PONE-D-21-11660

Rifampicin and Isoniazid drug resistance among patients diagnosed withpulmonary tuberculosis in southwestern Uganda

PLOS ONE

Dear Dr. Bazira,

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 fully major revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Christophe Sola, Pharm.D. Ph.D.

Academic Editor

PLOS ONE

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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: Partly

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

Reviewer #1: No

Reviewer #2: 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: Yes

Reviewer #2: Yes

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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: Yes

Reviewer #2: 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: While the study appears sound, the data presentation is flawed. Specifically according to table 3, 258 of the susceptible patients have a history of past TB, while in the results it is stated the contrary (266 new and 17 relapses). As a result after statistical analysis, the authors erroneously claim in line 160-161,

that "Newly diagnosed patients were more likely to have MDR than patients who had previously received treatment". Also in table 3, the number of MDR cases taken for statistical analysis is 8 for all variables. The study found a total of 19 MDR cases. It is not clear what the number 8 corresponds to; so is unclear the whole statistical analysis based in table 3.

I advise the authors to improve table 2 by putting only numbers of samples with mutations found for each of the mutations, instead of all (mutations found and not found). This will make the data presentation easier to follow. Also, in the table header please put mutation found/not found instead of susceptible/resistance.

The authors use age groups 18-37, 38-57, 58-77, >=78. The WHO standard forms use other age groups (15-24, 25-44, 45-64, >=65. The authors may consider reanalysing their data according to these groups to better check for age-resistance correlations.

Line 30 contains a typo error. Please put 5.9% instead of 9.5%

Reviewer #2: Surveillance for drug resistance TB is very important however the manuscript has many flaws that I have highlighted within the submitted manuscript. I also think the rationale for the work done is not well articulated and moreover the amount of work done is minimal. There will be much benefit if this method has been compared with at least one traditional method for DR determination. This will allow for comparison in terms of simplicity, sensitivity and specificity as well as cost.

Other comments

1. The manuscript needs a through language review

2. Lines 31-32: what is the reference data

3. Lines 44-49: very long sentence, loosing meaning

4. Lines 54-56: A major reason is that these are the backbone of TB treatment

5. Line 67: The authors indicated in the background that they find the methods, they used to assess DR as simple and straight forward. I do not agree that detection of mutations using melting curves is simpler in the Africa context

6. The authors should be mindful that the biomarkers (315 for KatG and -8 and -15 for inhA ) that they only used to analyse for INH detects less than 90% of isoniazid resistance in Uganda. This was not discussed as a limitation of the study.

PLoS One. 2018; 13(5): e0198091.Published online 2018 May 30. doi: 10.1371/journal.pone.0198091

7. The referenced article for the primers as indicated in the methodology section does not correspond to the cited article (Poudel et al. 2012). The cited article analyzed for DR conferring mutations among INH and RIF resistance phenotypes. There was no RT-PCR analysis nor the study designed primers for DR analysis.

8. I find setting P value less than 0.005 as indication of significance of difference in measured variable too high. I suggest the authors need to review the statistical analysis again. I think the burden of DR in TB is very crucial, that one needs to be critical in following leads

9. The authors need to reduce the amount of results repeated within the discussion. Also the discussion has too many speculations

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-11660R1

Rifampicin and Isoniazid drug resistance among patients diagnosed withpulmonary tuberculosis in southwestern Uganda

PLOS ONE

Dear Dr. Bazira,

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 23 2021 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: http://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,

Christophe Sola, Pharm.D. Ph.D.

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.

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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 #1: All comments have been addressed

Reviewer #2: (No Response)

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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 #1: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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 #1: Yes

Reviewer #2: 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 #1: Yes

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 #1: The authors may consider the following points:

Line 22-27: Consider rephrasing, like for example:

A total of 283 sputum samples (266 from newly diagnosed and 17 from previously treated patients), collected between May 2018 and April 2019 at four different TB diagnostic centres, were assessed for RIF and INH resistance using high-resolution melt curve analysis. The overall prevalence of monoresistance to INH and RIF was 8.5% and 11% respectively, while prevalence of MDR-TB was 6.7%.

29-31: Consider rephrasing, like for example:

Furthermore, among the newly diagnosed patients, prevalence of resistance to INH, RIF and MDR-TB was 8.6%, 10.2% and 6.4% respectively; while among the previously treated cases, these prevalence rates were 5.9%, 23.5% and 11.8%.

Line 29: “8.6% had resistance to INH” Please specify: Is this monoresistance or any resistance?

Line 134: Remove “suspects”. These patients are confirmed for TB as stated in the methods section, by smear microscopy and/or Xpert.

Lines 138-147: Please consider rephrasing, it is difficult to follow. Use either % or “per cent”. For example:

Monoresistance to rifampicin and isoniazid was found in 11% (95% CI: 0.077-0.150; p, 0.087) and 8.5% (95% CI: 0.056-0.123; p, 0.692) of all the patients, respectively. Resistance to RIF and INH among newly diagnosed patients was 10.2% and 8.6 %, while among previously treated patients, resistance to RIF and INH was 23.5% and 5.9 % respectively. Furthermore, 4.9% of the samples from newly diagnosed with INH monoresistance, were found to have mutations in the InhA region while 8.6% had mutations in the katG region, a condition that can lead to phenotypic isoniazid drug resistance (Palomino 2009). The overall resistance to both drugs (MDR-TB) was 6.7% (95% CI: 0.026-0.075; p, 0.982) among all patients (6.4 % among newly diagnosed and 11.8% among previously treated patients). Of the cases with MDR-TB, 6.4% had mutations in both the rpoB and KatG regions, while 3.9% had mutations in both the rpoB and InhA sites

Lines 216-217: This sentence is not correct: “settings with an MDR-TB prevalence of more than 3% especially among previously treated patients are considered as having a high MDR-TB burden”

Reviewer #2: line 41-43: the definition for acquired and primary resistance is wrong. Primary resistance a newly diagnosed case that was infected with a resistance strain and the reverse is true for acquired.

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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 #1: No

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.

Rifampicin and Isoniazid drug resistance among patients diagnosed withpulmonary tuberculosis in southwestern Uganda

PONE-D-21-11660R2

Dear Dr. Bazira

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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Kind regards,

Christophe Sola, Pharm.D. Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

lane 154-155 : "This relation, however, was not statistically significant ", please add to this sentence : "given the very low sample size of the previously treated patients sample"

Reviewers' comments: