Beyond Infection: The Interplay of Salivary Human Herpesvirus 6, Stress, and Host Factors in Major Depressive Disorder

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Since "major depressive disorder" is already included in the keywords, "MDD" may be redundant since it is an abbreviation for "major depressive disorder."
2. In the introduction, several statements regarding MDD are generic and extensively reported in the literature. 
3. The authors may consider updating the 2021 suicide statistics with more recent data, if available.
4. The rationale for including data on MDD from Canada prior to Thailand could be made clearer to improve the flow of the introduction.
5. A reference may be added to support the statement that evidence regarding the association between the HHV-6 and MDD is particularly limited in Thailand.
6. The authors may consider presenting the participants information in a table or in a more structured format to improve clarity and readibility. 
7. On line 172, extra space.
8. The abbreviation for MDD has already been defined (lines 164 and 172), as well as for the "HPA" axis (line 283).
9. There is inconsistency in the use of terminology across the table titles, where "major depressive disorder" is sometimes written in full (with abbreviation) (Table 4) and sometimes only as an abbreviation. 
10. In the author contribution section there is an extra comma (line 328).
11. The first square bracket in the reference list is bold. 
12. Although the authors acknowledged the limited number of MDD cases (n=52) as a limitation, this remains the key issue, given the imbalance between the cases and controls, as it likely reduced statistical power to detect an association, despite the large total sample size. 

Author Response

I sincerely very thank the reviewer for the excellent suggestions and for highlighting several important issues. I carefully considered each recommendation and revised the manuscript accordingly. Detailed point-by-point responses are provided below  in the attach file. .

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript addresses the relevant issue of identifying biological markers for MDD and investigates the potential role of the neurotropic virus HHV-6. The cross-sectional study design with a large sample size (N=2,403) is a definite strength. However, the work contains critical methodological flaws that undermine the validity of its main conclusions. A major revision of the manuscript is required.

 

General comments:

Abstract - two sentences in the abstract are redundant. The first states: "MDD was significantly associated with... higher stress and depressive symptom scores." The very next sentence states: "Multivariable analysis identified... stress, and depressive symptoms as independent predictors of MDD." This is essentially the same information repeated twice. Please merge these sentences or remove the duplication.

The descriptions of the questionnaires (ST-5, Q2, PHQ-9) in methods should be expanded. What do the score ranges signify? Please clarify the clinical interpretation of the scores (e.g., minimal, mild, moderate, severe) for the reader.

The MDD group includes only 52 participants, while the control group includes 2,351. This severe imbalance makes the regression analysis highly sensitive to outliers and can create an illusion of significance for rare factors. The reported lack of association (p = 0.309) may very well be a consequence of insufficient statistical power rather than a true absence of effect.

All tables should be placed immediately after their first mention in the text to improve readability.

Table 1 - It would be informative to see the proportion of HHV-6-positive and -negative cases within each subgroup (MDD / healthy) stratified by each sociodemographic variable presented. Please consider providing this as a supplementary table.

Table 5 presents over 20 separate interaction tests involving HHV-6. Given the large number of comparisons with no correction for multiple testing (e.g., Bonferroni correction), any statistically significant interactions are highly likely to be false-positive findings. Although the authors acknowledge this limitation in the discussion, they continue to interpret these interactions as meaningful effects in their conclusions. This is unacceptable.

How do the authors explain the clinically paradoxical finding that height is a significant predictor of MDD (p = 0.03, Table 4)? This contradicts biological plausibility and the existing literature. Please provide an explanation or address this as a likely statistical artifact.

A substantial number of participants in the control group have PHQ-9 scores indicative of depressive states. For example, 839 individuals appear to have scores consistent with mild depression according to the data in Table 1. How was the absence of MDD confirmed in these participants? Please clarify the exclusion criteria for the control group.

The use of saliva to detect HHV-6 fundamentally prevents any conclusions about the virus's association with MDD. HHV-6 permanently persists in the salivary glands, and its detection in 50.7% of participants merely reflects normal latent carriage, not pathological reactivation. Furthermore, PCR cannot distinguish between latent and active virus, and the presence of DNA in saliva provides no information about viral reactivation in the central nervous system. The authors mention this in their limitations, yet their discussion and conclusions contain speculative statements about neuroinflammation and viral dissemination to the brain that are not supported by the data. The conclusions must be significantly toned down, and this fundamental limitation must be discussed in much greater detail.

Author Response

I sincerely very thank the reviewer for the excellent suggestions and for highlighting several important issues. I carefully considered each recommendation and revised the manuscript accordingly. Detailed point-by-point responses are provided below  in the attach file. .

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I have carefully reviewed the revised manuscript and the authors' detailed responses to the previous round of comments. All of my concerns have been adequately addressed.

The authors have removed redundant text from the abstract, expanded the questionnaire descriptions with appropriate score interpretations, and acknowledged important limitations including low statistical power due to case–control imbalance and potential misclassification of controls. Furthermore, they appropriately toned down the discussion and conclusions to avoid speculative statements not supported by the data. A supplementary table stratifying HHV-6 positivity by subgroup is now provided.

I have no further concerns. The manuscript is acceptable for publication.