PONE-D-23-36124The drug susceptibility and gene-set enrichment analysis of M. tuberculosis isolates in Gansu Province, ChinaPLOS ONE

Dear Dr. Jia,

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Academic Editor

PLOS ONE

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 [Zhong Jia, Natural Science Foundation of Gansu Province (18JR3RA417)].

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 [Zhong Jia, Natural Science Foundation of Gansu Province (18JR3RA417)].

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Additional Editor Comments:

The manuscript has been reviewed by four reviewers, who identified significant concerns in the study's design and methodology. Mixing pulmonary and extrapulmonary isolates for analysis is problematic, as bedaquiline is intended for treating pulmonary multidrug-resistant tuberculosis (MDR-TB) according to CDC guidelines. This approach may lead to unreliable conclusions. The study also lacks comparison with existing research on mutations in the Rv0678, atpE, and pepQ genes, limiting the novelty and validity of the findings. Additionally, there is clear issues with the writing quality and data presentation, including unclear language, grammatical errors, and improper formatting of scientific terms. Some figures lack sufficient detail or resolution, making interpretation challenging.

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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?

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: Partly

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: No

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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: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

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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: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

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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: The drug susceptibility and gene-set enrichment analysis of M. tuberculosis isolates in

Gansu Province, China

The article seem to have a very good data that is not well presented starting from the title through the rest of the content. The abstract does not tell us clearly what the authors did, why, how and what did they conclude.

The methods regarding the bioinformatics section is very abstract and does not tell us how and why these list of databases and software are used.

Reviewer #2: Dear authors, as much I can evaluate your manuscript it can be accepted. However, at some of the area of your manuscript, I do not have expertise which needs to be evaluated by other reviewers.

Kindly consider the other reviewer comments seriously.

Reviewer #3: The authors describe that Bedaquiline is a new drug in the paper and carried out PCR and whole genome sequencing of isolates. This paper has multiple issues.

1. The authors are using pulmonary and extra pulmonary isolates together to interpret the data which does not make sense as the site of infection are different.

2. Bedaquiline has been found to be useful drug since 2016 not sure why the authors say it is a new drug.

3. The authors dont show any comparison with other studies for the mutations they found in Rv0678, atpE, and pepQ genes as there are multiple studies which have reported mutation in these genes from several isolates from different countries.

4. In MTB most of the genes show enrichment of of ABC transporters, two-component systems, and bacterial

secretion systems what new information are the authors adding in this study.

5. Bedaquiline may be used to treat adults with a confirmed diagnosis of pulmonary MDR TB as per the CDC guideline so why did the authors use extra pulmonary isolates in the study.

6. There are several grammatical errors in the paper for example M. tuberculosis is not italicized in the entire paper.

Reviewer #4: The study aimed to investigate various aspects of tuberculosis, including drug resistance, sensitivity, and genetic mutations, using samples collected from Lanzhou Pulmonary Hospital in Gansu, China, between February 2018 and July 2019. A total of 2,136 cases with M. tuberculosis strains were analyzed, following the Tuberculosis Diagnosis and Treatment Guidelines of the Chinese Medical Association.

Drug resistance detection was conducted using a proportionate approach for ten drugs, and drug sensitivity testing for bedaquiline was performed on 208 strains. DNA extraction and sequencing were carried out to identify drug-resistant gene sequences, with subsequent analysis to predict gene function and resistance mechanisms. Results indicated varying degrees of drug resistance, including multi-drug resistance and extensive resistance, among the samples. Mutations in specific genes, such as Rv0678, atpE, and PepQ, were associated with bedaquiline resistance. Additionally, pathway analysis revealed potential mechanisms underlying drug resistance.

1. Please use help of English speaker or any grammatical tools to improve the quality of the manuscript. Below are some examples which should be implemented to improve the quality. "Demonstrated a polymorphic distribution with different regions" - It's not clear what is meant by "polymorphic distribution. The phrase "were firstly predicted" should be "were predicted initially" or "were predicted first."

2. "Bedaquiline's, as a new drug, resistance range was identified" - This sentence is unclear. It could be rephrased as "The range of resistance to bedaquiline, a new drug, was identified."

3. Please elaborate “82.45% of the enzymes involved in carbohydrates”.

4. Write more clearly “the effects of drug-resistant tuberculosis cases” at Page 5 line 5?

5. Some images are not clear such as Figure 9 and 12

6. Figure 8 is not full please provide full image for pathway analysis.

7. “Note: A, B, C and D are amino acid comparison diagrams of atpE, Rv0678 and PepQ genes, respectively. 0 represents the amino acid sequence of the corresponding drug-resistant gene of the standard strain of M. tuberculosis. The other numbers represent the coding of the corresponding strain.” if it belongs to Figure 12 then modify it accordingly.

8. Data analysis methodology can be incorporated in more details for better understanding

9. The description of drug resistance categories (single drug resistance, multi-drug resistance, etc.) lacks clarity and standardization.

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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: Rehab Ahmed

Reviewer #2: Yes: Shaban Ahmad

Reviewer #3: No

Reviewer #4: No

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PONE-D-23-36124R1Phenotypic and genotypic analysis of drug resistance in M. tuberculosis isolates in Gansu, ChinaPLOS ONE

Dear Dr. Jia,

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 Sep 02 2024 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,

Salman Sadullah Usmani, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments:

There is still concern raised by one of the reviwer about the combined analysis of pulmonary and extra pulmonary isolates. It will be wise to show these analysis seperately, as the site of infections are different.

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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 #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?

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 #3: No

Reviewer #4: Yes

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3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: N/A

Reviewer #4: I Don't Know

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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 #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

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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 #3: 1. The author need to separately analyze pulmonary and extra pulmonary isolates as the site of infections are different and they cannot be mixed up and shown as they all are drug resistant. Show the results separately for pulmonary and extra-pulmonary isolates.

2. The authors need to show comparison of Bedaquiline mutations identified in their study with other WGS studies as Bedaquiline mutations have been also identified in other WGS studies. Please add this information is table2 showing what is unique and common mutations as compared to other studies

Reviewer #4: (No Response)

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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 #3: No

Reviewer #4: No

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[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.

Phenotypic and genotypic analysis of drug resistance in M. tuberculosis isolates in Gansu, China

PONE-D-23-36124R2

Dear Dr. Jia,

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,

Salman Sadullah Usmani, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

It seems that the authors have adequately responded to the third reviewer's query and provided a reasonable justification for their analysis. Despite this, the reviewer appears reluctant to accept the paper. However, it is worth noting that the other reviewers are satisfied with the updated version of the manuscript. After two rounds of revisions, I believe the manuscript is now satisfactory and suitable for publication.

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 #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?

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 #3: No

Reviewer #4: Partly

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3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: N/A

Reviewer #4: N/A

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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 #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

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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 #3: The author's have not cited the papers in Table 2 as they have reported the mutations in Bedaquiline.

The extraplumonary and pulmonary samples cannot be combined in the analysis as the author's themselves have written in the response the site of infection is different in pulmonary tuberculosis and in extrapulmonary tuberculosis.

Reviewer #4: (No Response)

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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 #3: No

Reviewer #4: Yes: Vinod Kumar

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