PONE-D-22-27870Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure in PakistanPLOS ONE

Dear Dr. Ahmad,

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. Please submit your revised manuscript by 1 July, 2023. 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.

Please include the following items when submitting your revised manuscript:

• 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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,

Atul Vashist, PhD

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. 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. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

3. Thank you for stating the following in the Acknowledgments/ Funding Section of your manuscript:

“IA received support from Swedish Research council (2020-06136) and Higher Education Commission Pakistan (8666/Punjab/NRPU/R&D/HEC/2017). FM, AA, SK and HA received support from University of Health Sciences, Lahore”

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“The author(s) received no specific funding for this work”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #2: No

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study highlights the drug resistance profiles and related gene mutations in M. tuberculosis in Pakistan based on phenotypic and genotypic studies. As M. tuberculosis drug resistance including MDR and XDR phenotypes are a serious concern worldwide, it is of great importance to look at the antibiotic resistance patterns for proper treatment and patient management. Therefore this report is a good compilation of work. However, I have some concerns as mentioned below, and upon clarifying some issues, the manuscript can be accepted for publication in PloS One.

Abstract:

-Please change the starting phrase in abstract to a correct way: "To treat tuberculosis is relatively a complicated procedure……

- in the abstract results section, please add the number and % of MDR and XDR cases.

Introduction:

-According to the reference 4, the first line anti-TB drugs for the treatment of non-MDR cases of TB (group 1) are Isoniazid, rifampicin, pyrazinamide and ethambutol as core drugs, streptomycin is no longer used routinely.

So why you included the latter in the anti-TB regimen? How you manage to use that as an injectable drug for duration of treatment? Please explain that in the discussion.

Methods:

-The information regarding the cases should well-explain at the beginning of methods section, for instance 1200 patients were confirmed TB cases, among them you found 64 drug-resistant cases and for comparison you used the same number naïve cases.

-What does (65-128) means?

-In Drug susceptibility testing (DST) section, please put the reference number together as [16-18]. However why you included 3 references for a single method?

Results:

-As I noticed the rate of MDR was high in this study, again is better when you are talking about resistance to Isoniazid and Rifampicin, add the word "simultaneous" to emphasis that they are MDR. I suggest to re-write the sentence as:

"Among these sixty-four isolates, 46 isolates (71.9%) were simultaneously resistant to rifampicin and isoniazid (MDR phenotype)."

-In naïve group you had also 4.6% MDR (with simultaneous resistance to rifampicin and isoniazid ), you did not explain in the discussion about these, since it seems that these are the cases of primary resistance. Have you investigated the primary and secondary resistance in this research project? This should be included as the information is very useful about the circulating resistant phenotypes in a community.

-you have bold the amikacin resistance in your study when is used for the treatment of MDR –TB cases. According to WHO the drug of choice for the treatment of first-line anti-TB treatment failure are aminoglycosides and fluoroquinolones. I did not find any statements in this section regarding the number and rate of XDR-TB, however in Table 2 it is mentioned.

What was your criteria for determining XDR strain? Since according to the WHO definition for XDR (resistance to fluoroquinolones and one of the injectable aminoglycosides), and you reported only one case with moxifloxacin.

-For XDR cases, please add a column in Table 1 and explain more in discussion section.

Reviewer #2: The authors in this study entitled “Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure in Pakistan” try to elucidate antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure and offer appropriateness of the erstwhile second line drugs and the efflux pump inhibitor verapamil against M. tuberculosis isolates associated with treatment failure. The manuscript targets an important issue; however, it needs to be substantially improved before consideration for publication. The authors should also perform a general proof reading for typographical errors in the manuscript. My comments are as below.

• WHO has recently changed the algorithm of Drug resistant TB treatment and redefined XDR-TB. Please discuss results according the new treatment guidelines. Even though the patients were collected in 2 sets one in 2016-17 and other recently. When were fresh samples collected? How were these patients treated? How long was the follow up done?

• What was the status of DST for these patients when diagnosed, when they were put on 1st line treatment. What were the results of the molecular tests for these patients, was MGIT-DST, Xpert or LPA done on these patients?

• The authors mention about DST being performed, but was it done as a part of diagnostic protocol for these patients or for the study; as majority of them were drug resistant. The major cause of treatment failure seems to be the non-identification of drug resistant cases upfront. A flow chart of the study will help clarify this, in accordance with STARD guidelines.

• What were the subjects´ characteristics i.e. HIV status? presence of diabetes? history of smoking or any other factors. This will help identify the risk factors of treatment failure and poor compliance for the purpose of predicting risk groups to improve treatment outcomes.

• The authors should mention strengths and limitations of the study, provide a proper conclusion, without overstating the results. Also, the Abstract does not really bring out the total work done. It should be rewritten.

Minor

• Please add line numbers

• Abstract, Pg 2, Para 2. 46+30, not adding up to 64, rewrite if isolates had resistance to more than 1 drugs.

• Pg. 3, Para 3. Not 512 and 528, its 511 and 526.

• Pg. 4, Para 1. inhA gene or inhA promoter region? If inhA promoter then mutations are not associated with AA change.

• Pg. 4, Para3. Guidelines for MDR-TB treatment have been updated in 2020 and the Definition of XDR-TB has been revised in 2020.

• Pg. 6, Para 2. "All culture positive MGIT 960 vials were ......performed" add reason or reference.

• Pg. 7 Para 3. Correct 0,5 to 0.5

• Pg. 8 Para1. Explain three biological replicates.

• Pg No.8 Para 2. Table S1 is list of primers.

• Pg. 9 Para 2. “Notably, serine 314 …… MDR/XDR isolates”. correct, 315

• Pg. 11 Para 2. “The mean MICs of rifampicin and isoniazid in MDR isolates were 5. 0 ug/ml and 1.5 ug /ml respectively” This detail should be moved to 'Materials and Methods' section.

• Pg. 24 Add abbreviation of ND and NA

• Pg. 24, Table footnote, change to NSM as used in table.

• Please give either Figure 1 or Table 1. The data is repeated.

• Figure 3A. What is the reason behind the low expression of Rv1258 in treatment failure resistant isolates?

**********

6. PLOS authors have the option to publish the peer review history of their article (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 #1: Yes: Azar Dokht Khosravi

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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.

PONE-D-22-27870R1Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure in PakistanPLOS ONE

Dear Dr. Ahmad,

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.

Please submit your revised manuscript by Oct 14 2023 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.

Please include the following items when submitting your revised manuscript:

• 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 you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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,

Atul Vashist, PhD

Academic Editor

PLOS ONE

Journal Requirements:

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

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

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors are advised to recheck the manuscript for typographical errors as they are still present throughout the manuscript. All standard tests should be quoted appropriately eg. Gene XPert MTB/RIF is written as Gene Expert through out; figure titles 'resistant' is spelled as ressitant . There are many spelling errors which should be corrected.

2. After revising the manuscript the Discussion portion does not seem homogenous. It should be rewritten.

**********

7. PLOS authors have the option to publish the peer review history of their article (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 #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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.

Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure

PONE-D-22-27870R2

Dear Dr. Ahmed,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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,

Atul Vashist, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

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

Reviewer #2: N/A

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (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 #2: No

**********