Comprehensive Diagnosis of Abnormal Vaginal Discharge Using qPCR-Based Microbial Dysbiosis Indices

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the  manuscript “Comprehensive diagnosis of abnormal vaginal discharge using qPCR-based microbial dysbiosis indices” by Vovko et al. authors developed novel dysbiosis indices for BV, VVC and AV and validated their performance against established reference methods. Overall, the study is well designed, the rationale is clear, and the manuscript is easy to follow. However, there are some methodological aspects that should be clarified or strengthened before the manuscript can be considered for publication.

 

Major comments:

1. Question about the model test and training set. Whether the dysbiosis indices were should be validated in an independent cohort? Whether this can affect the diagnostic performance?
2. Small number of AV cases. Only five participants are from AV group and reported sensitivity and specificity estimates for AV are based on a very small number of cases and should be interpreted with caution. The authors should acknowledge this limitation more in  Discussion section.
3. STI panel and interpretation. The Anyplex II STI-7 assay detects Mycoplasma genitalium, but it is not reported in Table 2. Whether there were positive cases? This should be reported even if there are no positive cases. In addition, whether Mycoplasma hominis can be reported among STI? This should be discussed in Discussion section.  
4. Please provide AUC values (with 95% confidence intervals) for all ROC analyses.
5. BV qPCR dysbiosis index development. The mathematical definition of the dysbiosis indices is not entirely clear. How the indices were calculated? 
6. For the BV+VVC category, Table 4 states that combined diagnosis was based on "G-L and ESS-T". Should this instead be "G-L and C-T"?
7. The manuscript briefly states that 10.9% of healthy controls had Nugent scores consistent with BV. However, asymptomatic BV is not discussed further in either the Results or Discussion sections. The authors may wish to comment on the prevalence of asymptomatic BV observed in their cohort and discuss whether inclusion of these individuals influenced the development and interpretation of the BV dysbiosis index.

Author Response

Thank you very much for taking the time to review our manuscript and for your valuable comments and suggestions.

Please find our detailed point-by-point responses to all reviewer comments, together with the corresponding revisions, in the attached Response to Reviewers document. The revised manuscript and the revised Supplementary Material have also been uploaded.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper is largely well-structured and well-written. There are, however, some important problems related to methodology that need to be discussed, such as modeling, validation approach, number of subjects, and possibility of over-fitting. Improving the paper on these aspects will definitely enhance its scientific weight.

1. The critical limitation lies in the absence of external or independent validation. The dysbiosis indices have been constructed using the ROC curves applied to training datasets and then assessed in the same study group. While there is mention of "validation" by the authors, it is important to note that this is internal validation and not independent validation. The authors should acknowledge this limitation explicitly.
2. Only 5 AV-positive subjects were identified in the entire study population (3 symptomatic and 2 controls). Despite this, the manuscript reports 100% sensitivity and 100% specificity
3. The study population consists of women from only two Slovenian clinics recruited between 2015 and 2016. Vaginal microbiome composition differs substantially across ethnic groups, sexual behaviour, hotmonal ststus. This the study generalizability is limited.
4.  The manuscript treats Mycoplasma hominis as an STI within the syndromic framework. However, M. hominis is frequently regarded as a commensal or opportunistic organism rather than a classical STI. What is the clinical relevance?
5. Profound english editing is required through the whole manuscript text

Author Response

Thank you very much for taking the time to review our manuscript and for your valuable comments and suggestions.

Please find our detailed point-by-point responses to all reviewer comments, together with the corresponding revisions, in the attached Response to Reviewers document. The revised manuscript and the revised Supplementary Material have also been uploaded.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing the comments. The manuscript went through the substantial improvement.