PONE-D-21-26334Molecular snapshot of drug-resistant  Mycobacterium tuberculosis  strains from the Plateau State, NigeriaPLOS ONE

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==============================Majority of reviewers have endorsed that it is a good study, but has some some major concerns, particularly the conclusions which should be based on hard data. I would suggest the authors to add a section of study limitation which should include beside other limitations, the phenotypic method (proportion method) of Drug resistance determination. 

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 paper is about clonal composition of Mtb isolates from central Nigeria hospitals. For lineage identification, the authors used several standard genotyping techniques: spoligotyping and MIRU-VNTR-typing. Also, each isolate was characterized by drug susceptibility testing. Then the authors compared the Mtb lineage composition from Nigeria to those from other West African countries available from the literature.

The paper is interesting. The methods are adequate to the aims and the declared results of this study. I support publishing this paper after several minor corrections.

The common problem of all studies of this kind is the relatively small sample size. The authors had analyzed 67 Mtb isolates. Figure 3 looks for me as a random snapshot of Mtb lineage compositions in different countries due to this problem rather than because of any internal differences between countries in terms of disease distribution history, history of human migrations or any human genetic differences. The authors have to add several clear statements on whether they believe themselves into this picture on Fig. 3 and what can support this believe. Or if it is just the current stay of the art that needs further studies, please say it explicitly.

Throughout the text the authors use the term ‘families’ for Mtb lineages (lines 38, 54, 58, 64, 180, 182, 230, 292, 297, 299, 300, 314, 353, 354, 360. Better use the terms ‘lineages’, or ‘clonal lines’, or ‘genotypes’ to avoid confusing with taxonomic families (i.e. Mycobacteriaceae).

I am a bit confused with the male-to-female ratio of the patients. In line 155 it is said that the ratio was 3:5. But in line 230: Males predominated in both Cameroon and T spoligotype families (80.9% and 78%). And in line 259: Males predominated (77.6%), irrespective of the genotype. So, there were more female patients in the group but males dominated. Explain the statistics.

Line 71 – decipher MDR and RR-TB when these terms are used for the first time.

Line 89 – remove the dot before the reference in “…identified. [13].”

Reviewer #2: Bakuła et al. applied spoligotyping and MIRU-VNTR to establish the genetic diversity of drug-resistant M. tuberculosis strains circulating in the Plateau State in Nigeria. The number of Mtb isolates included in this study was quite limited (67 strains) but allowed to identify the transmission rate and the dominant lineages present in the investigated area. Based on spoligotyping data and MIRU-VNTR analysis Authors found that all drug-resistant isolates

in the Plateau State belong to Euro-American lineages, mostly Cameroon and T clades. In contradiction to the previous reports, the TB-transmission rate was determined as low as <12%.

The manuscript is well written. The weak points of the presented story are a low number of isolates included in the study and 22 (not 24) loci included in the MIRU-VNTR analysis (due to a technical reason). However, these weaknesses do not affect the conclusions made by the authors.

Comments:

Abstract – the sentence “Most of the patients have developed TB due to a reactivation of infection or from a transmission event in the distant past” is speculation and should be excluded from the Abstract section. The increased number of isolates included in the study could affect the transmission rate determined by the authors.

67 drug-resistant strains isolated in a period of three months were enrolled in the analysis. It should be mentioned what % is it of all Mtb isolates, and DR-Mtb isolates, in this area in the time of one year.

Fig. 2 should be replaced with the dendrogram prepared based on the combined analysis, spoligotyping, and MIRU-VNTR, or at least spoligotyping data (SIT) should be included in the dendrogram description.

Fig. 3 each pie chart located in Fig. 3 should be described with a number of isolates (n=x).

Reviewer #3: The work entitled: Molecular snapshot of drug-resistant Mycobacterium tuberculosis strains from the Plateau State, Nigeria by Bakula et al., 2022 is intended the provide information about the molecular epidemiology of M tuberculosis strains in Nigeria based pn DR Mtb strains placed in a the West Africa context. Nigeria ranks 1st in Africa and 6th with the highest burden of TB. Spoligo and MLVA typing were used to disclosure Mtb clustered strains (20.9%) and recent transmission rate was estimated at 11,9%. Most of the strains were represented by two Clades (91%), Cameroon and T. Interestingly, TB cases came from reactivation of previous infection and no association was detected between Cameroon Clade and drug resistance.

Below, my comments.

I believe that results should be presented together instead of separately. I mean, a table with drug susceptibility profiles + both molecular epidemiologic markers + origins + ethnicities + languages + religions. I may suggest to show subtotals of Non MDR and MDR strains in Table 1. Both molecular markers complement each other and hence produce reliable results defining real clusters and correcting lineage origins plus having a global view of the epidemiologic situation. It is well know that both molecular markers are prone to convergent evolution, and Spoligotyping is more susceptible. In this way reading will be much more easy and analysis will be more afordable.

For example, in line 199 you say that two Haarlem strains had different SITs (Table 2), but they could be linked directly to each other with one deletion event….. it is well known that unique deletion events could not be linked with spoligo data alone and that a second and more robust marker is needed to verify this situation due to convergent evolution. What I see is a block of spacers deleted at the left side of the Haarlem spoligo signature, an event that is not possible to know if happened in a single step. I suggest to re-write this sentence.

It would have been highly appreciated a dendogram with all data suggested previously to be presented in one Table. This provides the most important and reliable information.

In page 11, line 230 you say that males predominate in Cameroon and T families and in line 259 (page 12) again irrespective of the genotype but previously (page 7, line 155) you said that male:female ratio was 3:5? could you please explain better what you really want to say.

Phrase in line 268 should be revisited for English writing.

You guys say that TB was developed due to reactivation of a previous infection rather than from a recent transmission event, based mainly on the low RTI and the clustering rate by epimol tools. However, sample size and the criteria to be included in the present study influence this low rate observed. I would like to add that your results show that the mean age of 67 enrolled patients was 35.2±10.8 years (range 15-58). When TB is present in patients around this age it is believed that should have been the result of TB transmission more than reactivation, that is expected in older people.

The biggest cluster, involving 17 strains, show that most of the strains came from the Northen and urban region of Nigeria. Strains 1, 2, 28, 33, 34, 43, 52, 58, 64 share at least resistance to IR and also they share the Hausa/Fulani ethnicity (Fig 2). These strains share a link plus the VNTR profile hence they might be transmitting in the population at a low rate. The arisen question is how is adherence to treatment?

The lack of treatment adherence selects for drug resistance appearance hence some patients that became infected with the transmitting susceptible strain will have the same strain type but now will became resistant. Classic molecular epidemiology surveys are needed to clarify this situation.

We must remember that drug susceptibility testing was based on proportion method rather than mutation detection which means that a population of bacilli could be evolving to develop resistance to any drug but may not be detected due to the test sensitivity unless the mutation conferring resistance will be prevalent in patient´s bacilli. It is well known that drug resistant strains mainly transmit at a lower rate than susceptible strains with rare exceptions. I conclusion, it may not be exactly the same strain but a very related one that arose by evolution from the parental strain.

I may suggest to say something about this.

Data supports partlaly the conclusions in the way they are written.

You should mention in Method which Statistical method you used.

All Data is avaiable but should be better presented.

The manuscript was wrote in standard english however some parts must be re-written.

Reviewer #4: This study is well written and provides interesting TB molecular epidemiology analyis in Nigeria.

However, I have some comments to improve it.

In the abstract section (line 31), you mentioned "a total of 20 spoligotypes", is this number corresponding to distinct spoligotypes ?

On line 29, you said "The study sample included 67 DR M. tuberculosis isolates"

Cote d’Ivoire is not always well written in the article.

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Reviewer #1: Yes: Oleg N. Reva, Dep. Biochemistry, Genetics and Microbiology; Centre for Bioinformatics and Computational Biology, University of Pretoria, Pretoria, South Africa.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Molecular snapshot of drug-resistant  Mycobacterium tuberculosis  strains from the Plateau State, Nigeria

PONE-D-21-26334R1

Dear Dr. Jagielski,

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Academic Editor

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