Impact of prebiotic (Lacto-N-neotetraose) and probiotic (B. infantis EVC001) supplementation on gut inammation and fecal pH in young infants suffering from severe acute malnutrition: ndings from a randomized controlled trial

Severe acute malnutrition (SAM) is associated with high levels of gamma Proteobacteria chronic gut inammation and poor gut health. In this study, we aimed to assess the effects of probiotic and/or prebiotic supplementation on gut inammation and fecal pH in young infants with SAM. regression analysis to of gut inammation in young infants with SAM. However, further studies are required to fully evaluate the role of this synbiotic intervention for sustained reduction in fecal pH in this study population. of of milk received as proportion of total feed family income, ongoing use or completed recommended dosage of between the selected time and mode of delivery β-coecient values the corresponding 95% condence (CI) to All statistical

Impact of prebiotic (Lacto-N-neotetraose) and probiotic (B. infantis EVC001) supplementation on gut in ammation and fecal pH in young infants suffering from severe acute malnutrition: ndings from a randomized controlled trial Abstract Background Severe acute malnutrition (SAM) is associated with high levels of gamma Proteobacteria chronic gut in ammation and poor gut health. In this study, we aimed to assess the effects of probiotic and/or prebiotic supplementation on gut in ammation and fecal pH in young infants with SAM.

Methods
This study was a single-blind RCT where infants aged between 2 and 6 months were randomized to receive either: probiotic (B. infantis EVC001), synbiotic (prebiotic, Lacto-N-neotetraose [LNnT] plus probiotic [B. infantis EVC001]), or placebo (Lactose) for 28 days followed by 28 days of post-supplementation follow up. Stool samples were collected at baseline and at day 10, day 28 and day 56. Fecal myeloperoxidase (MPO), as an indicator of gut in ammation and fecal pH were measured and the change in the levels of these biochemical parameters between the sample collection time points were calculated and denoted as ∆MPO and ∆pH. Kruskal-Wallis test was done to assess the effect of the supplementations on ∆MPO and ∆pH.
Multivariate quantile regression analysis was performed to analyze the association of the supplements with ∆MPO and ∆pH.

Results
In comparison to the placebo group at day 10, a signi cant decrease in ∆MPO was found for the probiotic group (β-coe cient: -18.44; 95% CI: -31.62 µg/ml, -52.47 µg/ml; p = 0.007) and for the synbiotic group (βcoe cient: -17.24; 95% CI: -30.94 µg/ml, -35.36 µg/ml; p = 0.015). This reduction in ∆MPO in comparison to the placebo group was sustained only in the synbiotic group at other time points. Decrease in ∆pH was observed only in the synbiotic group at day 10 and day 28, but not at day 56 in comparison to the placebo group.

Conclusion
Our ndings demonstrate that the use of this synbiotic supplementation may result in sustained effects in reduction of gut in ammation in young infants with SAM. However, further studies are required to fully evaluate the role of this synbiotic intervention for sustained reduction in fecal pH in this study population.

Trial registration
The trial is registered at ClinicalTrials.gov (NCT0366657). Registered on 12 September, 2018 (https://clinicaltrials.gov/ct2/show/NCT03666572) Background Severe acute malnutrition (SAM) is the most extreme and visible form of undernutrition, and is commonly de ned by a weight-for-length (WLZ) score of less than − 3 or by the presence of bilateral pedal edema, independent of anthropometric value (1). Recent reports suggest that on a global scale about 4 million infants of less than a months of age are affected with SAM (2)-(3).
Analysis of risk factors associated with malnutrition in those infants indicate that breastfeeding support may be an integral component of future management, however there is a desperate need for other nutritional intervention strategies for effective treatment of SAM (4)- (5). Although intervention strategies involving the use of therapeutic foods resulted in reduction of mortality in children with SAM, restricted rehabilitation of healthy growth remains a serious conundrum (5)- (6). Infancy represents a critical window of rapid development of the gastrointestinal system, a process which is accompanied by characteristic age speci c changes in the intestinal microbiota (7)-(8)- (9). A study conducted at the Dhaka Hospital of icddr, b in children with SAM demonstrated considerable immaturity of gut microbiota (10). Such alterations in the microbiota, a condition referred to as dysbiosis, have been found to be closely associated with intestinal in ammation (11).
Probiotics are refer to live microbial food ingredients that confer health bene ts to the physiology of the host, with the most studied probiotics belonging to the genera Lactobacilli and Bi dobacteria (12). A recent probiotic supplementation study conducted among breast-fed infants showed that infants supplemented with B. infantis EVC001, developed a persistent and stable population in the microbiome of the infant gut (13). Marked changes in the intestinal metabolome of these infants was also observed, with lower fecal pH and higher amounts of fecal lactate and acetate compared to the control group. This was accompanied by a reduction of certain gut pathogens by over 90% (14), indicating a possible therapeutic intervention against early gut dysbiosis. Prebiotics generally refer to oligosaccharide polymers that are not digested by the host system but are consumed by a probiotic bacterium (15). Human milk contains unique prebiotic oligosaccharides, known as human milk oligosaccharides (HMOs) which are not digested and subsequently utilized by most other by both the infant and majority of the bacteria constituting the infant gut microbiome. In fact, only Bi dobacterium longum subspecies infantis has the genetic and biochemical capability to consume all HMOs (16). The metabolism of HMOs by B. infantis results in production of short chain fatty acid molecules such as acetate and lactate, which are important fuel sources for enterocytes and also lower the gut pH to levels that are unfavorable to most opportunistic gut pathogens (16). Since B. infantis is able to consume all of the HMOs in human milk, it quickly dominates the microbiome of the breast-fed infant to levels of over 80% of the total gut microbiome once it is introduced (13). Infant formulas are now commercially available with certain HMOs added at very low levels (17). One trial with healthy formula-fed term infants supplemented with two HMOs (2'Fucosyllactose [2'FL] and Lacto-N-neotetraose [LNnT]), demonstrated an increase in Bi dobacteria in the gut microbiome, but not to the levels seen in healthy breast-fed infants who have been provided B. infantis (18). In a large, double-blind, placebo-controlled trial conducted among SAM children in Malawi (19) involving a cocktail of four different probiotic lactic acid bacilli (but not containing any Bi dobacterium species) and four prebiotic fermentable bers given in adjunct to the Ready-to-use-Therapeutic Food (RUTF), there was no effect on the recovery in weight for height, despite exhibiting reduced mortality in outpatient settings (19). These observations suggested to us that combinations of B. infantis and HMO at levels found in human milk would be the best option for remodeling the gut microbiome of the SAM infant.
Recent reports have shown intestinal in ammation to have a strong association with mortality in children with SAM (20). In addition, intestinal in ammation was found to be with subsequent faltering in linear growth in children (21). Although infant gut dysbiosis has only been correlated with enteric in ammation (22)-(23)- (24) conclusively demonstrated that when B. infantis EVC001 was provided to newborn breast-fed infants, the gut microbiome became dominated (> 85%) with B. infantis and levels of in ammatory cytokine biomarkers were signi cantly reduced relative to control infants who exhibited chronic enteric in ammation over the rst 60 days from birth. The in ammatory biomarker myeloperoxidase (MPO) has been shown to be unaffected by breast milk intake in malnourished children (25) and has demonstrated positive association with features of subclinical intestinal in ammation. However, levels of B. infantis in the gut microbiome of those infants were not determined.
A gradual increase in the infant fecal pH has been observed over the course of the past century, with a marked rise in fecal pH from 5.0 to 6.5 being observed (26). Fecal pH of children and is heavily in uenced by the abundance of Bi dobacterium which produce high concentrations short chain fatty acids and of acetate and lactate. Thus, a raised fecal pH is representative of a distinct change in the gut microbiome of infants even indicating early gut dysbiosis (26). Recent studies have demonstrated a signi cant positive association of fecal pH with severe acute malnutrition (27) and with stunted growth in early childhood (28).
The current study is a single-blind, randomized controlled trial (RCT) where our primary objective was to evaluate the effect of three supplementations, namely: placebo (lactose), probiotic (B. infantis EVC001) alone, and prebiotic plus probiotic (synbiotic of the HMO LNnT and B. infantis EVC001) on the change in gut in ammation and in fecal pH in SAM infants under six months of age. The safety and tolerability of this probiotic strain, B. infantis EVC001, has been evaluated elsewhere and this strain was safely consumed and well tolerated among infants of a similar age group (29). As an indicator of intestinal in ammation, we assessed fecal myeloperoxidase (MPO) since it is relatively unaffected by age or breastfeeding status (25)- (30).

Study Participants, Study Site and Ethics Statement
This short-term, single-blind, randomized controlled trial of prebiotic and probiotic was carried out at the Dhaka Hospital of the International Center for Diarrheal Disease Research, Bangladesh (icddr, b) between September 2018 and June 2019. This study was conducted among infants aged between 2 and 6 months suffering from SAM, who were admitted to the hospital with acute diarrhea and were transferred to the Nutritional Rehabilitation Unit (NRU) following acute phase management and stabilization. Infants suffering from septic shock or very severe pneumonia requiring assisted ventilation, acute kidney injury on hospital admission, jaundice or tuberculosis were not enrolled in the study. Moreover, participants screened with congenital defects interfering with feeding such as cleft palate or chromosomal anomalies were also excluded. Additional exclusion criteria included infants receiving ≥75% of nutrition from breast milk, as measured by the test weighing method (31) and a history of ongoing maternal antibiotic usage (for breastfeeding infants only).
All activities were conducted in consonance to the all the outlined ethical principles of the Declaration of Helsinki and in accordance to the guidelines of Good Clinical Practice. Due approval from the Institutional Review Board (IRB) of icddr, b was obtained and the trial is registered at ClinicalTrials.gov (NCT0366657). Written consents from the parents or legal guardians of the study participants were obtained prior to enrolment in the study. Strict adherence to CONSORT guidelines for reporting clinical trials was maintained all throughout the study (CONSORT checklist in Additional le 1).

Trial Arms
Upon transfer to the NRU, a total of 62 infants with SAM were randomized to receive one of the three interventions for 28 days. These interventions included: placebo (lactose, 625mg), B. infantis EVC001 (8 billion CFU/daily single serving), or B. infantis EVC001 plus HMO prebiotic (Lacto-N-neotetraose [LNnT], 1.6g/ 6 hourly feed at the NRU and 3.2g/day at community). The infants were also strati ed by age (2-3.9 months vs. 4-5.9 months) to ensure equal participation of infants by age groups. The study participants were followed up for a further 28 days after the respective interventions, making a total of 56 days of study duration for each participant.

Study Design and Data Collection
A summary of the study design is illustrated in Figure 1.
Infants in each trial arm were assigned to receive the standard of care diet (F-75) during the acute phase management of SAM, F-100 during hospital stay for nutritional rehabilitation, and standardized infant formula upon discharge. The interventions were available in powder format in sachets contained within feeding kits marked with unique code numbers that match the randomization code of each infant. The contents in the sachets were mixed with a teaspoon of infant formula or breast milk prior to oral administration to the infant.
Following enrolment in the study, anthropometric data from all study participants were collected by trained eld research assistants (FRAs). Participants were discharged from the hospital upon completion of nutritional rehabilitation and ful llment of hospital discharge criteria as described elsewhere (32). After discharge from NRU, participants were visited twice weekly by FRAs both during and after the intervention through the entire study period. During these home visits, participants were supplied with the respective interventions and the standardized infant formula for the time period until the next home visit. Proper storage and intake of the supplements and any antibiotic use during these home visits were also documented. Cases of hospital readmissions during the study period following possible serious adverse events were subsequently reported to the IRB of icddr, b. All the clinical record forms were de-identi ed by coded numbers to maintain the con dentiality of the study participants and to enable tracking throughout the study.

Collection of Fecal Samples and Laboratory Assessments
Stool samples were collected at baseline or prior to the start of supplementation, on day 10, on day 28 and on day 56. Stool samples collected were aliquoted in pre-labeled cryovials and ash frozen in liquid nitrogen within 20 minutes after defecation, as practiced in other studies (33). Aliquots were stored in −80•C freezers until analysis. In ammatory marker MPO was measured using commercially available ELISA kits (Alpco, Salem, NH, USA). Fecal pH was measured by using a portable pH meter (Hanna Instruments, USA) and by following a standard operating protocol described elsewhere (28). All the laboratory assays were carried out at icddr, b in Dhaka, Bangladesh.

Statistical Analysis
Categorical variables were represented as frequency and percentage of the total and quantitative variables were represented by median and interquartile range, due to the non-parametric distribution of the data.
Baseline indices as well as fecal MPO and fecal pH for each of the study groups at each time interval was compared using Kruskal-Wallis test and resultant p-values of less than 0.05 were considered to be statistically signi cant. Kruskal-Wallis test was done to compare ∆MPO and ∆pH between the three supplementation groups at different time points during the study period. P-values were adjusted using the Bonferroni-Holmes method and considered to be signi cant if p<0.05. If ∆MPO or ∆pH for any of the intervals between the three study groups was found to differ in a statistically signi cant manner (p<0.05), post-hoc Dunn's test was done to assess any possible statistical signi cant difference for each of the two interventions (probiotic and synbiotic) with respect to placebo.
Multivariate quantile regression was performed to analyze the association of the different supplements on the ∆MPO and ∆pH, where ∆MPO and ∆pH were selected as the nal outcomes. Covariates for the multivariable model were selected based on previous reports and literature survey (28)-(34)-(35)-(36) and included: age, sex, gestational age, baseline length, maternal age, presence of edema at baseline, birth order, percentage of breast milk received as proportion of total feed volume, family income, ongoing use or completed recommended dosage of antibiotics between the selected time points and mode of delivery of the infant. β-coe cient values and the corresponding 95% con dence intervals (CI) were used to determine the strength of the association. All statistical analyses were performed using SPSS version 20 (IBM Corp., Armonk, N.Y., USA) and STATA version 13.0 (college station, Texas).

Results
A total of 62 infants aged between 2 and 6 months of age with SAM at baseline completed the study, including the 28 days of supplementation and a further 28 days of post-supplementation follow up. Among these infants, 56.5% (n = 35) were aged between 4 and 6 months and the rest were between 2 and 4 months of age. Out of these 62 study infants, 56.5% (n = 35) infants were male, 51.6% (n = 32) infants were delivered by C-section, and 67.8% (n = 42) infants had bilateral pedal edema at baseline prior to the start of supplementation. 21 infants received placebo, 20 received only the probiotic (B. infantis EVC001) and the remaining 21 infants received the synbiotic supplementation (LNnT + B. infantis EVC001). Table 1 illustrates the baseline socio-demographic information and parameters of nutritional status collected from the study infants upon randomization to the three different supplementation arms. No signi cant statistical difference was found in the baseline socio-demographic and nutritional status between the study infants, indicating that the baseline characteristics were well-comparable among the study arms. Breast milk intake as represented by percentage of total feed was inadequate for infants in each of the study arms, indicating a distinct lack of breastfeeding practices among all the infants.  The median levels of fecal MPO and fecal pH measured at the different time points in infants randomized into the three intervention arms have been shown in Fig. 2 and Fig. 3, respectively. Before supplementation, no signi cant statistical differences in the fecal MPO and fecal pH were observed among the infants belonging to three arms. Following the supplementations, fecal MPO levels measured on day 10 and day 28 differed signi cantly (p < 0.05) between the infants enrolled in the three study arms (Fig. 2). The lowest MPO levels across each time point were found among infants receiving synbiotic supplementation. Infants who had received only the probiotic supplementation had higher fecal MPO levels during supplementation compared to the infants receiving the synbiotics, while a consistent rise in fecal MPO levels among the placebo group infants was observed. At day 56 of study, markedly lower levels of fecal MPO were observed among infants receiving either probiotic only (12.4 µg/ml) or synbiotic (11.8 µg/ml) and these results were statistically signi cant (p < 0.05) compared to those who had received placebo.
Fecal pH measured on day 10, day 28 and at day 56 among the infants irrespective of supplementation did not differ signi cantly (Fig. 3). However, infants receiving synbiotic supplementation had consistently lower fecal pH throughout the study post-baseline.
Change in the levels of fecal myeloperoxidase and in fecal pH (∆MPO and ∆pH) between the different sample collection time points All three study groups showed a sustained pattern in increased levels of fecal MPO in comparison to levels at baseline ( Table 2, Fig. 4) both at day 10 (∆MPO 1), at day 28 (∆MPO 2) and at day 56 (∆MPO 4). These increased levels in ∆MPO 1 and in ∆MPO 2 was the highest among the placebo group and lowest among the group that had received the synbiotic supplementation. This difference in increase in fecal MPO level compared to the baseline level among the three groups was statistically signi cant at day 10 (∆MPO 1; p = 0.04), but not at day 28 (∆MPO 2; p = 0.323). When post-hoc test was applied to determine any potential signi cance in difference of ∆MPO 1 for either the probiotic or synbiotic group with respect to placebo group (Table 2), statistically signi cant difference for ∆MPO 1 was found between synbiotic and placebo group (p = 0.034) but not between probiotic and placebo group (p = 0.539).  Fig. 4), the trend of rise in fecal ∆MPO was found in the placebo group. However, for the infants who had received the probiotic only, a marked decrease in ∆MPO 3 was observed and this difference was statistically signi cant among the three study arms (p = 0.038). However, upon a subsequent post-hoc analysis of ∆MPO 3, no statistically signi cant difference was found (Table 2), either between probiotic and placebo group (p = 0.057) or between synbiotic and placebo group (p = 1.00). The level of fecal MPO at day 56 compared to the baseline level, ∆MPO 4, was found to be lower for the synbiotic group in comparison to the groups that had received either the probiotic or the placebo. However this difference in ∆MPO 4 between the three study arms was not statistically signi cant (p = 0.342).
On the other hand, a persistent decrease in fecal pH both at day 10 and at day 28 compared to the level at baseline, i.e. -∆pH 1 and ∆pH 2, was observed for only the synbiotic group and the placebo group and (Table 3, Fig. 5), with the sharpest decrease in ∆pH 1 ∆pH 2 being observed in the group that had received the synbiotic supplementation. Although the difference in pH at day 10 compared to that at baseline (∆pH 1) was not statistically signi cant (p = 0.559), but at day 28 this difference in pH compared to that at baseline (∆pH 2) was statistically signi cant between the three study groups (p = 0.039). However upon a post-hoc analysis for ∆pH 2, no statistical signi cance was found (Table 3) either between probiotic and placebo group (p = 0.160) or between synbiotic and placebo group (p = 1.00).  Otherwise for the probiotic group there was no difference in fecal pH at day 28 and 56 compared to the baseline level, i.e. -∆pH 2 and ∆pH 4, respectively. On the other hand, a decrease in pH at day 56 compared to the baseline level, i.e. -∆pH 4 was observed for both the synbiotic and placebo group, although these changes were not statistically signi cant among the three groups (p = 0.208).

Association of the nutritional interventions on change in ∆MPO and in ∆pH
Both the probiotic (B. infantis EVC001) and the synbiotic (LNnT + B. infantis EVC001) resulted in reduction in the levels of ∆MPO 1 compared to the placebo group (  Data for ∆MPO at each interval has been represented as β-coe cient, 95% CI. However, this trend of reduction in the levels of MPO levels at day 28 compared to the baseline level (∆MPO 2) was not found with the group that had received only the probiotic supplementation (Table 4), where there was an increase in ∆MPO levels by 24.77 µg /ml compared to the placebo group (95% CI: -12.60 µg /ml, 17.55 µg /ml; p = 0.742). Subsequently, for the group that received the synbiotic supplementation, there was a sustained reduction in ∆MPO levels at day 28 compared to the baseline level (∆MPO 2), with a reduction by 15.31 µg /ml compared to the placebo group (Table 4), although this change was not found to be statistically signi cant at 95% con dence level (95% CI: -30.96 µg /ml, 33.0 µg /ml; p = 0.055). The change in MPO levels at day 56 of study compared to the levels at day 28 (∆MPO 3) was found to be lowered in both the intervened groups in comparison to the placebo group, with the ∆MPO being lowered by 47.94 µg /ml in the probiotic group (95% CI: -19.04 µg /ml, 94.55 µg /ml; p = 0.502) and by 24.39 µg /ml in the synbiotic group (95% CI: -16.99 µg /ml, 12.11 µg /ml; p = 0.737). Despite an overall reduction in the levels of ∆MPO 3 for both groups receiving the respective nutritional supplementations, the results were not statistically signi cant in comparison to the levels of ∆MPO 3 observed in the placebo group.
Subsequently, a reduction in the levels of ∆MPO 4 (MPO levels at day 56 compare to the baseline level) was observed for both the probiotic and synbiotic group in comparison to the placebo group, whereby a lowering of ∆MPO 4 by 3.58 µg /ml was observed in the probiotic group and by 12.99 µg /ml in the synbiotic group.
On the contrary, in comparison to the placebo group there was an increase in the levels ∆pH across all analyzed time points throughout the study for the group that had received only the probiotic supplementation (Table 5). However, these changes in the levels of ∆pH for the probiotic group were not statistically signi cant in comparison to the placebo group (Table 5). For the group that had received the synbiotic supplementation, there was a decrease in ∆pH 1 and ∆pH 2. In comparison to the placebo group, ∆pH 1 reduced by 0.24 units (95% CI: -1.02, 0.54, p = 0.535) and ∆pH 2 reduced by 0.10 units (95% CI: -0.86, 0.66, p = 0.794) in the synbiotic group, although these results were not statistically signi cant (Table 5). On the other hand, despite not being statistically signi cant, the levels of ∆pH 3 and ∆pH 4 for the synbiotic group was found to be increased by 0.26 units (95% CI: -0.33, 0.86, p = 0.374) and 0.30 (95% CI: -0.54, 1.13, p = 0.476) units, respectively in comparison to the placebo group (Table 5).

Discussion
This study addresses the effects of probiotic supplementation alone or in combination with a prebiotic in the change in status of gut in ammation and in fecal pH among young Bangladeshi infants with SAM. To our knowledge, this is the rst study exploiting an RCT to explore the role of such supplementations in the possible amelioration of gut in ammation and in the change in fecal pH among severely malnourished infants, through simple and non-invasive measurement of biochemical parameters.
Previous studies conducted among Bangladeshi children suffering from severe acute malnutrition have reported continual impairment in gut microbiota maturity, implying a de nite depletion of the bene cial microorganisms that usually colonize the human gut (10). This phenomenon, also referred to as "dysbiosis" has also been reported to lead to the development of chronic intestinal in ammation (37) and also resulting in a marked elevation of fecal pH (26).
In our study, levels of fecal MPO at baseline for the participants randomized into each of the study arms (Table 2) was markedly higher compared to the level of MPO (< 2 µg /ml) considered to be normal in tropical settings (38), thus indicating raised levels of intestinal in ammation in our study group. Data from the Bangladesh site of the MAL-ED birth cohort study (36) also indicate that among Bangladeshi infants of a similar age group of 2-6 months, similar trends in elevation of fecal MPO levels were found (38), despite these infants not suffering from SAM. In addition, we also report a general increasing trend in the levels of fecal MPO during the course of each of the respective supplementations.
A recently conducted study among breast-fed infants receiving the same probiotic (B. infantis EVC001) for 21 days reported a lowering of intestinal in ammation as measured by quantitation of fecal calprotectin and proin ammatory cytokines (39). This was found to positively correlate with increased colonization of B. infantis EVC001 and was negatively correlated with Enterobacteriaceae abundance. However, in this present analysis, we do not have the extent of colonization seen with healthy breast-fed infants (13). In our multivariate quantile regression analysis after adjusting for the above-mentioned covariates, only ∆MPO1 and ∆MPO 3 showed a lower β-coe cient value compared to that of the placebo group, indicating potential inconsistency in the sustainability of our probiotic supplementation in our study population. Subsequent regression analysis showed that ∆MPO at each time point was lowered for the prebiotic and probiotic group in comparison to the control group, thus indicating a sustained effect of both supplementations in reducing the intestinal in ammation, when given together in tandem. A continually lowered β-coe cient value for ∆MPO for this group nevertheless may indicate a newer strategy for a larger clinical trial to explore the role of this probiotic and prebiotic supplementation in amelioration of gut in ammation in such patients.
On the other hand, baseline fecal pH values for all the study participants were comparable to the levels reported in previous studies on fecal pH of children with SAM. This nding also corroborates with previous reports of immaturity in gut microbiota implying a certain depletion of short-chain fatty acid producing bacteria, in particular, Bi dobacterium (26)- (40) and inadequacy in production of short-chain fatty acids (SCFA) in stool and an elevated fecal pH. However, no sustained trend in change in fecal pH over the sample collection time points (∆pH) in either of the supplementation arms (synbiotic or probiotic) was observed throughout the study period in comparison to the placebo group. However, our regression analysis illustrated a decrease in the β-coe cient ∆pH 1 and in ∆pH 2 values for the synbiotic group in comparison to the placebo group, implying potential colonization of the probiotic, B. infantis EVC001 in the presence of LNnT during the course of the supplementation. An increase in the β-coe cient value for ∆pH 3 and ∆pH 4 in the synbiotic group may otherwise be a possible indication of the lack of sustainability of B. infantis EVC001 colonization of the gut. This in turn, implies for the potential implementation of a more extensive prebiotic and probiotic dosing for a sustained lowering of the fecal pH in such infants. However it must also be noted that unlike other studies involving the measurement of fecal pH (28), where measurement of fecal pH was done using fresh stool samples, in this study the measurement of fecal pH was done using thawed samples that were previously ash frozen and stored in -80 o C temperature.
B. infantis is involved in the selective degradation of HMO components, in particular, the Core-1 and Core-2 based HMOs (i.e. 80% of all HMOs) and glycoproteins with N-glycan linkages, but not glycoproteins with Oglycan linkages such as mucins (41)- (42). Insu cient breast milk intake by the study participants, particularly in the probiotic group, may have thus resulted in the inability of B. infantis EVC001 to effectively colonize the gut to high levels, possibly leading to reduced acetate and lactate production in stool and minimizing the ∆pH in the probiotic group, in comparison to the placebo group. The reduction in the values of ∆pH 1 and in ∆pH 2 in the placebo group may be indicative of possible natural colonization of the gut by lactic acid producing bacteria.

Limitations Of The Study
The small sample size underpowered the overall study ndings, despite some regularly observed positive effects of the interventions being observed at different time points throughout this pilot study. In addition, collection of morbidity data was not conducted in a systematic manner, leading to our inability of adjusting morbidity data of patients during the course of the study in our nal analysis. No supporting data delineating possible infections by characterized enteric pathogens was available in our study. Moreover, data regarding the impact of complementary feeding along with the lack of data on levels of acetate and lactate in the stool and on potential B. infantis EVC001 colonization during the course of supplementation, fails to endow us with the capability of assembling assumptions regarding the potency of such interventions.

Conclusion
This pilot study provides preliminary insights into the possible roles of the prebiotic, LNnT, and the probiotic, B. infantis EVC001, in the improvement of gut in ammation and fecal pH in young infants suffering from severe acute malnutrition. B.infantis, when given alone, was seen to result in an inconsistent trend in reduction of enteric in ammation compared to the placebo group, with substantial reduction in enteric in ammation only being observed during the post-supplementation follow up. However, no apparent lowering of fecal pH was observed in this group throughout the study period, possibly indicating the inability of B.infantis EVC001 to colonize the gut of these study participants which is consistent with the very low intake of human milk by these SAM infants.
However, when B. infantis EVC001 is given in conjunction with LNnT, it shows sustained effects in the reduction of intestinal in ammation throughout the study period. Reduction in fecal pH was only seen during the course of the intervention with B. infantis EVC001 plus LNnT and was not sustained after the supplementation. This in turn may imply a potential need for an increased dose of the supplementation or a longer duration of supplementation for a continued effect on the improvement of overall gut health status.
Abbreviations RCT: randomized controlled trial; SAM:severe acute malnutrition; WLZ:Weight for Length Z score, LNnT:Lacto-N-neotetraose; ELISA:Enzyme Linked Immunosorbent Assay; MPO:myeloperoxidase; CFU:colony forming units. Figure 1 Work ow of the study Figure 2 The p-value shown for each time point is a result of Kruskal-Wallis test using the median MPO values for each of the study arms at the respective time points.

Figure 3
The p-value shown for each time point is a result of Kruskal-Wallis test using the median fecal pH values for each of the study arms at the respective time points.