Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

This research aims to demonstrate the longstanding anecdotal knowledge that chronic adenoid disease with infection (adenoinitis) contributes to adenoid size and affects tubal function. It certainly proves a point in that direction, but there is a serious caveat in the study's design, namely, sample acquisition. Samples from the test and control groups are obtained in two different ways. My main concern is applying an antiseptic solution before obtaining the sample. As I understand it, the patient is in the supine position at the time the antiseptic solution is applied, and measures to prevent the solution from reaching the nasopharynx are not described, nor is there any acknowledgment of this in the manuscript. Another matter that needs to be addressed is the fact that one sample was effectively taken from "dead" tissue. I am sure that it is hard to assess how this affects the sample, but it needs to be acknowledged anyway.

References No. 23 and 24 seem to address similar clinical questions. Are those cohorts that are the same as in this article?

Once the above-mentioned  points are addressed, the article bears quite a clinical signifcance in the field. 

Author Response

We would like to thank the reviewer for the valuable insight and on-point suggestions for corrections!

Comment 1: My main concern is applying an antiseptic solution before obtaining the sample. As I understand it, the patient is in the supine position at the time the antiseptic solution is applied, and measures to prevent the solution from reaching the nasopharynx are not described, nor is there any acknowledgment of this in the manuscript.

We appreciate you highlighting this important  detail. The chlorhexidine-based antiseptic solution is applied strictly via targeted, moistened sponges to the perioral region, gums, and buccal mucosa. It is not administered as a wash, aerosol, or free-flowing liquid, which prevents the migration of the solution into the nasopharyngeal space. We have clarified this localized application technique in the Materials and Methods section (2.3.1)  

Comment 2: Another matter that needs to be addressed is the fact that one sample was effectively taken from "dead" tissue. I am sure that it is hard to assess how this affects the sample, but it needs to be acknowledged anyway.

Thank you for your observation. The adenoid tissue is swabbed immediately upon removal and its placement on the sterile field. While the negligible ischemia time ensures that the established mucosal biofilm architecture remains intact for 16S profiling, we agree that this methodological asymmetry compared to in vivo control swabs must be acknowledged. We have added a paragraph to the Limitations section of the Discussion explicitly acknowledging this ex vivo versus in vivo acquisition and its hypothetical impact on spatial biomass yield. We have also added a clinical justification to the Methods section (2.3.2) explaining the strict ethical and legal limitations regarding surgical tissue removal in healthy pediatric controls.

Comment 3: References No. 23 and 24 seem to address similar clinical questions. Are those cohorts that are the same as in this article?

The cohort in the present study builds directly upon the preliminary dataset utilized in our previous  studies  by incorporating 20 additional pediatric samples. This expanded cohort was subsequently evaluated utilizing more robust differential abundance methodologies (ANCOM-BC2 and DESeq2) to validate our initial findings with significantly higher statistical power. We have explicitly clarified this cohort relationship and the addition of the new samples in the Materials and Methods section (2.2).

Once again, thank you for your time!

The authors of the manuscript

Reviewer 2 Report

Comments and Suggestions for Authors

Overview

The present manuscript investigates the nasopharyngeal and adenoidal microbiota in a cohort of 78 pediatric patients, stratified into three groups: isolated adenoid hypertrophy (AH), adenoid hypertrophy with concurrent otitis media with effusion (AH+OME), and healthy controls.  It is noteworthy that microbial profiles between isolated AH and AH+OME exhibit striking similarity, indicating that the hypertrophied adenoid acts as a stable, shared dysbiotic reservoir, irrespective of middle ear fluid accumulation. Notwithstanding the fact that the study addresses a clinically relevant topic with sound high-throughput sequencing methodologies, there are several critical methodological, statistical and editorial shortcomings that must be addressed prior to publication.

Introduction

Terminological rigor is of paramount importance in this field. The manuscript displays inconsistency in the use of the terms "adenoid hypertrophy" (AH) and "adenotonsillar disease" (ATD). It is imperative that the terminology is harmonised throughout the text to prevent any potential confusion.

The concept of contextual framing is demonstrated here. It is imperative that the introductory narrative is re-framed in order to eliminate the awkward phrasing present in the opening sentences.

Material and Methods

 A methodological limitation is evident in relation to the collection of samples: ex vivo swabs were obtained from surgically excised adenoid tissue in the patient cohorts (AH and AH+OME), whereas in vivo transoral/transnasal swabs were collected from healthy controls. It is imperative that this asymmetry is explicitly acknowledged in the methodology, and that its potential impact on biomass yield and spatial tissue sampling is thoroughly discussed.

The concept of inclusion/exclusion contradiction. Item 6 of the Healthy Control inclusion criteria contains a critical logical contradiction, erroneously listing systemic antibiotic use within the preceding two weeks as an inclusion criterion instead of an exclusion criterion. This must be rectified without delay.

 The criteria defining OME duration require unification. It is imperative that the phrase "documented prolonged (4 months) effusion" is harmonised with "type B tympanogram recorded on at least two separate occasions over a 4-month period" across all subsections.

The following subheading sequence has been duplicated: It is notable that subheadings 2.5 and 2.6 bear an identical title, "Data analysis - 16S rRNA gene amplicon sequence processing." It is imperative that the sections are re-numbered and retitled in order to accurately reflect their content.

The manuscript contains a multitude of typographical errors that compromise its scientific integrity. It is incumbent upon the authors to rectify all instances of inaccuracy, including but not limited to: 'adnevid', 'adneoid', 'typmogram', 'endoscopicaly', 'resonance', 'grade II', 'weak', 'unstructured', and 'immediate'.

Discussion

The following text will address the topic of Eustachian tube pathophysiology and the role of biofilms in the maintenance of such conditions. The authors must expand on the implications of the lack of significant taxonomic divergence between isolated AH and AH+OME in ANCOM-BC2 analyses. .

The Anna Karenina Principle is a theoretical framework that offers a novel perspective on the study of microbial community dysbiosis. It posits that healthy nasopharyngeal states remain constrained and similar, whereas diseased states undergo stochastic, highly variable dysbiotic disruptions. This principle provides a novel lens through which to interpret the observed microbial community dysbiosis.

The following section will address the limitations of the study. A comprehensive elaboration is required on the methodological boundaries of the study.

The inherent resolution limits of 16S rRNA (V3-V4) amplicon sequencing preclude species-level identification and functional strain profiling.

The absence of functional transcriptomics, metagenomics, or direct measurement of bacterial virulence factors is a notable limitation.

The cross-sectional design is a methodological framework that hinders the ability to make causal inferences regarding the progression of diseases.

The absence of host mucosal immune response profiling is characterised by the lack of quantification of local secretory IgA or nasopharyngeal cytokine panels.

It is imperative that all extraneous internal comments, draft markers, and editorial annotations (e.g., placeholders [GS1] through [GS7]) are removed from the final submission.

The following AI Compliance Statement is hereby provided: It is imperative to ensure that the declaration pertaining to the utilisation of artificial intelligence, as stipulated at the conclusion of the manuscript, meticulously adheres to the established editorial policies of MDPI and COPE.

It is imperative that the formatting and sequential order of inline citations be thoroughly audited in order to ensure correct citation order and reference flow. It is imperative that all references adhere to a consecutive numerical sequence in accordance with the guidelines stipulated by the journal, thereby resolving any current discontinuities in the citation flow.

Author Response

First of all, we would like to thank the reviewer for the valuable insights into improving our manuscript !

Comment 1: Terminological rigor... The manuscript displays inconsistency in the use of the terms "adenoid hypertrophy" (AH) and "adenotonsillar disease" (ATD). It is imperative that the terminology is harmonised...

We have performed a comprehensive review of the text and standardized the terminology, strictly utilizing "adenoid hypertrophy" (AH) and "adenoidal disease" throughout the manuscript to eliminate any ambiguity regarding the palatine tonsils.

Comment 2: The concept of contextual framing... It is imperative that the introductory narrative is re-framed in order to eliminate the awkward phrasing present in the opening sentences.

The opening paragraph of the Introduction has been thoroughly revised. The narrative has been reframed to professionally describe the mucosal surface as a "highly dynamic, immunologically active junction" to better set the academic context.

Comment 3: A methodological limitation is evident in relation to the collection of samples: ex vivo swabs... whereas in vivo... It is imperative that this asymmetry is explicitly acknowledged.

We have added a dedicated segment to the Discussion (Limitations) acknowledging this ex vivoversus in vivo acquisition asymmetry and discussing its potential impact on spatial biomass yield. We also added clinical and ethical justification to the Methods section (2.3.2) explaining the infeasibility of surgical tissue removal in healthy pediatric controls.

Comment 4: Item 6 of the Healthy Control inclusion criteria contains a critical logical contradiction, erroneously listing systemic antibiotic use... as an inclusion criterion.

The antibiotic criteria has been removed from the inclusion list and properly placed in section 2.2.4 (Exclusion criteria).

Comment 5: The criteria defining OME duration require unification.

This discrepancy has been resolved. We have unified the OME definition across all sections to strictly read: "type B tympanogram recorded at least twice over a 4-month period."

Comment 6: The following subheading sequence has been duplicated (2.5 and 2.6).

The subheadings have been corrected. The final methodology section is now properly titled "2.6 Data analysis - statistics" to accurately reflect its content.

Comment 7: The manuscript contains a multitude of typographical errors...

We apologize for these oversights. A thorough proofreading has been conducted, and all typographical errors highlighted by the reviewer ('adnevid', 'typmogram', 'endoscopicaly', 'resonance', 'garde II', 'weak', 'unbstructed', 'immediate'), along with others, have been successfully corrected.

Comment 8: The authors must expand on the implications of the lack of significant taxonomic divergence between isolated AH and AH+OME in ANCOM-BC2 analyses [regarding Eustachian tube pathophysiology and biofilms].

Our discussion interprets ANCOM-BC2 findings. We explicitly discuss how the baseline pathogenic biofilm on the hypertrophic adenoid functions as a stable dysbiotic reservoir, which is functionally sufficient to sustain localized inflammation and Eustachian tube dysfunction without requiring a secondary, unique otopathogenic shift.

Comment 9: The Anna Karenina Principle... provides a novel lens through which to interpret the observed microbial community dysbiosis.

We appreciate this conceptual suggestion. The Anna Karenina Principle is detailed in our Discussion, explaining how the healthy nasopharynx exhibits highly diverse, individualized eubiotic configurations, whereas pathological adenoid hypertrophy constrains the ecosystem into a restricted, uniform dysbiotic bottleneck.

Comment 10: A comprehensive elaboration is required on the methodological boundaries of the study (16S resolution, lack of transcriptomics/metagenomics, cross-sectional design, lack of immune response).

We fully agree.  The final paragraphs of our Discussion to explicitly detail these exact boundaries. We have clearly stated the inherent resolution limits of 16S (V3-V4) sequencing regarding species-level profiling, acknowledged the lack of functional transcriptomics and metagenomics, noted the limitations of a cross-sectional framework, and highlighted the absence of localized IgA and specific inflammatory cytokine evaluations.

Comment 11: It is imperative that all extraneous internal comments, draft markers... are removed.

All internal draft markers and editorial annotations have been purged from the final manuscript.

Comment 12: The following AI Compliance Statement is hereby provided: It is imperative to ensure that the declaration... meticulously adheres to the established editorial policies of MDPI.

 The Acknowledgments section has been updated to mirror the exact standardized AI disclosure phrasing required by MDPI and COPE editorial policies.

Comment 13: It is imperative that the formatting and sequential order of inline citations be thoroughly audited...

A comprehensive audit of the citation flow has been completed. All inline references now appear in strict, consecutive numerical sequence according to the journal's guidelines.

Once again, thank you very much for your time and insight!

The authors of the manuscript

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

All my concerns were adressed. Thank you for the great work to improve the manuscipt.

I recommend the paper to be published in the corrected form.