PONE-D-24-12276Laboratory validation of a simplified DNA extraction protocol followed by a portable qPCR detection of M. tuberculosis DNA suitable for point of care settings.PLOS ONE

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Additional Editor Comments:

Thank you for submitting your well written manuscript to this journal. We believe that the comments from the reviewers are very constructive, should be fairly easy to respond to, and will improve the overall impact of the manuscript. We look forward to receiving the revised manuscript soon.

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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: Yes

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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: In the introduction

Please update data in the introduction (2021, 2022) with last up to date data if available.

Smear microscopy has low sensitivity but quite good specificity.

Real time PCR section

Explain why IS6110 region was selected to detect MTB.

You are using the Xpert Ultra cartridge. It is important to specify that from the beginning of the paper, and outlining its main difference with the regular MTB/Rif cartridge in terms of specificity and sensitivity

In the discussion.

It would be interesting to give a rough idea of how it would cost to test one individual when using the FTA + Q3-Plus. Obviously not doing a deep cost-effectiveness analysis but show how many individuals can be tested under routine practice by Xpert compared to FTA + Q3-plus.

I would recommend address more in details the trace results and elaborate more on the comparison/correlation of trace results vs FTA + Q3 plus.

In remote areas, it is usually the case that the level of competency of laboratory technicians is more limited. I suggest to mention how feasible it would be to implement this technology especially around the results interpretation, is there any language barrier to read the results,.

Please elaborate on the easiness for operating and what it really means for the laboratory technician if s/he were to use this technology. You mention it takes up to 4 hours using Q3-plus. I guess you are also including the extraction steps in those 4 hours, please specify in your text. Also please add details on how long the lab technician needs to have hands-on for samples handling and processing. For example for GeneXpert it could around 20 minutes hands-on (adding buffer, mixing for 10 + 5 minutes, load the sample in the cartridge, launch the test). Please indicate out of the 4 hours, how long is required for actual hands-on.

Please add some details on what it implies for the health system to end up with false negative and/or false positive results.

Please elaborate on the testing capacity of Xpert vs FTA+Q3-plus. How many samples can be tested in one run of 4 hours?

Reviewer #2: Current manuscript has described laboratory validation of a simplified DNA extraction protocol as a POC. Few issues that need to be discussed in the manuscript.

1. How affordable the assay will be as POC?

2. Have you compared the sensitivity, specificity of the children's specimens? Do you think this can be different?

3. Why only adults specimens had been chosen?

Thank you.

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Reviewer #1: No

Reviewer #2: No

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Laboratory validation of a simplified DNA extraction protocol followed by a portable qPCR detection of M. tuberculosis DNA suitable for point of care settings.

PONE-D-24-12276R1

Dear Dr. Costa,

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.

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Kind regards,

Shahriar Ahmed, MBBS, MHE, MPhil

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for submitting a revised version addressing all the comments from reviewers comprehensively. I would like to take this opportunity to congratulate all the authors. Thank you for submitting your manuscript to our journal and we look forward to working with you again in future.

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: 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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

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 #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

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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 #1: Many thanks for all the adjustments. All concerns were addressed.

Reviewer #2: Thank you for addressing and incorporating all the responses in the manuscript. This looks good now for going forward for publication.

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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: Yes: Dr Vibol IEM

Reviewer #2: No

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