Subjects and ethical approval

This is a multi-center, parallel randomized, controlled, double-blinded clinical intervention. Children of both sexes and aged 0–3 years who were outpatients and/or hospitalized with diarrhea were recruited between Dec. 2021 and Sep. 2022.

Inclusion, exclusion, and withdrawal criteria

Diagnostic criteria for watery diarrhea: Increased fecal frequency (≥4 times/day) [14] with watery feces (Bristol fecal score above type 6).

Inclusion criteria:

1.

Age: children 0–3 years old.

2.

Duration of diarrhea (the time of the previous bowel movement, before the stool consistency had returned to normal, was used as the endpoint of the course of diarrhea): more than 12 h and less than 72 h;

3.

No need for antibiotic treatment based on clinical diagnosis during recruitment;

Exclusion criteria:

1.

Nervous system dysplasia and severe organic diseases;

2.

Moderate and severe dehydration, serious diarrhea requiring Pediatric Intensive Care Unit (PICU) treatment, bloody stools;

3.

The same probiotics taken within one month before the diagnosis of this illness;

4.

Children are expected to receive antibiotic treatment during the trial.

Withdrawal criteria:

1.

Children without any clinical records for evaluation;

2.

Children taking drugs prohibited by the study, including hormones, immunosuppressive drugs, other probiotics, etc., during the treatment.

Allocation sequence generation and concealment

To ensure the randomization of participants in the study, a research staff, independent of the study, generated an allocation sequence. This sequence outlined how participants were assigned to different interventions and was created using the RAND function in Excel. Subsequently, another research staff, unaffiliated with the study, executed the central remote telephone randomization process. This step was implemented to prevent children’s guardians and trial personnel from knowing the forthcoming allocations until after the recruitment was confirmed. Children meeting the inclusion criteria were coded with random numbers and assigned into one of two groups, each consisting of 60 randomly assigned children.

Grouping and intervention

Recruited children were managed per the WHO guidelines [15]. Children in the intervention group (IG) received oral probiotics in addition to standard diarrhea management. The probiotic was given as a single sachet (Wecare Probiotics Co., Ltd., Production No.: SC10632050900407) containing BLa80 strain 5 × 109 CFU/sachet and was taken daily for seven consecutive days starting on the first day of clinical treatment. Children in the control group (CG) underwent standard therapy and were given a reference sachet (placebo) containing only maltodextrin. The probiotic and placebo had similar appearance, taste, and smell and were provided in identical sachets with identical labelling, expect for the subject-specific randomization number. The children’s parents and/or guardians, clinicians, laboratory personnel, data managers, and statisticians remained blinded to group assignments until the end of data analysis.

Data collection

After enrollment, the study staff performed assessments, recorded data on the clinical record form (CRF), and collected laboratory samples according to the protocol. During the trial period, the data of hospitalized children were recorded by the field workers. For discharged or outpatient children, parents took daily pictures of the child’s feces and sent them to the researcher for objective records. Fecal pictures were collected daily to confirm fecal type and evaluate treatment efficacy. Clinicians used the CRF to record the incidence of abdominal cramps, nausea, vomiting, fever, constipation, and low appetite in children during the treatment. The average daily Bristol fecal score was defined as the sum of the daily Bristol fecal score divided by the fecal frequency on a given day.

Fecal immune and inflammation biomarkers assessment

Fecal samples were collected from all children before and after the intervention to measure levels of sIgA, calprotectin, human beta-defensin 2 (HBD-2), and cathelicidin (LL-37) using commercial enzyme-linked immunosorbent assay kits (Shanghai Enzyme-linked Biotechnology Co., Ltd./mlbio).

Fecal microbiome analysis

A total of 158 fecal samples were collected for gut microbiome analyses, including 82 samples from 41 children in the IG before and after the intervention and 76 samples from 38 children in the CG. Genomic DNA from the samples was extracted using the CTAB/SDS method with the QIAamp Fast DNA fecal Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer’s instructions.

The isolated genomic DNA targeting the bacterial 16S rRNA gene V3–V4 region was amplified using the TransGen AP221-02 Kit (TransGen, Beijing, China). The library was sequenced on an Illumina NovaSeq platform, generating 250 bp paired-end reads. QIIME (Version 1.9.1) was used for calculating both alpha- (within sample) and beta- (between sample) diversity. Shannon, Simpson, Chao1, and ACE indices were used as indicators of the alpha diversity. Principal coordinate analysis (PCoA) based on Bray-Curtis distance was used to analyze β-diversity. Differential enrichment of the gut microbiome was analyzed using linear discriminant analysis effect size (LEfSe). To explore the functional profiles of the gut microbiome, Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was performed based on 16S information [16].

Efficacy judgement

According to the national pediatric diarrhea efficacy evaluation standards and similar studies [5, 14], the efficacy was assessed as follows:

Marked effectiveness: after 72 h of treatment, the frequency of diarrhea decreased to ≤2 times/day, and the fecal consistency and clinical manifestations returned to normal;

Normal Effectiveness: after 72 h of treatment, the frequency of diarrhea is reduced to ≥3, and <4 times/day, the fecal consistency is significantly reduced, and the clinical symptoms are basically disappeared;

Ineffectiveness: diarrhea without remission after treatment for 72 h;

Total efficiency = (number of marked effectiveness cases + number of normal effectiveness cases)/total number of cases × 100%.

Statistical analysis

All efficacy analyses were performed on both the intention-to-treat (ITT) dataset and the per-protocol dataset, comprising participants who adhered to the protocol, completed the clinical intervention, and provided all necessary demographic and clinical data, especially information on changes in stool consistency and frequency. SAS version 9.2 for Windows (SAS Institute Inc., Cary, NC, USA) was used for all analyses.

T test was used to compare normally distributed data. Wilcoxon rank-sum test was used for data without a normal distribution. χ2 test was used to compare differences in treatment efficacy between the two groups for countable data. The frequency of feces and average daily Bristol fecal score between the two groups before and after the intervention were compared using repeated measures analysis of variance (ANOVA). A p value less than 0.05 was considered statistically significant.

Sample size

In a previous study on the treatment of rotavirus enteritis using three combined strains [5], the duration of diarrhea in the CG and the IG was reported as 143.9 ± 19.8 h and 121.4 ± 13.7 h, respectively (a reduction of nearly 24 h). With β = 0.8, α = 0.05 (bilateral), the sample size for each group was calculated to be 50 subjects. Accounting for a 20% dropout rate, we selected a sample size of 120 subjects with 60 subjects in each group.