute pause, the same diet will be offered a second time until
 s/he refuses again. After a second two-minute pause, the diet will be offered a third time
 until refused again. After this third refusal, the feeding episode will be considered as
 'terminated'. The duration of the feeding (excluding the intervening 'rest periods') will be
 recorded by stopwatch, and the total duration of the feeding will be noted. This feeding
 episode will last for maximum 60 minutes. Measured volumes of plain water will also be given
 and the amount of water taken during this meal period of 60 minutes will be measured. The
 feeding episode will take place under the direct supervision of trained study personnel.
 Children will be considered as refusing further intake if they move their head away from the
 food, cry, clamp the mouth or clinch the teeth, or become agitated, spit out the food or
 refuse to swallow. The amount of MDCF/RUSF actually ingested will be calculated by
 subtracting the left over from the offered amount. Pre-weighed napkins will be provided; any
 food that is regurgitated, vomited or spilled will be swabbed, weighed and subtracted from
 the amount offered. The amount of consumed food (g), energy (kcal) and category of
 acceptability will be analyzed. The enrolled children will be monitored daily by Field
 Research Assistants for any possible side effects/adverse events (e.g. rash, urticaria due to
 food allergy or any significant changes in clinical status) for a week. If any side
 effects/adverse events are observed, they will be treated according to standard of care. A
 standardized production procedure will be followed to control the quality of RUSF and MDCF
 following international standard protocol. RUSF and MDCF will be prepared at the food
 processing laboratory. Preparation of food under different steps, that is, roasting, particle
 size reduction, homogeneous blending, and supplying to the nutrition centres will be
 monitored by icddr,b investigators. Food will be prepared everyday to ensure that no
 unexpected contamination and nutrient losses occur during preparation. Although raw food
 ingredients will be very carefully procured from the local market and stored in reasonable
 quantities, investigators will prepare, dispense, and feed the children the same day the MDCF
 and RUSF are prepared. Every child will be offered 25gm of the diet twice daily at the
 feeding center for the first 4 weeks. In the following month, the child will be offered 25gm
 of the diet at the feeding center and additional 25gm will be provided in a clean container
 to feed at home. In the third month, two separate containers containing 25gm diet will be
 provided every day to each enrolled child at participant's home.

 In this study nutritional status will be assessed through anthropometry, comparing with WHO
 growth reference standards. At the beginning of the study, information will be sought on the
 demographic characteristics (families' wealth, standard of housing, family structure and
 parental characteristics etc.), and FRAs will record the children's weight using a digital
 scale with 2g precision (Seca, model 728, Germany), length (using infantometer, Seca, model
 416, Germany), and mid upper-arm circumference to the nearest mm (using a non-stretch
 insertion tape). Anthropometrics will be done according to the standard procedures and all
 measurements will be taken thrice and the middle one will be recorded.

 All interventions in each study will be administered to children at the Mirpur health
 clinic/RADDA clinic or in Kurigram. Mothers/primary caregivers will be advised to maintain
 their child's current dietary and breastfeeding practices.

 Investigators will complete enrollment within 12 months and the follow up as well as data
 analysis within an additional 6 months. However, this trial on primary MAM will continue
 simultaneously with the other clinical trial on Post SAM-MAM that will have a longer period
 of duration.

 Recruitment, Screening and Consenting Census, screening, enrolment of study participants will
 be done in the catchment areas of the sites in Dhaka city and in Kurigram. Parents of
 children who meet the MAM criteria (for Primary MAM trial) will be approached about
 enrollment into the study. A Field Research Assistant will explain the study in detail,
 answer any questions from the parent(s), and invite the parent(s) to enroll the child in the
 study.

 At the beginning of the study, information will be sought on the demographic characteristics
 (families' wealth, standard of housing, family structure and parental characteristics etc.),
 and FRAs will record the children's weight using a digital scale with 2 g precision, length
 (using infantometer,), and mid-upper-arm circumference to the nearest mm. Study participants
 will be asked to come directly to the nutrition center for nutritional therapy.

 Preparation of MDCF 2 and RUSF Based on compatible combinations of complementary food
 ingredients identified in the Pre-POC study described above, MDCF2 as well as Rice-lentil
 RUSF will be produced at the icddr,b Food Processing facility in Mirpur and in Kurigram (to
 be established in both places) in sufficient quantities for clinical study. The two diets
 will be matched in energy density and micronutrient content. The energy density of MDCF is
 125 kcal/25 g (per serving), and caloric distribution is targeted to be 45-50 percent from
 fat and 8-10 percent from protein. Experiments in development of the MDCF prototypes and
 assessment of the organoleptic properties have been done during the Pre-POC clinical trial.
 After receiving the raw materials (rice/lentil/chickpea) the foreign materials/grains or
 seeds (if there is any) will be taken out, and then in an open pan the raw materials will be
 roasted. The temperature will be maintained at 120-130°C for roasting. Usually it takes 8-10
 minutes for roasting 100g of each raw material. Continuous stirring is essential to ensure
 roasting of single seeds/grains. After completion of roasting it will be kept aside for
 cooling and then will be grinned. Finally the pre-weighed premix powder will be added. The
 processing of whole green banana for inclusion in MDCF2 is different from the other
 ingredients. Green banana with skin will be placed in a deep pan in boiling water
 (100°C-110°C) and boiled for about 17-20 minutes until they are cooked and tender. The skin
 of the green banana will be peeled off and the edible white part would be taken and grated
 into small pieces. Then they will be taken in a pot and allowed to cool. The small pieces of
 banana will be smashed with spoon/hand crusher. The weights of all other ingredients will be
 recorded. Recipes will be produced in small batches by mixing all ingredients in an electric
 blender. A small amount (1 percent) of soy lecithin shall be added to the recipe in order to
 improve the consistency and prevent oil separation.

 Anthropometry The age of the child will be verified against documentation (birth certificate
 or immunization card, if available) or caregiver's report of the child's birth date. Length
 will be measured by a infantometer sensitive to 0.1 cm. Body weight will be measured by a
 balance sensitive to 2g. Length-for-age (LAZ), weight-for-length (WLZ) and Weight-for-Age
 (WAZ) Z-scores will be calculated following the Multicentre Growth Reference Study (MGRS) WHO
 (World Health Organization) growth standards. Edema will be examined by pressing the upper
 side of both feet for 3 seconds. Mid-upper arm circumference (MUAC) will be measured using
 MUAC tape (UK). Regular standardization of the measuring equipments will be done using
 standards.

 Analyses of plasma and fecal samples Plasma samples collected from this trial will be sent to
 Dr Jeffrey Gordon's lab in the Center for Genome Sciences and Systems Biology at Washington
 University in St. Louis. Advanced mass spectroscopic- and immunoassay-based methods will be
 used to obtain new knowledge about the role of gut microbiota immaturity and the effects of
 attempting acute repair of this immaturity with lead microbiota-directed complementary food
 (MDCF) on biomarkers and mediators of healthy growth. Comparisons will be made with the
 control group (i.e., those consuming reference RUSF standard). Targeted Ultra Performance
 Liquid Chromatography-Mass Spectrometry (UPLC-MS) and Gas Chromatography-Mass Spectrometry
 (GC-MS) will be used to profile analytes of specific interest in plasma and/or fecal samples
 including bile acids and short chain fatty acids (SCFAs); markers of mitochondrial function
 (e.g., β-hydroxybutyrate, acylcarnitines/acylCoAs, TCA cycle intermediates); amino acids in
 serum plus fecal samples [branch chain amino acids, tryptophan and tryptophan metabolites
 related to growth and inflammatory status, including those produced by bacterial tryptophan
 metabolism (e.g. indole acetic acid derivatives)]. Key mediators/biomarkers of linear growth
 (e.g., growth hormone and IGF-1), energy utilization (insulin, leptin), and bone biology
 [IL-6 (Interleukin-6), osteoprotegerin, the C-terminal peptide of type I collagen (CTX, a
 marker of osteoclast activity/bone resorption), and the amino-terminal propeptide of Type 1
 procollagen (P1NP, a marker of osteoblast activity/bone formation)], and systemic
 inflammation (CRP, AGP) will be quantified using established ELISA/Luminex assays. Proteins
 in blood will be identified using the SOMAlogic scan that permits identification of more than
 1300 different proteins. The proteomic study done on plasma samples from children with SAM in
 our previous Pre-POC trial done in Dhaka has already demonstrated a number of significant and
 clinically relevant associations between certain proteins and clinical phenotypes using the
 SOMAlogic scan.

 Information gathered will be used to select human fecal samples for transplantation into
 germ-free animals in the Gordon Lab in St Louis; these animals, who will be fed the diets of
 their human microbiota donors, will be used to further characterize the mechanisms that link
 MDCF prototypes, the gut microbiota, and host physiology/metabolism. In addition, these
 plasma and fecal biomarkers will be used to determine that MDCF promotes repair of microbiota
 immaturity and improves biological state in children.

 Sample Size Calculation In the pre-POC trial of different MDCFs, the baseline
 weight-for-length Z score of children who received MDCF2 was -2.2 and after one month of
 supplementation was -1.7. If it is considered the WLZ -2 at baseline and -1.7 at end line,
 pooled SD (Standard Deviation)as 0.53 then the sample size is 49 in each arm at 80% power and
 5% level of significance.

 With 20% attrition 62 children will be required to be enrolled in each arm. Therefore, for
 the Primary MAM trial, 62 children will receive MDCF2 and 62 children will receive
 Rice-lentil RUSF.

 Data Analysis The two groups of children with MAM will be compared at baseline and at
 different time points. All analytes mentioned in the section Analyses of plasma and fecal
 samples' will be compared between MDCF2 and RUSF groups.

 Data Safety Monitoring Plan (DSMP) Data collection tools for this study will include case
 report forms, laboratory worksheets and source documentation. Complete source documentation
 (study visits, laboratory reports, etc.) will be kept for each study participant in
 individual study charts. All laboratory specimens, reports, study data collection and
 administrative forms will be identified by coded number to maintain study participant
 confidentiality and to enable tracking throughout the study.

 Forms, lists, logbooks, appointment books, and any other listings that link study participant
 ID (Identification) numbers to other identifying information will be stored in a separate,
 locked file in an area with limited access. All information regarding study participants will
 be kept in password-protected computer files or in locked file cabinets that can be accessed
 only by authorized study personnel. Chart information and information from study records will
 not be released without written permission from the study participant's parent(s). However,
 records may be reviewed by representatives from the Research Review Committee and Ethical
 Review Committee of icddr,b.

 The study investigators are responsible for ensuring complete and accurate documentation for
 the study and for each study participant, including: medical records, records detailing each
 study participant's progress through the study, laboratory reports, Case Report Forms (CRFs),
 signed informed consent forms, correspondence with IRB (Institutional Review Board ), adverse
 event reports, and information regarding participant discontinuation and completion of the
 study. All required data will be clearly and accurately recorded in the CRFs by authorized
 study personnel. Only designated study-site personnel who have received appropriate training
 will record or change data in a CRF. The investigators are responsible for procuring the data
 and for quality of data recorded in the CRFs. Data entry and management will be performed at
 icddr,b.

 Ethical Assurance for Protection of Human rights The study will be started after obtaining
 IRB approval by the icddr,b Research Review Committee and Ethical Review Committee. Before
 enrolment in the study, informed written consent will be taken from the legal guardian of the
 study participants. The privacy, anonymity and confidentiality of data/information
 identifying the study participants will be strictly maintained. Personal identifications
 taken during enrolment and other study procedures will be kept under lock and key. None other
 than the study personnel will have access to information of personal identification and other
 sensitive information.

 Expected risks/adverse events for this protocol are those related to blood sample collection,
 fecal sample collection and feeding of microbiota directed complementary food. None of these
 qualify as a serious adverse event (SAE). Expected Adverse Events (EAEs) related to blood
 draw are as follows:

 -  discomfort

 -  pain

 -  introduction of infection

 -  bleeding

 -  fainting or bruising

 -  Precaution will be taken to avoid introduction of infection by disinfecting the
 site of venipuncture and using sterile equipment

 -  The risk of bleeding and bruising will be minimized by immediate application of
 pressure after venipuncture

 -  The participant (child) will be in the sitting or supine position during blood
 draws to avoid injuries from fainting

 All possible adverse events will be treated appropriately. These will include:

 -  Vomiting

 -  Diarrhoea

 -  Skin rash

 -  Urticaria from food allergy

 -  Abdominal distension

 -  Pain abdomen Assessment of Adverse Events Both serious and non-serious adverse events
 will be assessed for severity; relationship to study participation; actions taken; and
 outcomes. All SAEs will be reported to the ERC of icddr,b within 24 hours of the site's
 awareness of the event that will in turn be distributed to the sponsor. This will be
 done by direct telephone communication, fax or e-mail.

 Each category for AE assessment will be coded according to the following grading systems:

 Severity:

 1. Mild

 2. Moderate

 3. Severe

 Relationship to Study Participation:

 1. Definitely related: Clear cut temporal association, no other possible cause

 2. Possibly related: Less clear cut temporal association, other causes possible

 3. Unrelated: Independent of study, evidence exists that event is definitely related to
 another etiology

 Actions:

 None Remedial therapy (more than one dose of medicine required) Permanently discontinued from
 study participation Hospitalization Other Use of Animals Not applicable Collaborative
 Arrangements This project is a collaborative effort between the investigators in the
 Washington University School of Medicine and the International Centre for Diarrhoeal Disease
 Research, Bangladesh (icddr,b). The clinical work, field activities will be undertaken in
 Dhaka, Bangladesh, under the direct supervision of the Principal Investigator whereas the
 laboratory analyses will be conducted at the Gordon Lab at Washington University in St.
 Louis. All research units and the collaborating investigators have long histories of
 international collaborations. All investigators have communicated during development of this
 project and are committed to its successful implementation.

 Facilities Available icddr,b has a well equipped Food Processing Lab that will facilitate
 preparations of diet recipes. A number of project offices are located in Mirpur. More
 importantly, an excellent rapport has been established with the community. The community
 elite and elders are invited every year to a dissemination meeting so that they are aware of
 the research being conducted.

 Center for Genome Sciences, Washington University in St. Louis:
Inclusion Criteria:

 -  Parent(s) willing to sign consent form

 -  Child age 12-18 months and no longer exclusively breast fed

 -  WLZ (<-2 to -3) without bilateral pedal edema at the time of randomization

 -  Parent(s) willing to bring the child to the feeding center twice daily for 4 weeks for
 nutritional therapy, once daily for next 4 weeks and provide feeding once daily at
 home for 4 weeks and twice daily for next 4 weeks.

 -  The informed consent document will explicitly request permission to use the collected
 fecal samples for future studies, including but not limited to culturing component
 bacterial strains

 Exclusion Criteria:

 -  Medical conditions: Children with tuberculosis (diagnosis based on WHO 2014 guidelines
 which have been incorporated in the national TB control guidelines of Bangladesh). The
 guidelines depend upon the following five diagnostic principles (three out of five
 should be positive): 1. Specific symptoms of TB, 2. Specific signs, 3. Chest X-ray, 4.
 Mantoux test, and 5. History of contact. 10 or any congenital/acquired disorder
 affecting growth i.e. known case of trisomy-21 or cerebral palsy; children on an
 exclusion diet for the treatment of persistent diarrhea; having known history of soy,
 peanut or milk protein allergy

 -  Antibiotic use within the last 15 days

 -  Receiving concurrent treatment for another condition

 -  Severe anemia (<8mg/dl) will be assessed by Hemocue (Model no. Hemocue Hb 301)

 -  Failure to obtain informed written consent from parents or caretakers