PONE-D-22-28840High Proportion of RR-TB and mutations conferring RR outside of the RRDR of the rpoB gene detected in GeneXpert MTB/RIF assay positive pulmonary tuberculosis cases, in Addis Ababa, EthiopiaPLOS 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: Yes

Reviewer #2: Partly

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

Reviewer #1: I Don't Know

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: Introduction

Ethiopia is among the 30 high TB burden countries with an estimated annual TB incidence rate of 132 per 100 000. The number of people infected with TB is 151 000, 9 900 HIV positive TB incidence, 19 000 HIV negative TB mortality, 2 500 HIV positive TB mortality, 591 individuals diagnosed with DRTB, and 7 cases of laboratory-confirmed pre-XDR TB or XDR TB [9]. Include the years for which this data is referring to.

Ethical approval

Can authors include the ethical approval number and date when it was granted.

Inclusion and Exclusion Criteria

The inclusion criteria were clinical suspicion of new presumptive pulmonary TB strong enough to warrant a direct sputum investigation and a positive result confirmed by Gene Xpert MTM/RIF. Take note of the inconsistence in writing the name of the GeneXpert MTB/RIF assay throughout the manuscript.

GeneXpert MTB/RIF Assay

Briefly, sputum samples were subjected to processing by approximately mixing with 8ml of GeneXpert sample reagent (SR) containing NaOH [5 - 10%] and isopropyl alcohol [10 – 20%] into the sputum container in a ratio of 2:1 V.V. The mixture was vigorously mixed by shaking the tubes 20 times and incubated for 15 minutes at 20–30 °C. This step has been repeated in between the incubation time. Take note of the sentence tense used here

GeneXpert MTB/RIF Assay Performance Characteristics and Result Interpretation

Generally, results from GeneXpert MTB/RIF Assay were obtained and assessed with three result types namely, MTB Detected, MTB Not Detected, and Invalid/Error/No result. What of MTB detected with Rifampicin resistance (MTB/RR TB)?

Quality Assurance

The internal quality control of the GeneXpert MTB/RIF Assay System was validated using M. tuberculosis H37Rv, a non-RIF resistant and known RIF resistant strain stored in -20oC. Which strain is this and how often was this done ?

Ethical Considerations

Institutional Review Board approval has also been obtained from the Research and Ethics Review Committee of Saint Peter TB Specialized Hospital for the commencement of the study. Take note of the sentence tense used here

Sociodemographic Characteristics of Study Participants

Overall, a total of 216 participants who had enrolled in this study were considered persons with presumptive TB upon clinical suspicion and bacteriologically confirmed with GeneXpert MTB RIF Assay. Does Xpert detect presence of the bacteria or bacterial DNA?

Reviewer #2: Dear Author(s),

I find this piece of work very interesting and useful as I myself did a similar work in Nepal samples looking at the Rifampicin resistance profile. I have to admit that though we saw mutations outside the RRDR, we did not pursue it further, but we always thought that these profiles will be important for enhancing the DNA probes in future. So, it is an important piece of work.

These are my recommendations:

1. I would like to see more clarity on the study design especially pertaining to data related to Rifampicin resistance outside the RRDR.

2. I would like to see more clarity on Figure 1, Figure 2 and Table 3.

3. What was the mutation detected outside RRDR? How was it confirmed as missense mutation? How was it detected? Do we have any experimental data? I could not find any concrete evidence.

4. Do you have any data about any co-mutations on RRDR along with the RR mutation outside this region?

I would greatly appreciate your responses for these questions.

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Reviewer #1: Yes: Gerald Mboowa

Reviewer #2: No

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High Proportion of RR-TB and mutations conferring RR outside of the RRDR of the rpoB gene detected in GeneXpert MTB/RIF assay positive pulmonary tuberculosis cases, in Addis Ababa, Ethiopia

PONE-D-22-28840R1

Dear Dr. Akalu,

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,

Frederick Quinn

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 #1: 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

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

Reviewer #1: 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

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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

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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: In the revised manuscript, the authors have satisfactorily addressed all comments that I raised in the original version.

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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: Gerald Mboowa

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