Pilot of a Prebiotic and Probiotic Trial in Young Infants With Severe Acute Malnutrition
Pilot of a Prebiotic and Probiotic Trial in Young Infants With Severe Acute Malnutrition
Malnutrition is an ever-present problem worldwide. It is estimated that over 18 million
 children under the age of 5 are affected by the most extreme form of undernutrition, severe
 acute malnutrition (SAM). In spite of having standardized management protocols, in many
 hospitals, inpatient mortality reaches up to 30%. Infectious morbidity is common among
 survivors. Diarrhea, severe intestinal inflammation, low concentrations of fecal short-chain
 fatty acids (SCFAs), and severe systemic inflammation are significantly associated with
 mortality in SAM. Investigators of this study have earlier shown that the gut microbiota in
 children with SAM is immature and is causally related to SAM.

 Human milk contains between 10 and 20 g/liter of oligosaccharides (human milk
 oligosaccharides-HMOs) which is the third most abundant solid component after lactose and
 lipids. HMOs are resistant to gastrointestinal digestion in host infants, and thus the
 greater part of HMOs reached the colon and may act as prebiotics to shape a healthy gut
 ecosystem by stimulating the growth of useful microorganisms by acting as receptor analogs to
 inhibit the binding of various pathogens and toxins to epithelial cells. Probiotics are live
 organisms beneficial for a healthy life. The human digestive tract possesses a diverse
 microbial community throughout its extent, which supports their hosts generally for healthy
 living. Bifidobacterium spp. is dominant microbiota in infants who are exclusively breastfed
 and these infants are less likely to suffer from diarrhea. According to recent studies among
 the most common probiotics genera Lactobacillus and Bifidobacterium, the latter is more
 abundant in the gut. To carry out their functional activities, Bifidobacteria must be able to
 survive the gastrointestinal tract transit and persist, at least transiently, in the host.
 The population of Bifidobacteria in the gut community drastically decreases after weaning.
 Certain Bifidobacteria possess the metabolic capabilities to break down the HMOs.
 Consequently, it is observed that HMOs support the growth of select Bifidobacteria in the gut
 of the infant.

 Research done at icddr,b and Washington University indicates that gut microbes are related to
 undernutrition and that children with SAM have gut dysbiosis that mediates some of the
 pathologies of their condition. The standard of care in these children should be reinforced
 by an intervention that corrects the gut dysbiosis, improves weight gain during nutritional
 rehabilitation, and reduces infectious morbidity. Investigators do not have any published
 data on the microbiome response to probiotic supplementation (with and without prebiotics) in
 malnourished infants or preserving the microbiome with probiotics in non-malnourished
 children.

 A short-term pilot study should be conducted to evaluate the microbiome response to probiotic
 supplementation (with and without prebiotics) in malnourished populations to justify a larger
 study of clinical outcomes. Additionally, non-malnourished infants who are hospitalized for
 infectious conditions face challenges related to gut dysbiosis caused by antibiotic usage.
 Here the investigators will evaluate the ability of a probiotic intervention to rescue the
 microbiome of primarily breastfed non-malnourished infants.

 Intervention: Bifidobacterium longum subspecies infantis (EVC001) with and without prebiotic
 supplementation for 28 days.

 Objectives: To evaluate the microbiome response to probiotic supplementation (with and
 without prebiotics) in infants under 6 months with severe acute malnutrition and to compare
 the microbiome response with healthy infants with a probiotic.

 Methods: Single-blind RCT, stratified randomization will be based on infant age at the time
 of transfer to the Nutritional Rehabilitation Unit (NRU).

 3 treatment arms for infants with SAM

 1. Placebo (Lactose)

 2. Bifidobacterium infantis alone (Bif)

 3. Bifidobacterium infantis + prebiotic Lacto-N-neotetraose [LNnT] (Bif+prebiotic) Age at
 enrollment

 1. 2-3.9 months of age

 2. 4-5.9 months of age 1 open-label treatment arm for 18 non-malnourished primarily
 breastfed infants: Bifidobacterium infantis alone (Bif)

 Population:

 1. Group 1 (SAM): Infants between 2 and <6 months old with SAM as defined by
 weight-for-length Z score < -3 either sex, caregiver willing to provide consent for
 enrolment of the infant, caregiver willing to stay in the NRU for about 15 days,
 residence within 15 km from icddr,b

 2. Group 2 (non-malnourished): Non-malnourished infants (WLZ ≥ -1) <6 months old who are
 hospitalized for treatment with antibiotics for the infection, infants receiving at
 least 50% of nutritional intake from breast milk at the time of hospitalization, either
 sex, residence within 15 km from icddr,b

 Primary Outcome measures/variables:

 Bifidobacterium infantis colonization measured by qPCR during and after supplementation (with
 and without prebiotics)
Specific Objectives:

 To evaluate the microbiome response to probiotic supplementation (with and without
 prebiotics) in under 6 months infants with severe acute malnutrition and to compare the
 microbiome response with healthy infants supplemented with probiotic.

 Study Site: The study will be conducted at Dhaka Hospital of the International Centre for
 Diarrheal Disease Research, Bangladesh (icddr,b). The institution possesses well equipped
 clinical care as well as laboratory facilities capable of performing all the clinical tests.

 Study population:

 Group 1 (SAM) Group 2 (non-malnourished)

 Intervention:

 Bifidobacterium longum subsp. infantis (EVC001) with and without prebiotic supplementation
 for 28 days will be provided in the study.

 There are several studies in the developing world where prebiotic and probiotic were used in
 severe acute malnutrition and among at risk infants. Safety of Bifidobacterium longum subsp.
 infantis (EVC001) is evident from published literature as well as safety reports (Smilowitz
 et al., 2017; Sheridan et al., 2017; Grenov et al., 2017; Panigrahi er al., 2017; Kerac et
 al., 2009). The IMPRINT Study, a Phase I clinical trial, determined the safety and
 tolerability of supplementing breastfed infants with B. infantis (EVC001) (Smilowitz et al.,
 2017). Safety of prebiotic (LNnT) was evaluated by EFSA (European Food Safety Authority)
 Panel on Dietetic Products, Nutrition and Allergies (NDA) related food and panel concluded
 that LNnT is safe for infants. The International Scientific Association for Probiotics and
 Prebiotics (ISAPP) recommended that an attractive target for clinical interventions will lead
 to new approaches that combat malnutrition (Sheridan et al., 2017). A study held in Uganda
 using probiotic In hospitalized SAM children revealed that probiotic may have a role in
 follow up of hospitalized children with SAM in outpatient phase as it showed significant
 reduction of diarrheal duration (Grenov et al., 2017). In rural India significant reduction
 was observed for culture-positive and culture-negative sepsis and lower respiratory tract
 infections in infants with use of symbiotic (Kerac et al., 2009). The PRONUT study did not
 demonstrate any case of probiotic related bacteraemia . The response of microbiota to
 probiotics may differ by diet or geography.

 Trial arms:

 3 treatment arms for infants with SAM:

 -  Placebo (Lactose)

 -  B. infantis alone (Bif)

 -  B. infantis + prebiotic Lacto-N-neotetraose [LNnT] (Bif+prebiotic) Each arm will be
 assigned to receive the standard of care diet (F-75 during the acute phase treatment of
 SAM, F-100 during hospital stay for nutritional rehabilitation, and standardized infant
 formula upon discharge). Randomization for treatment allocation will be done in 1:1
 ratio using stratification by age (2-3.9 months vs. 4-6 months) to ensure equal
 participation of infants by age groups. Allocation will be achieved by using random
 permuted blocks size of 3. In total 54 participants will be enrolled with 18 infants
 (9+9) allocated in each treatment arm.

 Prebiotic will be used in the form of LNnT (Lacto-N-neotetraose, which is produced by
 fermentation and is chemically identical to LNnT present in human breast milk. LNnT contains
 a specific linkage that B. infantis can break down while other bacteria cannot. In the in
 vitro assay, when the growth media contains 50% or more of LNnT, growth of B. infantis is
 supported, but growth of Bacteroides and E.coli is inhibited. The distribution of the
 glycosylhydrolases needed to consume this structure are rare among Proteobacteria (including
 Campylobacter) as a whole, and absent among Bacteroides and therefore LNnT should not
 contribute to cross feeding, even in the absence of B. infantis engraftment.

 The aim of selecting the non-malnourished children is to see whether the disrupted microbiota
 of SAM infants recovers to higher level of B. infantis, similar to those levels seen in
 healthy breast fed infants who have an acute illness and are treated with an antibiotic.

 Sample size:

 For the pilot, we will have 18 infants per treatment arm, a total of 54 SAM infants. We will
 also enroll 18 non-malnourished infants for the open-label B. infantis treatment. A total of
 72 infants will be the study population for this pilot.

 Even if study participants drop or develop any hospital acquired infection or deteriorate, we
 have to complete 18 evaluable cases in each arm.

 Baseline data collection procedure: All SAM and non-SAM children within the defined age
 groups will be screened for study eligibility criteria by the study physician. Parents /
 attending care givers of those fulfilling the eligibility, in application of the inclusion
 and the exclusion criteria, will be invited to provide their consent for enrollment of their
 children in the study. Upon signing a written informed consent, after providing information
 about the study and its interventions, explaining possible benefits and risks, and voluntary
 nature of participation along with the right to withdraw children at any time after the
 initial consent without providing any reason, children will be enrolled by the study
 physician. Clinical Record Form will be used to collect relevant information such as medical
 history including nature and duration of illness, medication for current illness;
 socio-demographic characteristics such as age, sex, religion, parental age with education,
 parents occupation, fuel use and smoking history, monthly family income, number of
 siblings,etc. Information would also be collected about child's feeding practice such as
 history of breast feeding, formula or other complementary feeding, and immunisation status;
 family history of tuberculosis, recent respiratory tract infection of any family members and
 past medical history of child would be recorded.

 For long term follow up separate CRF will be used to collect information on weight, height,
 MUAC, feeding and co-morbidity history. Field Research Assistants will visit the houses of
 enrollees and will collect required information after taking written consent from parents or
 caregiver.

 Management:

 Group 1 (children with SAM): Eligible SAM infants will be treated for the stabilization phase
 in the Longer Stay Unit of the Dhaka Hospital of icddr,b. The standardized treatment protocol
 for children with SAM and diarrhea (developed at icddr,b and aligned to the WHO guidelines;
 Ahmed et al Lancet 1999) will be followed. Key elements include initially 2 hourly feeding
 with F-75 (the recommended starter formula, 130 ml/kg per day), folic acid, vitamin A,
 Ampicillin plus Gentamicin, temperature and blood glucose monitoring and control. Prior to
 initiation of antibiotics, baseline stool samples will be obtained (by fecal catheter if
 needed).

 The stabilization phase lasts on an average 4 days in our experience. Completion of
 stabilization phase is characterized by a satisfactory appetite, no need for nasogastric tube
 feeding, good general clinical condition, absence of diarrhea, dehydration, fever,
 hypothermia or tachypnea.

 Infants who are stabilized are transferred to the NRU upon receiving written consent from the
 caregiver. Stabilized infants will be randomized to the 3 treatment arms once they have
 completed the 5-7 day course of antibiotics. The Senior Dietician of the Hospital prepares
 the diet (F-100) and supplies to the NRU. F-100 is dispensed at 130 ml/kg per day every 2
 hours for the first 2 days, and thereafter given every 3 hours and gradually increased by 10
 ml at each serving until some feed is left. Breast feeding is encouraged in between the
 feeds. Amount of breast milk consumed will be estimated by test weighing of infants.

 Caregivers will be trained to provide psychosocial stimulation to the infants, an activity
 that is done routinely in the NRU. This is done through talking or singing to the infants,
 age specific physical activity and play with local toys.

 Measurement of breast milk intake by test weighing folloing standard published method (Islam
 et al., 2006):

 Collection of breast milk:

 Since this study will investigate the engraftment of Bifidobacteria which is linked with
 availability of HMOs in breast milk, we will collect 10 ml of breast milk under aseptic
 condition and will be analyzed for HMO content.

 This analysis will be performed in laboratories that have a track record of performing these
 assays.

 Additionally, we will also perform microbiota analysis of collected breast milk samples. This
 is because breast milk microbiota is likely to influence the colonization by other organisms
 and the intestinal mileage of infant.

 Breast milk will be collected from the breast between 0700 and 1100 hr, and women will be
 asked to have not to fed or expressed from the study breast for at least 2 hours prior to
 sample collection. Before sample collection, the breast will be cleaned with an iodine swab.
 A complete breast expression will be collected using a single-use, sterile, collection kit
 (Hygienikit®; Ameda, Cary, IL, USA) and an electric breast pump (Model SMR-B-R; Ameda-Egnell,
 Inc.). Milk will be immediately placed on ice.

 Bif (8 billion CFU/daily single dose-), Bif (8 billion CFU/daily single dose-) + Prebiotic
 (1gm/6hourly-4gm/day), and placebo (lactose 625mg) will be available in powder format in
 sachets contained within feeding kits marked with unique code numbers that match the
 randomization code of each infant. The sachet contents will be administered to an infant once
 every day for Bif and placebo sachets mixed with 5ml infant formula or breast milk and each
 preparation of 200ml F-100 one prebiotic (LNnT) sachet will be dissolved. Caregivers, but not
 study staff, will be blinded to treatment assignment.

 The dose and dispensing of LNnT will ultimately be derived upon the review of recent
 literature as well as experience of experts working in this field. The goal is to stimulate
 as much as possible the natural conditions of availability of HMOs in breast milk.

 The feeding kits (sachets) that have been assigned to randomized infants will be refrigerated
 or stored frozen until used while the participant is hospitalized. Feeding kits that have not
 yet been assigned to an infant will be stored in a hospital freezer with continuous
 temperature monitoring. The probiotic stored in the freezer (-20°C ± 5°C) can be used up
 until the expiration date printed on the sachet (1 year from date of packaging). It can be
 stored up to 31 days in the refrigerator (4°C ± 5°C). The prebiotic (LNnT) is stable at room
 temperature, but can be stored in the refrigerator or freezer with the probiotic.

 Group 2 (non-malnourished children): Eligible non-malnourished infants will be treated as per
 hospital protocol. In brief, they are admitted to the Longer Stay Unit or if necessary to the
 ICU. The hospital has defined treatment protocols for acute watery diarrhea, pneumonia and
 severe pneumonia, persistent diarrhea, severe sepsis, electrolyte disorders, and
 hospital-acquired infections. Children will be treated at the hospital per these protocols.
 Exclusively breastfed infants will continue on breast milk. Others are supplemented with
 modified infant formula every 2-3 hours. Amount of breast milk consumed will be estimated by
 test weighing of infants. Treatment with Bif will start on the day of completion of the
 antibiotic course and will be provided to infants in the same manner as Group 1. The sachet
 contents will be mixed with a teaspoon of infant formula or breast milk and fed to the
 infant.

 Even if study participants drop or develop any hospital acquired infection or deteriorate, we
 have to complete 18 evaluable cases in each arm.

 Discharge from Hospital:

 The discharge will be done if the infant with SAM has achieved a weight-for-length >-3, if
 possible ≥ -2 Z-score, and in case of edematous malnutrition when edema resolves and when the
 infant thriving and clinically well. Non-malnourished infants will be discharged when they
 fulfill the hospital discharge criteria.By then the caregivers will have been trained for
 food and supplement preparation and administration.

 Follow up:

 Infants with SAM will be followed up through home visits by Research Assistants twice every
 week until 8 weeks post randomization. Non-malnourished infants will be followed through home
 visits twice every week until the supplementation is complete and then once every week until
 6 weeks post randomization. During such visits, the rations of infant formula (for SAM
 infants) and supplements (both groups) will be replenished, actual intake of the diet will be
 observed or the amount given and the method of reconstitution verified, and morbidity
 recorded on a customized case record form. Because the B. infantis supplement should be kept
 cool to maintain optimum viability, Research Assistants will bring the supplement to home
 visits in insulated boxes or coolers with ice or frozen gel packs. Although the supplement
 will likely warm to room temperature between visits, it is not expected that the viability
 loss will significantly impact the colonization of B. infantis in the gut. A miniature fridge
 will be provided to each participant for the study period and each parent will be instructed
 to keep the supplement in these containers until use.

 Infants who have not yet achieved a weight-for-length ≥ -1 Z-score by 8 weeks post
 randomization (end of home visits) may continue to bring the infant to icddr,b for weighing
 and measuring and to pick up the formula rations on a routine basis until a weight-for-length
 ≥ -1 Z is achieved.

 A long term follow up will be carried out to assess the anthropometry of enrolled
 participants.

 Sample collection:

 Fecal samples will be collected as follows.

 -  Stool swabs (for microbiome analysis) (13times in SAM, 11 times in non-SAM)

 -  At admission, prior to antibiotic treatment

 -  After stabilization, prior to randomization and supplementation

 -  Day 2 of supplementation

 -  Day 4 of supplementation

 -  Day 7 of supplementation

 -  Day 10 of supplementation (or last day in NRU if discharged sooner)

 -  Day 14 of supplementation (in NRU or picked up by research assistant at home visit)

 -  Day 21 of supplementation (picked up by research assistant at home visit)

 -  Day 28 of supplementation (picked up by research assistant at home visit)

 -  1 week post supplementation (picked up by research assistant at home visit)

 -  2 weeks post supplementation (picked up by research assistant at home visit)

 -  3 weeks post supplementation (picked up by research assistant at home visit): SAM
 only

 -  4 weeks post supplementation (picked up by research assistant at home visit): SAM
 only

 -  Stool chunks or catheter fecal samples (for pH and inflammatory markers) in hospital
 [SAM infants only]:

 -  At admission, prior to antibiotic treatment

 -  After stabilization, prior to randomization and supplementation

 -  Day 7 of supplementation'

 -  Day 10 of supplementation (or last day in NRU if discharged sooner)

 -  Day 28 of supplementation (collected by research assistant at home visit)

 -  4 weeks post supplementation (collected by research assistant at home visit)

 -  Stool chunks or catheter fecal samples (for pH and inflammatory markers) in hospital
 [non-malnourished infants only]:

 -  At admission, prior to antibiotic treatment

 -  After start of supplementation, at hospital discharge

 -  Day 28 of supplementation (collected by research assistant at home visit)

 -  2 weeks post supplementation (collected by research assistant at home visit)

 Stool sample collection in the hospital:

 All children will be screened from Dhaka Hospital, icddr,b according to the above mentioned
 inclusion and exclusion criteria. Research staff will screen these children before
 administration of an antibiotic. Before collection of stool samples written consent will be
 taken from legal guardian.

 Stool should be freshly collected from each child. The samples must be immediately aliquoted
 into pre-labelled 2 ml cryovials and into the shipper (~-70⁰C) as quickly as possible after
 collection, ideally less than 15 minutes after collection. After collection and prior to
 aliquoting, the stool collection vessel should be maintained on (wet) ice. Ideally the
 cryovials would be filled to approximately 2/3 full. Two times in a week these cryopreserved
 samples will be transported to Biosafety Level Laboratory in icddr,b Dhaka Hospital for
 storage in -80⁰C freezer. In order to prevent thawing, vials will be transferred from the
 shipper to the -80⁰ freezer as quick as possible while transferring to freezer boxes. These
 freezer boxes will not be removed from the -80⁰ freezer until they are handed over to World
 Courier for shipment to USA.

 Measures to ensure best possible recovery of bacteria and their viability:

 1. The shippers should be fully charged before the collection day and a record will be
 maintained for the number of times they will be opened.

 2. The shippers need around 12 hours after adding liquid nitrogen to become fully charged.
 Therefore, we will re-charge (fill) the shippers at the end of each day and then pour
 off any remaining unabsorbed liquid nitrogen the next morning before using them.

 3. Shippers will be weighed before charging, after charging (ie. the next morning after
 pouring off any unabsorbed N2), and then again at the end of each day, to know how much
 liquid nitrogen remains in the canister. This will allow us to estimate how much liquid
 N2 is needed every time we recharge and how frequently this is needed.

 Stool swab collection in the Hospital and from the Community:

 Stool samples will be collected as mentioned in the time schedule. Once participants are
 discharged from the NRU, the stool swab will be collected within a two day window of the
 scheduled time.

 For collection of stool, child's/participant's primary caretaker will be informed one day
 before planned stool collection. A trained research staff will collect the first available
 fresh stool sample from the child on the day of the scheduled sampling.

 During collection in the Hospital and in the community, the research staff will be provided
 with the labeled stool swab tubes (containing stabilization buffer), individually wrapped
 swabs (with breakpoint), a cryogenic marker, and plastic bags.

 In the Hospital, the mothers/caretakers will be instructed to notify the research staff as
 soon as the child defecates. Then the research staff will do the following:

 -  Swab the baby's soiled diaper, collecting enough stool sample to entirely coat the head
 of the swab.

 -  Place the swab into the appropriately labeled green-topped collection tube and snap the
 swab head off at the breakpoint - the swab head will remain in the tube.

 -  Seal the tube tightly and shake vigorously to ensure distribution of the stool within
 the stabilization buffer.

 -  Using the provided cryogenic marker
 (https://www.thermofisher.com/order/catalog/product/4000221), write the date the stool
 was collected on the label on the tube.

 -  Repeat the swabbing with a second clean swab and the duplicate green-topped tube.

 -  Place green-topped tubes back in the provided plastic bags and store at room temperature
 until picked up by the health worker.

 Information about antibiotic intake will be collected for each participant during the study.
 At the time of each sample collection, we will ask mother or other caregiver for any illness
 during last 14 days, and whether treated. The nature of medicine will be verified by physical
 inspection of the medicine, the prescription and by asking the name or if the medicine was a
 powder that had to be reconstituted.

 Saliva sample collection from mother:

 We will do the "Secretor status phenotyping assay" in mothers' saliva to see the secretor
 status of mothers to detect HMO status as well as link with the Microbiota of infants.

 Saliva sample collection procedure

 1. Mothers' should wash the hands and rinse the mouth with cold water, and spit water out
 completely.

 2. They will remove the sampler being careful to touch only the handle.

 3. Mother will hold the handle Oracle sampling tube as a toothbrush and will insert in her
 mouth and will rub a sponge against her gums for one minute. Ensure that the sponge is
 completely saturated.

 4. without touching the sponge, they will insert the sampler into the original Oracol tube,
 sponge down, and will close the lid tightly.

 5. Mother will give the Oracle sampler tube back to the study staff (who will label and
 refrigerate the tubes if the samples will not be processed on the same day).

 Collection and transport of fecal samples from the community Each trained community health
 worker will be responsible for certain number of stool collections per month. She will
 contact mother over cell phone and arrive at the child's house early in the morning with a
 bench top nitrogen shipper and collect the stool swab.

 Initial collection of fecal samples should be made in a suitable sterile container after
 which smaller aliquots of fecal material should be transferred by the field worker (within 20
 minutes of defecation) into pre-labeled, sterile 2ml cryo-safe tubes. Tube labels should
 minimally include the Participant's Sample ID (SID) and the date of collection.

 Wearing clean, disposable latex gloves, the health workers will fill each 2 ml cryo-vial
 approximately one-half to two-thirds full using a sterile spatula and cap tightly. Any
 buffers, preservatives or additives will not be added to the sample. Precaution will be taken
 during use of screw cap vials and not overfilling them as this will minimize the potential
 for cross-contamination of samples during transport and storage.

 Then they will place the capped vials into liquid nitrogen pre-charged 'dry shippers'
 immediately to transport back to the laboratory. Although difficult, the specimens should be
 transferred to dry shippers within 20 minutes of defecation. In the laboratory, trained staff
 will empty the vials from the dry shipper into a bucket of dry ice to prevent thawing while
 sorting and transferring the vials to 9x9 freezer boxes for longer term storage and
 transport.

 Reporting:

 Information will be recorded on Stool Field Collection Form (SFC).

 Investigations:

 Tests performed at icddrb:

 -  determining fecal pH in hospital lab

 -  Testing of Myeloperoxidase and other biomarker levels in fecal samples

 Tests performed by Evolve Biosystems:

 -  Next Generation Sequencing, Metagenomics and q PCR analyses of samples

 -  Immune- related tests- Multiplex analysis of inflammatory and immune-related
 cytokines/analytes

 -  IL-10, TGF beta, IL-2, TNF alpha, IL-17, IL-22, IL-8, IL-6, IP-10

 -  ELISAs for sCD83, sTLR2 and fecal lipocalin 2

 Stool PH:

 Stool PH will be measured to assess the change from baseline in stool pH during
 supplementation.

 Myeloperoxidase:

 Myeloperoxidase is a specific marker of neutrophil activity. It was chosen because it is not
 elevated in breast milk or in the stools of breastfed children as are lactoferrin and
 calprotectin (Dorosko et al., 2008) and stool MPO levels have been correlated with disease
 activity in inflammatory bowel disease as assessed both by endoscopic and biochemical
 parameters (SAIKI T, 1998). MAL-ED study has already demonstrated the role of MPO as a marker
 of gut function (McCormick et al., 2017).

 We will measure this biomarker concentration by a commercial ELISA.

 Next Generation Sequencing: In recent years, several next-generation sequencing technologies
 have been established (Shendure & Ji, 2008) which further make it possible to analyze a large
 number of microorganisms in different environments (Tringe et al., 2005) and human body sites
 (Ding & Schloss, 2014, including the human gut (Methé et al., 2012).

 16S rDNA sequence analysis and metagenomics are two effective DNA sequencing techniques, and
 both have been used to study uncultivated gut microbial communities. 16S rDNA sequence
 analysis focuses on the sequencing of the conserved 16S rDNA gene present in all microbes
 (Woese & Fox, 1977) and has established a series of novel connections between intestinal
 microbiota composition and disease (Blaser et al., 2013). The research based on 16S rDNA
 sequence is a technique to reveal "who's there?" in a given microbial community, while
 shotgun metagenomic sequencing will be able to answer the complementary question of "what can
 they do?" (Lepage et al., 2012). Next generation sequencing will be performed to detect the
 engraftment of organism especially B. infantis after intervention in severely malnourished
 infants and comparing it with well nourished children.

 Metagenomics:

 Metagenomics was first described in 1998 by Handelsman and Rodon ( Handelsman et al., 1998)
 and became another DNA sequencing approach to study the complex gut microbial community. It
 aims to catalog all the genes from a community by the random sequencing of all DNA extracted
 from the sample (Qin et al., 2010). Firstly, the total DNA of all microorganisms is extracted
 from fecal samples. Before being sequenced, total DNA samples are randomly sheared by a
 "shotgun" approach. The comprehensive sequences are then analyzed to obtain either species
 profiles based on phylogenetic markers (16S rDNA) (Sunagawa et al., 2010) or genomic profiles
 based on whole genomes (Tringe et al., 2005). Metagenomics plays a role in understanding the
 human gut microbiome, including the diversity of the gut microbiome, identifying novel genes,
 and determining the etiology of functional dysbiosis (Wang et al., 2015). Baseline
 composition of gut microbiota (metagenomic analysis) and Bifidobacterium colonization
 (relative abundance) by metagenomic analysis during/after supplementation will be done.

 qPCR: Real time PCR will be done for quantitative detection of organism in DNA directly
 extracted from stool sample. B. infantis colonization by qPCR during and after
 supplementation (with and without prebiotics) will be done.

 Cytokines:

 Any cytokine that is produced locally as a result of gut inflammation may leak into the bowel
 lumen and appear in the stools. Large inflammatory infiltrate in the diseased mucosa of gut
 causes raised cytokines in stool. We will do cytokine assays to detect gut inflammation. We
 will measure in stool samples Interleukin-10 (IL-10), Transforming Growth Factor-β (TGF-β),
 Interleukin-2 (IL-2), Tumor Necrosis Factor-α (TNF-α), Interleukin-17 (IL-17), Interleukin-22
 (IL-22), Interleukin-8 (IL-8), Interleukin-6 (IL-6), Interferon Gamma-Induced Protein 10
 (IP-10).

 ELISAs for sCD83, sTLR2:

 A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is
 detectable in normal human sera (Hock et al., 2001). Toll-like receptors (TLRs) are a class
 of pattern recognition receptors (PRRs) that play a key role in innate immunity and trigger a
 specific immune response. sTLR2 has been detected in human fluids, such as plasma, breast
 milk, saliva and amniotic fluid as well as in supernatant of cultured monocytes (LeBouder et
 al., 2003; Kuroishi et al., 2007; Dulay et al., 2009) We will isolate sCD83 and sTLR2 from
 stool samples of malnourished infants.

 Fecal lipocalin 2:

 This glycoprotein is expressed in several cells and, has been associated with primary and
 secondary granules of human neutrophils (Roudkenar et al., 2007). Upregulation of Lcn-2 is
 observed in tissue damaging conditions such as infection, kidney injury, cancer, ulcerative
 colitis and burn injury where production of free radicals occurs (Chassaing et al., 2012).
 Lipocalin-2 correlates less tightly with the neutrophil markers, it may provide a
 complementary marker of epithelial cell disruption or damage ( Prata et al., 2016). We will
 do the test for both the malnourished and well nourished infants to detect enterocyte damage.

 Breast milk microbiota and HMO analysis:

 There are several study where breast milk analysis (Williams et al., 2017) and bacterial
 community in breast milk (Hunt et al., 2011) were assessed. In this study Microbiota analysis
 will be done with 16S rDNA sequence analysis. HMO measurement will be done to know LnNT
 level.

 Data processing Procedure:

 -  Questionnaires will be visually scanned soon after interview & marked for omissions,
 inconsistencies or mistakes that will be addressed immediately

 -  SPSS will be used & data will be entered after creating a template for each data entry
 file with appropriate logical and consistency checks

 -  Data will be continuously entered as the data are being generated in hospital and
 laboratory

 -  Data will be validated by a series of logical and range checks, producing summary
 statistics and tables

 -  In addition 50 % CRF (and all variables) will be checked by investigator(s).

 -  Data will be immediately copied on the hard disks of two computers as soon as data
 verification will be completed.

 Sample Size Calculation and Outcome (Primary and Secondary) Variable(s):

 For the pilot study, we will enrol 18 SAM infants per treatment arm, and additional 18
 non-malnourished children, thus a total of 72 infants.

 Data Analysis Data will be entered in to the pre-tested Clinical Record Forms (CRFs) using
 SPSS (20.0 version, Armonk, NY) and finally cleaned with repeated check. To know the outcome
 of prebiotic and probiotic supplementation in our study children for normally distributed
 data we will perform ANOVA and in case of non-normal distribution of measurements, equivalent
 non-parametric tests like Kruskal-Wallis H test will be used and appropriate post hoc test
 will be done for the sub-group analysis, if required. A probability of less than 0.05 will be
 considered as statistically significant and strength of association will be determined by
 calculating odds ratio (OR) and 95% confidence intervals (CI). If any death occurs we will
 perform verbal autopsy and evaluate the associated factors of death by the same analytic
 plan.

 Data Safety Monitoring Plan (DSMP) Data safety Monitoring will be rigorously performed
 throughout the course of the study. All forms will be reviewed by study physician after
 enrolment and data collection followed by supervisor for completeness, legibility, and
 internal consistency. Administration of prebiotic and probiotic in hospital will be in
 presence of study physician. All aspects of specimen accession, processing and interpretation
 will also be performed according to SOPs.

 Collaborative Arrangements icddr,b will perform the study in collaboration with Evolve
 BioSystems, Inc. USA , a private enterprise established in California by internationally
 reputed academicians researching into infant gut microbiome and its critical association with
 human breast milk.
Inclusion Criteria:

 Group 1 (SAM):

 -  Infants between 2 and <6 months old with SAM as defined by weight-for-length < -3 Z
 and/ or bilateral pedal edema

 -  either sex

 -  caregiver willing to provide consent for enrolment of the infant

 -  caregiver willing to stay in the Nutritional Rehabilitation Unit for about 15 days

 -  residence within 15 km from icddr,b

 Group 2 (non-malnourished):

 -  Non-malnourished infants (WLZ ≥ -1) <6 months old who are hospitalized for treatment
 with antibiotics for infection (infants who come with a history of antibiotic intake
 for 3 days or more will be eligible; the last dose of such an antibiotic will have to
 be taken within last 24hours, the antibiotic intake should be documented by the
 verification of a prescription, the bottle of antibiotic or asking the caregiver about
 the name of antibiotic or its price and how it is reconstituted)

 -  infant receiving at least 50% of nutritional intake from breast milk at the time of
 hospitalization

 -  either sex

 -  residence within 15 km from icddr,b

 Exclusion Criteria:

 -  Septic shock or very severe pneumonia requiring assisted ventilation

 -  acute kidney injury on admission

 -  congenital defects interfering with feeding such as cleft palate

 -  chromosomal anomalies

 -  jaundice

 -  tuberculosis

 -  presence of bilateral pedal edema ongoing maternal antibiotic usage for breastfeeding
 infants

 Group 1 (SAM) additional exclusion criteria:

 Infants receiving ≥75% of nutrition from breast milk

 Group 2 (non-malnourished) additional exclusion criteria:

 Infants receiving <50% of nutrition from breast milk