PONE-D-24-38581Linking genetic and phenotypic bedaquiline resistance in Mycobacterium tuberculosis strains from GeorgiaPLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: The above manuscript is well written and adds to the currently emerging BDQ resistance literature. The study shows that among the isolates from Georgia, BDQ resistance was mainly due to the mutations in Rv0678 supporting the other reports elsewhere and WHO. It also introduces the concern that the same mutations in some isolates might lead to phenotypic resistance and some may not.

Minor comments:

please make sure everywhere in the manuscript units of measurement are properly mentioned.

for example, micron symbol should be of universal symbol, microgram is not 'yg' (page 10),

missing units for MIC.

Page 7. please mention the reference to the literature for your MIC baseline. was the MIC defined at 90% or 99%? line 197: Sensititre mycoTB: please mention the catalogue number, company, country.

line 199: when classifying TB resistance wondering what you classify samples with MIC >0.06 but <0.12?

Line 204: please define routine? if done on sensititre or LJ medium or MGIT? how were the MIC cut off established? any references would be helpful since from the literature, it doesnt seem to clear on setting these cut offs. my personal concern also is with the pDST and the methods used differs so much that may be there is a need of a an strict universal pDST standard for BDQ.

line 216: REF? is reference missing?

line 224: Incomplete phrase missing information here: "Additional mutations in genes with- # were detected in <90% of reads".

lines 254, 255, 268: ul or ug?

Table 2: please mention BDQ MIC (ug/ml) in the MIC column.

Table 2: please provide reference for e MIC cut-off values for all different drugs mentioned here.

Line 320: do you mean the role of "Rv0678" and not "BDQ' mutations in poor treatment outcome?

Fig. 1: please change to N=, as per universal standards. mentioning N36, N15 etc., gives the impression of sample number rather than total numbers.

Also please reconsider this figure design. it is understandable but the standard flow chart probably will be better.

Fig. 2 resolution is poor. Cant read anything on the X-axis.

Reviewer #2: The manuscript by Maghradze et al is a useful contribution to the field. It is well written, easy to follow and draws sound conclusions. Methods seem appropriate for the study. I only have some brief comments.

I wouldn’t say the main resistance mechanism in vitro is atpE. Whilst these mutants can be isolated, several studies report and over representation of rv0678 mutants as being the dominant mechanism in vitro. Maybe reporting that atpE is high level resistance is relevant?

In figure 2, maybe an additional indicating whether the strain is susceptible, intermediate or resistant to bedaquiline. There is the MIC data, and the are described in the text, but may make it easier to read

There are 9 strains in table 1 with MIC of 0.25 or 0.5, yet 12 mentioned in the text variants are identified. Of the three with no SNPs in target genes was WGS performed?

The link to patient data is really nice, showing the influence of these mutations on outcomes

I think the statement “If confirmed, this would be similar to the situation with katG, for which the mutation Ser315Thr is rarely observed in the laboratory due to its high fitness cost in vitro despite being, by far, the most clinically relevant isoniazid resistance-conferring mutation due to its high fitness in vivo” is misleading, and should be altered or removed. I would argue that the disconnect with S315T is because in the lab any LOF mutation will provide resistance katG yet these mutants are non-viable in host due to the loss of catalase function. S315T is predominant in vivo, because it is one of the few mutations that preserves catalase function whilst also preventing the activation of INH. Again, also see comment above about frequency of atpE mutations.

Reviewer #3: Bedaquiline (BDQ) is a novel antituberculosis agent, that is pivotal to the potent multidrug regimens for the treatment of drug-resistant tuberculosis (TB). Although the genes associated with BDQ resistance has been described, the mutations that are associated with increased minimum inhibitory concentrations (MICs) have not been fully characterized. As such, the manuscript by Maghradze et al., that describes genomic correlates of BDQ resistance, provides useful information of interest to the field. Overall, the methods and results are presented clearly. A few detailed comments are bulleted below:

• Small inconsistencies in reporting number of programmatically determined BDQ-R isolates that were reclassified as susceptible or borderline:

o In the Results section, reports 3/15 initially classified as resistant that had a MIC<0.06 and no mutations, resulting in them being reclassified as susceptible. Also reports 2/15 initially classified as phenotypically resistant with MIC=0.12 and no resistance mutations. So should be 5/15 (?)

o In Discussion reports 6/15 identified through phenotypic testing turned out to be BDQ susceptible “based on repeated testing using both... pDST, as well as based on the genotypic data”

� Unclear where this 6th isolate comes from. Presumably it is the one with a mutation at 4% allele frequency, but they specifically did not include AF in their inclusion criteria for mutations, so this is incredibly unclear.

• 3 isolates found to have MIC=0.25-0.5 (classified as phenotypically resistant) but no mutation in target genes

o They only look in the target genes atpE, pepQ, Rv0678. While these are the genes previously associated with resistance, I’m curious as to why they didn’t choose to investigate mutations in other genes associated with the MOA of bedaquiline or with the efflux pump

• I understand why they would choose to only show treatment outcomes for those classified as phenotypically BDQR, however I think it could have strengthened the paper to demonstrate the difference in treatment outcomes between the resistance groups. In addition, as the authors note, the study and sample size preclude any meaningful discussion of clinical outcomes.

• States that six of the phenotypically BDQR patients had a previous TB episode, but reports the percentage as 46.2% (of 12 total patients, 6 would be 50%). Then in the table, only lists 5 patients with known TB history and 1 with missing data.

• Unclear if the 3 patients without known outcomes are still undergoing treatment, or if the data was truly unavailable, making it difficult to understand the cure rate.

• Included mixed-lineage isolates in their analyses and tree. This might not be as big of an issue for the BDQR mutation calling since they include multiple mutations at different allele frequencies for a few of their samples, but they should not be in the tree.

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Reviewer #1: Yes:  Priya Banada

Reviewer #2: Yes:  Matthew McNeil

Reviewer #3: No

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Linking genetic and phenotypic bedaquiline resistance in Mycobacterium tuberculosis strains from Georgia

PONE-D-24-38581R1

Dear Dr. Gagneux,

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,

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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: 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

Reviewer #3: Yes

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

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: 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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: The authors have considered most of the suggestions and have answered the concerns raised by the reviewers to my satisfaction. they have presented a rebuttal that due to small sample size statistical analysis was not performed. I do not have the right expertise in the stastics to make any further comments.

I am happy to recommend this manuscript for publication in PlosOne.

Reviewer #2: The authors have addressed my previous comments. The manuscript will be a a valuable contribution to the field.

Reviewer #3: I do not have any further comments. The authors have addressed all my concerns from the original submission.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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