J Clin Tuberc Other Mycobact Dis 21 (2020) 100182 Contents lists available at ScienceDirect J Clin Tuberc Other Mycobact Dis journal homepage: www.elsevier.com/locate/jctube A pilot study on the genetic diversity of Mycobacterium tuberculosis complex T strains from tuberculosis patients in the Littoral region of Cameroon Benjamin D. Thumamo Pokama,b,⁎, D. Yeboah-Manub, P.M. Teyimc, P.W. Guemdjomd, B. Waboa, A.B.D. Fankepa, R.E. Okonub, Anne E. Asuquoe a Department of Medical Laboratory Science, University of Buea, Cameroon b Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana c Douala Tuberculosis Reference Laboratory, Littoral Region, Cameroon d Department of Public Health, University of Buea, Cameroon e Department of Medical Laboratory Science, College of Medicine, University of Calabar, Calabar, Nigeria A R T I C L E I N F O A B S T R A C T Keywords: Background: The re-emergence of tuberculosis (TB) worldwide, compounded by multi-drug resistance (MDR) of Tuberculosis the causative agents constitutes a major challenge to the management of the disease. Rapid diagnosis and ac- Xpert MTB/RIF curate strain identification are pivotal to the control of the disease. This pilot study investigated the genetic Rifampicin resistance diversity of Mycobacterium tuberculosis complex (MTBC) strains from TB patients in the Littoral region of UgandaI sublineage Cameroon as well as their resistance to rifampicin (RIF). Cameroon Patients and methods: This was a cross sectional hospital-based study carried out between January and December 2017 and including 158 isolates from sputum smear positive individuals [105 (66.5%) males and 53 (33.5%) females]. Sputum samples were tested using Xpert MTB/RIF, followed by culture on Lowenstein–Jensen medium. Isolates were further subjected to molecular characterization using IS6110 typing, deletion analysis and spoligotyping. Results: Thirteen (8.8%) of the 147 isolates with susceptibility results available were resistant to RIF. Drug resistance occurred in 5/50 (10%) female compared to 8/97 (8.2%) male (OR, 0.81; 0.25–2.62; p = 0.764), and there was no significant difference across the age ranges (p = 0.448). On the other hand, RIF resistance was associated (OR, 0.18, 95%CI, 0.05–0.69; p = 0.023) with previously treated patients [(4/14 (28.6%)] compared to new ones [9/133 (6.8%)]. The 150 identified lineages included among others 54 (36%) Cameroon, 18 (12%) UgandaI, 32 (21.3%) Haarlem, 17 (11.3%) Ghana, 9(6%) West African 1, 7(4.7%) Delhi/CAS, 4 (2.7%) LAM and 3 (2%) UgandaII. Of the 150 isolates, the major cluster was the Cameroon SIT 61, with 43(28.7%) isolates. Six (35.3%) of the 17 UgandaI sub-lineage were RIF resistant (OR, 9.58; 95%CI, 2.74–33.55, p = 0.001). Conclusion: The cosmopolitan Littoral region presents with a wide Mycobacterium tuberculosis (MTB) strains diversity and the UgandaI sub-lineage likely associated with RIF resistance. Understanding the spread of this clade through surveillance will enhance TB control in the region. 1. Introduction Conventional diagnostic methods for Mycobacterium tuberculosis (MTB) are slow and/or lack sensitivity [7], resulting to large proportion of TB Tuberculosis (TB) remains a major cause of illness and death cases as well as drug resistant TB remaining undiagnosed and leading to worldwide, especially in Africa [1], where drug resistant TB transmis- continuous transmission. sion results from failure to implement proper TB control programs, TB control efforts were for a long time hampered by the lack of including inadequate care as well as ineffective management of TB accurate point of clinical care tests for detection of MTB and drug re- cases ranging from administration of improper regimens to failure to sistance [8], thereby delaying the initiation of TB second-line at the ensure treatment completion by patients [2–4]. Early diagnosis of TB early stage of treatment. However, the problem has been mitigated by resistance through rapid drug susceptibility testing is important for the development and endorsement by the WHO of the Gene Xpert® management of multidrug resistance tuberculosis (MDR-TB) [5,6]. MTB/RIF assay, a rapid molecular assay which concurrently determines ⁎ Corresponding author at: Department of Medical Laboratory Science, Faculty of Health Sciences, University of Buea, P.O.Box 63, Buea, Cameroon. E-mail address: thumamo@yahoo.fr (B.D. Thumamo Pokam). https://doi.org/10.1016/j.jctube.2020.100182 Available online 01 September 2020 2405-5794/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). B.D. Thumamo Pokam, et al. J Clin Tuberc Other Mycobact Dis 21 (2020) 100182 MTB and rifampin resistance (RR) which serves as a surrogate marker out between January and December 2017 and including one hundred for MDR–TB [9]. Moreover, the test can be performed with minimal and fifty-eight (158) isolates obtained from TB patients attending technical expertise and results available within 2 h, thereby allowing various TB care centers across the Littoral region of Cameroon. The the early commencement of disease management [10]. The sensitivity region is subdivided into four divisions: Wouri, Moungo, Nkam and and specificity of Xpert MTB/RIF in detecting TB have been shown to be Sanaga-Maritime with their respective capitals at Douala, Nkongsamba, 88% and 99% respectively, while the the sensitivity and specificity in Yabassi and Édéa. Douala is the economic capital of the country and is a detecting RR were 95% and 98% respectively [11]. densely populated and cosmopolitan city with foreigners as well as The emergence of MDR-TB fueled by poor TB control especially in people coming from the other 9 regions of the country seeking for Africa as a result of limitation to funding, laboratory capacity, erratic business and employment. Hence the presence of some crowded slums. drug supplies, qualified personnel, and facilities, [12] added to the One part of an unprocessed sputum specimen from each participant Human Immunodeficiency Virus/ acquired immunodeficiency syn- was analyzed using GeneXpert MTB/RIF assay v 4.3 (Cepheid Inc., drome (HIV/AIDS) global pandemic [13], has contributed to the dra- Sunnyvale, CA, USA) according to manufacturer’s instructions. Briefly, matic increase in the TB burden worldwide. The rpoB mutations gen- one mL of sputum samples in a 1:2 ratio of sample reagent processing erally found in rifampicin-resistant MTB strains are located in a region solution (isopropanol and NaOH) was used [9]. The treated sample at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, incubated at room temperature for 15 min was transferred to the car- referred to as Rifampicin-resistance-determining region (RRDR) [14]. tridge and loaded into the GeneXpert instrument with subsequent fully The drug-resistant TB requires accurate diagnosis to guide therapy and automated processing. The other part of the sputum sample was further interrupt transmission of resistant strains in communities [15]. decontaminated using the N-acetyl L-cysteine–sodium hydroxide Genetically diverse lineages and sublineages of MTB have evolved (NALC/NaOH) method, cultured using Lowenstein–Jensen (L-J) [16], and seven major lineages have been shown to be adapted to medium and incubated at 37 °C for 6–8 weeks. Positive slants were human sub-populations in diverse geographic settings with different reconfirmed by Acid Fast Bacilli (AFB) microscopy following Ziehl- variation in virulence [17,18]. Recently, lineage 8 likely restricted to Neelsen staining technique. the African Great Lakes and associated MDR has been discovered [19]. Therefore, some lineages occur globally as lineages 2 and 4 probably 2.2. Genotyping MTBC isolates due to high virulence, while others show geographical restriction as lineages 5 and 6, mainly restricted to West Africa. It thus appears that The preserved isolates from Cameroon in glycerol were shipped to specific lineages have different propensities to transmit and develop the Bacteriology Laboratory of the Noguchi Memorial Institute for drug resistance [20]. The molecular techniques have allowed the Medical Research (NMIMR) - Ghana, where approval from the Scientific identification and tracking of individual strains of MTB [21], providing Technical Committee and Institutional Review Board of the NMIMR an insight into the prevalence and transmission of Mycobacterium tu- was obtained for the genotyping of the isolates. The obtained DNA from berculosis complex (MTBC) [22]. This in turn may help improve TB heat-killed mycobacterial cells suspensions (95 °C for 50 min) were control and patient management strategies [23]. subjected to molecular analyses. IS6110, a 1361-bp long, belongs to a family of insertion sequences (IS) of the IS3 category and have been utilized as targets in the iden- 2.3. IS6110 amplifications, deletion analyses and spoligotyping tification of MTB by Polymerase Chain Reaction (PCR). The reliability, sensitivity, and specificity of PCR have been shown to be dependent on PCR detection of the insertion sequence was carried out to confirm the amplification of DNA with primers specific to different target se- the MTBC as described previously [31], and the products were elec- quences in the genome [24]. It is highly conserved and has been used trophoresed on 2% agarose gels and visualized under UV light following for the molecular epidemiological analysis of clinical isolates [25]. ethidium bromide staining. However, IS6110 based diagnosis has been shown to be limited by the The Large Sequence Polymorphisms (LSPs) typing assay identifying presence of low copy number or absence of the IS6110 repetitive se- regions of difference (RD) 1, 4, 9, 12, 702, 711 was carried out on the quence. [25–27]. Spoligotyping on the other hand has some major mycobacterial DNA. Lineage-defining LSPs were detected by PCR using advantages over standard IS6110 typing, requiring minimal quantities the primers described earlier [31] and identifying RD 1, 4, 9, 12, 702, of DNA [21] and thus can be used directly on clinical specimens 711 using the reactions described previously [31]. Distinct lineages without the need for prior culture. It can be valuable in countries which within the MTBC in the study were grouped as previously defined [18], do not routinely culture specimens considering its ability to type iso- and finally, the spoligotyping was carried out following manufacturer’s lates using small amounts of DNA. However, several samples may be instructions (Isogen Bioscience, The Netherlands) on a membrane using required before successful typing can be performed on sputum speci- the 43-spacer [21]. M. tuberculosis H37Rv, and M. bovis BCG DNAs mens [28]. were used as parallel positive controls and distilled water as a negative In Cameroon, TB incidence, mortality and MDR/RR-TB rate in 2018 control. were 186 (121–266), 31 (18–47) and 3.5 (1.7–6.0) for 100,000 popu- lation respectively [29]. Although changes in TB notification data 2.4. Data analysis might indicate successful TB control in the country, there are also strong indications that TB transmission is still ongoing [30]. However, All the data were entered into an Excel sheet and the spoligotype there is a paucity of data on the MTBC circulating strains as well as the patterns in a binary format were analyzed (Supplementary material I) possible transmission of antimycobacterial drugs resistant lineages in using the SpolDB4 database/MIRU VNTR plus [32]. SPSS version 20 population across the Littoral region of Cameroon. This study therefore analyzed the association between the variables using Chi square and evaluated the genetic diversity of MTBC and the associated rifampicin Fisher exact test. Values of p (two sided p-values) less than 0.05 were resistant sublineages from TB patients in the Littoral region of Ca- considered significant at 95% confidence interval. meroon which can provide a basis for TB control in the study area. 3. Results 2. Methods and patients 3.1. Rifampicin resistance by gender, age and previous treatment 2.1. Study design and area, specimen collection and culture Of the 158 isolates included in this study, 11 (7%) were obtained This was a prospective cross sectional hospital-based study carried through L – J culture and not subjected to the Xpert MTB/RIF test. Of 2 B.D. Thumamo Pokam, et al. J Clin Tuberc Other Mycobact Dis 21 (2020) 100182 Table 1 RIF susceptibility by gender, age and treatment status of patients. No. Tested Rifampicin Odds ratio (95% CI) P value Sensitive Resistant Gender 0.81(0.25 – 2.62) 0.764 Male 97 89 (91.8) 8 (8.2) Female 50 45 (90) 5 (10) Total 147 134 13 Age group (years) 0.448 15–24 31 30 (96.8) 1 (3.2) 0.29(0.04–2.31) 0.302 25–34 42 37 (88.1) 5 (11.9) 1.64(0.5–5.33) 0.520 35–44 36 31 (86.1) 5 (13.9) 2.08(0.63–6.81) 0.308 45–54 24 22 (91.7) 2 (8.3) 0.93(0.19 –4.47) 0.642 ≥ 55 14 14 (100) 0 (0) 0.367 Total 147 134 13 Treatment Status 0.18(0.05–0.69) 0.023 New 133 124 (93.2) 9(6.8) Previously treated 14 10 (71.4) 4 (28.6) Total 147 134 13 the 147 RIF susceptibility results obtained using the Xpert MTB/RIF, 13 Eighteen (12%) of the 150 isolates including 15 sublineages (3 Ca- (8.8%) were resistant and 134 (91.2%) were sensitive. One hundred meroon, 1 Delhi/CAS, 3 Haarlem, 2 LAM, 3 UgandaI, and 3 West and twenty-four (92.5%) of the 134 RIF sensitive were new cases. Of African 1) did not have a SIT number, while 3 isolates with SIT numbers the 13 resistant cases, 3 (23%) occurred in relapsed patients, 1(7.7%) 1548, 847 and 852 had no identified sublineages. MDR contact, 1 (7.7%) with treatment failure and 8 (61.5%) among newly diagnosed patients. Though not significant (OR, 0.81; 95%CI, 3.3. Rifampicin resistance and sublineages association 0.25–2.62; p = 0.764), resistance occurred in 5/50 (10%) female compared to 8/97 (8.2%) male. The age groups 25 – 34 and 35 – 44 The distribution of the sublineages according to RR is shown in recorded [5/42 (11.9%) and [5/36 (13.9%)] resistant cases (OR,1.64; Fig. 2. Six (46.2%) of 13 RIF resistant isolates were UgandaI sublineage, 95%CI, 0.5–5.33; p = 0.520 vs OR,2.08; 95%CI, 0.63–6.81; p = 0.308) 4 (31%) Cameroon, 1(8%) Dehli/CAS and 2(15%) with no sublineage. respectively. Overall, there was no significant difference across the age The distribution of sublineages and RR in various quarters of Douala ranges (p = 0.448). There was a significant association (OR, 0.18, and across cities within the Littoral region of Cameroon is shown in 95%CI 0.05–0.69; p = 0.023) among previously treated patients [(4/14 Supplementary material II (and map in Supplementary material III), (28.6%)] compared to new ones [9/133 (6.8%)] (Table 1). based on patients’ referral hospital location. The six patients with RIF resistance associated with the UgandaI 3.2. Genotypes distribution sublineage were reported as either treatment failure (1), relapse (2), MDR at first month of evaluation (2) or MDR contact (1), harboring Eight (5.1%) of the 158 isolates were IS6110 negative and the three T2 [2 SIT317 and 1 SIT 52], one T1 (SIT 244), and two undefined identified 150 isolates subdivided into 8 sublineages included 54 (36%) SIT. All the terms used were based on the World Health Organisation Cameroon, 32 (21.3%) Haarlem, 18 (12%) UgandaI, 17 (11.3%) Ghana, (WHO) definition [33] as treatment failure including a patient who is 9(6%) West African 1, 7(4.7%) Delhi/CAS, 4 (2.7%) LAM, 3 (2%) sputum smear or culture positive at 5 months or later after the initiation UgandaII and 6 (4%) with no sublineage (Fig. 1). of anti-TB treatment. Table 2 shows the distribution of the 150 isolates classified into 31 identified Shared International Types (SIT). The Cameroon with SIT 61 3.4. Rifampicin resistance association with SIT and sublineages represented the major cluster [43/150 (28.7%) isolates], followed by Haarlem SIT 50, Ghana SIT53, UgandaI SIT 52 and Delhi/CAS SIT 46 The association between RR with SIT and sublineages is shown in with 21(14%), 12(8%), 10(6.7%) and 6(4%) isolates respectively. Table 3. Of the 67 isolates with SIT numbers, 9(13.4%) were RR. Four SIT [3 SIT 61 and 1 SIT 850] did not have RR results, and 3 sensitive No sublineage (4%) 6 Cameroon sublineage were without SIT number. Equally, 1 Dehli/CAS UgandaII (2%) 3 without SIT number was sensitive, 1 SIT 244 of UgandaI sublineage was without RR result and 3 (2 resistant and 1 sensitive) UgandaI sub- LAM (2.7%) 4 lineages had no SIT number. All the SIT of the Ghana, Haarlem, West Delhi/CAS (4.7%) 7 African 1, LAM and UgandaII sublineages as well as the 3 SIT (1548, West African 1 (6%) 9 847 and 852) with no identified sublineages exhibited no RR. Among the Cameroon sublineage, 4/40 (10%) SIT 61 (p = 0.488) Ghana (11.3%) 17 were RIF resistant. One (100%) SIT 244, 2 (100%) SIT 317 and 1/10 UgandaI (12%) 18 (10%) SIT 52 (OR, 0.04; 95%CI, 0.0–0.79; p = 0.04) within the UgandaI sublineage (p = 0.02) were RIF resistant. One (16.7%) of the 6 Haarlem (21.3%) 32 Dehli/CAS was RIF resistant Cameroon (36%) 54 Of the 147 sensitivity tests performed, 2 (1.4%) resistant and 10 (68%) sensitive strains had no sublineage. All the 3 (100%) UgandaII, 3 0 10 20 30 40 50 60 Frequency (100%) LAM, 9 (100%) West African 1, 30 (100%) Haarlem and 16 (100%) Ghana sublineages were sensitive to RIF. The number ex- Fig. 1. Spoligotype distribution of the isolates in the Littoral Region of hibiting RR included 6/17 (35.3%) UgandaI (OR, 9.58; 95%CI, Cameroon. 2.74–33.55, p = 0.001), 4/50 (8%) Cameroon (OR, 085, 95%CI, 3 Sublineages B.D. Thumamo Pokam, et al. J Clin Tuberc Other Mycobact Dis 21 (2020) 100182 Table 2 Distribution of genotypes, sublineages, shared international types (SITs) and spoligotypes patterns of 150 Mycobacterium tuberculosis complex isolates in the Littoral region of Cameroon. No sublineage, n of rapidly detecting resistant strains of MTB, but the GeneXpert does = 2, 15% not detect isoniazid (INH) resistance [34]. Nevertheless, some evalua- Cameroon, n = 4, tion studies have shown that using Xpert MTB/RIF instead of smear 31% microscopy as currently practiced in several developing countries can Delhi/CAS, n= 1, tremendously improve TB detection [36,37]. The large scale im- 8% plementation and widespread extension policy of the use of Xpert MTB/ RIF in Cameroon, is an asset in the rapid diagnosis of TB in the country, especially as culture is not carried out routinely due to limited facilities. In 2018, Noeske and coworkers [38] reported GeneXpert based RR of 1.6% among new cases of TB in 10 regions of Cameroon with regional variations ranging from 0 to 3.3%. The higher prevalence in our study could be explained by the fact that our data was generated from both retreatment and newly diagnosed TB patients. The diagnosis of ex- tensively drug-resistant (XDR) TB using the Xpert MTB/XDR assay for INH and second-line drug resistance currently under clinical evaluation UgandaI, n = 6, trial, will be an added value in the rapid detection of XDR-TB strains 46% [39], which magnitude is largely unknown in the country. Fig. 2. Distribution of spoligotype sublineages associated with RIF resistance. RIF resistance in this study was not associated with gender.as shown in similar studies [35,40]. It has been shown in general that there was 0.25–2.91; p = 0.53) and 1/7(14.3%) Dehli/CAS (OR, 1.78; 95%CI, no evidence of either sex being more at risk of MDR/RR-TB in 81% (86/ 0.2–16.02; p = 0.484) sublineages. 106) of countries using sex-disaggregated data of TB patients reported to WHO [41]. In an earlier report, Coovadia et al. [34] showed that males had an increased odd of being RIF mono-resistant as compared to 4. Discussion females, and that RIF resistance is more likely to occur in the 25–29 years’ age category. It has been shown in a neighboring country This study carried out in the Littoral region of Cameroon has shown that significantly higher proportion of TB patients aged above 45 years a RR of 8.8%, similar to a research carried out in South Africa [34] but had RR compared with patients below 45 years [42], in contrast to our higher than a previous one in Zambia [35]. RR is regarded as a proxy study which found no age related association. for MDR-TB. Currently available molecular assays have the advantage 4 B.D. Thumamo Pokam, et al. J Clin Tuberc Other Mycobact Dis 21 (2020) 100182 Table 3 Association between Rifampicin resistance with SITand sublineages. No. Tested Rifampicin Odds ratio (95% CI) P-value Sensitive n (%) Resistant n (%) SIT Cameroon 0.979 SIT 403 1 1 (100) 0 (0) 0.915 61 40 36 (90) 4 (10) 0.512 838 3 3 (100) 0 (0) 0.761 839 1 1 (100) 0 (0) 0.915 844 1 1 (100) 0 (0) 0.915 850 1 1 (100) 0 (0) 0.915 Total 47 43 (91.5) 4 (8.5) UgandaI 0.02 SIT 244 1 0 (0) 1 (100) 0.286 3 1 1 (100) 0 (0) 0.714 317 2 0 (0) 2 (100) 0.659 52 10 9 (90) 1 (10) 0.04(0.0–0.79) 0.04 Total 14 10 (71.4) 4 (28.6) Delhi/CAS SIT 46 6 5 (83.3) 1 (16.7) Total 67 58 (86.6) 9 (13.4) Sublineages 0.005 UgandaI 17 11 (64.7) 6 (35.3) 9.58(2.74–33.55) 0.001 Cameroon 50 46 (92) 4 (8) 0.85(0.25–2.91) 0.53 Delhi/CAS 7 6 (85.7) 1 (14.3) 1.78(0.2–16.02) 0.484 UgandaII 3 3 (100) 0 (0) 0.756 LAM 3 3 (100) 0 (0) 0.756 West African 1 9 9 (100) 0 (0) 0.606 Haarlem 30 30 (100) 0 (0) 0.071 Ghana 16 16 (100) 0 (0) 0.362 No sublineage 12 10 (83.3) 2 (16.7) 2.25(0.44–11.61) 0.601 Total 147 134 (91.2) 13 (8.8) This study has shown that previously treated patients were sig- study in the Adamaoua region of Cameroon reported the ubiquitous T nificantly infected with RIF resistant strains unlike a study carried out family and in addition, noted the significant presence of the H1 family, in Ethiopia who found no association compared to new patients [43], suggesting an adaptation of these strains to the local population fol- but was in line with a neighboring country - Nigeria where a significant lowing their introduction through migration. These observations have proportion of RR-TB was found among previously treated TB patients been noted elsewhere [49,56]. In our study, M. africanum (MAF) re- [42]. Several studies have linked history of previous TB treatment as a presented 6% with 2% belonging to SIT 101. This corroborates with the strong risk factor for MDR-TB [44,45], calling for a surveillance findings of a previous study carried in the same study area which strategy to be implemented in order to control the dissemination of identified 2.74% MAF1 represented by AFRI_2 lineages [53]. The con- MDR strains by patients on retreatment. Improvement of treatment tribution of MAF to TB disease has substantially decreased from 56% adherence as well as laboratory capacities and introduction of mole- over the past four decades, accounting for only 9% of cases about cular diagnostic tools detecting the various TB genotypes will be re- 20 years ago in the country [48]. In neighboring Nigeria, it accounted quired for an efficient control not only of MDR dissemination, but for 12% a decade ago [49]. equally MDR associated lineages to prevent the likely spread of MDR- This study has shown the UgandaI sublineage as well as SIT 52 RR strains in the country. within the UgandaI sublineage likely associated with RIF resistance. The predominant sublineage in our study was the Cameroon family, This is contrary to a study carried out in Uganda which showed through which has earlier been shown not only to be prevalent in the country, a cluster analysis no significant association between drug resistance and but largely implicated in different pocket of TB transmission [46]. The lineages, especially among the T2 family [57]. Previous studies in Ca- spoligotype 61 especially which lacks spacers 23, 24, and 25 in the meroon have shown no statistical link between drug resistance and direct repeat (DR) region represented about 29% of all the isolates (and MTBC genotypic families [46,47]. It could have been hypothesized in- about 80% of the Cameroon family in this study) has been shown to be stead an association of drug resistance with the most prevalent Ca- widely prevalent Cameroon [46,47,48], Nigeria [31,49], Chad [50], meroon sublineage (especially SIT61) present in the country. Equally, and within the west African region [51,52]. this lack of association has been demonstrated recently in a neighboring The Cameroon family (36% in this study) seems well established country [31]. The Uganda genotype of MTB has been shown not only to since its designation and description in the west region of Cameroon be the prevalent (up to 70% of isolates) cause of PTB in Uganda [58], (more than 40%) nearly two decades ago [48]. A similar study as ours but equally associated with extrapulmonary TB [59]. Kigozi et al. [60] carried out recently in the Littoral region specifically in Douala the city in a study assessing RR in MTB isolates from Uganda found that lineage capital, recorded 54% of the Cameroon family with 51% of SIT61 [53], 4/sub-lineage Uganda accounted for 36% of the rifampicin-resistant thereby buttressing the clustering of pulmonary TB in this city [54]. isolates with 24% being UgandaII and 11% UgandaI. This is lower The other families in this study such as the H (lineage H1 and H3), T compared to our findings, where all the six patients with RIF resistance (lineage T1, T2 and T5) and U with lineage U and U (Likely H) have associated with the UgandaI sublineage had pulmonary TB. Also in been previously described in the country [46,55], as well as in Douala their study, patients were either on treatment failure, relapse, MDR at [53]. The latter authors recorded in line with our study that, the first month of evaluation or MDR contact. Noeske et al. [61] in a non- Haarlem sublineage, especially SIT 50 was the second most pre- molecular based study, recorded 12% MDR strains in the Littoral region dominant one encountered. Koro Koro and colleagues [46] in their with low positive treatment outcome rates in retreatment patients with 5 B.D. Thumamo Pokam, et al. J Clin Tuberc Other Mycobact Dis 21 (2020) 100182 MDR-TB. Although TB drug resistance has been linked both to the 5. Conclusion quality of control programs as well as socioeconomic status, the in- trinsic factors prompting its emergence and expansion remain unclear The Xpert MTB/RIF does provide a rapid surrogate MDR detection [62,63]. However, there is evidence of genotypic linkage of MTB strains and thus a timely management of TB patients, preventing the associated driving the epidemiology of drug resistant TB isolated from patients in increased morbidity and mortality in the study area. The cosmopolitan different geographical region and suggesting and adaptation of various Littoral region presents with a wide MTB strains diversity, with the lineages to particular genetic, cultural or environmental characteristics predominant being the Cameroon family and the UgandaI sub-lineage of the host [64]. This might be an evolutionary trend which will require likely associated with RIF resistance. Clear mapping and understanding further careful investigation to measure the real impact of this asso- the current trend of dissemination of the UgandaI sublineage is essential ciation in the Littoral region of Cameroon. The emergence and spread of for the control and development of the drug resistance associated with this drug resistant MTB lineage originally absent in this region as shown this clade, which appears to constitute a flash point in the study area, by a previous study [53], could be associated with immigration, clinical country and sub-region. and demographic factors, as well as evolution of MTB strains. Under- standing the mechanisms shaping transmission and regardless of whe- Authors contributions ther the patients acquired the infection elsewhere or from their current locality or from a reactivated disease contracted in their native country BDTP, DYM conceived and designed the experiment. BDTP, PMT, can provide an insight into the potential approaches for TB control in REO performed the laboratory experiment. BDTP, PWG, BW, ABDF this setting [65,66]. Although a hypothesis suggesting that lineages not analyzed the data. BDTP, DYM, PWG, BW drafted the manuscript and previously described in a defined population earlier could be in- wrote the paper. PMT, ABDF, AEA substantially revised the manuscript. troduced by immigrants, paucity of information and data on various All the authors read and approved the final version. drug resistant genotypes especially in Africa makes this assumption difficult to prove. The introduction of new MTB strains that more Ethical statement transmissible and virulent and more prone to develop drug resistance has been associated with migration, (especially movement of popula- This research was approved by the Scientific Technical committee tion to bigger cosmopolitan cities in search of better health care facil- and Institutional Review Board of the Noguchi Memorial Institute for ities as well as employment opportunities) and can drive the current Medical Research (NMIMR), University of Ghana, Legon, Accra – Ghana changing TB situation [67]. In the current context of globalization and [FWA 00001824; IRB 00001276; NMIMR-IRB CPN 007/16-17; IORG population movement, this is a particular challenge in the control of 0000908]. drug resistant TB strains dissemination. Douala our study area, being not only the economic capital of the country with attendant high po- Declaration of Competing Interest pulation overcrowded slums, is additionally the main entry point in Cameroon with a seaport and the busiest airport of the country bringing The authors declare that they have no known competing financial in foreigners. This can thus further explain the introduction of the interests or personal relationships that could have appeared to influ- UgandaI sublineage in this setting. ence the work reported in this paper. The circulating MTBC strains surveillance in a locality is important for understanding TB epidemiology. MTB strain identification can Acknowledgement contribute to the better control of the disease [68]. Newer strategies including specific active surveillance especially of relapsed and re- This work was supported by the Bill and Melinda Gates Foundation treatment patient should be elaborated for efficient control of the surge to B.D.T.P, under the Postdoctoral and Postgraduate Training in of MDR and associated lineages in the region. The prevalence of the Infectious Diseases Research awarded to the Noguchi Memorial UgandaI genotype likely associated with RR in the Littoral region of Institute for Medical Research-NMIMR (Global Health Grant number Cameroon might suggests a recent introduction as well as result of poor OPP52155). The funder had no role in study design, data collection and treatment adherence considering that all the patients involved were analysis, decision to publish, or preparation of the manuscript. either on retreatment or MDR contacts. Furthermore, this genotype has not been previously associated with drug resistance in the country. Appendix A. 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