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

Nigeria ranks 1st in Africa and 6th globally with the highest burden of tuberculosis (TB). However, only a relatively few studies have addressed the molecular epidemiology of Mycobacterium tuberculosis in this country. The aim of this work was to analyze the genetic structure of drug-resistant (DR) M. tuberculosis population in the Plateau State (central Nigeria), with the results placed in the broader context of West Africa. The study sample included 67 DR M. tuberculosis isolates, recovered from as many TB patients between November 2015 and January 2016, in the Plateau State. The isolates were subjected to spoligotyping and MIRU-VNTR typing. A total of 20 distinct spoligotypes were obtained, split into 3 clusters (n = 50, 74.6%, 2-33 isolates per cluster) and 17 (25.4%) unique patterns. The Cameroon clade was the largest lineage (62.7%) followed by T (28.3%), LAM (3%), and Haarlem (3%) clades. Upon MIRU-VNTR typing, the isolates produced 31 profiles, i.e. 7 clusters (n = 43, 64.2%, 2-17 isolates per cluster) and 24 singletons. A combined spoligotyping and MIRU-VNTR typing analysis showed 20.9% of the cases clustered and estimated the recent transmission rate at 11.9%. In conclusion, two lineages, namely Cameroon, and T accounted for the majority (91%) of cases. No association was observed between the most prevalent Cameroon lineage and drug resistance, including multidrug resistant (MDR) phenotype, or any of the patient demographic characteristics.

Introduction

Tuberculosis (TB) in Africa has a long history of counteracting and overlapping events. Anatomically modern humans have originated in the easternmost region of the African continent and so could be the case for a causative agent of human TB. The discovery of the increasing number of Mycobacterium tuberculosis lineages restricted to East Africa supports this notion [1]. Nonetheless, most of the globally prevalent M. tuberculosis lineages have originated, evolved, and adapted to local human populations on other continents and only historically recently have been brought back to the African continent. This is especially the case of the Euro-American Lineage (Lineage 4), which had hypothetically emerged a few millennia ago in the Eurasian heartland and subsequently spread in all directions but mainly westwards [2]. The ancestral Lineage 4 types were then brought to most of Africa in the 18th and 19th centuries, and started to evolve locally thereafter. Consequently, several genetic lineages were identified and named after their countries of first isolation, such as Uganda, Ghana, Cameroon etc. They were initially proposed upon distinct spoligotyping and Variable Number of Tandem Repeats (VNTR) typing patterns. More recently, the whole-genome sequencing (WGS)-based studies confirmed their identity and supported the existence of more clonal lines within Lineage 4, such as the Congo [3]. Unlike autochthonous Mycobacterium africanum, first described in Senegal in 1968 [4], newly imported strains of M. tuberculosis demonstrated increased pathogenic capacities against the naïve local population in the sub-Saharan Africa. This seemingly contrasts with a belief that a history of TB exposure provides resistance to the disease. It appears, however, that current M. tuberculosis strains circulating at least in West Africa descended from the Euro-American lineage, which followed a historical local evolution influenced by adaptation to humans and occasional human migration [1]. Thus, the higher susceptibility of black populations, compared to Caucasian people, to modern TB clades can be explained by the fact that TB has been endemic in Europe for a much longer period [5, 6].

Nigeria holds one of the top positions on the list of 30 high TB burden countries. It ranks 6th for the global TB crisis, with an estimated 440,000 new TB cases and 154,000 TB-related deaths in 2019 [7]. Nigeria is also among countries with the highest global multidrug resistant/rifampicin resistant (MDR/RR) TB and TB/HIV coinfection rates, approaching 11 and 23 per 100,000 population, respectively [7]. Noteworthy, the level of MDR-TB may be higher than officially reported [8].

Nigeria is marked with a high level of ethnic and language diversity. In particular, the Afro-Asiatic language family includes Hausa, Ngas, Goemai, and Mwaghavul languages (northern region), while Niger–Congo language family includes Berom, Yoruba, Tarok, and Tiv languages (southern and central regions). Islam is the predominant religion of the Hausa people, while Christianity and local religions are practiced in other ethnic groups. This ethnic and religious stratification may be worth taking into consideration when interpreting molecular typing data of M. tuberculosis and formulating epidemiological surveillance policies.

Molecular epidemiology of human TB in Nigeria has been investigated in a limited number of studies, mainly based on isolates collected in 2014 or before [8-14]. Most of these works have explored M. tuberculosis variability at a regional level [8–10, 12]. Only once has the genetic diversity of the Nigerian M. tuberculosis population been evaluated with a widely accepted combination of spoligotyping and 24-locus MIRU-VNTR typing [11]. All the remaining studies have used either spoligotyping only [8, 9, 13, 14] or spoligotyping in combination with a 5- or 12-locus MIRU-VNTR typing [10, 12]. The Cameroon lineage consistently predominated, and usually was followed by M. africanum [8, 10–13]. In central Nigeria, sublineage L4.6.2 (Cameroon linegae) was mostly identified [13]. Across the studies, the overall recent transmission of TB was high and accounted from 38% up to 61.1% of the cases, without major regional differences, and with clearly delineated MDR-TB clusters [10-12].

This study was largely motivated by the paucity of research on molecular epidemiology of M. tuberculosis in Nigeria. Its advantage lies in the combined use of spoligotyping and MIRU-VNTR typing on drug-resistant (DR) M. tuberculosis. Moreover, for the first time, the association between TB spoligotype signatures and patients’ ethnicity and language groups in Nigeria was determined. Additionally, the obtained phylogenetic results were placed in a wider spatiotemporal context of West Africa.

Materials and methods

Study sample

The study sample included 67 M. tuberculosis isolates, resistant to at least one first-line drug, recovered from as many pulmonary TB patients admitted to three different hospitals in central Nigeria, over a 3-month period (November 2015-January 2016). The hospitals were: (i) Plateau State Specialist Hospital TB DOTs Center; (ii) Jos University Teaching Hospital MDR-TB Clinic; (iii) Ministry of Health TB Prevention and Control Center in Jos, Plateau State, and covered Plateau State. The total number of M. tuberculosis isolates collected in the study period was 45 for the Plateau State Specialist Hospital TB DOTs Center and 145 for Jos University Teaching Hospital MDR-TB Clinic. Data on the total M. tuberculosis isolates from the Ministry of Health TB Prevention and Control Center in Jos was unavailable. Also, not for all M. tuberculosis isolates were data on their drug resistance profiles obtainable.

Since no data on patient management and long-term treatment outcomes were available, they were excluded from the analysis.

Primary isolation, culturing, and species identification were performed with standard mycobacteriological methods [15].

All methods were carried out in accordance with the guidelines and recommendations of University of Jos Ethics Committee. All experimental protocols were approved by University of Jos Ethics Committee. All data were anonymized prior the study, therefore the need for consent was waived by the Ethics Committee.

Drug susceptibility testing

Drug susceptibility testing (DST) for first-line drugs was performed using the standard 1% proportion method on the Löwenstein-Jensen medium, following the WHO protocols [16]. The M. tuberculosis H37Rv reference strain was used as a control. The critical concentrations for specific drugs were as follows: isoniazid (INH), 0.2 mg/L; rifampicin (RIF), 40 mg/L; ethambutol (EMB), 2 mg/L; streptomycin (STR), 4 mg/L.

DNA isolation

Extraction of genomic M. tuberculosis DNA was done using the cetyl-trimethyl ammonium bromide (CTAB) method, as described elsewhere [17]. The purified DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and quantified with the NanoDropTM 2000 Spectrophotometer (ThermoFisher Scientific, Waltham, USA). The DNA samples were diluted to the required concentration (ca. 10 ng/μL) and stored at –20°C until used.

Spoligotyping

Spoligotyping was performed using commercial kits (Ocimum Biosolutions, India) and following the published protocol [18]. All profiles were assessed by two independent researchers. SITVIT2 database [19; http://www.pasteur-guadeloupe.fr:8081/SITVIT2/) was used to ascertain Spoligotype International Type (SIT) numbers for the isolates studied.

MIRU-VNTR-typing

Genotyping at 24 MIRU-VNTR loci was performed, essentially as described previously [20] using agarose gels. MIRU-VNTRplus online resource (https://www.miru-vntrplus.org/) was used for phylogenetic analysis of the MIRU-VNTR profiles. A PCR failure (i.e. multiple bands or no bands) was permanently observed for Mtub39 and QUB-11b. Consequently, those two loci were excluded from the analysis.

Data analysis

A spoligotyping cluster was defined as two or more isolates sharing identical spoligotypes. The same criteria, i.e. exact match at 22 VNTR loci was applied for a MIRU-VNTR cluster. A minimum spanning tree was drawn based on the spoligotyping data with the SpolTools programme [21]. A dendrogram was constructed based on 22-locus MIRU-VNTR typing data, by using MIRU-VNTRplus software (https://www.miru-vntrplus.org/). The Hunter Gaston discriminatory index was calculated using the formula described earlier [22]. The Recent Transmission Index (RTI) was calculated using the “n-1” formula, i.e. , where N is the number of clustered cases, C is the number of clusters, and SS is the sample size [23].

A chi-square test was used to detect any significant difference between the two groups. Yates corrected χ2 and P-values were calculated with 95% confidence interval at MEDCALC (http://www.medcalc.org/calc/odds_ratio.php) online resource.

Results

Socio-demographic characteristics

The mean age of 67 enrolled patients was 35.2±10.8 years (range 15–58). Male-to-female ratio was ca. 4:1. Three-fourths (77.6%) of the patients were from urban areas, whereas the others (22.4%) came from rural locations (S1 Table in S1 File). Most (77.6%) of the patients came from north part of the Plateau State (S1 Table in S1 File).

In general, patients belonged to eight ethnic groups, with main ethnicities being Hausa, Berom, and Ngas (S1 and S2 Tables in S1 File). Hausa and Berom patients (n = 48) were all from the northern region and all but 3 (93.7%) lived in urban areas. In contrast, patients from the central region mostly (90.9%) dwelled in rural areas and most (63.6%) of them belonged to the Ngas ethnic group (S1 and S2 Tables in S1 File).

Half (52.2%) of the isolates met the definition of MDR-TB. Among isolates resistant to more than one drug, the most common drug resistance profile was INH+RIF+STR+EMB (17.9%), followed by INH+RIF+EMB (16.4%), and INH+RIF (10.4%). Monoresistance was detected in 38.8% of the isolates, and mostly (80.8%) concerned STR (Table 1).

Table 1

Drug susceptibility profiles of 67 isolates under the study.

	DST profile*	No. of isolates (n/%) n = 67
Non-MDR n = 32 (47.8%)	INH	1/1.5
	RIF	2/3
	EMB	2/3
	STR	21/31.3
	RIF+EMB	2/3
	INH+EMB	1/1.5
	INH+STR	3/4.5
MDR n = 35 (52.2%)	INH+RIF	7/10.5
	INH+RIF+EMB	11/16.4
	INH+RIF+STR	5/7.4
	INH+RIF+EMB+STR	12/17.9
	Total	67/100

*isoniazid (INH), rifampicin (RIF), ethambutol (EMB), streptomycin (STR).

In total, 20 spoligotyping profiles were identified, including 3 types shared by 2, 15, or 33 isolates, and 17 unique profiles. All 67 isolates belonged to the Lineage 4. The largest clusters were SIT61 (Cameroon) and SIT53 (T), represented by 33 (49.2%) and 15 (22.4%) isolates, respectively (Table 2). Five unique profiles could not be found in SITVIT2 and were thus designated as orphan/new types, although application of the SITVIT2 decision rules and expert assessment permitted to assign 3 such isolates to the Cameroon (n = 2) and T (n = 1) lineages. The remaining two isolates with undefined clonal line status were designated as L4-unclassified. Only two lineages (Cameroon and T) accounted for 91% of the isolates (62.7% and 28.3%, respectively).

Table 2

Spoligotype-based diversity of 67 M. tuberculosis isolates under the study.

SIT	Lineage	43-signal spoligoprofile	No. of isolates (n/%)
61	Cameroon	■■■■■■■■■■■■■■■■■■■■■■□□□■■■■■■■□□□□■■■■■■■	33/49.2	42/62.7
838		■■■■■■■■■■■■■■■■■■■■■■□□□■■■■■■■□□□□■■■■□■■	1/1.5
2970		■■■■■■■■■■■■■■■■■■□■■■□□□■■■■■■■□□□□■■■■■■■	1/1.5
3400		■■■■■■■■■■■■■■■□□□□■■■□□□■■■■■■■□□□□■■■■■■■	1/1.5
115		■■■■■■■■■■■■■■□■■■■■■■□□□■■■■■■■□□□□■■■■■■■	1/1.5
2550		■■■■■■■■■■■■□■■■■■■■■■□□□■■■■■■■□□□□■■■■■■■	1/1.5
852		■□□□□□□□□□□□□□□□■■■■■■□□□■■■■■■■■□□□□■■■■■■■	1/1.5
2830		■■■■■■■■■■■■■■■■■■■■■■□□□■□■■■■■□□□□■■■■■■■	1/1.5
Orphan5new		■■■■■■■■■■■■■■■□□■■■■■□□□■■■■■■■□□□□■■■■■■■	1/1.5
Orphan1new		■■■■■■■■■■■■■■■■■■■■■■□□□■■■■■■■■□□□□■■■■□□□	1/1.5
53	T	■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□□□□■■■■■■■	15/22.3	19/28.3
774		■■■■■■■■■■■■■■■■■■■■■■■■□■■■■■■■□□□□■■■■■■■	1/1.5
1580		■■■■■■■■■■■■■■■■■■■■■■□□■■■■■■■■□□□□■■■■■■■	1/1.5
1913		■■■■■■■■■■■■■■□■■■■■■■□■■■■■■■■■□□□□■■■■■■■	1/1.5
Orphan7new		■■■■■■■■■■■■■■□■■■■■■■□□■■■■■■■■□□□□■■■■■■■	1/1.5
49	Haarlem	■■■■■■■■■■■■■■■■■■■■■■■■■■■■■■□■□□□□■■■□■■■	1/1.5	2/3
316		■■■■■■■■■■■■■■■■■■■■■■■■□□□□□□□■□□□□■■■□■■■	1/1.5
17	LAM	■■□■■■■■■■■■□■■■■■■■□□□□■■■■■■■■□□□□■■■■■■■	2/3	2/3
Orphan8new	L4-unclass.*	■■□■■■■■■■■■■■■■□□■■■■■■■■■■■□■■□□□□■■■□■■■	1/1.5	2/3
Orphan3new		■■■■■■■■■■■■■■■■■■■■□□□□□□□□□□□□□□□□■■□□■■■	1/1.5

*L4-unclass.– L4-unclassified; assignment to the Lineage in accordance with an expert assessment.

A schematic view of the evolutionary relationships between spoligotypes is shown in Fig 1. The network was built based on the general knowledge of the evolution within Lineage 4, in particular the ancestral state of the SIT53 and taking into consideration that M. tuberculosis CRISPR/DR locus is evolving by loss of spacers. Although the picture is simplified it gives an adequate view of the sequence of events behind evolution of the Nigerian spoligotypes, and a local historical evolution of the Cameroon lineage.

Fig 1

Spoligoforest of 67 M. tuberculosis isolates from the Plateau State, Nigeria, illustrating the potential evolutionary relationships of the spoligotypes identified in this study.

Two Haarlem strains had different SITs (Table 2) and due to their single deletion event difference, they could not be directly linked to each other. In addition, they differed in other variables; hypothetically ancestral SIT49 was a STR-resistant strain, isolated from a Hausa patient based in the northern urban area, while SIT316 was a RIF-resistant strain from a Ngas patient dwelling in the central rural area. Therefore, these isolates could be at most, only distantly related. In contrast, LAM SIT17 isolates were both from patients living in urban northern area, yet of different ethnicities (Hausa and Ngas). The strains also exhibited different drug resistance profiles (INH+RIF+EMB vs. INH+RIF+STR).

Whereas the MDR rate was somewhat higher among T compared to Cameroon isolates (57.9% vs. 47.6%, P = 0.4), the proportion of MDR isolates among two major types, i.e. SIT61 and SIT53 was almost identical (45.5% vs. 46.7%) (S3 Table in S1 File).

Concerning the geographical distribution, the Cameroon lineage was more prevalent in the center of the Plateau State, compared to north (81.8% vs. 63.5%, P = 0.2), while T lineage was more common in the north (28.8% vs. 9.1%, P = 0.2). The northern sample was somewhat more diverse in terms of HGDI than the central one (S4 Table in S1 File). No significant difference was found between urban and rural subgroups, except for slightly higher prevalence of SIT61 in the urban setting and higher diversity in the rural setting (S5 Table in S1 File).

The MDR isolates were more frequent in the Berom than Hausa population (59.1% vs. 50%; P = 0.5) (S6 and S7 Tables in S1 File). The Cameroon genotypes were found at a similar frequency in the two ethnic groups, but the T lineage and SIT53 type were more common among the Berom people (P = 0.3 and 0.5, respectively).

In addition to the ethnic subdivision, the isolates were grouped according to the language family and religion (S7–S9 Tables in S1 File). The T clonal line was more prevalent in the Niger-Congo language family (38.5% vs. 22.0%; P = 0.1). Two LAM isolates were from Hausa and Ngas patients that belonged to the Afro-Asiatic language family. Monoresistance was more prevalent among Moslems (42.3% vs. 22.0%, P = 0.08) (S7 Table in S1 File), but the two religious groups did not differ significantly with regard to the genotype distribution (S9 Table in S1 File).

As for the age stratification, patients infected with the Cameroon lineage isolates had a mean age of 34.4±10.4 years (range 15–58), and those excreting T clonal line isolates had a mean age of 38.4±11.5 years (range 19–58). The proportion of young patients (≤35 years old) was higher in Cameroon than T group (55.8% vs. 36.8%, P = 0.17).

Males predominated in both Cameroon and T spoligotype lineages (80.9% and 78%).

MIRU-VNTR typing

A total of 7 MIRU-VNTR types were described, shared by 2 to 17 isolates, totaling 43 (64.2%) isolates in clusters. The remaining 24 (35.8%) isolates harbored unique patterns. None of the VNTR types were associated with a particular ethnic group or place of living. The two largest clusters comprised of 17 and 11 isolates, representing different regions and two major language families, i.e. Niger-Congo and Afro-Asiatic (Fig 2).

Fig 2

Dendrogram based on the 22 VNTR loci for 67 M. tuberculosis isolates from the Plateau State, Nigeria.

Abbreviations: I–isoniazid; R–rifampicin; E–ethambutol; S–streptomycin; SIT—Shared International Type; 22 MIRU loci order: MIRU 2-4-10-16-20-23-24-26-27-31-39-40, Mt-04, ETR-C, Mt-21, ETR-A, Mt-29, Mt-30, ETR-B, Mt-34, QUB-26, QUB-4156c.

Combined analysis

A combined spoligotyping and MIRU-VNTR analysis resolved 53 (79.1%) unique patterns, and yielded a HGDI of 0.995. The HGDIs for spoligotyping and MIRU-VNTR alone were of 0.713 and 0.905, respectively. The clusters (6 in total) comprised a maximum of 3 isolates, producing the overall clustering rate of 20.9% and RTI of 11.9%. However, only one cluster (n = 2; 3%) contained isolates of the same resistance profile (STR-resistant) and recovered from patients of the same ethnicity (Hausa) and place of living (urban area in the northern part of the Plateau State).

Discussion

Association of genotypes with phenotypic and patient-related characteristics. TB transmission

In previous studies from Cameroon and Nigeria, male gender and age between 25 and 34 years were significantly associated with the Cameroon lineage [10, 14, 24]. Here, a proportion of younger patients was clearly higher among those infected by the Cameroon (55.8%) compared to the T lineage bacilli (36.8%). This difference was not significant, possibly due to a small sample size. Males predominated (77.6%), irrespective of the genotype.

No relationship was observed between the Cameroon clonal line and drug resistance in the previous studies [12, 14, 24]. In the present one, somewhat higher MDR rate was found for the T lineage compared to Cameroon, but the difference was insignificant. Thus, the increasing prevalence rate of the Cameroon clonal line in this and some previous studies cannot be attributed to its capacity to acquire MDR phenotype. In the first study that described this lineage, in Cameroon, it was noted that the clonal line was distributed homogeneously across the entire Ouest province and the reasons for its selection and dissemination were unknown [25]. The authors hypothesized the M. bovis BCG vaccination, which is common practice in Cameroon, may play a role in the selection of Cameroon lineage isolates. In vivo virulence studies may be useful to understand reasons behind dissemination the of this and other African genotypes.

Monoresistance was quite high (38.8%) and STR-monoresistance dominated (31.3%). However, STR-monoresistant isolates were located in different spoligotyping clusters (Fig 2). Furthermore, resistance to STR was not associated significantly with any patient characteristic. Thus, STR-monoresistant TB in the Plateau State is rather related to noncompliance or inadequate treatment than spread of a particular clone.

So far, only three studies have estimated the proportion of TB transmission in Nigeria, based on a combination of spoligotyping and MIRU-VNTR typing [10-12]. Using a 5-, 12- and 24-locus set, the RTI was calculated at 61.1%, 48.1%, and 38%, respectively. In this study, the RTI was importantly lower, (11.9%), which is even more surprising given the missing results from the two VNTR loci (22/24). In addition, clustered isolates were mostly isolated from patients of different socio-demographic characteristics and whose strains exhibited different resistance profiles. All this may indicate that patients developed TB due to reactivation of a latent infection or transmission in a distant past, rather than due to a recent transmission event. This might be somewhat surprising in the light of the patients’ age (mean age 35.2±10.8 years). It thus seems that TB transmission in our group was somewhat underestimated, and that the criteria used for clustering, as a proxy for TB transmission, were too restrictive. As for the other countries in central/west Africa, the RTI reported, and established with the same methodology, was much higher, calculated at 42.6%, 35.8%, and 26.8% in Congo, Burkina Faso, and Benin, respectively [26-28]. Contrastingly, in a study from Cameroon, no MIRU-VNTR clusters were observed, and thus no transmission was assumed [29].

M. tuberculosis phylogeography in Nigeria and West Africa

While large-scale population structure is deeply rooted in time, the frequent everyday transborder movements can influence the spread of the particular clonal clusters and spoligotypes. To place our results in a wider phylogeographic context, we compared the main genotype lineages from this study with those observed in West Africa, and included dynamic changes, whenever available (Fig 3 and S10 Table in S1 File). The main SIT in this study was SIT61 (Cameroon lineage). In the first articles on M. tuberculosis strain typing in Africa it was noted that this predominant spoligotype in Cameroon was limited to West African countries (Benin, Senegal, and Ivory Coast) and to the Caribbean area [25]. SIT61 is the prototype of the Cameroon clonal line whose signature is deleted signals 23–25 and 33–36. The Cameroon lineage is one of the most known African genotypes of M. tuberculosis. Previously, based on spoligotyping, this clonal line was misnamed as LAM-CAM. More recently, MIRU-VNTR and WGS analysis demonstrated that it is a separate clonal line within the large and heterogeneous Euro-American lineage. Phylogeographically, the Cameroon strains are circulating in different African countries, and have been found on other continents, due to cross border migration. In SITVIT2, 1,095 isolates are assigned to the Cameroon lineage and the most numerous (ca. 40%) are samples from Nigeria and Cameroon itself. An increase of the Cameroon clonal line has been reported over years in Chad [30, 31] and Benin [27, 32] (S10 Table in S1 File). Similarly, in Guinea, 132 (71.7%) strains were genotypically clustered, and the Cameroon lineage was detected to be spreading significantly faster (p<0.001) than the background rate of the whole spoligotype population [33]. Overall, our meta-analytical approach showed that the prevalence of the Cameroon lineage was high in Cameroon, Nigeria, Burkina Faso, Benin, and Ghana. The increasing trends for this clonal line were reported in Nigeria and Chad, while decreasing or low ratio in the western part of West Africa, e.g. in Ivory Coast, Gambia, Mali, and Guinea (Fig 3).

Fig 3

Geographic distribution of the main M. tuberculosis spoligotype lineages in Nigeria and other parts of West Africa (see S10 Table in S1 File for details and references).

For each setting we show the years of surveillance and number of isolates studied. Free map: USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/.

The second main SIT in this study was SIT53 (T lineage). According to our global analysis, SIT53 showed an increase in Cameroon and Chad, and a very high rate in Niger and Ghana (Fig 3). However, one should keep in mind that SIT53 is a heterogeneous group of distantly related strains otherwise found on different branches of the L4 tree based on genome-wide SNPs [34]. The SIT53 populations from diverse geographical locations most likely represent unrelated branches that evolved from ancestral SIT53 over historically long time [34]. In this sense, SIT53 populations in different parts of Africa may represent unrelated genotypes.

Minor groups in this study represented well-known global LAM and Haarlem clades. SIT17 belongs to the RD-Rio sublineage of the LAM clade and it was hypothesized that this genotype derived from SIT20 by single spacer deletion, originated in South America [35]. The published MIRU-VNTR-based phylogenetic analysis of the LAM lineage demonstrated that SIT17 isolates were present in Brazil, Venezuela, and Sierra-Leone. Interestingly, SIT17 was also described in Cameroon 20 years ago [25] and the identification of SIT17 in the present study could reflect its endemic circulation, albeit at a low rate. The same may be true for the Haarlem SIT49 spoligotype that was also described in other country-wide study from Nigeria (5/549 isolates [13]), a study from Ivory Coast (2/194 isolates; [36]), and from Burkina Faso (1/72 isolates; [37]).

The M. africanum isolates were not included in this work. However, the prevalence of M. africanum has been decreasing in recent decades due to its reduced virulence [38]. According to previous reports from Nigeria, M. africanum was detected with a prevalence of 33% and 13% [10, 11]. More recently, only 3% of TB in Nigeria was shown to be due to M. africanum [14]. On the other hand, the low recovery of M. africanum might be explained by difficulties in isolating this species in routine practice [37].

Our own analysis of the geographic distribution of the causative agent of human TB in West Africa showed that the prevalence of M. africanum was low in Ivory Coast, Niger, and Guinea, and decreasing in Cameroon (Fig 3). Furthermore, M. africanum was not detected in Chad. In contrast, a high proportion of M. africanum was reported in Gambia, Mali, Burkina Faso, and Ghana, and in some parts of Nigeria (Fig 3). It appeared that there is no apparent pattern of M. africanum geographical distribution across West Africa. Thus, specific features of the national TB control programs and ethnic background may play a role in shaping the dissemination pattern of M. africanum.

The common problem with TB epidemiological studies is their small sample size. Thus, the distribution of M. tuberculosis lineages shown in Fig 3 may be just a random snapshot of lineage compositions in different countries. In this sense, Fig 3 presents the current state of knowledge that needs further studies.

Certain limitations of the study have to be mentioned. Firstly, study sample included 67 isolates originated from only one state of Nigeria. Therefore, the results could not be extrapolated to the entire country. Secondly, since only the conventional DST method was applied, the acquired genetic resistance could not be tracked under the study. Thirdly, a substantial improvement to the study would be a scrupulous clinical interview aiming at a better understanding of transmission routes of M. tuberculosis infection. Finally, of particular importance would be data concerning management and treatment outcomes of the patients.

Conclusions

To conclude, this work describes the genetic diversity of DR M. tuberculosis strains circulating in the Plateau State, Nigeria, with a combination of spoligotyping and 22-loci MIRU-VNTR-typing. There are three major findings from the study. First, all DR M. tuberculosis isolates were of the Euro-American lineage, and belonged to a limited number of spoligotype-defined lineages, mostly Cameroon and T clades (91%). The minor spoligotypes represented otherwise globally widespread Haarlem and LAM clonal lines. Second, the combination of spoligotyping and MIRU-VNTR typing resulted in a TB transmission rate of 11.9%, suggesting that recent transmission only scarcely contributes to the persistence of DR-TB in the Plateau State. Third, no association was observed between MDR or STR-monoresistance and Cameroon lineage. Thus, an increasing prevalence of this clonal line, observed here and in previous studies, cannot be associated with an increased acquisition of drug resistance. Further in vivo studies are needed to elucidate the pathobiological reasons behind the dissemination of M. tuberculosis lineages in Nigeria.

(DOCX)

Click here for additional data file.

15 Feb 2022

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Reviewer #2: Yes

Reviewer #3: Partly

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

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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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10 Mar 2022

Warsaw,

21st February, 2022

To:

Sarman Singh, MD, FRSC, FRCP

Academic Editor

PLOS ONE

Dear Sarman Singh,

I would like to thank you, as well as your expert Reviewers, for helping us to improve our manuscript (PONE-D-21-26334). You will find below our answers (highlighted in grey).

Editor:

1. Majority of reviewers have endorsed that it is a good study, but has 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.

We appreciate a positive feedback from the Editor. Now we have added section regarding study limitations in the “Discussion”. Please see below.

“Certain limitations of the study have to be mentioned. Firstly, study sample included 67 isolates originated from only one state of Nigeria. Therefore, the results could not be extrapolated to the entire country. Secondly, since only the conventional DST method was applied, the acquired genetic resistance could not be tracked under the study. Thirdly, a substantial improvement to the study would be a scrupulous clinical interview aiming at a better understanding of transmission routes of M. tuberculosis infection. Finally, of particular importance would be data concerning management and treatment outcomes of the patients.”

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.

We would like to thank the Reviewer for this positive comment.

1. 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.

We agree with this comment. However some of the studies shown in Fig. 3 were not so small (N>300-500). The following paragraph was added to the revised version of the manuscript in the “Discussion” section. Please see below.

"The common problem with TB epidemiological studies is their small sample size. Thus, the distribution of M. tuberculosis lineages shown in Fig. 3 may be just a random snapshot of lineage compositions in different countries. In this sense, Fig. 3 presents the current state of knowledge that needs further studies.”

2. 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).

The suggested correction has been made in the revised manuscript and supplementary tables.

3. 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.

We thank the Reviewer for pointing out this error. In the study there were 15 females (22.4%), and 42 males (77.6%). We have now corrected this mistake, as follows (changes are marked in bold).

“Male-to-female ratio was ca. 4:1.”

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

The correction has been made.

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

We have corrected this.

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.

We appreciate the Reviewer's positive feedback

1. 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.

As suggested by the Reviewer, we have now removed this sentence from the “Abstract”.

2. 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.

We agree with the Reviewer, that data regarding percentage of all M. tuberculosis isolates collected in the sampled area (and during the sampling period), along with isolates’ drug resistance profiles, would be of much value for the study. However, the data on the total number of M. tuberculosis isolates collected in the study period from the Ministry of Health TB Prevention and Control Center in Jos were unavailable. Furthermore, drug resistance profiles were unavailable for most of the isolates. Thus, initially we decided not to include those data in the manuscript.

We have now commented on this in the “Materials and Methods” section. Please see below.

“The total number of M. tuberculosis isolates collected in the study period was 45 for the Plateau State Specialist Hospital TB DOTs Center and 145 for Jos University Teaching Hospital MDR-TB Clinic. Data on the total M. tuberculosis isolates from the Ministry of Health TB Prevention and Control Center in Jos was unavailable. Also, not for all M. tuberculosis isolates were data on their drug resistance profiles obtainable.”

3. 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.

The suggested correction has been made. Please see Fig. 2.

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

The suggested correction has been made. Please see Fig. 3.

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 on 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.

As suggested by the Reviewer 1, we have incorporated spoligotyping and MIRU-VNTR typing analysis into Figure 2. Therefore, now all the data are presented together in one figure. Furthermore, we have added the subtotals of non MDR and MDR strains in Table 1, as requested.

1. 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.

As requested by the Reviewer, we have rephrased the sentence. Please see below.

“Two Haarlem strains had different SITs (Table 2) and due to their single deletion event difference, they could not be directly linked to each other.”

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.

We have already addressed this issue. Now all the data are combined in Figure 2. If requested, we can add Supplementary Excel File, with all the raw data.

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.

Male to female ratio was wrongly calculated in the manuscript. We are sorry about it. The study included 15 females (22.4%), and 42 males (77.6%). Therefore, the predominance of males in the Cameroon and T families is not surprising.

We have now corrected the error, as follows (changes are marked in bold).

“Male-to-female ratio was ca. 4:1.”

Phrase in line 268 should be revisited for English writing.

We thank the Reviewer for this comment. We have now reparsed the sentence (see below):

“The authors hypothesized the M. bovis BCG vaccination, which is common practice in Cameroon, may play a role in the selection of Cameroon lineage isolates.”

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.

We thank the Reviewer for this valuable comment. We have now rephrased the Discussion section. Please see below (changes are marked in bold).

“All this may indicate that patients developed TB due to reactivation of a latent infection or transmission in a distant past, rather than due to a recent transmission event. This might be somewhat surprising in the light of the patients’ age (mean age 35.2±10.8 years). It thus seems that TB transmission in our group was somewhat underestimated, and that the criteria used for clustering, as a proxy for TB transmission, were too restrictive.”

The biggest cluster, involving 17 strains, show that most of the strains came from the Northern 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?

We agree with the Reviewer that data regarding history of treatment is of great importance in studies like ours. Sadly, data on patients’ clinical management were either fragmentary (precluding reconstruction of treatment history) or completely absent. We have added now the following sentences in the “Materials and Methods” and “Discussion” section.

“Since no data on patient management and long-term treatment outcomes were available, they were excluded from the analysis.”

“Finally, of particular importance would be data concerning management and treatment outcomes of patients.”

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.

As suggested by the Reviewer, we have now commented on this in the paragraph regarding study limitations in the “Discussion” section. Please see below:

“Secondly, since only the conventional DST method was applied, the acquired genetic resistance could not be tracked under the study”

Data supports partially 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.

We thank the Reviewer for these concluding remarks. We have addressed them carefully, as described above. Furthermore, the description of statistical methods used in this study is depicted in the Material and Methods section. Please see below.

“A chi-square test was used to detect any significant difference between the two groups. Yates corrected χ2 and P-values were calculated with 95% confidence interval at MEDCALC (http://www.medcalc.org/calc/odds_ratio.php) online resource.”

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

However, I have some comments to improve it.

We would like to thank the Reviewer for this positive comment.

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

Yes. This number corresponds to distinct spoligotype patterns. We have rephrased the sentence for better clarity. Please see below (changes are marked in bold).

“A total of 20 distinct spoligotypes were obtained, split into 3 clusters (n=50, 74.6%, 2-33 isolates per cluster) and 17 (25.4%) unique patterns”.

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

As stated in the Materials and Methods section, the study sample included 67 M. tuberculosis isolates, resistant to at least one first-line drug. Therefore, the sentence “The study sample included 67 DR M. tuberculosis isolates" is correct.

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

We thank the Reviewer for bringing our notice to that. We have now corrected the “Ivory Coast” name through the manuscript.

Submitted filename: Rebuttal.docx

Click here for additional data file.

29 Mar 2022

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

PONE-D-21-26334R1

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12 May 2022

PONE-D-21-26334R1

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

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