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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Nutr.</journal-id>
<journal-title>Frontiers in Nutrition</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Nutr.</abbrev-journal-title>
<issn pub-type="epub">2296-861X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
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</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fnut.2025.1606264</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Nutrition</subject>
<subj-group>
<subject>Systematic Review</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>The effect of probiotics on the diarrhea and constipation outcomes in children: an umbrella review of systematic reviews and meta-analyses</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author" equal-contrib="yes">
<name><surname>Wang</surname> <given-names>Qizheng</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="author-notes" rid="fn0001"><sup>&#x2020;</sup></xref>
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<contrib contrib-type="author" equal-contrib="yes">
<name><surname>Ren</surname> <given-names>Tailiang</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="author-notes" rid="fn0001"><sup>&#x2020;</sup></xref>
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<contrib contrib-type="author" corresp="yes">
<name><surname>Wang</surname> <given-names>Haijun</given-names></name>
<xref ref-type="aff" rid="aff3"><sup>3</sup></xref>
<xref ref-type="corresp" rid="c001"><sup>&#x002A;</sup></xref>
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<contrib contrib-type="author" corresp="yes">
<name><surname>Lin</surname> <given-names>Xiaofei</given-names></name>
<xref ref-type="aff" rid="aff1"><sup>1</sup></xref>
<xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
<xref ref-type="corresp" rid="c001"><sup>&#x002A;</sup></xref>
<uri xlink:href="https://loop.frontiersin.org/people/3026457/overview"/>
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<aff id="aff1"><sup>1</sup><institution>Department of Pediatrics, Huai&#x2019;an Maternal and Child Health Care Hospital Affiliated to Yangzhou University</institution>, <addr-line>Huai'an</addr-line>, <country>China</country></aff>
<aff id="aff2"><sup>2</sup><institution>Department of Pediatrics, The Huai'an Maternity and Child Clinical College of Xuzhou Medical University</institution>, <addr-line>Jiangsu</addr-line>, <country>China</country></aff>
<aff id="aff3"><sup>3</sup><institution>Department of Pediatrics, Lianshui County People&#x2019;s Hospital, Affiliated Hospital of Kangda College, Nanjing Medical University</institution>, <addr-line>Jiangsu</addr-line>, <country>China</country></aff>
<author-notes>
<fn fn-type="edited-by" id="fn0002">
<p>Edited by: Philippe G&#x00E9;rard, Institut National de recherche pour l&#x2019;agriculture, l&#x2019;alimentation et l&#x2019;environnement (INRAE), France</p></fn>
<fn fn-type="edited-by" id="fn0003">
<p>Reviewed by: Hussain Ahmad, Islamia University of Bahawalpur, Pakistan</p>
<p>Tiziana Maria Mahayri, Academy of Sciences of the Czech Republic (ASCR), Czechia</p></fn>
<corresp id="c001">&#x002A;Correspondence: Xiaofei Lin, <email>linfei7774@aliyun.com</email>; Haijun Wang, <email>wanghaijun619366@163.com</email></corresp>
<fn fn-type="equal" id="fn0001"><p><sup>&#x2020;</sup>These authors have contributed equally to this work</p></fn>
</author-notes>
<pub-date pub-type="epub">
<day>18</day>
<month>07</month>
<year>2025</year>
</pub-date>
<pub-date pub-type="ecorrected">
<day>26</day>
<month>08</month>
<year>2025</year>
</pub-date>
<pub-date pub-type="collection">
<year>2025</year>
</pub-date>
<volume>12</volume>
<elocation-id>1606264</elocation-id>
<history>
<date date-type="received">
<day>04</day>
<month>04</month>
<year>2025</year>
</date>
<date date-type="accepted">
<day>04</day>
<month>07</month>
<year>2025</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x00A9; 2025 Wang, Ren, Wang and Lin.</copyright-statement>
<copyright-year>2025</copyright-year>
<copyright-holder>Wang, Ren, Wang and Lin</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<sec id="sec1">
<title>Background</title>
<p>The existing literature on the effects of probiotics on diarrhea and constipation outcomes remains inconsistent. Therefore, this umbrella review of systematic reviews and meta-analyses aims to provide a concise and definite understanding in relation to the effect of probiotics on diarrhea and constipation in children.</p>
</sec>
<sec id="sec2">
<title>Methods</title>
<p>A comprehensive systematic search was carried out in on Scopus, PubMed, Embase, Web of Science, and Google Scholar up to December 2024. The overall effect size was calculated using random effect model. Also, subgroup analyses were performed regarding age group, health condition, single or multi-strain probiotics.</p>
</sec>
<sec id="sec3">
<title>Results</title>
<p>This umbrella study comprises a systematic review of 35 studies. Our findings illustrated that probiotics reduce odds [odds ratio (OR)&#x202F;=&#x202F;0.51; 95% confidence interval (CI): 0.27, 0.94] and risk of diarrhea incidence [relative risk (RR)&#x202F;=&#x202F;0.54; 95% CI: 0.40, 0.71] compared to control group, meaningfully. Also, it is successful in reducing diarrhea duration [weighted mean difference (WMD)&#x202F;=&#x202F;&#x2212;1.85; 95% CI: &#x2212;2.83, &#x2212;0.86] and [standardized mean difference (SMD)&#x202F;=&#x202F;&#x2212;0.94; 95% CI: &#x2212;1.32, &#x2212;0.56] significantly. Moreover, probiotics supplementation resulted in decreased stool frequency (WMD&#x202F;=&#x202F;&#x2212;0.21; 95% CI: &#x2212;0.37, &#x2212;0.04). Probiotics prevent diarrhea by about 36% (RR&#x202F;=&#x202F;0.64; 95% CI: 0.63, 0.65(, and significantly improved diarrhea treatment (SMD&#x202F;=&#x202F;&#x2212;0.49; 95% CI: &#x2212;0.59, &#x2212;0.38). Also, the analyses revealed that probiotics significantly impact on constipation (OR&#x202F;=&#x202F;1.17, 95% CI: 1.01&#x2013;1.37).</p>
</sec>
<sec id="sec4">
<title>Conclusion</title>
<p>This meta-analysis supports the potential role of probiotics in relation to diarrhea and constipation outcome in children. Probiotic supplementation contributed to a declined risk and odds of diarrhea incidence. Also, probiotic supplementation was accompanied with decreased diarrhea duration.</p>
</sec>
</abstract>
<kwd-group>
<kwd>probiotics</kwd>
<kwd>diarrhea</kwd>
<kwd>constipation</kwd>
<kwd>systematic review</kwd>
<kwd>meta-analysis</kwd>
</kwd-group>
<counts>
<fig-count count="5"/>
<table-count count="1"/>
<equation-count count="0"/>
<ref-count count="78"/>
<page-count count="13"/>
<word-count count="7954"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Nutrition and Microbes</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="sec5">
<title>Introduction</title>
<p>Gastrointestinal issues are fairly common throughout childhood and adulthood, affecting an estimated 8&#x2013;25% of the general population (<xref ref-type="bibr" rid="ref1">1</xref>). These percentages may vary based on the specific gastrointestinal condition, and the age of the individuals affected (<xref ref-type="bibr" rid="ref2">2</xref>). Gastrointestinal disorders, including diarrhea and constipation, are common health concerns worldwide. Diarrhea is a significant global gastrointestinal issue, causing around 500,000 deaths annually in children under five (<xref ref-type="bibr" rid="ref3">3</xref>). The World Health Organization (WHO) defines diarrhea as the passage of three or more loose or watery stools within a 24-h period (<xref ref-type="bibr" rid="ref4">4</xref>). The disruption of intestinal microflora is a hallmark of diarrhea and can be triggered by various factors, including antibiotic use, infectious agents, and poor nutrition (<xref ref-type="bibr" rid="ref5">5</xref>). Diarrhea can cause dehydration and electrolyte imbalances in children and adults, leading to serious consequences such as growth stunting in children, malnutrition, and recurrent enteric infections (<xref ref-type="bibr" rid="ref6">6</xref>). This impairment is linked to a heightened risk of mortality (<xref ref-type="bibr" rid="ref3">3</xref>, <xref ref-type="bibr" rid="ref7">7</xref>).</p>
<p>Also, constipation, characterized by infrequent and painful bowel movements, abdominal discomfort, and fecal incontinence, poses a major challenge in pediatric and adults healthcare worldwide. The prevalence of this condition is estimated to be between 0.7 and 29.6% worldwide (<xref ref-type="bibr" rid="ref8">8</xref>). It is a common concern among both children and adults, often causing significant physical discomfort in affected individuals, along with psychological impacts (<xref ref-type="bibr" rid="ref9">9</xref>). Constipation arises from a combination of factors, including genetic predisposition, disrupted intestinal motility, low dietary fiber and fluid intake, insufficient physical activity, and a diminished urge to defecate (<xref ref-type="bibr" rid="ref10">10</xref>). Given these impacts of diarrhea and constipation on health and well-being, finding effective interventions is crucial. This is where probiotics come into play, offering a promising approach to managing and alleviating these gastrointestinal issues.</p>
<p>Probiotics have gained widespread recognition for their role in promoting gut health, particularly in preventing and managing gastrointestinal disorders like diarrhea and constipation. Their effectiveness is primarily linked to their ability to restore microbial balance, enhance gut barrier function, and modulate immune responses (<xref ref-type="bibr" rid="ref11">11</xref>). Probiotics, defined by the World Health Organization as &#x201C;live microorganisms that, when consumed in adequate amounts, confer health benefits to the host,&#x201D; have demonstrated efficacy in managing gastrointestinal disorders such as diarrhea and constipation (<xref ref-type="bibr" rid="ref12">12</xref>, <xref ref-type="bibr" rid="ref13">13</xref>). Probiotics exert their effects through a variety of mechanisms, including competitive inhibition of pathogenic bacteria, enhancement of mucosal barrier integrity, modulation of local and systemic immune responses, and production of antimicrobial compounds such as bacteriocins and short-chain fatty acids (<xref ref-type="bibr" rid="ref14">14</xref>). The therapeutic efficacy of probiotics is strain-dependent. <italic>Lactobacillus rhamnosus</italic> GG enhances intestinal barrier function and stimulates the production of anti-inflammatory cytokines, while Saccharomyces boulardii has been shown to inhibit pathogen adhesion and increase enzyme activity that aids in nutrient absorption (<xref ref-type="bibr" rid="ref15">15</xref>).</p>
<p>Numerous meta-analyses have evaluated the therapeutic effects of probiotics on diarrhea and constipation in the pediatric population (<xref ref-type="bibr" rid="ref7">7</xref>, <xref ref-type="bibr" rid="ref9">9</xref>, <xref ref-type="bibr" rid="ref16 ref17 ref18">16&#x2013;18</xref>). However, their findings remain inconsistent (<xref ref-type="bibr" rid="ref9">9</xref>, <xref ref-type="bibr" rid="ref19 ref20 ref21">19&#x2013;21</xref>), likely due to differences in statistical approaches and heterogeneity in study designs. To address these discrepancies, we applied a uniform statistical methodology to synthesize the evidence and provide a more definitive assessment of the effects of probiotic supplementation on diarrhea- and constipation-related outcomes in children.</p>
</sec>
<sec sec-type="methods" id="sec6">
<title>Methods</title>
<p>The present meta-analysis was conducted in accordance with the PRISMA guidelines (<xref ref-type="bibr" rid="ref22">22</xref>). The protocol for this study has been documented in the International Prospective Register of Systematic Reviews (PROSPERO).</p>
<sec id="sec7">
<title>Search strategy</title>
<p>A comprehensive systematic search was conducted on scientific databases, including PubMed, Scopus, EMBASE, Web of Science, and Google Scholar, covering the period from inception until December 2024. The search strategy was developed using a combination of MeSH terms and keywords. (&#x201C;Probiotics&#x201D; OR &#x201C;Probiotics&#x201D; [tiab] OR &#x201C;probiotic&#x201D; [tiab] OR &#x201C;lactobacillus&#x201D; OR &#x201C;lactobacillus&#x201D; [tiab] OR &#x201C;Bifidobacterium&#x201D; [tiab]) AND (&#x201C;stool consistency&#x201D; OR &#x201C;stool frequency&#x201D; [tiab] OR &#x201C;diarrhea&#x201D; [tiab] OR &#x201C;constipation&#x201D; [tiab]) AND (&#x201C;pediatric populations&#x201D; [tiab] OR &#x201C;children&#x201D; [tiab]) AND (&#x201C;systematic review&#x201D; [tiab] OR &#x201C;meta-analysis&#x201D; [tiab]).</p>
</sec>
<sec id="sec8">
<title>Inclusion and exclusion criteria</title>
<p>The PICO criteria for this umbrella meta-analysis were defined as follows: Population/Patients (P: both individuals under 18&#x202F;years old receiving probiotic treatment); Intervention (I: administration of probiotics); Comparison (C: a control or placebo group); and Outcome (O: prevention of diarrhea, diarrhea incidence, duration of diarrhea, constipation, stool frequency and stool consistency). This umbrella review incorporated systematic reviews and meta-analysis that examined the impact of probiotic supplementation on diarrhea and constipation, specifically those that provided effect sizes (ESs) along with their respective confidence intervals (CIs). Conversely, the review excluded studies of an <italic>in vitro</italic>, <italic>in vivo</italic>, or ex vivo nature, as well as case reports, observational studies, quasi-experimental studies, and controlled clinical trials. Furthermore, the search was restricted to articles published in the English language.</p>
</sec>
<sec id="sec9">
<title>Methodological quality assessment</title>
<p>The methodological quality of the included articles was evaluated using the A Measurement Tool to Assess Systematic Reviews (AMSTAR) 2 questionnaire, which was administered by two independent researchers (<xref ref-type="bibr" rid="ref23">23</xref>). The AMSTAR2 checklist is classified into four distinct quality categories: &#x201C;critically low quality,&#x201D; &#x201C;low quality,&#x201D; &#x201C;moderate quality,&#x201D; and &#x201C;high quality.&#x201D;</p>
</sec>
<sec id="sec10">
<title>Study selection and data extraction</title>
<p>Two independent reviewers, conducted a screening of the articles in accordance with the established eligibility criteria. Initially, the titles and abstracts of the articles were evaluated. Subsequently, the full texts of the remained articles were evaluated to determine their eligibility for inclusion in the current umbrella meta-analysis. Any discrepancies were discussed. The extracted data encompassed the outcomes, specifically ESs and CIs, along with details such as the name of the first author, year of publication, the geographical location, number of included studies in each meta-analysis, total sample sizes, and the outcome.</p>
</sec>
<sec id="sec11">
<title>Data synthesis and statistical analysis</title>
<p>The pooled ES and its associated 95% CI were estimated using random-effects models implemented via the restricted maximum likelihood (REML) approach (<xref ref-type="bibr" rid="ref24">24</xref>). The Cochran-Q test and the I<sup>2</sup> index were utilized to evaluate the heterogeneity within the meta-analysis. A significant level of heterogeneity in the data was established when I<sup>2</sup> exceeded 50% or when the Cochran-Q test yielded a significant result (<italic>p</italic>&#x202F;&#x003C;&#x202F;0.10) (<xref ref-type="bibr" rid="ref24">24</xref>). Subgroup analysis using predetermined variables&#x2014;type of ES (WMD or SMD), health status, and single or multi-strain probiotics&#x2014;helped identify potential sources of heterogeneity. A sensitivity analysis was performed to evaluate the impact of excluding a specific study on the overall ES. For outcomes that included a minimum of 10 studies, both Egger&#x2019;s and Begg&#x2019;s tests were applied, alongside a visual assessment of funnel plots, to explore the presence of small study effects (<xref ref-type="bibr" rid="ref25 ref26 ref27">25&#x2013;27</xref>). All statistical analyses were conducted using STATA version 16.0 (Stata Corporation, College Station, TX, US). A <italic>p</italic>-value of less than 0.05 was deemed significant.</p>
</sec>
</sec>
<sec sec-type="results" id="sec12">
<title>Results</title>
<sec id="sec13">
<title>Study selection</title>
<p>According to systematic search on above mentioned databases, 956 records were identified. Then, 163 duplicates were removed to screen the title and abstract of remained studies thoroughly. Afterward, 793 records were excluded and 43 studies were evaluated using full-text. Finally, eight studies were excluded by reason: studies that have used probiotics in combination with other compounds (<italic>n</italic>&#x202F;=&#x202F;2), studies that assessed the effect of synbiotics (<italic>n</italic>&#x202F;=&#x202F;3), studies with other languages (<italic>n</italic>&#x202F;=&#x202F;1), and irrelevant studies (<italic>n</italic>&#x202F;=&#x202F;2). In the end, a total of 35 studies met our specified inclusion criteria. A summary of the study selection process is provided in <xref ref-type="fig" rid="fig1">Figure 1</xref>.</p>
<fig position="float" id="fig1">
<label>Figure 1</label>
<caption>
<p>PRISMA flow diagram.</p>
</caption>
<graphic xlink:href="fnut-12-1606264-g001.tif">
<alt-text content-type="machine-generated">Flowchart of a study selection process for a meta-analysis. It starts with 956 records identified through database searching. After removing duplicates, 793 records remain. 750 articles are excluded based on title and abstract. Forty-three full-text articles are evaluated for eligibility, with eight articles excluded for reasons such as assessing probiotics with drugs or synbiotics, different languages, or irrelevance. Finally, 35 studies are included in the meta-analysis.</alt-text>
</graphic>
</fig>
</sec>
<sec id="sec14">
<title>Study characteristics</title>
<p>In the present systematic review, a total of 35 systematic reviews were included (<xref ref-type="table" rid="tab1">Table 1</xref>). All these studies were published between 2002 and 2024. The following are the number of meta-analyses for the outcomes across included studies: Diarrhea (prevention-RR): <italic>n</italic>&#x202F;=&#x202F;4, Diarrhea (prevention-OR): <italic>n</italic>&#x202F;=&#x202F;1, Diarrhea (Incidence-OR): <italic>n</italic>&#x202F;=&#x202F;6, Diarrhea (Incidence-RR): <italic>n</italic>&#x202F;=&#x202F;9, Diarrhea (Treatment-SMD): <italic>n</italic>&#x202F;=&#x202F;3, Diarrhea duration: <italic>n</italic>&#x202F;=&#x202F;19, Constipation: <italic>n</italic>&#x202F;=&#x202F;3, and, Stool frequency (WMD): <italic>n</italic>&#x202F;=&#x202F;3. The age range of included children and adults was &#x003C;18&#x202F;years old. The most used probiotics were <italic>Lacticaseibacillus. acidophilus, Lacticaseibacillus. reuteri, Lacticaseibacillus. casei, Lacticaseibacillus. Bulgaricus, Streptococcus thermophilus, S. boulardii, Bifidobacterium, B. longum</italic> and mix of probiotics (<italic>Bifidobacterium, Lacticaseibacillus</italic>, and <italic>Streptococcus</italic>).</p>
<table-wrap position="float" id="tab1">
<label>Table 1</label>
<caption>
<p>Study characteristics of included studies.</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th align="left" valign="top">References</th>
<th align="center" valign="top">Location</th>
<th align="center" valign="top">No. of studies in meta-analysis</th>
<th align="center" valign="top">No. of participants in meta-analysis</th>
<th align="center" valign="top">Age (year)</th>
<th align="center" valign="top">Intervention</th>
<th align="center" valign="top">Health condition</th>
<th align="center" valign="top">Outcomes</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="middle">Higuchi et al. (<xref ref-type="bibr" rid="ref54">54</xref>)</td>
<td align="center" valign="middle">Japan</td>
<td align="center" valign="middle">7</td>
<td align="center" valign="middle">878</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle">Probiotic</td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Higuchi et al. (<xref ref-type="bibr" rid="ref54">54</xref>)</td>
<td align="center" valign="middle">Japan</td>
<td align="center" valign="middle">14</td>
<td align="center" valign="middle">1761</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle">Probiotic</td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Liang et al. (<xref ref-type="bibr" rid="ref55">55</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">1,465</td>
<td align="center" valign="middle">&#x003C;1</td>
<td align="center" valign="middle">Probiotic</td>
<td align="center" valign="middle">Infant</td>
<td align="center" valign="middle">Diarrhea incidence (RR) (NS)</td>
</tr>
<tr>
<td align="left" valign="middle">Fu et al. (<xref ref-type="bibr" rid="ref56">56</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">8</td>
<td align="center" valign="middle">1,051</td>
<td align="center" valign="middle">&#x003C;7</td>
<td align="center" valign="middle"><italic>S. boulardii</italic></td>
<td align="center" valign="middle">Children with diarrhea</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Cheng et al. (<xref ref-type="bibr" rid="ref57">57</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">6</td>
<td align="center" valign="middle">698</td>
<td align="center" valign="middle">&#x003C;12</td>
<td align="center" valign="middle"><italic>L. acidophilus</italic></td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">Diarrhea duration (NS)</td>
</tr>
<tr>
<td align="left" valign="middle">Cheng et al. (<xref ref-type="bibr" rid="ref57">57</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">9</td>
<td align="center" valign="middle">1765</td>
<td align="center" valign="middle">&#x003C;12</td>
<td align="center" valign="middle">Mix</td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Huang et al. (<xref ref-type="bibr" rid="ref31">31</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">6</td>
<td align="center" valign="middle">668</td>
<td align="center" valign="middle">&#x003C;6</td>
<td align="center" valign="middle"><italic>L. reuteri, L. casei, S. boulardii, L. rhamnosus</italic></td>
<td align="center" valign="middle">Children with diarrhea</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2191; OR)</td>
</tr>
<tr>
<td align="left" valign="middle">Huang et al. (<xref ref-type="bibr" rid="ref31">31</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">10</td>
<td align="center" valign="middle">2,223</td>
<td align="center" valign="middle">&#x003C;6</td>
<td align="center" valign="middle"><italic>L. reuteri, L. rhamnosus, L. acidophilus, S. boulardii</italic>, Mix</td>
<td align="center" valign="middle">Children with diarrhea</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Wu et al. (<xref ref-type="bibr" rid="ref58">58</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">12</td>
<td align="center" valign="middle">1907</td>
<td align="center" valign="middle">&#x003C;5</td>
<td align="center" valign="middle"><italic>L. rhamnosus, L. acidophilus L. sporogenes, L. reuteri, S. boulardii</italic>, Mix</td>
<td align="center" valign="middle">Acute diarrhea</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Szajewska et al. (<xref ref-type="bibr" rid="ref59">59</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">23</td>
<td align="center" valign="middle">3,450</td>
<td align="center" valign="middle">&#x003C;15</td>
<td align="center" valign="middle"><italic>S. boulardii</italic></td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Li et al. (<xref ref-type="bibr" rid="ref60">60</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">517</td>
<td align="center" valign="middle">&#x003C;3</td>
<td align="center" valign="middle"><italic>L. rhamnosus</italic></td>
<td align="center" valign="middle">Acute pediatric diarrhea (&#x003E;3&#x202F;days)</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; OR)</td>
</tr>
<tr>
<td align="left" valign="middle">Li et al. (<xref ref-type="bibr" rid="ref60">60</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">475</td>
<td align="center" valign="middle">&#x003C;3</td>
<td align="center" valign="middle"><italic>L. rhamnosus</italic></td>
<td align="center" valign="middle">Acute pediatric diarrhea (&#x003E;4&#x202F;days)</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; OR)</td>
</tr>
<tr>
<td align="left" valign="middle">Li et al. (<xref ref-type="bibr" rid="ref60">60</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">7</td>
<td align="center" valign="middle">973</td>
<td align="center" valign="middle">&#x003C;5</td>
<td align="center" valign="middle"><italic>L. rhamnosus</italic></td>
<td align="center" valign="middle">Acute pediatric diarrhea</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Yang et al.(<xref ref-type="bibr" rid="ref17">17</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">12</td>
<td align="center" valign="middle">925</td>
<td align="center" valign="middle">15.86</td>
<td align="center" valign="middle">Mix</td>
<td align="center" valign="middle">Children with acute diarrhea</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Fang et al. (<xref ref-type="bibr" rid="ref61">61</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">358</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle">Lactobacillus</td>
<td align="center" valign="middle"><italic>Helicobacter pylori</italic> patients</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Szajewska et al. (<xref ref-type="bibr" rid="ref62">62</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">15</td>
<td align="center" valign="middle">3,820</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. rhamnosus</italic></td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Patro-Go&#x0142;a et al. (<xref ref-type="bibr" rid="ref63">63</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">220</td>
<td align="center" valign="middle">&#x003C;5</td>
<td align="center" valign="middle"><italic>L. reuteri</italic></td>
<td align="center" valign="middle">Acute gastroenteritis</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Patro-Go&#x0142; et al. (<xref ref-type="bibr" rid="ref63">63</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">2</td>
<td align="center" valign="middle">160</td>
<td align="center" valign="middle">&#x003C;5</td>
<td align="center" valign="middle"><italic>L. reuteri</italic></td>
<td align="center" valign="middle">Acute gastroenteritis with diarrhea</td>
<td align="center" valign="middle">Stool frequency (NS)</td>
</tr>
<tr>
<td align="left" valign="middle">Harris et al. (<xref ref-type="bibr" rid="ref16">16</xref>)</td>
<td align="center" valign="middle">Australia</td>
<td align="center" valign="middle">11</td>
<td align="center" valign="middle">835</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. reuteri</italic>, Mix</td>
<td align="center" valign="middle">Patients with functional constipation</td>
<td align="center" valign="middle">Constipation (&#x2191; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Harris et al. (<xref ref-type="bibr" rid="ref16">16</xref>)</td>
<td align="center" valign="middle">Australia</td>
<td align="center" valign="middle">14</td>
<td align="center" valign="middle">965</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. reuteri</italic>, Mix</td>
<td align="center" valign="middle">Patients with functional constipation</td>
<td align="center" valign="middle">Stool frequency (NS)</td>
</tr>
<tr>
<td align="left" valign="middle">Ianiro et al. (<xref ref-type="bibr" rid="ref64">64</xref>)</td>
<td align="center" valign="middle">Italy</td>
<td align="center" valign="middle">6</td>
<td align="center" valign="middle">919</td>
<td align="center" valign="middle">3.82</td>
<td align="center" valign="middle"><italic>Bacillus clausii</italic></td>
<td align="center" valign="middle">Acute diarrhea</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Ianiro et al. (<xref ref-type="bibr" rid="ref64">64</xref>)</td>
<td align="center" valign="middle">Italy</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">689</td>
<td align="center" valign="middle">4.02</td>
<td align="center" valign="middle"><italic>Bacillus clausii</italic></td>
<td align="center" valign="middle">Acute diarrhea</td>
<td align="center" valign="middle">&#x2193; Stool frequency</td>
</tr>
<tr>
<td align="left" valign="middle">Jin et al. (<xref ref-type="bibr" rid="ref21">21</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">382</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. casei, L. rhamnosus, Bifidobacterium</italic></td>
<td align="center" valign="middle">Functional constipated children</td>
<td align="center" valign="middle">Constipation (RR) (NS)</td>
</tr>
<tr>
<td align="left" valign="middle">Xu et al. (<xref ref-type="bibr" rid="ref28">28</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">21</td>
<td align="center" valign="middle">7,225</td>
<td align="center" valign="middle">&#x003C;12</td>
<td align="center" valign="middle">Bifidobacterium</td>
<td align="center" valign="middle">Pediatric antibiotic-associated diarrhea</td>
<td align="center" valign="middle">Prevention of diarrhea (&#x2193; OR), Incidence diarrhea (&#x2193; OR)</td>
</tr>
<tr>
<td align="left" valign="middle">Huang et al. (<xref ref-type="bibr" rid="ref9">9</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">267</td>
<td align="center" valign="middle">5.62</td>
<td align="center" valign="middle">Mix</td>
<td align="center" valign="middle">Constipated children</td>
<td align="center" valign="middle">Stool consistency (NS)</td>
</tr>
<tr>
<td align="left" valign="middle">Lau et al. (<xref ref-type="bibr" rid="ref65">65</xref>)</td>
<td align="center" valign="middle">US</td>
<td align="center" valign="middle">4</td>
<td align="center" valign="middle">888</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle">Mix</td>
<td align="center" valign="middle">Inpatients and outpatients</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Urbanska et al. (<xref ref-type="bibr" rid="ref66">66</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">256</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. reuteri</italic></td>
<td align="center" valign="middle">Diarrhoeal diseases</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Szajewska et al. (<xref ref-type="bibr" rid="ref67">67</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">5</td>
<td align="center" valign="middle">445</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. rhamnosus</italic></td>
<td align="center" valign="middle">Antibiotic-associated diarrhea</td>
<td align="center" valign="middle">Prevention of diarrhea (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Ahmadi et al. (<xref ref-type="bibr" rid="ref68">68</xref>)</td>
<td align="center" valign="middle">Iran</td>
<td align="center" valign="middle">17</td>
<td align="center" valign="middle">1,149</td>
<td align="center" valign="middle">&#x003C;6</td>
<td align="center" valign="middle"><italic>L. rhamnosus, L. casei, L. acidophilus, L. reuteri, S. boulardii</italic></td>
<td align="center" valign="middle">Acute rotavirus diarrhea</td>
<td align="center" valign="middle">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="middle">Wanke et al. (<xref ref-type="bibr" rid="ref69">69</xref>)</td>
<td align="center" valign="middle">Poland</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">1,043</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle"><italic>L. rhamnosus</italic></td>
<td align="center" valign="middle">Healthcare-associated diarrhea</td>
<td align="center" valign="middle">Prevention of diarrhea (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="middle">Li et al. (<xref ref-type="bibr" rid="ref29">29</xref>)</td>
<td align="center" valign="middle">China</td>
<td align="center" valign="middle">3</td>
<td align="center" valign="middle">217</td>
<td align="center" valign="middle">&#x003C;18</td>
<td align="center" valign="middle">Mix</td>
<td align="center" valign="middle"><italic>Helicobacter pylori</italic> patients</td>
<td align="center" valign="middle">Diarrhea incidence (&#x2193; OR)</td>
</tr>
<tr>
<td align="left" valign="top">Li et al. (<xref ref-type="bibr" rid="ref29">29</xref>)</td>
<td align="center" valign="top">China</td>
<td align="center" valign="top">2</td>
<td align="center" valign="top">151</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top">Mix</td>
<td align="center" valign="top"><italic>Helicobacter pylori</italic> patients</td>
<td align="center" valign="top">Constipation (OR) (NS)</td>
</tr>
<tr>
<td align="left" valign="top">Szajewska et al. (<xref ref-type="bibr" rid="ref70">70</xref>)</td>
<td align="center" valign="top">Poland</td>
<td align="center" valign="top">11</td>
<td align="center" valign="top">2,444</td>
<td align="center" valign="top">&#x003C;6</td>
<td align="center" valign="top">Lactobacillus</td>
<td align="center" valign="top">Acute gastroenteritis</td>
<td align="center" valign="top">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="top">Videlock et al. (<xref ref-type="bibr" rid="ref71">71</xref>)</td>
<td align="center" valign="top">US</td>
<td align="center" valign="top">10</td>
<td align="center" valign="top">1,246</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top">Probiotics</td>
<td align="center" valign="top">Antibiotic-associated diarrhea</td>
<td align="center" valign="top">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="top">Salari et al. (<xref ref-type="bibr" rid="ref72">72</xref>)</td>
<td align="center" valign="top">Iran</td>
<td align="center" valign="top">19</td>
<td align="center" valign="top">3,787</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>L. acidophilus, L. rhamnosus, L. paracasei, L. casei, S. boulardii, S. thermophilus</italic>, Mix</td>
<td align="center" valign="top">Acute diarrhea</td>
<td align="center" valign="top">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="top">Szajewska et al. (<xref ref-type="bibr" rid="ref73">73</xref>)</td>
<td align="center" valign="top">Poland</td>
<td align="center" valign="top">2</td>
<td align="center" valign="top">823</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>L. rhamnosus</italic></td>
<td align="center" valign="top">Healthcare-associated diarrhea</td>
<td align="center" valign="top">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="top">Kale-Pradhan et al. (<xref ref-type="bibr" rid="ref74">74</xref>)</td>
<td align="center" valign="top">US</td>
<td align="center" valign="top">4</td>
<td align="center" valign="top">585</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top">Lactobacillus</td>
<td align="center" valign="top">Antibiotic-associated diarrhea</td>
<td align="center" valign="top">Prevention of diarrhea (RR) (NS)</td>
</tr>
<tr>
<td align="left" valign="top">Szajewska et al. (<xref ref-type="bibr" rid="ref75">75</xref>)</td>
<td align="center" valign="top">Poland</td>
<td align="center" valign="top">4</td>
<td align="center" valign="top">1,305</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>S. boulardii</italic></td>
<td align="center" valign="top"><italic>Helicobacter pylori</italic> patients</td>
<td align="center" valign="top">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="top">Chmielewska et al. (<xref ref-type="bibr" rid="ref76">76</xref>)</td>
<td align="center" valign="top">Poland</td>
<td align="center" valign="top">2</td>
<td align="center" valign="top">106</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>L. reuteri</italic></td>
<td align="center" valign="top">Acute infectious diarrhea</td>
<td align="center" valign="top">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="top">Szajews et al. (<xref ref-type="bibr" rid="ref77">77</xref>)</td>
<td align="center" valign="top">Poland</td>
<td align="center" valign="top">7</td>
<td align="center" valign="top">876</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top">Lactobacillus</td>
<td align="center" valign="top">Acute infectious diarrhea</td>
<td align="center" valign="top">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="top">Szajewsk et al. (<xref ref-type="bibr" rid="ref78">78</xref>)</td>
<td align="center" valign="top">Poland</td>
<td align="center" valign="top">4</td>
<td align="center" valign="top">473</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>S. boulardii</italic></td>
<td align="center" valign="top">Acute infectious diarrhea</td>
<td align="center" valign="top">&#x2193; Diarrhea duration</td>
</tr>
<tr>
<td align="left" valign="top">Johnston et al.(<xref ref-type="bibr" rid="ref19">19</xref>)</td>
<td/>
<td align="center" valign="top">6</td>
<td align="center" valign="top">707</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>Lactobacillus, S. boulardii</italic></td>
<td align="center" valign="top">Antibiotic-associated diarrhea</td>
<td align="center" valign="top">Diarrhea incidence (&#x2193; RR)</td>
</tr>
<tr>
<td align="left" valign="top">D&#x2019;Souza et al. (<xref ref-type="bibr" rid="ref50">50</xref>)</td>
<td align="center" valign="top">UK</td>
<td align="center" valign="top">9</td>
<td align="center" valign="top">623</td>
<td align="center" valign="top">&#x003C;18</td>
<td align="center" valign="top"><italic>Lactobacillus, S. boulardii</italic>, Mix</td>
<td align="center" valign="top">Antibiotic associated diarrhea</td>
<td align="center" valign="top">Diarrhea incidence (&#x2193; OR)</td>
</tr>
<tr>
<td align="left" valign="top">Huang et al. (<xref ref-type="bibr" rid="ref18">18</xref>)</td>
<td align="center" valign="top">US</td>
<td align="center" valign="top">25</td>
<td align="center" valign="top">1917</td>
<td align="center" valign="top">&#x003C;5</td>
<td align="center" valign="top">Mix</td>
<td align="center" valign="top">Acute diarrhea</td>
<td align="center" valign="top">&#x2193; Diarrhea duration</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="sec15">
<title>Risk of bias assessment</title>
<p>The risk of bias for included studies was assessed using AMSTAR questionnaire. Detailed results are presented in <xref ref-type="supplementary-material" rid="SM1">Supplementary Table 1</xref>.</p>
</sec>
<sec id="sec16">
<title>Probiotics supplementation on prevention of diarrhea</title>
<p>The utilized random effect model revealed that probiotics significantly reduced the relative risk of diarrhea by 36% compared to the control group (RR&#x202F;=&#x202F;0.64; 95% CI: 0.63, 0.65, <italic>p</italic>&#x202F;&#x003C;&#x202F;0.001) without heterogeneity (<italic>I</italic><sup>2</sup>&#x202F;=&#x202F;0.0%, <italic>p</italic>&#x202F;=&#x202F;0.498) (<xref ref-type="fig" rid="fig2">Figure 2</xref>). This finding is highlighting potential of probiotics as an effective preventive strategy. However, one studies which have reported odds ratio (OR) for preventing diarrhea, were included in our systematic review (RR&#x202F;=&#x202F;0.34; 95% CI: 0.28, 0.41, <italic>p</italic>&#x202F;&#x003C;&#x202F;0.05) (<xref ref-type="bibr" rid="ref28">28</xref>). Moreover, sensitivity analysis demonstrated that no study could affect the pooled effect size. Furthermore, no evidence for publication bias based on Begg&#x2019;s (<italic>p</italic>&#x202F;=&#x202F;0.999).</p>
<fig position="float" id="fig2">
<label>Figure 2</label>
<caption>
<p>Mean difference and 95% CIs presented in forest plot of the studies on the effects of probiotics on prevention of diarrhea.</p>
</caption>
<graphic xlink:href="fnut-12-1606264-g002.tif">
<alt-text content-type="machine-generated">Forest plot displaying effect sizes (ES) and confidence intervals (CI) for four studies on a vertical axis. Studies are listed with their effect sizes: Kale-Pradhan et al (2010), Wanke et al (2014), Szajewska et al (2015), Agamennone et al (2018), and an overall effect. Most weight is on Agamennone et al (2018) with ES of 0.64 (CI 0.63, 0.65). The plot includes a dashed line at ES of 1 and notes weights from random effects analysis.</alt-text>
</graphic>
</fig>
</sec>
<sec id="sec17">
<title>Probiotics supplementation on incidence of diarrhea (OR)</title>
<p>The pooled effect size revealed that probiotic intervention significantly reduced the odds of diarrhea incidence by 49% compared to the control group (OR&#x202F;=&#x202F;0.51; 95% CI: 0.27, 0.94 <italic>p</italic>&#x202F;=&#x202F;0.032; <italic>I</italic><sup>2</sup>&#x202F;=&#x202F;93.3%, p-heterogeneity &#x003C;0.001) (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). Moreover, single-strain probiotics could exert beneficial effects in relation to reducing the OR of diarrhea incidence based on subgroup analysis (<xref ref-type="supplementary-material" rid="SM1">Supplementary Table 2</xref>). Furthermore, no single study effect was seen to affect the overall effect size of diarrhea incidence (OR). Furthermore, no evidence for publication bias was seen based on Begg&#x2019;s (<italic>p</italic>&#x202F;=&#x202F;0.904).</p>
<fig position="float" id="fig3">
<label>Figure 3</label>
<caption>
<p>Mean difference and 95% CIs presented in forest plot of the studies on the effects of probiotics on incidence diarrhea based on OR <bold>(A)</bold>, and RR <bold>(B)</bold> analysis.</p>
</caption>
<graphic xlink:href="fnut-12-1606264-g003.tif">
<alt-text content-type="machine-generated">Forest plots comparing studies. Panel A shows studies by D'Souza et al. (2002) to Huang et al. (2021) with an overall effect size of 0.51 (CI: 0.27, 0.94), I-squared 93.3%. Panel B includes studies from Johnston et al. (2006) to Higuchi et al. (2024), with an overall effect size of 0.54 (CI: 0.40, 0.71), I-squared 88.7%. Both panels note weights from random effects analysis, indicating variations and respective confidence intervals of each study.</alt-text>
</graphic>
</fig>
</sec>
<sec id="sec18">
<title>Probiotics supplementation on incidence of diarrhea (RR)</title>
<p>The analysis of the impact of probiotics on diarrhea incidence (RR) involving 8,595 children. Probiotic administration has significantly decreased risk of diarrhea incidence by 46% compared to control group (RR&#x202F;=&#x202F;0.54; 95% CI: 0.40, 0.71, <italic>p</italic>&#x202F;&#x003C;&#x202F;0.001) (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Besides, significant between-study heterogeneity has been detected (<italic>I</italic><sup>2</sup>&#x202F;=&#x202F;88.7%, and <italic>p</italic>&#x202F;&#x003C;&#x202F;0.001). Additionally, both single-strain and multi-strain probiotics were associated with positive effects in reducing diarrhea incidence (RR) (<xref ref-type="supplementary-material" rid="SM1">Supplementary Table 2</xref>). Based on sensitivity analysis, no significant changes have been identified following removing one single study. Begg&#x2019;s test has shown no significant publication bias (<italic>p</italic>&#x202F;=&#x202F;0.09).</p>
</sec>
<sec id="sec19">
<title>Probiotics supplementation on duration of diarrhea</title>
<p>The overall effect of probiotic supplementation on duration of diarrhea was analyzed across 18 studies with 19 ESs. The pooled analysis revealed a ES of &#x2212;1.12 (95% CI: &#x2212;1.48 to &#x2212;0.76; <italic>p</italic>&#x202F;&#x003C;&#x202F;0.001; <italic>I</italic><sup>2</sup>&#x202F;=&#x202F;90.9%, <italic>p</italic>&#x202F;&#x003C;&#x202F;0001) (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Subgroup analysis demonstrated that both single- and multi-strain probiotics are effective in reducing the period of diarrhea. Based on subgroup analysis both SMD analysis (SMD&#x202F;=&#x202F;&#x2212;0.94; 95% CI: &#x2212;1.32, &#x2212;0.56, <italic>p</italic>&#x202F;&#x003C;&#x202F;0.001) and WMD analysis (WMD&#x202F;=&#x202F;&#x2212;1.85; 95% CI: &#x2212;2.83, &#x2212;0.86, <italic>p</italic>&#x202F;&#x003C;&#x202F;0.001) demonstrated that probiotics are effective in reducing the period of diarrhea (<xref ref-type="supplementary-material" rid="SM1">Supplementary Table 2</xref>). The effects of probiotics on acute diarrhea and diarrheal diseases were stronger than in other subgroups (<xref ref-type="supplementary-material" rid="SM1">Supplementary Table 2</xref>). In addition, sensitivity analysis revealed that no significant change has been detected following removing one single study. Begg&#x2019;s and Egger&#x2019;s tests, and visual inspection of funnel plot pointed to significant publication bias (<italic>p</italic>&#x202F;&#x003C;&#x202F;0.005).</p>
<fig position="float" id="fig4">
<label>Figure 4</label>
<caption>
<p>Mean difference and 95% CIs presented in forest plot of the studies on the effects of probiotics on duration of diarrhea <bold>(A)</bold>, and diarrhea treatment <bold>(B)</bold>.</p>
</caption>
<graphic xlink:href="fnut-12-1606264-g004.tif">
<alt-text content-type="machine-generated">(A) A forest plot showing effect sizes (ES) and 95% confidence intervals (CI) for multiple studies, with weights from random effects analysis. The overall ES is -1.12 (-1.48, -0.76), with significant heterogeneity (I-squared = 90.9%, p = 0.000).(B) A forest plot illustrating ES and 95% CIs for three studies. The overall ES is -0.49 (-0.59, -0.38), with no significant heterogeneity (I-squared = 0.0%, p = 0.406). Weights are from random effects analysis.</alt-text>
</graphic>
</fig>
</sec>
<sec id="sec20">
<title>Probiotics supplementation on diarrhea treatment</title>
<p>Probiotic administration significantly improved diarrhea treatment (SMD&#x202F;=&#x202F;&#x2212;0.49; 95% CI: &#x2212;0.59, &#x2212;0.38, <italic>p</italic>&#x202F;&#x003C;&#x202F;0001) (<xref ref-type="fig" rid="fig4">Figure 4B</xref>).</p>
</sec>
<sec id="sec21">
<title>Probiotics supplementation on constipation</title>
<p>The overall analysis revealed that probiotics significantly impact on constipation (OR&#x202F;=&#x202F;1.17, 95% CI: 1.01 to 1.37; <italic>p</italic>&#x202F;=&#x202F;0.043, <italic>I</italic><sup>2</sup>&#x202F;=&#x202F;0.0%, <italic>p</italic>&#x202F;=&#x202F;0.583) (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Three meta-analyses (<xref ref-type="bibr" rid="ref16">16</xref>, <xref ref-type="bibr" rid="ref21">21</xref>, <xref ref-type="bibr" rid="ref29">29</xref>) demonstrated limited and inconsistent evidence supporting probiotic use for pediatric functional constipation. While some strains showed modest improvements in stool frequency, significant heterogeneity in study design, dosage, and outcome measures restricted definitive conclusions. These findings underscore the importance of strain- and condition-specific research before recommending probiotics for functional constipation in children.</p>
<fig position="float" id="fig5">
<label>Figure 5</label>
<caption>
<p>Mean difference and 95% CIs presented in forest plot of the studies on the effects of probiotics on constipation <bold>(A)</bold>, and stool frequency <bold>(B)</bold>.</p>
</caption>
<graphic xlink:href="fnut-12-1606264-g005.tif">
<alt-text content-type="machine-generated">Figure A shows a forest plot with three studies, each with effect sizes and confidence intervals. The overall effect size is 1.17. Weights are from random effects analysis. Figure B illustrates another forest plot with three studies. The overall effect size is -0.21, sourced similarly. Both plots include a diamond indicating the overall effect size and confidence interval on a logarithmic scale.</alt-text>
</graphic>
</fig>
</sec>
<sec id="sec22">
<title>Probiotics supplementation on stool frequency and consistency</title>
<p>Probiotics significantly affected stool frequency with a WMD of &#x2212;0.21 (95% CI: &#x2212;0.37 to &#x2212;0.04; <italic>p</italic>&#x202F;=&#x202F;0.015; <italic>I</italic><sup>2</sup>&#x202F;=&#x202F;0.0%, <italic>p</italic>&#x202F;=&#x202F;0.924) (<xref ref-type="fig" rid="fig5">Figure 5B</xref>), but had no effect on stool consistency (WMD&#x202F;=&#x202F;&#x2212;0.07; 95% CI: &#x2212;0.21, 0.06, <italic>P</italic> &#x02C3;0.05).</p>
</sec>
</sec>
<sec sec-type="discussion" id="sec23">
<title>Discussion</title>
<p>The present umbrella of systematic reviews and meta-analysis attempted to summarize the available data evaluating the effect of probiotics on diarrhea and constipation outcomes among children. Accordingly, probiotic supplementation significantly reduced the RR values for diarrhea incidence and prevention in children compared to the control group. This outcome is consistent with a Cochrane review, which concluded that probiotics are effective in preventing antibiotic-associated diarrhea (AAD) in children, with specific strains showing increased effectiveness (<xref ref-type="bibr" rid="ref30">30</xref>). Also, probiotic supplementation was associated with a reduced odds of diarrhea incidence in children specifically. In addition, probiotics were able to shorten the duration of diarrhea in study subjects. Focusing on pediatric populations, a meta-analysis demonstrated that the duration of diarrhea in children receiving probiotics was significantly shorter than in control groups, and the length of hospital stay was also reduced (<xref ref-type="bibr" rid="ref31">31</xref>). All these findings point to beneficial effects of probiotics as an adjunctive approach in managing diarrhea across different age groups.</p>
<p>The types of diarrhea included across the analyzed studies varied and encompassed AAD, infectious diarrhea, and cases where the etiology was not explicitly stated. This heterogeneity may influence the pooled estimates, as the efficacy of probiotics is known to differ depending on the underlying cause (<xref ref-type="bibr" rid="ref30">30</xref>). For example, probiotics such as <italic>Lactobacillus rhamnosus</italic> and <italic>Saccharomyces boulardii</italic> have demonstrated greater efficacy in managing infectious and antibiotic-associated diarrhea, respectively (<xref ref-type="bibr" rid="ref32">32</xref>). Clinicians should consider probiotic strains with well-established efficacy, such as <italic>Lactobacillus rhamnosus</italic> and <italic>Saccharomyces boulardii</italic>, administered at dosages typically ranging from 10<sup>9</sup> to 10<sup>10</sup> colony-forming units (CFU) per day, consistent with doses used in clinical trials (<xref ref-type="bibr" rid="ref33">33</xref>, <xref ref-type="bibr" rid="ref34">34</xref>). Accordingly, the variability in diarrhea type should be considered when interpreting the overall results, and future meta-analyses may benefit from stratifying outcomes based on etiology to enhance clinical relevance.</p>
<p>Based on the available evidence, some probiotic strains were more effective in improving gastrointestinal outcomes in children. <italic>Lactobacillus rhamnosus</italic> GG and Saccharomyces boulardii were consistently associated with significant reductions in the duration and frequency of acute diarrhea, particularly in cases of viral or antibiotic-associated etiology (<xref ref-type="bibr" rid="ref33">33</xref>, <xref ref-type="bibr" rid="ref34">34</xref>). These findings are supported by several high-quality randomized controlled trials with low risk of bias. In contrast, the evidence for constipation was more variable, with <italic>Bifidobacterium lactis</italic> and <italic>Lactobacillus casei rhamnosus</italic> showing moderate effectiveness in improving stool frequency and consistency (<xref ref-type="bibr" rid="ref35">35</xref>). However, the quality of evidence for constipation-related outcomes was generally lower, often limited by small sample sizes and heterogeneous outcome measures.</p>
<p>Moreover, probiotics resulted in decreased stool frequency in children. While, Dong et al., demonstrated improved defecation frequency following probiotic treatment in children (<xref ref-type="bibr" rid="ref36">36</xref>). This discrepancy is justified by smaller number of included studies and lower sample size in Dong et al.&#x2019;s study (<xref ref-type="bibr" rid="ref36">36</xref>), which may affect the statistical power to detect significant changes. Also, this issue shed the light that microecological differences between children and adults may lead to variations in constipation-related outcomes.</p>
<p>Furthermore, odds of diarrhea incidence were significantly affected by single-strain probiotics. Whereas both single-strain and multi-strain probiotics effectively reduced diarrhea duration, as well as the risk of diarrhea incidence. It is worth noting that while single -strain probiotics demonstrated beneficial effects on diarrhea-related outcomes and stool frequency, they were also shown to increase stool consistency in constipated patients. Given this, caution is warranted in interpreting these findings. Accordingly, Schnadower et al. demonstrated that probiotics single-strain (<italic>Lactobacillus rhamnosus</italic>) was ineffective in the treatment of acute enteritis in children (<xref ref-type="bibr" rid="ref37">37</xref>). Similarly, Szyma&#x0144;ski et al. reported no significant changes in relation to diarrhea symptoms following <italic>Lactobacillus reuteri</italic> administration (<xref ref-type="bibr" rid="ref38">38</xref>). However, Zhang et al. highlighted that enhanced diversity of microbiota can improve constipation-related measures significantly. This underscores the effectiveness of multi-strain probiotics in addressing constipation (<xref ref-type="bibr" rid="ref39">39</xref>). Previous studies have demonstrated strain-specific effects of probiotics in different health status (<xref ref-type="bibr" rid="ref40 ref41 ref42">40&#x2013;42</xref>). For example, in constipated children some probiotics have been known as discriminative species such as <italic>Bacteroides</italic> and <italic>Bifidobacterium longum</italic> species (<xref ref-type="bibr" rid="ref43">43</xref>).</p>
<p>Probiotic administration resulted in significant improvements in reducing the duration and frequency of diarrhea in children. This effect was particularly significant in cases of acute viral diarrhea. The therapeutic efficacy of probiotics is also strain-dependent, with specific strains such as <italic>Lactobacillus rhamnosus</italic> and <italic>Saccharomyces boulardii</italic> showing the greatest benefit (<xref ref-type="bibr" rid="ref44">44</xref>). Furthermore, while single-strain and multi-strain formulations have been used, current evidence suggests that clinical outcomes are more influenced by individual characteristics and strain viability than by the number of strains present (<xref ref-type="bibr" rid="ref45">45</xref>). In addition, probiotic supplementation serves as an adjunct to standard diarrhea management protocols.</p>
<p>The relatively modest impact of probiotics on constipation observed in pediatric populations may be attributed to several factors. Constipation is a multifactorial disorder influenced by diet, hydration, physical activity, gut motility, and psychosocial elements, which probiotics alone may not fully address (<xref ref-type="bibr" rid="ref35">35</xref>). Additionally, the heterogeneity of probiotic strains studied, differences in dosage, and variability in intervention duration complicate the interpretation of efficacy (<xref ref-type="bibr" rid="ref46">46</xref>). The mechanisms by which probiotics might alleviate constipation&#x2014;such as modulation of gut microbiota composition, enhancement of short-chain fatty acid production, and improvement of intestinal transit&#x2014;may require longer treatment periods or higher doses than those employed in existing trials (<xref ref-type="bibr" rid="ref47">47</xref>). Furthermore, outcome measures in constipation studies often vary widely, and subjective symptom reporting can affect the reliability of results. Finally, individual differences in baseline microbiota composition may influence response to probiotic supplementation, underscoring the need for personalized approaches in future research (<xref ref-type="bibr" rid="ref48">48</xref>).</p>
<p>Several underlying mechanisms have been proposed through which probiotics influence gastrointestinal disorders such as diarrhea and constipation. Probiotics are known to modulate gut microbiota composition, helping to restore microbial balance. It has been shown that probiotics compete with pathogenic bacteria for adhesion sites which may affect the incidence of infectious diseases causing diarrhea and alleviating constipation (<xref ref-type="bibr" rid="ref49">49</xref>). On the other hand, probiotics stimulate the intestinal immune cells and commensal microflora to regulate immune responses by activation of regulatory T cells (Tregs) (<xref ref-type="bibr" rid="ref50">50</xref>, <xref ref-type="bibr" rid="ref51">51</xref>). Also, probiotics can improve the integrity of the intestinal barrier through strengthening tight junctions. This action prevents the adhesion of pathogenic bacteria to mucosal epithelial cells, and prevents translocation of harmful pathogens and toxins (<xref ref-type="bibr" rid="ref49">49</xref>, <xref ref-type="bibr" rid="ref51">51</xref>). Moreover, probiotics were effective in alleviating inflammation state associated with diarrhea (<xref ref-type="bibr" rid="ref52">52</xref>). It seems that each of these mechanisms may be possible through various strains. For example, <italic>Saccharomyces boulardii</italic> and <italic>lactobacilli</italic> modify immune pathways to remove the pathogens (<xref ref-type="bibr" rid="ref50">50</xref>). <italic>Lactobacilli</italic> increases the production of immunoglobulins in the gut which contribute to production of interferons. It has been demonstrated that <italic>Lactobacillus rhamnosus GG (LGG)</italic> could produce antimicrobial substances to inhibits the growth of <italic>Escherichia coli</italic>, <italic>streptococci</italic>, and <italic>Clostridium difficile</italic> (<xref ref-type="bibr" rid="ref50">50</xref>).</p>
<p>Although probiotics are generally considered safe for use in children, especially in healthy children, long-term safety data remain limited. Most clinical trials have focused on short-term administration during acute illness, and few studies have evaluated long-term or repeated use of probiotics. Rare adverse events, such as infections in immunocompromised individuals, highlight the need for cautious use in vulnerable populations (<xref ref-type="bibr" rid="ref33">33</xref>, <xref ref-type="bibr" rid="ref34">34</xref>). Furthermore, the variability in probiotic formulations and lack of close regulatory oversight may lead to inconsistent safety profiles. Adherence to probiotic supplementation can also affect clinical efficacy, particularly in chronic conditions such as constipation where long-term administration may be necessary. Factors that influence adherence include: formulation palatability, dosing frequency, and caregiver education (<xref ref-type="bibr" rid="ref53">53</xref>). Ensuring user-friendly formulations and clear instructions can improve adherence and optimize outcomes. Future research should prioritize long-term safety monitoring and explore strategies to increase adherence in pediatric populations.</p>
<p>As a strength, this study is a high-level research synthesis method that integrates findings from multiple meta-analyses in relation to effects of probiotics on diarrhea and constipation outcomes. So, this study provides more comprehensive overview of probiotics evidence, reliable conclusions and bias-minimized assessment. Moreover, this study included larger population to analyze the effects of probiotics in children. However, this study had some limitations too. First, several factors including: various type of preparation, purity, storing methods and cold chain principles (maintaining appropriate temperature conditions during storage and transportation to preserve the viability of live microorganisms) may result in different outcomes. Second, the number of studies on diarrhea caused by radiation, chemotherapy, HIV, and <italic>Clostridium difficile</italic>&#x2013;associated diarrhea was insufficient to reach a confirm conclusion. Third, it is important to highlight changes in the intestinal flora, which were not reported in the included studies. Forth, included studies were heterogeneous in term of sex distribution.</p>
</sec>
<sec sec-type="conclusions" id="sec24">
<title>Conclusion</title>
<p>This umbrella of meta-analysis supports the potential role of probiotics in relation to diarrhea and constipation outcome in children. Probiotic supplementation contributed to a declined risk and odds of diarrhea incidence compared to control group. Additionally, they were able to shorten the duration of diarrhea and did not increase stool frequency and consistency too. Furthermore, subgroup analysis revealed that both single and multi-strain probiotics were able to reduce the diarrhea duration and the risk of diarrhea incidence. Therefore, further investigations on special types of the probiotics, their purity, and their combination with prebiotics may be much helpful.</p>
</sec>
</body>
<back>
<sec sec-type="data-availability" id="sec25">
<title>Data availability statement</title>
<p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/<xref ref-type="supplementary-material" rid="SM1">Supplementary material</xref>.</p>
</sec>
<sec sec-type="author-contributions" id="sec26">
<title>Author contributions</title>
<p>QW: Conceptualization, Writing &#x2013; original draft, Software, Funding acquisition, Visualization, Resources, Writing &#x2013; review &#x0026; editing, Investigation, Project administration, Validation, Formal analysis, Methodology, Supervision, Data curation. TR: Investigation, Resources, Visualization, Supervision, Funding acquisition, Conceptualization, Software, Validation, Formal analysis, Project administration, Writing &#x2013; review &#x0026; editing, Data curation, Methodology, Writing &#x2013; original draft. HW: Conceptualization, Project administration, Validation, Data curation, Resources, Supervision, Methodology, Writing &#x2013; original draft, Funding acquisition, Software, Investigation, Visualization, Formal analysis, Writing &#x2013; review &#x0026; editing. XL: Validation, Formal analysis, Project administration, Visualization, Supervision, Data curation, Methodology, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing, Funding acquisition, Conceptualization, Software, Investigation, Resources.</p>
</sec>
<sec sec-type="funding-information" id="sec27">
<title>Funding</title>
<p>The author(s) declare that no financial support was received for the research and/or publication of this article.</p>
</sec>
<sec sec-type="COI-statement" id="sec28">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="sec99">
<title>Correction note</title>
<p>This article has been corrected with minor changes. These changes do not impact the scientific content of the article.</p>
</sec>
<sec sec-type="ai-statement" id="sec29">
<title>Generative AI statement</title>
<p>The authors declare that no Gen AI was used in the creation of this manuscript.</p>
</sec>
<sec sec-type="disclaimer" id="sec30">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec sec-type="supplementary-material" id="sec31">
<title>Supplementary material</title>
<p>The Supplementary material for this article can be found online at: <ext-link xlink:href="https://www.frontiersin.org/articles/10.3389/fnut.2025.1606264/full#supplementary-material" ext-link-type="uri">https://www.frontiersin.org/articles/10.3389/fnut.2025.1606264/full#supplementary-material</ext-link></p>
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</sec>
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