Saccharomyces boulardii CNCM I-745 for Prevention of Antibiotic-Associated Diarrhea and Clostridioides difficile in China: Systematic Review and Meta-Analysis

Probiotics are commonly used for patients receiving antibiotics to prevent antibiotic-associated diarrhea (AAD). Randomized, controlled trials (RCTs) conducted in China are often not included in probiotic reviews and meta-analyses due to the difficulties in accessing Chinese journals and translating the Chinese language. Our aim is to evaluate the efficacy and safety of S. boulardii CNCM I-745 in AAD prevention trials done in China. A literature search was conducted using PubMed, Google Scholar and Chinese databases (CNKI, CMB) (from inception to June 2023) for RCTs assessing S. boulardii CNCM I-745 for the prevention of AAD and Clostridioides difficile infections (CDI). Inclusion criteria: RCT done in China, new prescription of antibiotic, randomized to S. boulardii and control groups. Random-effect or fixed-effects models were used depending upon the degree of heterogeneity and the risk of bias for each study was determined. This review was registered with Prospero (PROSPERO CRD 429831). The literature search found 361 articles, which were screened and 46 (8,201 participants) were included in the analysis. S. boulardii CNCM I-745 significantly reduced the incidence of AAD (RR=0.43, 95% C.I. 0.40,0.48, P < 0.0001) and CDI (RR=0.30, 95% C.I. 0.10, 0.87, P = 0.03). S. boulardii CNCM I-745 had similar degrees of efficacy for both the prevention of AAD in trials done in China and for trials done in other countries. Saccharomyces boulardii CNCM I-745 was well tolerated and effective in trials in China for both the prevention of AAD and CDI.


Introduction
Antibiotic-associated diarrhea (AAD) is a common complication of antibiotic use resulting from the disruption of the normally protective intestinal microbiome.While some factors that disrupt the intestinal microbiome are well-known (such as antibiotic exposure, advanced age, diet, and health status) [1], other factors relating to geography and ethnicity are less well documented, but may have important implications for microbiome-based clinical therapies [2].The World Health Organization (WHO) defines acute diarrhea as ≥3 loose or liquid stools per day for ≥3 days and lasting <14 days [3].Antibiotic-associated diarrhea is defined as diarrhea occurring while on antibiotics or within 8 weeks of antibiotics [1].The prevalence of AAD in patients receiving antibiotics can be quite high, ranging from 5-37% in adults and may be higher in children (11-40%) [1].The onset of AAD typically occurs during antibiotic administration, but delayed onset AAD may occur in 10-20% for up to 8 weeks after antibiotics have been discontinued [1].While the severity of AAD is typically mild-moderate in severity, 16% of cases are more severe, requiring further antibiotic treatments and 10-35% may be due to Clostridioides difficile infection (CDI), which can cause healthcare-associated outbreaks.Consequences of AAD include dehydration (especially in young children), higher mortality and morbidity, increased lengths-ofstays for inpatients (4-24 days), higher risk of colectomy (1-9%), higher healthcare costs and 25-28% of those with CDI develop recurrent form of the infection [1,4].
China is the second largest consumer of antibiotics, most commonly azithromycin, clindamycin and erythromycin [5,6,7].However, only 25-39% of antibiotics prescribed in China are given appropriately [5].The prevalence of AAD in China ranges from 14-35%, depending upon age, hospitalization status and co-morbidities [4,8].Efforts prevent AAD include antibiotic stewardship programs and the adjunctive use of probiotics during antibiotic use [9,10].A survey of 138 pediatric practitioners in China found 31% gave probiotics along with antibiotics, mainly due to concerns about antibiotic resistance or development of AAD [6].Specific types of probiotics have been recommended in clinical guidelines for the prevention of AAD, but not all probiotic strains have shown efficacy [10,11,12].Guidelines for probiotic use to prevent AAD in children focused on Asia-Pacific populations had strong recommendations for S. boulardii CNCM I-745 and L. rhamnosus GG [13].These two stains were also recommended for the prevention of AAD in adults in the Asia-Pacific region [14].Indeed, the efficacy for probiotics is strain-specific and each strain or multi-strained blend must be assessed separately to determine efficacy for AAD prevention [15].Prior meta-analyses have found Saccharomyces boulardii CNCM I-745 and Lactobacillus rhamnosus GG to have the strongest evidence for the prevention of AAD, but often have not included RCTs published in non-English languages, due to translation problems and limited access to Chinese journals [16,17,18,19,20].
Saccharomyces boulardii CNCM I-745 is a strain of probiotic that has a long history of use and has demonstrated significant efficacy and safety for a variety of diseases ranging from prevention of AAD and CDI, treatment of pediatric acute gastroenteritis, to reduction of side-effects of H. pylori eradication therapies [17,20,21,22,23].S. boulardii CNCM I-745 reaches high levels in the intestine within 2-3 days, is cleared from the colon within 5 days and can be given concurrently with antibiotics, as the yeast is not susceptible to antibiotics [24].The ability of S. boulardii CNCM I-745 to be therapeutic is due to multiple mechanisms-of-action: direct inhibition of pathogen growth or destruction of pathogenic toxins, interference with pathogen/ toxin attachment on enteric cell surfaces, improving intestinal cell health, reduction of water secretion, immune system regulation and restoring the normally protective microbiome barrier layer [14,21,25].S. boulardii CNCM I-745 has a remarkable safety profile in that this strain has been given to a wide variety of patient types (children, adults and the elderly, hospitalized inpatients and outpatients, patients with acute and chronic conditions) and data gathered since its introduction in Europe in the 1950s has shown few adverse reactions, most were thirst and constipation [22].Genetic transfer of antibiotic-resistant genes has not been observed with this strain [26].In rare cases (1/5.6 million users), immunocompromised patients or inpatients with central catheters have developed fungemia [23].
Our aim in this study is to determine its efficacy and safety of Saccharomyces boulardii CNCM I-745 for the prevention of AAD and CDI in trials done in China using a comprehensive literature search to uncover trials not previously found in non-Chinese databases.

Literature search
The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed [27].The PRISMA checklist is provided (Supplementary Table S1).Recent recommendations for reporting probiotic meta-analyses were also followed [28].The project and protocol were prospectively registered with PROSPERO database of systematic reviews [www.Secondary searches of grey literature included reference lists, authors, reviews, meeting abstracts websites and clinicaltrials.govfor unpublished trials.A recursive search was also performed, using the bibliographies of all obtained articles.

Study selection
Inclusion criteria included: randomized, controlled clinical trials (RCTs) in children or adults receiving new prescription of antibiotics, comparison of S. boulardii versus control interventions and published in peer-reviewed journals.Only formulations of S. boulardii CNCM I-745 fulfilling the standard definition (must be living microbe, of adequate dose and conferring a health effect on the host) were included [29].This definition excludes dead or heat-killed microbes and prebiotics.As bacterial and fungal taxonomies shift over time, the most current strain designations are presented in this review and strain identification was confirmed with the original authors or the manufacturer whenever possible.Trials with the primary outcome of prevention of AAD or CDI were included.Studies were included only if performed in China.
Exclusion criteria included: study not done in China, nonhuman studies, early phase 1 or 2 safety or mechanism of action studies, no control group, probiotic or AAD not well-described, clinical trials for the treatment of existing diarrhea, primary outcome was for the eradication of H. pylori, other probiotic strain(s) contained in the probiotic formulation, non-China setting, reviews and duplicate reports.Cross-over trials were excluded due to the potential for effect carry-over after short wash-out periods used in these trials and direct probiotic-probiotic studies were excluded if no non-probiotic control group was included.

Data extraction
The literature was searched independently by two coauthors (LM, LT).Data from all RCTs were extracted using a standardized data extraction form (Supplementary data, Form S1) initially completed by one co-author and then each study was independently scored by the other co-author following the standard methods for systematic reviews and meta-analysis [27,30].Any disagreements were discussed until consensus was reached.The data extracted included PICOS data: (1) population (neonate, pediatric or adults, age range and country), (2) intervention (type of probiotic or controls used, daily doses, formulation, duration and follow-up times), (3) comparisons (type of control group either placebo or open, unblinded), (4) Primary outcomes (incidence of AAD or CDI), (5) secondary outcomes (stool frequency, length of hospitalization, cost data, safety data and clinical characterization of AAD).In addition, data on potential confounding factors were collected: type of antibiotic given, type of control, study quality, setting (inpatient or outpatient), dose and duration of probiotic.For missing data not reported in the published article, we attempted to contact the author or co-authors to obtain the missing data.

Intervention
The intervention was the yeast probiotic strain Saccharomyces boulardii CNCM I-745 (Yihuo ™ or Bioflor™, Biocodex, France).If the strain designation was not reported in the published study, the strain was verified with the manufacturer or website.The intervention may be given in oral administration as capsules or sachets.The duration of the intervention was typically given for the duration of the antibiotic(s), but may have been continued after the antibiotics are discontinued.

Outcomes
The primary outcomes include the efficacy for the reduction of AAD incidence and the reduction of CDI incidence.The outcome measures included: the number of patients developing AAD (diarrhea defined as at least two loose/watery stools in young children or ≥3 loose/watery stools in older children and adults for 48 hours, not caused by rotavirus, with an onset while on antibiotics or within 8 weeks of antibiotic cessation or CDI (AAD with positive C. difficile toxin result).Secondary outcomes included: daily stool frequency at the end of the intervention, length of hospitalization, clinical presentation of AAD (measured by duration of AAD, severity of AAD symptoms and treatment effectiveness rating), costs of medical care and frequency of adverse events.Severe AAD was defined differently in the RCTs, but typically included "diarrhea with electrolyte disorders, severe dehydration, acidosis or other symptoms of toxicity".Outcomes may be documented by patients/parents using daily diaries or by healthcare providers if hospitalized.

Risk of bias
Each included RCT was reviewed for quality and risk of bias and scored independently by both co-authors using standard methods [31].The risk of bias (RoB) assessed with the RoB 2.0 tool and was graded (high, some concerns, or low) for each of five domains of bias (randomization process, deviations from intended interventions, missing outcome data, measurement of outcome, selection of reported result) and a summary table and figure of bias was generated [32].

Statistical Analysis
As recommended by experts, inclusion of studies in our meta-analysis also required at least two RCTs using a common Volume 9; Issue 1 J Dig Dis Hepatol, an open access journal ISSN: 2574-3511 outcome measure [28].Statistical analysis and generation of forest plots of pooled summary estimates was performed using Stata software version 16 (Stata Corporation, College Station, Texas) with meta-analysis modules [33].Dichotomous outcomes were assessed using relative risks (RR) and 95% confidence intervals (C.I.) and continuous outcomes were assessed using standardized mean difference (SMD) and 95% C.I. using standard methods [31].The significance level was set at P-value < 0.05.Heterogeneity across trials was evaluated using the I 2 statistic, 0% indicating none and ≥50% indicating a high degree of heterogeneity across the trials [33].Random-effects models were used for the meta-analysis if significant heterogeneity was found (I 2 >50%), otherwise fixed-effects models were used.Publication bias was assessed using funnel plots and the Egger's test [33].Subgroup analysis was used to explore sources of heterogeneity and was assessed with the Cochrane Q test [31].A priori subgroup analyses based on factors that might influence the magnitude of efficacy estimates were planned for the following: (a) age, (b) daily dose S. boulardii, (c) risk of bias, (d) indication for antibiotic use (upper respiratory disease, gastrointestinal, etc.), (e) type of patient (inpatient/outpatients), (f) time of intervention initiation from onset of diarrhea, (g) rural versus urban setting, (h) route antibiotic given (IV or oral) and (i) extent of blinding.Trials were pooled in the meta-analysis if a common outcome was used and there were at least two trials within each sub-group.Sequential sensitivity analysis was done to explore the extent outcomes were dependent upon a particular trial.
The types of antibiotics used varied, with most RCTs (n=23, 50%) using different types of antibiotics while four RCTs enrolled patients given only a single type of antibiotic (9%), but the types of antibiotics were not reported in 19 (41%) trials (Table 1).Most of the antibiotics were given for upper respiratory infections (38, 83%) or mixed types of infections (5, 11%) or not reported (n=3, 6%).The duration of antibiotics ranged from 5 to 21 days and most (26, 56.5%) were given only intravenously while a few RCTs (n=3, 6.5%) enrolled patients given either IV or oral antibiotics.The antibiotic route was not reported in 17 (37%) of the RCTs.

Primary outcome: prevention AAD
Efficacy to prevent AAD was reported all included trials (n=46), as shown in (Table 2).However, the reported definitions for AAD varied (Supplementary Table S2) with 5 (11%) not reporting how AAD was defined.Most RCTs (n=41, 89%) did report their definition of AAD in their papers, typically representing a change in stool consistency, increased frequency of watery/loose stools for longer than one day, which was associated with antibiotic use or shortly afterwards.The incidence of AAD ranged from 3/100 to 38/100 (mean 15.0 ± 7.2/100) in S. boulardii groups compared to 9/100 to 56/100 (mean 35.9 ± 10.7/100) in controls.The pooled RR for AAD was significantly lower for S. boulardii compared to controls (RR= 0.43, 95% C.I. 0.40, 0.48, P<0.0001,I 2 =5.5%), as shown in (Figure 2).

Secondary outcomes
Several secondary outcomes had sufficient data for analysis: daily stool frequency at study end, clinical characterization of AAD (duration, severe AAD, effectiveness rating) and safety.Other a priori secondary outcomes were infrequently reported (rural versus urban and cost-effectiveness) and could not be analyzed.

Safety
Many (23/46, 50%) of the RCTs provided data on adverse reactions or tolerance (Table 2), while no data on safety was provided in 23 (50%) of the RCTs.Twenty-one RCTs only provided a statement that the "probiotic was well-tolerated" or "no adverse events were noted", while two RCTs provided more detailed data on mild adverse reactions [44,58].Guo et al reported abdominal pain (19% in S. boulardii group and 11% in controls, P = 0.22), abdominal distension (16% and 11%, respectively, P = 0.42) and nausea (13% and 8%, respectively, P = 0.40).Tang et al. noted only mild allergic reactions in two (3%) of those given S. boulardii and in 3 (4%) of controls (P>0.05)[58].No serious adverse events were reported in any of the trials.

Age
As the enrollment age ranges were not mutually exclusive, only three age sub-groups could be analyzed: neonates (under 14 days old), pediatric (1 month to 16 years old) and elderly (>65 years old).As shown in (Supplementary Figure S5), S. boulardii significantly prevented AAD in all three age groups, with no significant differences by age groups (X 2 =0.8, P = 0.67).

Study Quality
All the RCTs were assessed for risk of bias (Supplementary Table S3).Of the 46 RCTs, two (4.3%) were considered an overall low risk of bias (Supplementary Figure S7), 84.8% had 'some concerns' about bias and five (10.9%) were considered high risk bias (when more than one bias domain was rated as high risk).The only individual domain in the 46 RCTs commonly rated high-risk was for the lack of double-blinded design (96%).Other individual domains were rated as low risk.When five high risk trials for prevention of AAD were excluded [42,43,49,52,57], the reduction of AAD risk was similar (RR=0.42,95% CI 0.38, 0.46, P<0.001,I 2 =0.5%), as shown in (Supplementary Figure S8).

Other sub-groups
Some sub-groups could not be analyzed as the groups had similar characteristics or were infrequently reported.For example, respiratory tract infections were the most common indication for antibiotics (94% trials), most trials were in children (96%), 96% had open controls and all patients were hospitalized.Some data was not reported in sufficient numbers of trials (for example, 33% of the trials did not report intervention initiation time and only four trials reported the length of hospitalization (Supplementary Table S4).None of the trials reported if the hospital patients were from urban or rural healthcare facilities and none reported cost or cost-saving data.

Discussion
Our meta-analysis found S. boulardii CNCM I-745 was significantly effective for the prevention of AAD and the prevention of CDI in trials done in China and was well-tolerated.S. boulardii CNCM I-745 effectively reduced AAD in hospitalized children and adults receiving the types of antibiotics typically prescribed for respiratory tract infections in China (cephalosporins, penicillins, macrolides, etc.), which are well known risk factors for AAD and CDI [80].S. boulardii CNCM I-745 was shown to not only significantly reduce the incidence of AAD and CDI, but also reduce the duration of AAD and the daily stool frequency.S. boulardii was well tolerated in the included trials and safety has been thoroughly documented in patients receiving antibiotics in comprehensive meta-analyses and reviews [17,21,22].Antibiotic resistance is another safety concern associated with antibiotic use and one study reported a reduction in the antibiotic resistance gene load when H. pylori eradication therapy was combined with S. boulardii CNCM I-745 [81].Healthcare providers and policy makers should be aware of the high rate of AAD in their patients treated with antibiotics in China and strive to implement strategies to reduce AAD.
As the intestinal microbiome is influenced by ethnicity and geography, the question often arises if probiotic efficacy is also affected by these factors [2].The effects of differences in diet, genetic make-up, nutritional status, healthcare access and living conditions are not commonly explored in probiotic reviews.One method of controlling for these confounding factors is to limit the meta-analysis to one country or to assess the efficacy using subgroups of different geographic regions [17,82].We showed S. boulardii significantly reduced the risk of AAD (RR=0.43) in China, which was similar to studies done in other countries and in different populations.In a meta-analysis, Szajewska et al. [17] reported S. boulardii significantly reduced the risk of AAD regardless of where the trial was conducted: trials done in Europe (RR=0.46)or in USA (RR=0.46),as shown in (Supplementary Figure S9).Guo et al. [19] reviewed different types of probiotics for the prevention of AAD in children and pooled data from nine RCTs using S. boulardii.These nine trials were done in different countries (China, France, Turkey, India and Poland) but were not assessed by country sub-groups but nevertheless found a significant reduction of AAD (RR=0.36,95% C.I. 0.24, 0.54, P<0.0001) for the nine trials.In a network meta-analysis with 10 different probiotic types, Cai et al. [18] reported a significant reduction in AAD from 11 RCTs using S. boulardii (RR=0.41,95% C.I. 0.29, 0.57, P<0.05) and a significant reduction in CDI from three RCTs (RR= 0.35, 95% C.I. 0.15, 0.85, P<0.05), but did not report in which countries the trials were conducted.

Study Strengths
Use of Chinese databases (CNKI and CBM) for the literature search revealed 42 trials that would have been undetected if only the most common literature databases (PubMed, Google Scholar, etc.) had been used.We also followed recently published guidelines for reporting meta-analyses involving probiotics [28].We used rigorous inclusion criteria to include trials that adequately described the probiotic intervention and outcome measures.Subgroup analysis was done to examine factors that might influence the estimates of efficacy.As geographic region factors might influence the efficacy of a probiotic intervention, one strength was that we limited trials done in one country (China), although we acknowledge vast differences in regions within China exist.Another strength was that we limited our meta-analysis to one specific type of probiotic (S. boulardii CNCM I-745), as the efficacy of probiotics has been shown to be both strain-specific and disease-specific [15].

Study limitations
Geographic factors in China that might affect probiotic efficacy were not documented in the included trials (such as differences in diet, health status, urban/rural, etc.).Future trials may benefit in exploring these factors.Significant heterogeneity was observed in some outcomes and sub-group analyses failed to explain the possible sources of the heterogeneity.Another limitation is that some trials failed to report specific confounder data, so that not all proposed sub-group analyses could be performed.Several meta-analyses found the efficacy of a probiotic is better when the probiotic is started within 48 hours of the antibiotic [83,84], and while many RCTs in China reported the intervention was started at the same time as the antibiotics, the specific times were rarely Volume 9; Issue 1 J Dig Dis Hepatol, an open access journal ISSN: 2574-3511 stated.We excluded 17 trials with a primary outcome of H. pylori eradication, but S. boulardii has been shown to significantly reduce AAD in patients treated with antibiotics for H. pylori infections [21].In a meta-analysis by McFarland et al. [85], S. boulardii CNCM I-745 significantly reduced the risk of AAD associated with H. pylori eradication therapies in eight RCTs (RR=047, 95% C.I 0.37, 0.60).Even though the 46 trials in this meta-analysis were limited to those done in China, the robustness of the efficacy estimates from trials done in other countries may show the efficacy of S. boulardii CNCM I-745 may be generalized to other populations.

Future studies
To improve the quality of future RCTs, it is recommended to provide a complete description of the method of randomization, consider using a placebo control and to follow subjects postantibiotics to detect late-onset cases of AAD.In addition, RCTs need to report safety data as this is an important clinical consideration when using probiotics.Most studies did not compare their study results with other trials using S. boulardii CNCM I-745 and this should be included in the discussion of future papers.This study shows the value of utilizing resource rich literature databases, especially in non-English language studies.

Conclusion
In summary, the results of this meta-analysis of 46 randomized, controlled trials in China demonstrated that S. boulardii CNCM I-745 was effective in both the prevention of AAD and CDI and was well tolerated.The use of Chinese databases for literature searches revealed an untapped resource to find important clinical trials and offers clinicians and policy-makers guidance for an effective strategy to prevent complications of antibiotic use.

Disclosures
crd.york.ac.uk/PROSPERO/] on May 25, 2023 (CRD 429831).The following databases were searched [PubMed, Google Scholar, and Chinese databases (Chinese Biomedical Literature (CBM) and Chinese National Knowledge Infrastructure (CNKI)] from database inception to June 2023 to identify randomized Volume 9; Issue 1 J Dig Dis Hepatol, an open access journal ISSN: 2574-3511 controlled trials (RCTs) of probiotic use (limited to Saccharomyces boulardii CNCM I-745) to prevent antibiotic-associated diarrhea (AAD), including Clostridioides difficile infections (CDI) using randomized controlled trials done in China.The search terms were used: (antibiotic-associated diarrhea OR C. difficile AND Saccharomyces boulardii AND China).No language restrictions were imposed and publications in Chinese were translated into English.

Figure 2 :
Figure 2: Forest plot of efficacy for prevention of AAD: S. boulardii compared to controls.DL, Der Simonian-Laird.

Figure 3 :
Figure 3: Forest plot of efficacy for prevention of CDI: S. boulardii compared to controls.DL= Der Simonian-Laird.

Figure 4 :
Figure 4: Forest plot of efficacy for stool frequency/day: S. boulardii compared to controls.SMD, standardized mean difference; IV, Inverse variance.Clinical characteristics of AADThree measures were used to determine if S. boulardii influenced the severity of AAD (duration of AAD, frequency of severe AAD and effectiveness rating).Data was collected from 232 cases of AAD in S. boulardii treated patients and 454 cases of AAD in the controls.

Figure 5 :
Figure 5: Forest plot of efficacy for reduction of AAD duration in days: S. boulardii compared to controls.SMD, standardized mean difference; IV, inverse variance.

Table 1 :
Study population characteristics of 46 included trials for the prevention of AAD. 1 Dose/day may vary by age of patient; 2 On abx + days extended post-antibiotics;3Li J (treatment AAD arm excluded);4Yu M (two study arms excluded, on for treatment of AAD by S. boulardii and another with probiotic blend).

Table 3 :
Efficacy of S. boulardii on prevention of secondary AAD: stool frequency and duration of AAD, and stool frequency/day, AAD, antibiotic-associated diarrhea; na, not applicable; nr, not reported; Ref, reference; Sb, Saccharomyces boulardii CNCM I-745, Std dev, standard deviation.

Table 4 :
Clinical characteristics of AAD in 232 cases of AAD in S. boulardii and 454 cases of AAD in controls; %, percentage; AAD, antibiotic-associated diarrhea; n, number in numerator; N, total number in denominator, nr, not reported.