PONE-D-23-09720Phenotypic and genotypic drug susceptibility patterns of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Central and Southern EthiopiaPLOS ONE

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Additional Editor Comments:

I have also provided independent comments due to the difficulty in finding a second reviewer. I would like to see answers to all the provided comments with special attention to either performance of discordance testing if the strains are still available or consideration of the limitation of the MGIT testing performance if no repeat testing is possible for the strains that had resistant results and a select set of the S strains. 

Comments 

The authors have used convenience sampling to collect a set of septum samples from newly diagnosed MTB infected subjects. The manuscript is well prepared and written but I have a few comments with the last being the most substantive. From the supplemental data set, there were 36 DR strains, but only 10 with a genotypic explanation. I suggest providing the data set in a .csv file in future so that it is ease to review. Further there were 25 strains that tested as INH-R in MGIT 0.1 ug/mL while only of those 9 had KatG mutations and 1 mixed genotype KatG/WT was detected. No inhA mutations were detected, leaving 14 strains with discordant results. AhpC-oxyR intergenic region was not tested. The streptomycin resistance was also not verified molecularly although since the drug is little used and has minimal overlap with other injectables, it is likely not significant to the subjects treatment, but I would have preferred to see these strains molecularly confirmed.

One MDR strain was identified by both genotypic and phenotypic testing. Generally, the definition of MDR TB requires both INH and RIF resistance, so only 1/250 strains tested were MDR. The other 5 strains might be better called something else, maybe polyresisent or another term in common use.

The WHO Technical manual for drug susceptibility testing of medicines for treatment of tuberculosis (2018) now recommends a 0.1 ug/mL for INH. Often the 0.4 mg/mL INH concentration has been used and is useful in that it identified INH highly resistant strains. We have found the 0.4 ug/mL concentration correlates better with the occurrence of the katG mutation. This same document indicates that all phenotypic methods for EMB resistance produce inconsistent results and it is not recommended. I would suggest that without genotypic evidence of EMB resistance, this data not be emphasized. You might list this poor test performance in the limitations.

No discussion is included of the proficiency of the MGIT testing laboratory. Does it participate in a TB DST qualification program on a regular basis?

There are numerous reports of mixed strains that have a WT and MUT phenotype. Not sure how often it is seen in Africa but its common in Asia. Not sure I would use the word surprising in the results, especially since you discuss others seeing mixed signals in the discussion.

Lines 290 to 292 – it is not clear which strains were tested with the MTB-DRplus assay. Was it all strains, if 7 strains or 1, perhaps indicate in table with a subscript. Part of the confusion is the definition of MDR. The authors are also confused since they use both a singular and plural form of isolate(s) in these lines.

I am not convinced that table 3 is necessary given the amount of data in it and it well described in the text, but if it is kept, I suggest using the mutation code for amino acid change (column 3) and not the names of the probes from the testing kits. WT/ can be added for the mixed strain.

In line 318-319, the authors talk about the performance of MTBDRplus assay but do not indicate that any repeat discordance MGIT testing was done. Slight differences in drug inoculation and bacterial inoculums produce results indicating resistance that can be in error. Repeated MIGT INH testing as a discordance evaluation is advised for the purposes of a manuscript like this, if you want to discuss test performance.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

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

Reviewer #1: Yes

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Reviewer #1: No

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Reviewer #1: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors provide an overview of pDST and gDST results from a cross-sectional study of TB isolates from Central/South Ethiopia.

1. The major finding of the study was noted that there was discordance between pDST and gDST for INH (14 isolates), and the authors concluded that INH-R TB was found in Ethiopia that was not detected by LPA. However this conclusion was not supported by any discordance testing therefore it is unknown whether these difference were due to improper MGIT DST performance or actual mutations not found on the LPA. Therefore this is a major flaw of the study, and discordancy testing (if not possible to be performed) should be addressed.

2. Why was MGIT DST performed only for the low concentration (0.1 ug/mL) and not the 0.4 ug/mL concentration? Considering the 100% of INH mutations to be katG, it would have been useful to test this higher concentration.

3. There were no inhA mutations detected-this is a bit unusual considering low-level INH resistance is commonly found. The authors should include this in the discussion.

4. Second-line LPA testing was only performed on the MDR isolate (N=1)-however to fully understand the resistance profiles of the isolates tested it would have been useful to employ this testing on all isolates (or at least those with any resistance) to look at FQ monoresistance and correlate with MGIT 2nd line DST.

5. Supplementary material was not able to be reviewed as the file would not download (It was in a .SAV format)

6. The demographic information, while useful to review was not the main focus of the study and can be put as a supplemental Table.

7. Kindly check spelling of "heteroresistance" and "MGIT" throughout manuscript.

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Phenotypic and genotypic drug susceptibility patterns of Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients in Central and Southern Ethiopia

PONE-D-23-09720R1

Dear Dr. Tilahun

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication once the two comments below are addressed and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

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Additional Editor Comments (optional):

I thank you for your responses to the reviewers comments. Other than a few small remaining clarifications, the responses are accepted.

On line 450, I'd suggest removing the words "due to contamination",

On line 451 I'd suggest a sentence more like "resource constraints prevented us from

conducting repeated INH MGIT on the the strains with discordant results to confirm INH resistance. These constraints also prevented us from....."

Reviewers' comments: