Real-Time Breath Diagnostics: Linking Molecular Pathways, Measurement Technologies, and Clinical Translation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript is, to date, the most thorough review on breath diagnostics that I have assessed. It not only provides an extensive overview of the sources of various VOCs and their potential links to physiological and pathological states, but also delves deeply into topics like the “Particle Phase Transport of Low Volatility Metabolites and Nonvolatile Compounds” and “Assay Standardization, Metrology, and Model Robustness.” The latter, in particular, is seldom referred in other review articles.  However, I recommend incorporating a discussion on induced volatolomics in the “Future Directions” section, as this emerging technology offers significant advantages over current breath diagnostic methods. Relevant references include but are not limited to: J. Breath Res. 13 (2019) 032001; Angew. Chem. Int. Ed. (2019) 58, 17563–17566; Nature Nanotechnology 15 (2020) 792–800; Cell Biomaterials (2026) 2, 100421.

Author Response

Dear Editors and Reviewers,

Thank you for the careful evaluation of our manuscript and for the constructive comments provided by the reviewers. We have revised the manuscript accordingly. All newly added or modified text in the revised version is highlighted in red to facilitate review.

Below, we provide a point-by-point response to each reviewer's comment.

Reviewer 1

Comment: Incorporate a discussion on induced volatolomics in the “Future Directions” section, citing recent work (J. Breath Res. 13 (2019) 032001; Angew. Chem. Int. Ed. (2019) 58, 17563–17566; Nat. Nanotechnol. 15 (2020) 792–800).

Response:

We agree that induced volatolomics is an important emerging molecular strategy that can improve specificity by turning breath readouts into pathway-focused functional assays. We have added a new paragraph to Section 6 (Future Directions: Integration Into Clinical Workflows) that introduces induced volatolomics and the exogenous volatile organic compound probe concept, and we discuss how enzyme-activated or pathway-targeted substrates can generate volatile reporters with reduced sensitivity to diet, ambient exposure, and baseline inflammation. We also added a translational perspective on dose and kinetic characterization, background metabolism and microbiome contributions, and regulatory implications when the probe, sampling interface, and algorithm together define a single assay. The reviewer suggested references have been incorporated (new References [101–103]). We also cited a recent example of phenyl-β-D-glucuronide-induced breath analysis for pan-cancer warning, which illustrates the direction toward portable, pathway-anchored implementation (new Reference [107]).

We thank the reviewers again for their helpful feedback, which improved the manuscript.

Sincerely,

Velmurugan Thavasi and co-authors

Reviewer 2 Report

Comments and Suggestions for Authors

In this work, the authors analyzed the literature about real-time breath sampling and analytical technologies, including the biological origins of breath signals and the main sources of noise and variability. The manuscript is clear and detailed, and the literature findings are well described. The presented schematics are interesting and meaningful. This work also proposes relevant strategies for future research directions, and the conclusions are consistent with the evidence presented in the review. Overall, the work is coherent and can be considered suitable for publication. However, a minor revision, as outlined below, is required.

• Please consider other recent revisions about this subject, as for example:
  • https://doi.org/10.1016/j.bj.2023.100623
  • https://doi.org/10.3390/biomedicines11113029
  • https://doi.org/10.1016/j.cca.2023.117692

and others in literature, and highlight the advances of this work in relation to previously published reviews.

Author Response

Dear Editors and Reviewers,

Thank you for the careful evaluation of our manuscript and for the constructive comments provided by the reviewers. We have revised the manuscript accordingly. All newly added or modified text in the revised version is highlighted in red to facilitate review.

Below, we provide a point-by-point response to each reviewer's comment.

Reviewer 2

Comment: Consider additional recent reviews in the field (e.g., 10.1016/j.bj.2023.100623; 10.3390/biomedicines11113029; 10.1016/j.cca.2023.117692) and highlight the advances of this work relative to previously published reviews.

Response:

We appreciate this suggestion and agree that it is important to clarify how the present review complements existing review articles. We have added a concise positioning paragraph in Section 6 that cites these recent reviews and explains how our manuscript differs in scope and emphasis. Specifically, we note that prior reviews provide valuable inventories of candidate breath biomarkers and analytical summaries, while our review is organized around molecular pathway logic and clinical translation for real-time measurement. We emphasize mechanistic mapping from pathways to analytes, the role of kinetics and breath fraction control, and the practical requirements for reproducibility, model robustness, and device algorithm governance in clinical deployment. The recommended reviews have been added to the reference list and cited in the new paragraph (new References [104–106]).

 

We thank the reviewers again for their constructive feedback, which improved the manuscript.

 

Sincerely,

Velmurugan Thavasi and co-authors
Corresponding author: velmu@ou.edu