Age-stratified Evaluation Of Saccharomyces boulardii In A Pediatric Randomized Controlled Trial And Adult Observational Study of Infectious Enteritis

Summary

This protocol evaluates the age-specific clinical efficacy and safety of Saccharomyces boulardii as an adjunct therapy for infectious enteritis, using a randomized controlled trial in pediatric patients and a real-world observational study in adults.

Abstract

Infectious enteritis remains a significant global health burden, particularly among children under five years of age. Saccharomyces boulardii (S. boulardii), a probiotic yeast, has been widely studied for its role in reducing diarrhea; however, direct comparisons of its effects across age groups remain limited. This study aims to evaluate and compare the efficacy and safety of S. boulardii in pediatric and adult patients with infectious enteritis. A double-blind, randomized, placebo-controlled trial was conducted in 186 children with acute diarrhea, while a retrospective observational study included 270 adult patients. All participants received standard rehydration therapy; the intervention groups additionally received S. boulardii. The primary outcome was diarrhea duration. Secondary outcomes included stool frequency, length of hospital stay, recurrence rate, and adverse events. In pediatric patients, S. boulardii significantly reduced diarrhea duration (2.1 vs. 3.9 days in rotavirus-positive cases), hospital stay (3.2 vs. 4.8 days), and stool frequency (4.2 vs. 6.7 stools/day) compared to placebo (all p < 0.05). In adult patients, S. boulardii was associated with shorter diarrhea duration (2.7 vs. 5.4 days), reduced hospital stay (3.6 vs. 5.2 days), higher symptom resolution rates (77.8% vs. 15.8%), and lower recurrence (8.1% vs. 21.7%; all p < 0.01). Adverse events were mild and comparable between groups. S. boulardii appears to be a safe and effective adjunct therapy for infectious enteritis, with age-related differences in clinical outcomes. S. boulardii appears to be a safe and effective adjunct therapy for infectious enteritis in both pediatric and adult populations. Within each cohort, improvements were observed in clinical outcomes, including diarrhea duration, hospitalization, and recurrence. Observed differences between pediatric and adult cohorts are descriptive and reflect separate analyses conducted under different study designs; therefore, no direct statistical comparisons between age groups were performed.

Introduction

Infectious enteritis is an inflammatory condition of the small intestine caused by bacterial, viral, or parasitic pathogens and represents a major global health burden affecting both children and adults. Transmission commonly occurs through contaminated food or water and via person-to-person spread, making the disease highly contagious^1,2. Children under 5 years of age are disproportionately affected, with infectious enteritis contributing significantly to morbidity and mortality in low- and middle-income countries^3,4. According to the World Health Organization, diarrheal diseases account for approximately 525,000 deaths annually among children under 5 years, particularly in low- and middle-income countries. Rotavirus and norovirus are the most common viral causes of infectious enteritis worldwide⁵. Severe or prolonged cases may present with complications such as inflammation, bloody stools, and immunosuppression⁶.

Probiotics such as Saccharomyces boulardii exert therapeutic effects through mechanisms including inhibition of pathogen adhesion, modulation of host immune responses, and reduction of intestinal inflammation. These effects contribute to enhanced host defense and faster clinical recovery^5,6. With the passage of time, awareness of probiotics as a therapeutic modality and the number of randomized clinical trials have increased substantially⁷. Although infectious enteritis affects both children and adults, disease presentation and outcomes differ markedly, with viral etiologies predominating in children and bacterial or mixed infections more common in adults^7,8,9. These distinctions indicate that therapeutic responses may vary by age and require comparative evaluation.

Rotavirus remains the leading cause of severe acute gastroenteritis in infants and young children worldwide. Clinical manifestations range from mild, self-limiting diarrhea to severe dehydration requiring hospitalization⁷. Transmission occurs primarily via the fecal–oral route, either directly or through contaminated food and water⁸. According to the World Health Organization, diarrheal diseases account for approximately 525,000 deaths annually among children under 5 years of age, particularly in low- and middle-income countries^3,9,10. Rotaviruses were first described in 1973 in the duodenal mucosa of six infants with acute gastroenteritis and are now considered the most common causative agents of illness¹¹. Infection with rotavirus occurs in both children and adults and usually produces mild symptoms¹². Data from India account for approximately 20% of cases¹³. Rotavirus infection is most common in infants, with a hospitalization rate of approximately 40%¹⁴. According to current statistics, India accounts for 23% of diarrheal deaths¹⁵. In India, rotavirus infection affects approximately 11.37 million children aged <5 years¹⁶. Transmission occurs via the fecal–oral route, both directly and indirectly, through contaminated food and water¹⁷. The clinical presentation ranges from subclinical infection to severe disease¹⁸. The incubation period is approximately 24–72 h, and the illness typically resolves within 3–7 days¹⁹.

Norovirus is the most common cause of acute gastroenteritis outbreaks across all age groups and is particularly prevalent in adult populations²⁰. Characterized by acute-onset vomiting and watery diarrhea, norovirus infection is highly contagious and requires a very low infectious dose. Transmission occurs via fecal–oral routes, contaminated surfaces, food, water, and aerosolized particles during vomiting²¹. After rotavirus, it is considered the second most common cause of illness worldwide. Norovirus infection was first identified in 1968²². This virus is highly contagious and is transmitted via fecal–oral routes through contaminated food and water, similar to rotavirus infection in young children and infants²³. Norovirus incidence is highest in young children in low-income countries²⁴. It is commonly detected in stool samples and is a major cause of sporadic outbreaks²⁵. Table 1 and Table 2 summarize the epidemiological and clinical differences between rotavirus and norovirus infections. Outbreaks can spread rapidly in clinical settings such as clinics and nursing homes due to the high infectivity and low infectious dose²⁶. According to literature reviews and recent data, approximately 185,000 cases of gastroenteritis occur globally, of which 18% are attributable to norovirus²⁷. Data from India remains limited, particularly in the northeast regions. The prevalence of norovirus infection in India ranges from 1.4% to 44%, with a recent study from Kolkata reporting approximately 6% in children under 5 years²⁸.

S.No	Feature	Rotavirus	Norovirus
1.	Mean Age of Infection	34.74 months	29.65 months
2.	Symptom Severity	More severe with higher hydration	Less severe, more vomiting
3.	Fever Prevalence	Higher (44.3%)	Lower (45%)
4.	Duration of Symptoms	Longer	Shorter
5.	Coinfections	Often with bacterial pathogens	Less common, no significant impact

Table 1: Comparison of rotavirus and norovirus in children. A comparative overview of the clinical and epidemiological characteristics of rotavirus and norovirus infections in children is provided. Variables include mean age, symptom severity, fever prevalence, illness duration, and coinfection frequency.

S.No	Feature	Rotavirus	Norovirus
1.	Virology	Double stranded RNA virus	Single Stranded RNA virus
2.	Common in adults	Less common, usually mild or asymptomatic	Very common, leading cause of adult gastroenteritis
3.	Transmission	Fecal-oral route, through food/water, surfaces	Fecal-oral route, highly contagious through air,food and  water
4.	Infectious Dose	Moderate	Extremely low (10-100 viral particles can cause infection)
5.	Incubation period	1-3 days	12-48 hours
6.	Duration of illness	3-8 days	1-3 days
7.	Common Symptoms	Watery diarrhea, vomiting, fever, abdominal pain	Acute vomiting, watery diarrhea,nausea, cramping
8.	Severity in adults	Mild to moderate more severe in immunocompromised	Often self-limiting but may cause outbreaks in adults.
9.	Vaccination	Available (oral, live-attenuated) but not used in adults	No vaccine currently available

Table 2: Comparison of norovirus and rotavirus in adults. Virological features, transmission patterns, clinical manifestations, and disease severity of rotavirus and norovirus infections in adult populations are outlined.

Saccharomyces boulardii has been used clinically since the mid-20th century and is supported by multiple randomized controlled trials and meta-analyses demonstrating its efficacy in acute gastroenteritis²⁹. Reported benefits include reduced duration of diarrhea, decreased stool frequency, shorter hospital stays, and a favorable safety profile in both children and adults³⁰. It is a yeast-based probiotic and is widely available as a dietary supplement³¹. Large prospective placebo-controlled clinical trials have further elucidated its mechanisms of action and supported the development of new therapeutic applications³².

The efficacy of S. boulardii in reducing the duration and severity of diarrhea is well established. However, these studies are typically limited to either pediatric or adult populations, with few directly comparing outcomes across age groups^28–32. Given the differences in etiological agents, immune responses, and clinical outcomes between children and adults, evaluating age-specific therapeutic responses is critical. Compared with bacterial probiotics such as Lactobacillus spp., S. boulardii offers advantages, including resistance to antibiotics and greater stability in the gastrointestinal tract. Thus, although S. boulardii is recognized as an effective adjunct therapy, the age-dependence of its therapeutic benefits remains unclear.

S. boulardii has been used since 1950 in Europe and has been evaluated in multiple clinical trials³³. Several clinical guidelines support the safety and efficacy of probiotics in the treatment of acute gastroenteritis. S. boulardii is among the most commonly used probiotics in clinical practice and is recommended by various clinical experts³⁴. It is widely used for the prevention and treatment of diarrhea, including rotavirus and norovirus infections in children³⁵. Clinical studies have demonstrated that it reduces the duration of diarrhea in acute enteritis and rotavirus infection, with a favorable safety profile and minimal adverse effects²³. A meta-analysis involving 1,282 patients per cohort showed that pediatric patients demonstrated faster symptom resolution, whereas adult patients showed lower recurrence rates. Additionally, studies from West Africa have reported clinical improvement in children with acute diarrhea following S. boulardii treatment, including normalization of stool frequency by the second day of therapy³². The strain has also demonstrated efficacy in rotavirus infection by reducing stool frequency and shortening disease duration in infants compared with controls^{20,21,22,23,24}. Other studies have reported a decreased incidence of rotavirus infection in hospitalized neonates, highlighting its preventive potential³⁰. S. boulardii is considered a safe treatment option in children30. Proposed mechanisms of action include inhibition of bacterial toxins, anti-inflammatory effects, and stimulation of the intestinal mucosa^26,29,35.

The dual-design approach was adopted to balance methodological rigor and real-world applicability. A randomized controlled trial was feasible in pediatric patients, whereas ethical and practical constraints necessitated a retrospective observational design in adults. Cross-cohort comparisons are therefore exploratory and descriptive.

To address the limited availability of studies directly comparing age-specific responses to S. boulardii, a dual-design approach was adopted. A randomized controlled trial was conducted in pediatric patients, whereas a retrospective observational design was used in adults due to ethical and practical constraints. Importantly, the two cohorts were analyzed independently. Any observed differences between pediatric and adult outcomes are presented descriptively and are not intended for direct statistical comparison or inferential interpretation.

Protocol

All procedures involving human participants were conducted in accordance with institutional and national ethical standards. The pediatric randomized controlled trial was approved by the Ethics Committee of Harbin Fourth Hospital, Harbin, China (Approval No.: KY2026-06). Written informed consent was obtained from the parents or legal guardians of all pediatric participants prior to enrolment. The adult retrospective observational cohort study was approved by the Ethics Committee of Harbin Fourth Hospital, Harbin, China (Approval No.: KY2026-06), with informed consent waived due to the study's retrospective nature.

1. Study design and cohort identification

1. Identifying the study population
   1. Retrieve hospital medical records of pediatric and adult patients diagnosed with acute infectious enteritis between January 2020 and January 2022. A total of 186 pediatric patients were identified; 10 were excluded due to incomplete data, resulting in 176 participants for analysis.
   2. Confirm infectious enteritis using clinical assessment and stool testing. Collect fresh stool samples in sterile stool containers within 24 h of admission. Perform rotavirus testing using a rapid stool antigen immunochromatographic assay according to the manufacturer’s instructions.
2. Defining study arms
   1. Analyze pediatric and adult cohorts independently due to differences in study design (randomized controlled trial vs retrospective observational study). Do not perform statistical tests to compare outcomes between age groups. Interpret any cross-cohort comparisons descriptively only.
   2. Assign pediatric patients to either the intervention group (S. boulardii) or the placebo group using a randomized allocation schedule.
   3. Assign adult patients to the intervention group if they received S. boulardii as part of routine clinical care.
   4. Identify a historical adult control group matched 1:1 by age, sex, and diagnosis that did not receive S. boulardii.
      ​NOTE: Pediatric and adult datasets are analyzed separately and are not pooled for statistical comparison.

2. Participant eligibility criteria

1. Inclusion criteria
   1. Pediatric cohort: Obtain written informed consent from parents or legal guardians prior to enrolment. Include children aged 6–48 months admitted with acute infectious diarrhea confirmed by clinical history and stool examination. Confirm diarrhea onset within 72 h prior to hospital admission. Verify that participants can tolerate oral rehydration therapy and are medically stable.
   2. Adult cohort: Include patients ≥18 years presenting with ≥3 unformed stools in the previous 24 h and symptom duration ≤5 days. Confirm infectious etiology, excluding Clostridioides difficile. Obtain a waiver of informed consent due to the retrospective nature of the study.
2. Exclusion criteria
   1. Exclude pediatric patients with severe malnutrition (weight-for-height Z-score < −3), gross blood in stool, exclusive breastfeeding status, or recent antibiotic use within 14 days.
   2. Exclude participants with chronic gastrointestinal disease, immunodeficiency, or severe systemic illness.
   3. Exclude adult patients with prior intestinal surgery, immunosuppressive therapy, recent probiotic use within two weeks, or inability to provide consent.
      NOTE: Data collection may be paused after cohort identification and resumed prior to statistical analysis.

3. Sample size determination

1. Calculating pediatric sample size
   1. Set alpha at 0.05 and power at 80%. Calculate the sample size to detect a mean difference of 1.5 days in diarrhea duration with a standard deviation of 2.0 days.
   2. Increase sample size by 10% to account for potential dropout, resulting in 90 participants per group.
2. Determining adult sample size
   1. Estimate a minimum of 250 participants per group to detect a 15% difference in symptom resolution.
   2. Include 270 adults in both intervention and historical control groups to ensure adequate statistical power.

4. Intervention and control procedures

1. Administer Saccharomyces boulardii at a dose of 250 mg twice daily (bid) for 5 days using oral sachet formulations. Dissolve the sachet contents in water or milk at room temperature prior to administration. Administer the preparation orally after feeding to improve tolerance. Provide standard oral rehydration therapy (ORT) concurrently according to WHO guidelines^23,24,25.
   1. Use commercially available formulations containing Saccharomyces boulardii or equivalent strains approved for clinical use.
   2. Dissolve sachet contents in water or milk at room temperature immediately prior to oral administration.
2. Pediatric control group
   1. Administer placebo sachets containing inert maltodextrin (250 mg per sachet) orally twice daily (bid) for 5 days, matching the dosing schedule of the intervention group. Dissolve the placebo in water or milk prior to administration and administer after feeding. Ensure that placebo sachets are identical in appearance, taste, weight, and packaging to the Saccharomyces boulardii sachets to maintain blinding.
   2. Provide standard oral rehydration therapy.
   3. Provide oral rehydration using WHO low-osmolarity oral rehydration salts. For patients requiring intravenous hydration, administer 0.9% sodium chloride solution or Ringer’s lactate solution according to dehydration severity and physician assessment. Adult intervention group
3. Adult intervention group
   1. Identify adult patients who received S. boulardii as part of routine clinical care. Document concurrent treatments, including oral or intravenous rehydration and antibiotic therapy.
4. Adult control group
   1. Identify matched adult patients who received standard supportive care without S. boulardii. Perform matching based on age, sex, and diagnosis. Acknowledge that potential confounding factors, including antibiotic use, comorbidities, and disease severity, may not have been fully controlled.
   2. Record treatment details from electronic medical records.
5. Assessment and handling of confounding variables in the adult cohort
   1. Extract data on potential confounding variables from electronic medical records, including antibiotic use (type and duration), baseline disease severity (frequency of diarrhea, presence of dehydration, and need for intravenous fluids), comorbidities (e.g., diabetes and chronic gastrointestinal disease), and duration of symptoms prior to admission.
   2. Compare baseline characteristics between intervention and control groups to assess balance after matching.
   3. Perform stratified analyses based on key confounders, including antibiotic co-therapy (yes/no) and disease severity (mild vs moderate).
   4. Conduct multivariable logistic regression analysis to evaluate the association between Saccharomyces boulardii use and clinical outcomes (e.g., symptom resolution and recurrence) while adjusting for identified confounders.
   5. Report adjusted odds ratios (aOR) with 95% confidence intervals (CI).
   6. Acknowledge that residual confounding may persist due to the retrospective nature of the study.

5. Blinding and randomization

1. Pediatric randomization
   1. Generate the pediatric randomization sequence using statistical software with block randomization. Maintain allocation concealment using sequentially numbered, opaque, sealed envelopes prepared by an independent pharmacist.
2. Blinding
   1. Blind investigators, caregivers, and outcome assessors to group allocation in the pediatric trial. Blind data analysts to treatment allocation during statistical analysis for both cohorts.
      NOTE: Do not perform blinding in the adult retrospective cohort.
      CAUTION: Handle biological samples using appropriate biosafety protocols. Wear personal protective equipment (gloves, lab coat, mask). Dispose of contaminated materials according to institutional biomedical waste guidelines.

6. Outcome assessment

1. Primary outcome
   1. Measure duration of diarrhea as the number of days from the first intervention dose to the last unformed stool, followed by 24 h without diarrhea.
2. Secondary outcomes in pediatric patients
   1. Record stool frequency per 24 h until resolution. Measure duration of hospitalization in days.
   2. Record incidence of vomiting during treatment. Assess clinical cure by Day 5. Document all adverse events.
3. Secondary outcomes in adult patients
   1. Assess symptom resolution by Day 7. Record recurrence of diarrhea within 14 days.
   2. Measure hospital stay duration. Evaluate the impact of concomitant antibiotic therapy. Document adverse events from electronic medical records.
4. Diagnostic assessment
   1. Collect fresh stool samples before probiotic or placebo administration whenever possible.
   2. Test pediatric stool samples for rotavirus using a rapid antigen immunochromatographic assay. Record the result as rotavirus-positive or rotavirus-negative.
   3. For adult patients, extract stool test results, clinical diagnosis, and pathogen category from electronic medical records.

7. Statistical analysis

1. Pediatric data analysis
   1. Perform statistical analysis using statistical softwares.
   2. Express continuous variables as mean ± standard deviation (SD). Assess normality of continuous variables using the Shapiro–Wilk test. Analyze non-normally distributed data using the Mann–Whitney U test.
   3. Compare continuous variables using an independent sample t-test. Compare categorical variables using the chi-square test or Fisher’s exact test where appropriate.
2. Adult data analysis
   1. Present categorical variables as frequencies and percentages. Calculate odds ratios (OR) with 95% confidence intervals (CI). Perform comparisons using the chi-square test.
   2. Perform multivariable logistic regression analysis to adjust for potential confounders, including antibiotic use, baseline disease severity, and comorbidities.
   3. Report adjusted odds ratios (aOR) with 95% confidence intervals (CI).
   4. Conduct subgroup analyses stratified by antibiotic co-therapy status.
   5. Consider p < 0.05 as statistically significant.
3. Significance threshold
   1. Define statistical significance as p < 0.05. Missing data were handled using complete-case analysis. Report effect estimates with 95% confidence intervals where applicable.

Results

Table 3 summarizes the demographic and clinical baseline characteristics of the pediatric participants (n = 176). The mean age of children receiving S. boulardii was 23.0 ± 12.3 months, compared to 21.2 ± 11.8 months in the placebo group. The sex distribution was comparable between groups (S. boulardii: 47 males/43 females; placebo: 51 males/35 females). Chi-square analysis of sex distribution showed no statistically significant difference (χ² = 0.63, p = 0.428), confirming t...

Discussion

Importantly, outcomes from pediatric and adult cohorts should not be directly compared. The pediatric study was a randomized controlled trial, whereas the adult cohort was a retrospective, observational cohort. Therefore, any differences observed between cohorts are descriptive and may reflect differences in study design, population characteristics, or underlying disease etiology rather than true comparative treatment effects. The study is presented in a structured protocol format to enhance reproducibility and transpare...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Computer with statistical software	IBM; R Foundation for Statistical Computing	SPSS v25.0; R v4.3.1	Licensed statistical software Purpose: Data analysis
Econorm	Beijing Hanmi Pharmaceutical Co., Ltd., Beijing	CNCM I-745	Commercial strains Purpose: NA
Electronic medical record system	Hospital information system	Institutional system	Secure digital patient database Purpose: Data extraction (adult cohort)
Florastor	Biocodex, France	(S. boulardii CNCM I-745)	Commercial strains Purpose: NA
IBM SPSS Statistics version 25.0	IBM Corp., Armonk, NY, USA	Version 25.0	Used for statistical analysis, including chi-square tests, odds ratio calculation, and significance testing
Intravenous fluid (0.9% sodium chloride)	Baxter Healthcare / Fresenius Kabi	As per supplier	Sterile isotonic saline solution Purpose: Intravenous rehydration
Oral rehydration salts (ORS)	World Health Organization formulation	WHO standard ORS sachet	Low-osmolarity ORS (Na + 75 mmol/L) Intravenous rehydration Purpose: Oral rehydration therapy
Personal protective equipment (PPE)	3M / equivalent	Gloves, masks, lab coats	Disposable protective gear Purpose: Biosafety compliance
Placebo sachets (maltodextrin)	Beijing Hanmi Pharmaceutical Co., Ltd., Beijing, China.	Custom-prepared	250 mg per sachet, identical in appearance to active drug Purpose: Control intervention
R software version 4.3.1	R Foundation for Statistical Computing, Vienna, Austria	Version 4.3.1	Used for statistical analysis, subgroup analysis, and multivariable regression modeling
Randomization software	R software / GraphPad Prism	v4.3.1 / v10	Computer-generated block randomization Purpose: Allocation sequence
Rotavirus antigen rapid test kit	Abbott Diagnostics / SD Biosensor	SD BIOLINE Rota/Adeno	Immunochromatographic assay Purpose: Rotavirus detection
Saccharomyces boulardii CNCM I-745 sachets	[Beijing Hanmi Pharmaceutical Co., Ltd., Beijing, China.	[Insert catalog/product code]	250 mg lyophilized probiotic yeast per sachet Purpose: Intervention
Stool collection container (sterile)	Thermo Fisher Scientific / equivalent	120 mL sterile container	Leak-proof, sterile Purpose: Stool sample collection
Stool transport container/bag	BD Biosciences / equivalent	Specimen transport kit	Biohazard-labeled, sealed transport Purpose: Sample handling