A randomized, double-blind, placebo-controlled parallel group trial was conducted in three Government centers as well as some private sectors, including Aditya Multi-Specialty Hospital, Guntur; Government Medical College and General Hospital, Srikakulam; and Aarupadai Veedu Medical College & Hospital (AVMCH), Pondicherry, from July 2022 to December 2022. The study was carried out in accordance with Good Clinical Practices as required by the International Conference on Harmonization. A written, informed consent was provided by the parents or legal guardians of all children participating in the study at the time of the child's enrolment. The study protocol (CW/97/BCG_ADRH/II/MAR/21) was approved by the institutional ethics committee (IEC) of each hospital and the trial was registered prospectively with the Clinical Trial Registry of India (CTRI) with the registration number CTRI/2022/06/043239 registered on 14/06/2022.

The sample size was determined based on the baseline stool frequencies reported in an earlier publication (34). Considering the mean stool frequency value of 8.62 ± 3.17 with 80% power, alpha = 0.05, significance level and correlation of 0.4, the total sample size was determined to be 96 participants. Allowing a drop-out rate of 15%, the total sample size for recruitment was finalized as 110 children, in a 1:1 ratio between the two treatment groups (55 per treatment group).

The randomization sequence table was prepared by an independent statistician using randomization software (SAS-9.4). The randomization was performed as per a computer-generated randomization schedule using block randomization method with a block size of 6.

To improve the blinding and concealment of allocation, the placebo and probiotic were coded centrally with randomization numbers as per the computer-generated randomization schedule. The participants, clinical staff, and data analysts were unaware of trial group assignments. The children enrolled in this study were randomly assigned to receive either the probiotic or the placebo. Sealed envelopes with the randomization codes were provided to the principal investigator to be opened in case of emergency.

Children between the ages of 1 and 10 years with clinical symptoms of acute diarrhea, as per the WHO 2005 definition, were enrolled into the study. Acute moderate diarrhea was defined as the passage of unusually loose or watery stools at least three times in the previous 24-h period, with mild or moderate signs of dehydration (the children had moderate dehydration with symptoms of restlessness and irritability, sunken eyes, and feeling thirsty, as per the WHO classification) (35, 36). The children/parents were informed about the purpose of the study by the principal investigator and the study protocol was also well explained to the parents or legal guardians who were willing to comply with all its procedures.

Exclusion criteria included the requirement for antibiotics or intravenous fluid treatment, and clinical signs of severe dehydration or severe diarrhea that requires treatment other than ORS. The children who were immunodeficient, malnourished, suffering from severe chronic illness or consuming any commercially available probiotics, or had undergone recent surgeries were also excluded from the study. In addition, children hospitalized for pneumonia, acute gastroenteritis, or having bilious vomiting, hematochezia, pancreatic dysfunction, or insufficiency as judged by medical history were also excluded from the study.

The probiotic and placebo were provided by Sami-Sabinsa group of companies. Each sachet of the probiotic contained 4 × 10⁸ spores of W. coagulans MTCC 5856, while the placebo contained maltodextrin. Investigational products were manufactured, handled, and stored in accordance with applicable good manufacturing practice (GMP) and with the approved protocol.

The study participants were instructed to consume a one gram sachet containing W. coagulans MTCC 5856 (4 × 10⁸ spores) or a matching placebo dissolved in approximately 10 ml of freshly boiled and cooled drinking water twice daily along with ORS and Zn supplementation therapy. The probiotic sachets contained 0.03 g of W. coagulans MTCC 5856 equivalent to 4 × 10⁸ spores and 0.97 g of maltodextrin, while placebo contained 1 g of maltodextrin. The appearance of the probiotic and placebo sachets and their contents were indistinguishable. The subjects were monitored for a duration of 5 days, which involved three visits (days 1, 3, and 5) followed by telephonic contact with the parent/guardian, 3 days after the final visit to enquire about the overall well-being of the study participant. All the participants were supplied with a diary for the parent or legal guardian to complete. They were required to fill in the diary relating to study treatment, perceived efficacy scales, and stool record. Empty vials and sachets were recorded for compliance with the study.

The primary efficacy endpoints were the mean duration of diarrhea (expressed in hours), as counted from the time of randomization up to recovery (the first normal stool as recorded as <5 according to Bristol score as normalization of stool) and mean diarrhea frequency per day after the initiation of treatment. Frequency of stools was defined as the sum of the frequency of stools and the frequency of diapers with stools. The secondary end points were the perceived efficacy of the treatment, recorded every day by caregivers as frequency of diarrhea and dehydration status (Table 1).

The Bristol stool chart was assessed for each stool passing based on the entries made by parents in the subject diaries given to them to record diarrhea frequency and duration of diarrhea as well as investigational product compliance daily. The criteria for the evaluation of stool were explained to the parents and the children. The Bristol stool chart scale is a 7-point scale that ranges from type 1 for the hardest to type 7 for the softest stool. The stool consistency ≥5 on the Bristol stool scale is considered to be loose or liquid stool, which is indicative of diarrhea along with other symptoms (37). The caregivers were also asked to assess the signs of dehydration including pinch analysis on abdomen skin and were instructed to record their score on the perceived efficacy scale questionnaire. As an exploratory end point, Rota virus and Adenovirus were screened at baseline to evaluate the efficacy of the study treatment in children with confirmed viral diarrhea.

The study participants were monitored for safety data throughout the course of the study. All the children underwent general physical examination including recording of vitals such as temperature, pulse, and respiratory rate at baseline and on day 5. The occurrences of any adverse events and/or serious adverse events were monitored throughout the study period.

The normality of the quantitative variables was analyzed using the Shapiro–Wilks test, and the data are represented as means, standard deviation (SD), or median, and range based on their distribution. The differences within the group at different time points were compared by repeated measures analysis of variance (ANOVA) and Dunnett's multiple comparison. For categorical variables, the frequency and percentage of the population were presented. A descriptive comparison was provided to differentiate the treatment effect between the treatment groups. For continuous variables, the Mann–Whitney U test was performed for the comparative analysis between treatment groups for non-normally distributed data and the results were presented as mean and p-value. Comparative analysis was performed using the Wilcoxon signed rank test for the non-normally distributed data within the group and the results were presented as mean and p-value. All the statistical analysis was performed by STATA Software version 16.0 by an independent statistician blinded to the study groups. The level of significance was defined as a p-value less than 0.05.

A total of 210 children diagnosed with acute diarrhea were prescreened and 110 children, who met the inclusion criteria, were randomized to receive W. coagulans MTCC 5856 (n = 54) or a placebo (n = 56) (Figure 1). The baseline demographic characteristics were well balanced between the two groups. The mean age of the children was 5.55 ± 2.57 years (5.78 ± 2.76 in the probiotic and 5.34 ± 2.38 in the placebo groups) and the mean weight was 21.44 ± 8.34 kg (22.19 ± 8.41and 20.71 ± 8.28) with an average BMI of 17.83 ± 8.37 (18.89 ± 11.36 and 16.80 ± 3.52) in W. coagulans MTCC 5856 and placebo, respectively. Among the study participants, 55.45% were boys and 44.54% were girls. A total of 34 (30.90%) children were positive for adenoviruses in stool (18 in the probiotic group and 16 in the placebo group). Rotavirus was found in the stools of 23 (20.90%) children; 10 in the active group and 13 in the placebo group, while 9 children were positive for both viruses (5 in the probiotic group and 4 in the placebo group). The details of the demographic characteristics are presented in Table 2.

The primary outcome parameters were the analysis of mean duration and frequency of diarrhea from randomization to recovery. The children in the probiotic group showed a mean recovery (normal stool with Bristol stool chart score <5) after 51.43 ± 20.76 h, whereas the children in the placebo group showed recovery after 62.74 ± 24.51 h of treatment, which was significantly better (p = 0.012) in the probiotic supplemented children (Figure 2A). The median recovery and confidence interval (CI) were 50.43 (45.76, 57.09) h for the W. coagulans MTCC 5856 group and 67.65 (56.17, 69.30) h for the placebo group. The net reduction in diarrhea duration was 17.22 h (95% CI: 1.3–21.40, p = 0.012), calculated as 67.65–50.43 h. A Kaplan–Meier curve, generated to compare the total duration of diarrhea between groups, showed a statistically significant difference between treatment groups with a log rank p-value of 0.0024 and hazard ratio of 0.55 (95% CI: 0.37, 0.82) (Figure 2B). In the probiotic supplemented group, 53/54 (98.14%) children recovered from diarrhea in 5 days, while 50/56 (89.28%) children recovered in the placebo group. The cumulative recovery was better in the W. coagulans MTCC 5856 group compared with the placebo group.

The mean frequency of diarrhea per day did not differ significantly between groups. A gradual decrease in the frequency was observed in both treatment groups. (Table 3). Repeated measure ANOVA within the group showed a significant improvement in both W. coagulans and the placebo groups from day 3 onward.

The difference between the dehydration status was evaluated as per WHO classification. The dehydration status improved (reduction in score) in all children with time and was found to be statistically significant in children supplemented with W. coagulans MTCC 5856 (2.37 ± 1.56 on day 1–0.00 on day 5) compared with the placebo (2.16 ± 1.58 to 0.09 ± 0.29, p = 0.026 on day 5) (Table 4). The perceived efficacy score as recorded by the caregivers also improved in both groups and was significantly better in the probiotic group on day 5 (3.69 ± 0.75 to 0.00 in the probiotic group and 3.73 ± 0.59 to 0.23 ± 0.54 in the placebo group, p < 0.001).

The children who were positive for rotavirus or adenovirus at baseline took a longer duration for recovery in both groups, although the difference did not reach statistical significance. The median duration of diarrhea was shorter in the W. coagulans MTCC 5856 supplemented group (59.95 ± 18.88 h in rotavirus and 58.82 ± 21.42 h in adenovirus positive children) compared with the placebo (70.11 ± 17.57 h and 62.86 ± 26.89 h in rotavirus and adenovirus positive children, respectively), but the difference was not significant. The median recovery and confidence interval were 48.50 (43.15, 55.84) h for the W. coagulans MTCC 5856 group and 62.00 (52.50, 68.51) h for the placebo group. The net reduction in diarrhea duration was 13.50 h (95% CI: 0.2–22.30, p = 0.03), calculated as 62.0–48.50 h in rotavirus negative children. The median recovery and confidence interval in adenovirus negative children was 48.50 (41.07, 54.39) h for the W. coagulans MTCC 5856 group and 66.50 (54.26, 71.46) h for the placebo group. The net reduction in diarrhea duration was 18.00 h (95% CI: 1.9–26.60, p = 0.007), calculated as 66.50–48.50 h. Probiotic supplementation reduces the recovery time in children positive for the virus, but it was not significant most likely due to the small numbers in the present study (Table 5).

Vital signs were also measured as a part of the safety analysis, and no abnormal or out-of-range values were observed. Further, no changes associated with the treatment in safety measurements including adverse events and/or serious adverse events were reported in both groups (Table 6). The vital signs like diastolic and systolic blood pressures (67.28 ± 7.95 /102.15 ± 10.90 and 70.21 ± 9.05/ 102.77 ± 10.82), pulse rate (94.80 ± 9.68 and 95.29 ± 9.69), and body temperature (97.99 ± 0.73 and 98.17 ± 0.58) in the probiotic and placebo groups, respectively, were normal in both groups at the end of the study (Table 7).