Dear Dr. Hasan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers have recommended publication, but also suggest significant revisions to your manuscript.  Therefore, I invite you to respond to the reviewers' comments and revise your manuscript.

Please submit your revised manuscript by Aug 16 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Fumihiro Yamaguchi

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.-->--> -->-->Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at -->-->https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and -->-->https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf-->--> -->-->2. During our evaluation of the documents provided, we noted that your ethics approval letter did not cover the entire date range for participant recruitment and data collection. Before we can proceed further with the submission, please provide the ethics approval extension document(s) for the study. If the document is in another language, please also provide an English translation. Please note that if this document is not included when your manuscript is resubmitted, it may be rejected.-->--> -->-->3. Thank you for stating in your Funding Statement: -->-->TB Healthcare Biotechnology, China provided reagents and CDC, China provided support for the study. Uzair Abbas was supported by Higher Education Commission, Pakistan- Grand Challenges Fund Grant number -913, which also provided research support for this study.  -->--> -->-->Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now.  Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. -->-->Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.-->--> -->-->4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.-->?>

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1:  Avoid Causal Language:

As this is a cross-sectional observational study, please avoid phrases like “LTBi confers protection” or “LTBi reduces severity.” Use terms such as “associated with” or “suggests a possible protective effect.”

Multivariate Analysis:

Include logistic regression to control for confounders such as age, sex, comorbidities, vaccination status, and time since infection. This is essential for substantiating your key conclusion.

Clarify LTBi Definition:

While the X.DOT-TB assay is explained, consider clarifying its comparability to standard QFT in the main Methods (not just Supplementary). It would also help to justify using only this assay for LTBi classification.

BCG Vaccination Interpretation:

BCG scar status was not associated with COVID-19 outcomes, but the Discussion still overemphasizes the “trained immunity” angle from BCG. Please reconcile this more clearly with your data.

Immunological Findings Interpretation:

The significance of raised OAS1 and SOCS3 in LTBi-positive COVID-19 cases is underexplained. Are these markers known to mediate antiviral responses in the TB context? Please elaborate or temper the discussion if speculative.

Discussion Length and Focus:

The Discussion repeats findings and prior literature excessively. Please condense and focus on the novel aspects of your findings.

Reviewer #2:  In this interesting paper the authors report a possible interaction between COVID-19 disease severity and LTBI status. They also observe limited or no effect of BCG status. This interaction may (partially) explain the epidemiology of SARS-Cov-2 in the Authors country.

I have one point regarding the interpretation of the results. The authors state that LTBi may protect against SARS-CoV-2 and reduce the severity of COVID-19 disease. I agree that the data presented appears to show that the severity of COVID-19 disease is reduced in persons with LTBi but I do not think the data presented convincingly shows protection against SARS-CoV-2 infection. Notably in figure 3 E compares IgG levels in a small number of HC who were either LTBi- (n=30) or LTBi+ (n=30), a significant proportion of the LTB+ve patients are positive for anti-RBD antibodies. The difference between the groups in figure 3E does not look significant to me and if I understand correctly both groups appear to have been approximately equally likely to have been infected with SARS-CoV-2 (and presumably had mild symptoms). If I am correct I would ask the authors to limit their claim of protection to protection from serious disease not protection from infection throughout the paper (introduction, discussion and conclusions). Notably also in the conclusions section of the abstract “We suggest that the immune reactivity to Mtb is associated with an increased protection against SARS-CoV-2 as indicated by both the lower frequency of COVID-19 and the reduced severity of disease in LTBi positive individuals.” delete “lower frequency of COVID-19” from this statement.

Minor point CODID should be COVID in the title “Higher numbers of M. tuberculosis-specific T cells in HC than in CODID 19 LTBi positive individuals.”

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Reviewer #1: No

Reviewer #2: Yes:  RM Anthony

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Dear Dr. Hasan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

The reviewers have recommended publication, but also suggest significant revisions to your manuscript.  Therefore, I invite you to respond to the reviewers' comments and revise your manuscript.

==============================

Please submit your revised manuscript by Dec 12 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Fumihiro Yamaguchi

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

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Reviewer #2: The authors have revised their paper and moderated the claims with respect to protection of infection and the potential protective effect of BCG. All the points raised have been addressed.

Reviewer #3: This study compares the Mycobacterium tuberculosis–specific cellular immune response in patients with COVID-19 versus healthy individuals. The authors conclude that this immune response is significantly more pronounced in healthy individuals compared with COVID-19 patients.

Major Comments

1. 1. The lack of interferon production by T cells indicates a decrease in cellular immunity, which may also be attributable to the higher prevalence of chronic comorbidities in the patient group compared with healthy controls. This should be clearly reflected in the discussion.

2. 2. The selection of patients and controls may be subject to biases, which are not sufficiently explained in the Materials and Methods section. For example, the timing of COVID-19 patient recruitment varies considerably after infection, which could substantially influence cellular immune responses.

3. 3. The fact that some patients were hospitalized while others were managed on an outpatient basis likely contributed to heterogeneous immune responses. This should be acknowledged and discussed as a limitation of the study.

4. 4. In the Methodology section, the process of recruiting both patients and controls requires a more straightforward explanation.

Minor Comments

5. 1. In the Abstract, the three introductory sentences are not entirely coherent with the primary study objective.

6. 2. The following sentence in the Abstract is unclear: "Therefore, MTB infection in active TB patients, or individuals with latent MTB infection, are likely to experience immune modulation that may alter the consequence of SARS-CoV-2 infection?"

7. 3. The authors should clarify what the messenger RNA sequences for OAS1, MAVS, SOCS1, and SOCS3 represent.

8. 4. The description of the mRNA sequences analyzed should be expanded, and the primers used for real-time PCR should be specified.

9. 5. For the method 'X. In the DOT-TB assay for screening LTBi, the antigens employed should be described.

10. 6. In the phrase 'As per manufacturer's instructions', the manufacturer of the kit must be explicitly named.

11. 7. In the Results section, subheadings currently present the outcomes rather than the study objectives.

12. 8. The entire manuscript requires a comprehensive revision of the English language, including correction of grammatical errors.

Reviewer #4: Overall, this is a well written revised manuscript with a few minor suggestions for revision;

INTRODUCTION:

- Rephrase 'with 677 million cases and 6.9 million deaths reported worldwide to 10

March 2023 ---' as 'with 677 million cases and 6.9 million deaths reported worldwide by 10

March 2023 ---'

- Define 'TST' at first use of term

- Rephrase 'Currently, studies on the association between SARSCoV-2 and MTB are both scare' as 'Currently, studies on the association between SARSCoV-2 and MTB are both scarce' ---

METHODS:

- You state that 'All 128 individuals in the COVID-19 group had been confirmed through a positive SARS-CoV-2 PCR

test'.

- Further, was genotyping performed to ascertain the SARS-CoV-2 strain that was associated with COVID-19 at that time?

- Further, for SARS-CoV-2 PCRs, did you use in-house or commercial kits?

- For RT-PCR on PBMCs, did you mean you did 'reverse-transcriptase real-time PCR' or just 'reverse-transcriptase PCR'? Please clarify.

- Are the primer sequences in-house, i.e., designed by you, or you used primer sequences published in literature?

DISCUSSION:

- In the limitations section, or somewhere in the discussion, comment on the fact that the phrase 'TB infection' is currently evolving from being understood as a dichotomy to a continuum, meaning the LTBi individuals could actually have been having TB disease? Just a thought.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes:  David P Kateete

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To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures 

You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. 

NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications.

Individuals with latent tuberculosis in a high TB endemic country show mild COVID-19

PONE-D-25-25202R2

Dear Dr. Hasan,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Fumihiro Yamaguchi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

**********

Reviewer #3: The authors have fully addressed the reviewers’ comments and the article can be accepted for publication in its revised version

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #3: No

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