PONE-D-24-51052Microbiome and pediatric leukemia, diabetes, and allergies: systematic review and meta-analysisPLOS ONE

Dear Dr. Gallant,

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Academic Editor

PLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: After reviewing the manuscript titled "Microbiome and pediatric leukemia, diabetes, and allergies: systematic review and meta-analysis", here are several weaknesses and areas for improvement:

The manuscript effectively addresses an important research area but could be improved in several aspects:

1. While the manuscript includes a robust meta-analysis, the included studies exhibit substantial heterogeneity in design, microbiome measurement methods, and timing of microbiome assessment. This variability weakens the strength of the conclusions, as the lack of uniformity may introduce biases or confounders that are not adequately accounted for.

2. Limiting the included studies to English-only publications may introduce language bias, potentially excluding relevant high-quality research published in other languages.

3. Although the study discusses confounders such as antibiotic use, breastfeeding, and delivery mode, these factors are not uniformly addressed across the included studies. A more rigorous subgroup analysis or adjustment for these confounders would enhance the reliability of the findings.

4. The meta-analysis focuses exclusively on alpha diversity metrics (e.g., Shannon Index). While this is an important measure, other diversity indices and taxonomic composition metrics, such as beta diversity, could provide deeper insights into microbiome changes.

5. Many studies included are cross-sectional. A greater emphasis on longitudinal studies would provide a better understanding of causality and temporal dynamics of the microbiome in disease progression.

6. Although funnel plots were used to assess publication bias, these may not fully capture biases inherent to microbiome research, such as selective reporting of positive findings. This should be discussed further.

7. There is variability in the demographic details reported across studies, including ethnicity, socioeconomic factors, and geographic location. These factors significantly impact microbiome composition and should be more systematically accounted for.

8. Some results, particularly for T1DM, fail to reach statistical significance, yet are discussed as trends. This could mislead readers into overestimating the strength of these associations.

9. The clustering analysis comparing disease-related microbiome profiles with delivery mode and breastfeeding status is intriguing but appears speculative without robust statistical validation. Including additional data to support these claims would improve the scientific rigor.

10. The manuscript highlights the potential for microbiome-based interventions but fails to provide detailed, evidence-based recommendations or an actionable framework for implementing such strategies in clinical practice.

Addressing these issues would enhance the manuscript's clarity, scientific rigor, and impact. Future iterations should strive for a more standardized approach to data inclusion and analysis, and further explore longitudinal, diverse, and multi-factorial datasets to strengthen the findings.

Reviewer #2: This manuscript offers an insightful and timely exploration of the relationship between gut microbiome diversity and pediatric conditions like acute lymphoblastic leukemia (ALL), type 1 diabetes mellitus (T1DM), and allergic disorders. It’s clear that a lot of thought and effort went into this work, and it adheres to rigorous scientific standards. The authors followed PRISMA guidelines carefully, and the inclusion and exclusion criteria are well-justified. The findings—particularly the link between reduced microbiome diversity and conditions like ALL and eczema—are compelling and well-supported by the data. I also appreciate the use of subgroup analyses and sensitivity tests to address key confounders, like antibiotic use or insulin therapy, which strengthens the validity of the conclusions. That said, while trends were observed for T1DM and asthma, the lack of statistical significance here deserves a little more reflection on the possible limitations.

The statistical approach was thoughtful and thorough. The random-effects model used for the meta-analysis was appropriate given the variability across studies, and funnel plots helped confirm that publication bias wasn’t an issue. Using the Shannon Index as a measure of alpha diversity was a smart choice, as it’s both widely recognized and relevant. I did wonder, though, whether incorporating meta-regression might have added even more depth—especially for teasing apart how factors like study design or age at diagnosis could influence the results. Additionally, the wide confidence intervals in some cases, such as for T1DM, suggest there might be more to discuss regarding the power or variability of certain analyses.

In terms of writing, the manuscript is clear and easy to follow, though a few areas might benefit from some streamlining. The Methods and Results sections, in particular, are pretty dense with statistical details, which might make them tough for readers who aren’t deeply familiar with meta-analysis. Simplifying some of this language or adding a brief summary in plain terms could make these sections more approachable. The Discussion is well thought out and comprehensive, but some of the transitions—especially between microbiome findings and the interplay with genetic or environmental factors—could flow a bit more smoothly. A little more context around some of the more technical methods, like clustering analysis or the use of the ComplexHeatmap package, would also help make this work more accessible to a wider audience.

The authors have done an excellent job ensuring data transparency. They clearly state that all data used are from publicly available sources, with summary statistics compiled and available upon request. Even for studies where data had to be estimated from graphical representations, the process is well-documented, which is reassuring. This level of transparency is commendable and aligns with best practices in systematic reviews.

Overall, this study makes a strong contribution to the growing body of research on the microbiome’s role in childhood diseases. The connections drawn between early-life exposures, microbiome diversity, and disease risk are fascinating and highlight exciting possibilities for future research. The idea of using microbiome-based interventions, like probiotics or dietary modifications, as preventative strategies is particularly intriguing. I’d encourage the authors to dive a little deeper into the practical challenges and limitations of applying these findings clinically. For instance, how feasible is it to use microbiome diversity as a screening tool, or how realistic are these interventions in a real-world healthcare setting?

In summary, this is an impressive piece of work that brings meaningful insights to an important area of research. A few tweaks to improve accessibility, clarify some transitions, and expand on the clinical implications would make it even stronger. I look forward to seeing how this research develops and its potential impact on pediatric care.

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Reviewer #1: No

Reviewer #2: No

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Microbiome and pediatric leukemia, diabetes, and allergies: systematic review and meta-analysis

PONE-D-24-51052R1

Dear Dr. Gallant,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Fahrul Nurkolis

Academic Editor

PLOS ONE

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