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 significant 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.
Table 1
Socio-demographic and nutritional parameters collected from the study infants randomized to the three different treatment arms.
Baseline Indices | Placebo (n = 21) | Probiotic (n = 20) | Synbiotic (n = 21) | p-value |
Male sex (%) | 11 (52) | 11 (55) | 13 (62) | 0.816 |
C-section (%) | 11 (52) | 13 (65) | 8 (38) | 0.231 |
Gestational age (weeks) | 37 (36, 40) | 37 (36, 38) | 38 (35, 39) | 0.653 |
Birth weight (kg) | 2.6 (1.9, 3) | 2.7 (2.3, 3.2) | 3.0 (2.5, 3.5) | 0.347 |
Breast milk percentage (as percentage of total feed) | 21.0 (12.6, 28.3) | 19.7 (7.9, 28.2) | 16.5 (12.3, 27.5) | 0.833 |
Maternal age (years) | 23 (18, 28.5) | 25 (20, 29.3) | 23 (19, 25.5) | 0.600 |
Monthly Family Income (USD) | 179 (119, 238) | 179 (143, 211) | 191 (143, 327) | 0.384 |
Baseline WLZ | -4.1 (-4.3, -3.4) | -3.8 (-3.9, -3.6) | -3.6 (-4.3, -3.4) | 0.597 |
Presence of bilateral pedal edema at baseline | 14 (67%) | 12 (60%) | 16 (76%) | 0.542 |
Footnotes: |
Data represented as median and interquartile range. The p-value shown for each time point is a result of Kruskal-Wallis test comparing each of the baseline indices for each of the study arms. |
Levels of fecal myeloperoxidase and fecal pH at each time interval for each of the three study arms
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 significant 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 significantly (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 significant (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 significantly (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 significant 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 significance in difference of ∆MPO 1 for either the probiotic or synbiotic group with respect to placebo group (Table 2), statistically significant difference for ∆MPO 1 was found between synbiotic and placebo group (p = 0.034) but not between probiotic and placebo group (p = 0.539).
Table 2
Change in fecal MPO levels within the different time points throughout the study.
Interval considered for evaluation | Change in fecal MPO levels (µg/ml) |
Placebo (n = 21) | Probiotic (n = 20) | Synbiotic (n = 21) | p-value for comparison between three groups | p-value for placebo vs probiotic | p-value for placebo vs synbiotic |
∆MPO 1 | 20.11 (-1.19, 35.02) | 7.95 (-4.36, 20.53) | 3.04 (-12.66, 10.12) | 0.040 | 0.539 | 0.034 |
∆MPO 2 | 8.28 (-18.12, 27.70) | 7.30 (-2.89, 32.15) | 0.46 (-7.73, 10.24) | 0.323 | --- | --- |
∆MPO 3 | 1.24 (-8.39, 13.00) | -8.58 (-27.44, -2.03) | 0.33 (-6.48, 5.15) | 0.038 | 0.057 | 1.00 |
∆MPO 4 | 17.06 (-13.18, 23.70) | 17.35 (-7.58, 14.30) | 2.53 (-10.01, 8.63) | 0.342 | ---- | ---- |
Footnotes: |
Data represented as median and interquartile range. |
∆ MPO 1 = MPO at day 10– MPO at baseline |
∆ MPO 2 = MPO at day 28 – MPO at baseline |
∆ MPO 3 = MPO at day 56 – MPO at day 28 |
∆ MPO 4 = MPO at day 56 – MPO at day baseline |
The level of fecal MPO at day 56 of study compared to the level at day 28, i.e.-∆MPO 3 (Table 2, 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 significant among the three study arms (p = 0.038). However, upon a subsequent post-hoc analysis of ∆MPO 3, no statistically significant 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 significant (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 significant (p = 0.559), but at day 28 this difference in pH compared to that at baseline (∆pH 2) was statistically significant between the three study groups (p = 0.039). However upon a post-hoc analysis for ∆pH 2, no statistical significance was found (Table 3) either between probiotic and placebo group (p = 0.160) or between synbiotic and placebo group (p = 1.00).
Table 3
Change in pH within the different time points throughout the study.
Interval considered for evaluation | Change in fecal pH |
Placebo (n = 21) | Probiotic (n = 20) | Synbiotic (n = 21) | p-value for comparison between three groups | p-value for placebo vs probiotic | p-value for placebo vs synbiotic |
∆pH 1 | -0.50 (-0.95, 0.25) | -0.20 (-0.70, 0.10) | -0.60 (-1.10, 0.05) | 0.559 | --- | --- |
∆pH 2 | -0.70 (-1.30, 0.15) | 0.00 (-0.98, 0.38) | -1.10 (-1.20, 0.10) | 0.039 | 0.16 | 1.00 |
∆pH 3 | 0.20 (-0.30, 0.60) | 0.00 (-0.58, 0.38) | 0.10 (-0.25, 0.95) | 0.649 | --- | --- |
∆pH 4 | -0.50 (-0.90, -0.25) | 0.00 (-0.65, 0.38) | -0.70 (-1.40, 0.25) | 0.208 | --- | --- |
Footnotes: |
Data represented as median and interquartile range. |
∆ pH 1 = pH at day 10 – pH at baseline |
∆ pH 2 = pH at day 28 of – pH at baseline |
∆ pH 3 = pH at day 56– pH at day 28 |
∆ pH 4 = pH at day 56– pH at baseline |
A small rise in the pH at day 56 compared to the level at day 28 (∆pH 3) was observed in the placebo and in the synbiotic groups (Table 3, Fig. 5), although these change were not statistically significant (p = 0.649). For the probiotic group, a decrease in pH from the baseline level was only observed at day 10, i.e. - ∆pH 1. 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 significant 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 (Table 4). For the group that had received only the probiotic, the ∆MPO 1 was found to be lowered by 18.44 µg/ml compared to the placebo group (95% CI: -31.62 µg /ml, -52.47 µg/ml; p = 0.007) while for the group that had received the synbiotic, ∆MPO 1 was reduced by a margin of 17.24 µg/ml compared to the placebo group (95% CI: -30.94 µg /ml, -35.37 µg/ml; p = 0.015).
Table 4
Results of multivariate quantile regression showing association of supplements on ∆fecal MPO throughout the study.
Intervention Group | ∆MPO 1 (µg/ml) | ∆MPO 2 (µg/ml) | ∆MPO 3 (µg/ml) | ∆MPO 4(µg/ml) |
β-coefficient, 95% CI | p-value | β-coefficient, 95% CI | p-value | β-coefficient, 95% CI | p-value | β-coefficient, 95% CI | p-value |
Probiotic (n = 20) | -18.44 (-31.62, -52.47) | 0.007 | 24.77 (-12.60, 17.55) | 0.742 | -47.94 (-19.04, 94.54) | 0.502 | -3.58 (-16.87, 9.81) | 0.590 |
Synbiotic (n = 21) | -17.24 (-30.94, -35.37) | 0.015 | -15.31 (-30.96, 33.00) | 0.055 | -24.39 (-16.99, 12.11) | 0.737 | -12.99 (-27.42, 1.44) | 0.077 |
Footnotes: |
Data for ∆MPO at each interval has been represented as β-coefficient, 95% CI. |
∆ MPO 1 = MPO at day 10– MPO at baseline |
∆ MPO 2 = MPO at day 28 – MPO at baseline |
∆ MPO 3 = MPO at day 56 – MPO at day 28 |
∆ MPO 4 = MPO at day 56 – MPO at day baseline |
Multivariate quantile regression performed adjusting for possible co-variates based on literature survey and previous reports and includes: 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. |
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 significant at 95% confidence 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 significant 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. These decreases in ∆MPO 4 were not statistically significant at 95% confidence level for both the probiotic group (95% CI: -16.87 µg /ml, 9.71 µg /ml; p = 0.590) and the synbiotic group (95% CI: -27.42 µg /ml, 1.44 µg /ml; p = 0.077).
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 significant 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 significant (Table 5). On the other hand, despite not being statistically significant, 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).
Table 5
Results of multivariate quantile regression showing association of supplements on ∆fecal pH throughout the study.
Intervention Group | ∆pH 1 | ∆pH 2 | ∆pH 3 | ∆pH 4 |
β-coefficient, 95% CI | p-value | β-coefficient, 95% CI | p-value | β-coefficient, 95% CI | p-value | β-coefficient, 95% CI | p-value |
Probiotic (n = 20) | 0.15 (-0.60, 0.90) | 0.690 | 0.56 (-0.13, 1.29) | 0.131 | 0.18 (-0.40, 0.76) | 0.529 | 0.47 (-0.30, 1.24) | 0.226 |
Synbiotic (n = 21) | -0.24 (-1.02, 0.54) | 0.535 | -0.10 (-0.86, 0.66) | 0.794 | 0.26 (-0.33, 0.86) | 0.374 | 0.30 (-0.54, 1.13) | 0.476 |
Footnotes: |
Data for ∆pH at each interval has been represented as β-coefficient, 95% CI. |
∆ pH 1 = pH at day 10 – pH at baseline |
∆ pH 2 = pH at day 28 of – pH at baseline |
∆ pH 3 = pH at day 56– pH at day 28 |
∆ pH 4 = pH at day 56– pH at baseline |
Multivariate quantile regression performed adjusting for possible co-variates based on literature survey and previous reports and includes: 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. |