Abstract
Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and an important cause of human tuberculosis in West Africa that is rarely observed elsewhere. Here we genotyped 613 MTBC clinical isolates from Ghana, and searched for associations between the different phylogenetic lineages of MTBC and patient variables. We found that 17.1% (105/613) of the MTBC isolates belonged to M. africanum, with the remaining belonging to M. tuberculosis sensu stricto. No M. bovis was identified in this sample. M. africanum was significantly more common in tuberculosis patients belonging to the Ewe ethnic group (adjusted odds ratio: 3.02; 95% confidence interval: 1.67–5.47, p<0.001). Stratifying our analysis by the two phylogenetic lineages of M. africanum (i.e. MTBC Lineages 5 and 6) revealed that this association was mainly driven by Lineage 5 (also known as M. africanum West Africa 1). Our findings suggest interactions between the genetic diversity of MTBC and human diversity, and offer a possible explanation for the geographical restriction of M. africanum to parts of West Africa.
Author Summary
Tuberculosis remains one of the main global public health problems. Human tuberculosis is caused by bacteria known as the Mycobacterium tuberculosis complex (MTBC). The MTBC includes a variant called Mycobacterium africanum, which causes up to half of all tuberculosis cases in West Africa. For reasons unknown, M. africanum does not occur in other parts of the world. To explore the possible reasons for this geographic restriction of M. africanum, we analysed a large collection of bacterial strains isolated from tuberculosis patients in Ghana. We genetically characterized these bacterial isolates and collected relevant socio-demographic and epidemiological data. We found tuberculosis patients infected with M. africanum were more likely to belong to the Ewe ethnic group, compared to patients carrying other MTBC bacteria. The Ewes are indigenous inhabitants of coastal regions in West Africa that have previously been shown to harbour a high prevalence of M. africanum. Our findings support the hypothesis that different variants of MTBC have adapted to different human populations, and offer a possible explanation for the geographical restriction of M. africanum to West Africa.
Introduction
Tuberculosis (TB) remains the leading cause of adult death by a single infectious disease world-wide [1]. Despite the high mortality caused by TB, only 5% to 10% of infected immunocompetent individuals progress from initial infection to active disease [1]. In 2013, an estimated 9.0 million new cases and 1.5 million deaths due to TB occurred; with 30% of the global burden of TB occurring in Africa, an indication of the strong association with HIV/AIDS [1].
TB is caused by a group of closely related bacteria referred to as the Mycobacterium tuberculosis complex (MTBC). MTBC comprises M. tuberculosis sensu stricto and M. africanum which are the main agents of TB in humans, and several variants adapted to various domestic and wild mammal species, including M. bovis, M. caprae, M. microti and M. pinnipedii [2]. MTBC relevant to human disease has been classified into seven main phylogenetic lineages [3]–[4]: Lineages 1 to 4 together with Lineage 7 are collectively known as M. tuberculosis sensu stricto, whereas Lineage 5 and 6 are also known as M. africanum West Africa I and II, respectively [5].
Because MTBC harbours limited genetic diversity compared to other bacteria [6], for a long time the assumption was that host and environmental factors were the only relevant determinants driving the course of TB infection. However, recent studies have challenged this dogma. Indeed, experimental infection models have shown that MTBC strains differ in virulence and immunogenicity [7], and epidemiological studies have demonstrated that in addition to host and environmental factors, strain diversity contributes to the variable outcome of TB infection and disease in clinical settings [8].
The MTBC lineages adapted to humans exhibit a strong phylogeographic population structure [4]. This together with the finding that the MTBC most likely originated in Africa and accompanied human migrations over millennia [9] has led to the proposal that the different lineages of human-associated MTBC might have locally adapted to different human populations [10]. Support for this notion comes from the observation that in metropolitan settings, MTBC lineages tend to transmit preferentially among sympatric (as opposed to allopatric) host populations [11], and that this sympatric host-pathogen association is perturbed by HIV co-infection [12].
Previous work showed that in Ghana, human TB is caused by six out of the seven MTBC lineages, with 20% of all cases attributed to Lineages 5 and 6 [13] (i.e. M. africanum West-Africa I and West-Africa II, respectively). M. africanum is highly restricted to West-Africa for reasons unknown [5], [10]. One possibility could be that M. africanum has adapted to particular human populations in that region of the world. To address this possibility, we performed a retrospective molecular epidemiological study of MTBC in Southern Ghana. We combined bacterial genotyping with detailed demographic and epidemiological patient data and sought for associations between host factors and the main MTBC lineages prevailing in Ghana.
Methods
Ethics statement
All study protocols including oral and written consent format used for this study were approved by the Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research, Legon-Ghana (NMIMR; Federal wide Assurance number FWA00001824) and from the Ethikkommission Beider Basel (EKBB) in Basel, Switzerland. The standard procedure for sampling as outlined by the National Tuberculosis Program (NTP) for the routine management of TB in Ghana was used in the study. Written (in the case of literate participants) or oral (for illiterates) informed consent was sought from all participants before inclusion in the study. For minors (below sixteen years of age) consent was sought from their parents/guardians before enrolment into the study. In the case of minors between sixteen and eighteen years, in addition to parental consent, assent was sought from them before enrolment into the study. As per the guidelines of the IRB of NMIMR, information confidentiality was strictly adhered to. In addition, objectives and benefits of the study were explained to all participants.
Study setting and patients' characteristics
The study was conducted from July 2007 to August 2011. All patients were diagnosed as sputum Acid-Fast-Bacilli-positive pulmonary TB cases by microscopy. The patients were recruited before treatment initiation from five main health facilities; Korle-Bu Teaching Hospital in the Greater Accra region, Agona Swedru Government Municipal Hospital, Winneba Government Hospital, St Gregory Catholic Clinic from the Central Region and Effia-Nkwanta Regional Hospital from Western Region of Ghana. Information on age, sex, nationality, ethnicity, employment status, previous history of TB, crowding, substance abuse and duration of symptoms were obtained from the patients with a structured questionnaire. Patients with missing information or culture-negative status were excluded from analysis. Ethnicity was classified in line with Ghana demographic data 2010 [14]. Patient origin was defined by place of residence.
Isolation of mycobacterial species and genotyping
Sputum samples obtained were decontaminated using 5% oxalic acid [15] and inoculated on two pairs of Lowenstein Jensen (LJ) slants; one supplemented with 0.4% sodium pyruvate to enhance the isolation of M. africanum and M. bovis, and the other with glycerol for the growth of M. tuberculosis sensu stricto. The cultures were incubated at 37°C and were read weekly for growth for a maximal duration of 16 weeks. MTBC strains were identified by detection of insertion sequence IS6110 as previously described [16]. Classification into the main phylogenetic lineages of MTBC was achieved by large sequence polymorphism typing identifying regions of difference (RD) [2] in a stepwise manner. Firstly, all isolates were screened for RD9. RD9-undeleted strains were further sub-typed for the “Cameroon” strain family (known to be most dominant Lineage 4 sub-lineage in Ghana) by screening for deletion of RD726 [11]. Isolates identified as RD9-deleted were subsequently sub-typed for Lineage 5 and 6 using RD711 and RD702 flanking primers, respectively. All lineages identified were confirmed by TaqMan real time PCR (TaqMan, Applied Bio systems, USA) using probes targeting lineage-specific SNPs as reported [17].
Spoligotyping
All MTBC isolates were typed by spoligotyping [18]. This was performed according to the manufacturer's instructions, using commercially available kits (Isogen Bioscience BV Maarssen, The Netherlands). Spoligotyping patterns were defined according to SITVITWEB database [19] (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE). SITVITWEB assigned shared types numbers were used whenever a spoligotyping pattern was found in the database while families and subfamilies were assigned based on the MIRU-VNTRplus database (http://www. miru-vntrplus.org) [20]. Shared types were defined as patterns common to at least two or more isolates. All patterns that could not be assigned were considered orphan spoligotypes.
Data entry, management and analysis
Information from the structured questionnaire was double entered using Microsoft Access and validated to remove duplicates and data entry inconsistencies. Multivariable logistic regression models were used to compare patient characteristics associated with M. africanum compared to M. tuberculosis sensu stricto. All statistical analyses were performed in STATA 10.1 (Stata Corp., College Station, TX, USA).
Results
Tuberculosis patients and their characteristics
A total of 622 TB patients were included in this study. Age of patients ranged from 8 to 77 years with a median age of 35 years (Table 1). Overall, 208/622 (33.4%) were females with median age of 33 years; the remaining 414 (66.6%) were males with a median age of 36. Twenty-nine out of the 622 patients (4.6%) were children (age<16 years). Most patients originated from Greater Accra Region (325 cases, 52.3%), followed by 268 cases (43.1%) from Central Region with the remaining twenty-nine patients (4.6%) from Western Region of Ghana. Out of the 622 patients, 596 (95.8%) were Ghanaians, 21 (3.3%) were Liberians, 2 Togolese (0.3%) and 1 (0.2%) each of Nigerian, Ivorian and Gambian origin, respectively. Most of the patients were of Akan ethnicity (N = 427, 68.7%), followed by Ga (N = 104, 16.7%), Ewe (N = 71, 11.4%) with the remaining ethnicities accounting for 3.2% (N = 20). In terms of education, 436 patients (70.1%) were illiterates, 44 (7.1%) primary education, 132 (21.2%) had up to secondary education, and the remaining 10 (1.6%) tertiary education. More than half of the study population (N = 324, 52%) consumed alcohol on a regular basis, while only 44 (7%) smoked.
Table 1. Characteristics of patients included in the study.
Variable | N = 622 | % |
Sex | ||
Male | 414 | 66.6 |
Female | 208 | 33.4 |
Age | ||
Years 08–25 | 124 | 20.0 |
Years 26–40 | 282 | 45.3 |
Years 41–77 | 216 | 34.7 |
Residency | ||
Rural | 117 | 18.8 |
Urban | 505 | 81.2 |
Region | ||
Greater Accra | 325 | 52.3 |
Central | 268 | 43.1 |
Western | 29 | 4.6 |
Ethnicity | ||
Akan | 427 | 68.7 |
Ewe | 71 | 11.4 |
Ga | 104 | 16.7 |
Other | 20 | 3.2 |
Religion | ||
Christian | 564 | 90.7 |
Muslim | 37 | 5.9 |
Pagan | 21 | 3.4 |
Level of Education | ||
No education | 436 | 70.1 |
Primary school | 44 | 7.1 |
Secondary | 132 | 21.2 |
Tertiary | 10 | 1.6 |
Alcohol Intake | ||
Yes | 324 | 52.1 |
No | 298 | 47.9 |
Smoking Status | ||
Smokers | 44 | 7.1 |
Non smokers | 578 | 92.9 |
Crowding(1-4 pers) | 195 | 31.4 |
(>5 pers) | 427 | 68.6 |
Occupation | ||
Farmer | 45 | 7.2 |
Others | 577 | 92.8 |
Prevalence of MTBC lineages and sub-lineages
MTBC isolates were obtained from all 622 TB patients. Upon genotyping, 9 of these (1.4%) produced ambiguous results and were thus excluded from further analysis. Hence, a total of 613 isolates were used for further analysis. Based on LSP and SNP typing, we identified six out of the seven human-associated MTBC lineages in our study sample (Table 2). The dominant lineages were Lineage 4 with 483 cases (78.8%), Lineage 5 (N = 86, 14.0%) and Lineage 6 (N = 19, 3.1%). Eleven isolates (1.8%) belonged to Lineage 1, 10 to Lineage 2 (includes Beijing; 1.6%), and the remaining 4 isolates to Lineage 3 (0.7%). Among the 483 Lineage 4 isolates, 313/483 (65.0%) belonged to the sub-lineage of Lineage 4 known as the ‘Cameroon family’. No M. bovis was identified in our sample.
Table 2. Genotyping profiles of 613 M. tuberculosis complex isolates from Ghana.
Species | SNP | RD9 | RD726 | RD711 | RD702 | Spoligotyping profile | Sub-lineagea | SIT | No | % |
MTBss | L1 | Undel | Undel | ND | ND | ▪□□▪▪▪▪□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪□▪▪▪▪▪▪▪▪▪▪ | EAI | 340 | 8 | 1.3 |
MTBss | L1 | Undel | Undel | ND | ND | ▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪□□▪▪▪▪▪□▪▪▪ | EAI | 1 | 0.2 | |
MTBss | L1 | Undel | Undel | ND | ND | ▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪□▪▪▪▪▪▪▪▪▪▪ | EAI | 342 | 1 | 0.2 |
MTBss | L1 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪□▪▪▪▪▪▪▪▪▪▪ | EAI | 236 | 1 | 0.2 |
MTBss | L2 | Undel | Undel | ND | ND | □□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□▪▪▪▪▪▪▪▪▪ | Beijing | 1 | 10 | 1.6 |
MTBss | L3 | Undel | Undel | ND | ND | ▪□□▪□□□□□□□□▪▪▪▪▪▪▪▪▪▪□□□□□□□□□□□□▪▪▪▪▪▪▪▪▪ | DEHLI/CAS | 2 | 0.3 | |
MTBss | L3 | Undel | Undel | ND | ND | ▪▪▪□□□□▪▪▪▪▪▪▪□▪▪▪▪▪▪□□□□□□□□□□□□□▪▪▪▪▪▪▪▪▪ | DEHLI/CAS | 1 | 0.2 | |
MTBss | L3 | Undel | Del | ND | ND | ▪▪▪□□□□▪□▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□□□□□□□□▪▪▪▪▪▪▪▪▪ | DEHLI/CAS | 1092 | 1 | 0.2 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 61 | 226 | 36.8 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪□□▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 772 | 20 | 3.2 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 115 | 7 | 1.1 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪▪□▪▪ | Cameroon | 838 | 3 | 0.4 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 26 | 4.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪□▪▪▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪□▪▪▪▪▪▪□▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪□▪▪□▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪□▪□▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 2 | 0.3 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 1141 | 1 | 0.2 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪□▪▪▪ | Cameroon | 403 | 1 | 0.2 |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 2 | 0.3 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪□□□▪▪▪□□▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 2 | 0.3 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪□□□□□▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 3 | 0.4 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪□▪▪□▪ | Cameroon | 2 | 0.3 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪□▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□□▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪□□▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□□▪▪▪▪▪▪▪ | Cameroon | 2 | 0.3 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪□□□□▪▪□▪▪□▪ | Cameroon | 3 | 0.4 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪□▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 2 | 0.3 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□□□▪▪▪□□□□▪▪▪▪▪▪▪ | Cameroon | 3 | 0.4 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪▪□□▪□▪ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Del | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪□□□□▪□□□□□□ | Cameroon | 1 | 0.2 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 53 | 26 | 4.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪□□▪▪▪▪ | Ghana | 65 | 4 | 0.7 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 504 | 7 | 1.1 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 118 | 12 | 1.9 |
MTBss | L4 | Undel | Undel | ND | ND | ▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 804 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 462 | 4 | 0.7 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 44 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 86 | 12 | 1.9 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 167 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 373 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 393 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | □□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | Ghana | 272 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪□▪▪□▪ | Ghana | 4 | 0.7 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□▪□□□□▪▪▪▪▪▪▪ | Haarlem | 1652 | 4 | 0.7 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□□□□□□▪▪▪▪▪▪▪ | Haarlem | 1498 | 6 | 0.9 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪□□□□▪▪▪▪▪▪▪ | Haarlem | 50 | 15 | 2.4 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪□□□□□□▪□□□□▪▪▪▪▪▪▪ | Haarlem | 45 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪□□□□▪▪▪▪▪▪▪ | Haarlem | 655 | 3 | 0.4 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□▪□□□□▪▪▪▪▪▪▪ | Haarlem | 47 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□▪□□□□▪▪▪□▪▪▪ | Haarlem | 62 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□□□□□▪▪□▪▪□▪ | Haarlem | 2 | 0.3 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□▪□□□□▪▪▪□□▪▪ | Haarlem | 1 | 0.2 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□□□▪▪□▪▪▪□□□□▪▪▪▪▪▪▪ | LAM | 306 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪□□□□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | LAM | 1 | 0.2 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | LAM | 42 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪□□□▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | LAM | 33 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪□□□□□□□□▪▪▪▪▪▪□▪▪□□□□▪▪▪▪▪▪▪▪□□□□▪▪□▪▪▪▪ | 70 | 7 | 1.1 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪□□□□▪▪□□□□▪ | Uganda I | 2 | 0.3 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪□▪▪▪ | Uganda I | 52 | 4 | 0.7 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪□□□□▪ | Uganda I | 244 | 1 | 0.20.4 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪□▪▪▪ | Uganda I | 848 | 3 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪□□▪▪ | Uganda I | 2 | 0.3 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪□□▪□▪ | Uganda I | 78 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪□□□□▪▪□□□□▪ | Uganda I | 1 | 0.2 | |
MTBss | L4 | Undel | Undel | ND | ND | □□□□□□□□□□□□□□□□□□□□□□□□▪▪▪▪▪▪▪▪□□□□▪▪□□□□▪ | Uganda I | 125 | 1 | 0.2 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪□□□□ | Uganda II | 51 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪□□▪□□▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪□□□□▪▪□□▪□▪ | Uganda II | 2 | 0.3 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪□□□□□□▪▪▪▪▪□▪▪▪▪▪▪▪▪□▪□□□□▪▪▪▪▪▪▪ | Uganda II | 3 | 0.4 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | S | 1223 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪□▪▪▪▪▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | S | 1211 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | X | 119 | 2 | 0.3 |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪□□□□□□□□□▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪□□□□ | 200 | 7 | 1.1 | |
MTBss | L4 | Undel | Undel | ND | ND | ▪▪□▪▪▪▪□□□□□▪□▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | 2 | 0.3 | ||
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪□□□□▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | 2 | 0.3 | ||
MTBss | L4 | Undel | Undel | ND | ND | ▪□□▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪□□□□▪▪▪▪▪▪▪ | 1 | 0.2 | ||
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪□□□□□□□▪□▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | 1 | 0.2 | ||
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪□□□□□□□▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪□□□□ | 4 | 0.7 | ||
MTBss | L4 | Undel | Undel | ND | ND | ▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪ | 1 | 0.2 | ||
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 331 | 17 | 2.8 |
Mafric | L5 | Del | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□▪▪□▪▪▪▪▪□□▪□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 1 | 0.2 | |
Mafric | L5 | Del | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□▪▪□▪▪▪▪▪□□□□□▪□□□□□□□□□□□□□▪▪▪ | WA I | 1 | 0.2 | |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□□□▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 1 | 0.2 | |
Mafric | L5 | Del | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 319 | 16 | 2.6 |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 438 | 9 | 1.5 |
Mafric | L5 | Del | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 860 | 1 | 0.2 |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□▪▪▪▪▪▪▪▪□□□□□▪□□□□□□□□□□□□□▪▪▪ | WA I | 1592 | 2 | 0.3 |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪□□□□□▪▪▪▪□□□□□□□□▪▪□□□□□▪▪▪▪▪□□□▪▪▪▪ | WA I | 1 | 0.2 | |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪▪▪□▪▪▪▪▪□▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪□▪ | WA I | 1 | 0.2 | |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪□□▪□▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪□▪ | WA I | 1 | 0.2 | |
Mafric | L5 | Del | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□▪▪▪▪▪▪▪▪□□□□□▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 3 | 0.4 | |
Mafric | L5 | Del | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□▪▪□▪▪▪▪□□□□□□▪□▪▪▪□□□▪▪□□□□▪□▪ | WA I | 1 | 0.2 |
Sub-lineage as defined by the MIRU-VNTRplus database, Undel = not deleted, Del = deleted, ND = Not done.
All isolates were further sub-typed using spoligotyping (Table 2). We detected a total of 117 spoligotypes, 485/613 isolates (79%) had previously defined shared type number (SIT). The remaining 128 isolates could not be defined by the SITVIT database and thus were defined as ‘orphan’. In addition to Cameroon sub-lineage, seven additional sub-lineages were identified among Lineage 4 based on spoligotyping; Ghana (N = 75, 15.5%), Haarlem (N = 37, 7.7%), Uganda I (N = 15, 3.1%), Uganda II (N = 7, 1.4%), LAM (N = 5, 1.0%), S (N = 4 (0.8%), and X (N = 2, 0.4%).
Association between MTBC lineages and patient characteristics
Table 3 illustrates the association of socio demographic and behavioural factors with the main MTBC lineages present in our study sample. Using multivariable logistic regression model analysis, we found that individuals of Ewe ethnicity were significantly more likely to present with TB caused by M. africanum as opposed to M. tuberculosis sensu stricto irrespective of their place of residence (adjusted odds ratio (adjOR) = 3.02; 95% confidence interval (CI): 1.67–5.47, P<0.001) (Table 3, S1 Fig.). This association was independent from other risk factors. Moreover, we found TB caused by M. africanum to be associated with smoking (adjOR = 2.02; 95% CI: 0.95–4.27) when compared to M. tuberculosis sensu stricto. However, this association was only borderline statistically significant (P = 0.07). No significant associations between MTBC lineages and other patient variables were found. Because M. africanum comprises two phylogenetic distinct lineages (i.e. MTBC Lineages 5 and 6), we performed a stratified analysis by lineage. Using multivariate logistic regression model analysis, we observed a significant association between Ewe ethnicity and Lineage 5 (adjOR) = 2.79; 95% CI: 1.47–5.29, P<0.001). This association was independent from other risk factors (Table 4). Interestingly, based on univariate analysis, we also saw an association between Ewe ethnicity and Lineage 6 (adjOR = 4.03; 95% CI: 1.33–10.85). However, because of the limited number of Lineage 6 isolates (n = 18) multivariate analyses could not be performed to confirm the independence of this association.
Table 3. Risk factors for TB caused by M. africanum compared to M. tuberculosis sensu stricto.
Risk factor | %(n) Mafr | %(n) MTBss | OR (95%CI) | adjOR (95%CI)a |
(n = 102) | (n = 511) | |||
Sex (male) | 68% (69) | 66% (338) | 0.93 (0.59–1.47) | |
Age category | ||||
years 08–25 | 17% (17) | 21% (105) | reference | |
years 26–40 | 53% (54) | 44% (223) | 1.50 (0.83–2.70) | |
years 41–77 | 30% (31) | 36% (183) | 1.05 (0.55–1.98) | |
Rural residency | 20% (20) | 18% (93) | 1.10 (0.64–1.88) | |
Region | ||||
Accra | 55% (56) | 52% (267) | reference | reference |
Central | 42% (43) | 43% (218) | 0.94 (0.61–1.45) | 0.97 (0.60–1.56) |
Western | 3% (3) | 5% (26) | 0.55 (0.16–1.88) | 0.44 (0.12–1.63) |
Ethnicity | ||||
Akan | 58% (59) | 71% (359) | reference | reference |
Ewe | 23% (23) | 9% (48) | 2.91 (1.65–5.14)* | 3.02 (1.67–5.47)* |
Ga | 15% (15) | 17% (89) | 1.03 (0.56–1.89) | 0.97 (0.51–1.83) |
other | 5% (5) | 3% (15) | 2.03 (0.71–5.79) | 2.35 (0.77–7.13) |
Religion | ||||
Christian | 92% (94) | 90% (462) | reference | |
Muslim | 7% (7) | 6% (29) | 1.18 (0.50–2.79) | |
Pagan | 1% (1) | 4% (20) | 0.25 (0.03–1.85) | |
Educational level | ||||
No education | 74% (75) | 70% (356) | reference | |
Primary school | 6% (6) | 7% (38) | 0.75 (0.30–1.83) | |
Secondary | 21% (21) | 23% (117) | 0.85 (0.50–1.44) | |
Alcohol | 57% (58) | 52% (263) | 1.23 (0.81–1.90) | |
Smoking | 11% (11) | 6% (32) | 1.81 (0.88–3.72) | 2.02 (0.95–4.27)† |
Crowding (>5 pers)b | 63% (64) | 70% (359) | 0.71 (0.45–1.10) | |
Occupation farmer | 9% (9) | 7% (35) | 1.32 (0.61–2.83) |
Table 4. Risk factors for Risk factor for TB caused by Lineage 5 compared to M. tuberculosis sensu stricto.
Risk factor | %(n) Lineage 5 | %(n) MTBss | OR (95%CI) | adjOR (95%CI)a |
(n = 84) | (n = 511) | |||
Sex (male) | 59% (58) | 66% (338) | 1.41 (0.69–1.88) | |
Age category | ||||
years 08–25 | 18% (15) | 21% (105) | reference | |
years 26–40 | 51% (43) | 43% (223) | 1.35 (0.72–2.54) | |
years 41–77 | 31% (26) | 36% (183) | 0.99 (0.5–1.96) | |
Rural residency | 19% (16) | 18% (93) | 1.06 (0.59–1.91) | |
Region | ||||
Accra | 54% (45) | 52% (267) | reference | |
Central | 42% (36) | 43% (218) | 0.98 (0.61–1.57) | |
Western | 4% (3) | 5% (26) | 0.68 (0.2–2.36) | |
Ethnicity | ||||
Akan | 61% (51) | 70% (359) | reference | reference |
Ewe | 20% (17) | 9% (48) | 2.49 (1.33–4.66)** | 2.79 (1.47–5.29)** |
Ga | 14% (12) | 17% (89) | 0.95 (0.49–1.86) | 0.85 (0.43–1.69) |
other | 5% (4) | 3% (15) | 1.88 (0.6–5.88) | 1.64 (0.53–5.34) |
Religion | ||||
Christian | 93% (78) | 90% (462) | reference | |
Muslim | 6% (5) | 6% (29) | 1.02 (0.38–2.72) | |
Pagan | 1% (1) | 4% (20) | 0.29 (0.04–2.24) | |
Educational level | ||||
No education | 70% (59) | 70% (356) | reference | |
Primary school | 7% (6) | 7% (38) | 0.95 (0.39–2.35) | |
Secondary + | 23% (19) | 23% (117) | 0.98 (0.56–1.71) | |
Alcohol | 62% (52) | 52% (263) | 1.53 (0.95–2.45)† | 1.62 (0.99–2.68)† |
Smoking | 11% (9) | 6% (32) | 1.8 (0.82–3.91) | 1.54 (0.68–3.50) |
Crowding (>5 pers)c | 63% (53) | 70% (359) | 0.72 (0.44–1.16) | |
Occupation farmer | 11% (9) | 7% (35) | 0.61 (0.28–1.32) | 0.64 (0.29–1.45) |
Discussion
Our retrospective molecular epidemiological investigation of MTBC clinical isolates from Southern Ghana revealed that i) the Cameroon sub-lineage of Lineage 4 is the dominant cause of human TB in this region, ii) 17.1% of human TB is caused by M. africanum, iii) TB patients infected with M. africanum were more likely to smoke, and iv) to belong to the Ewe ethnic group.
Our finding that the Cameroon sub-lineage causes 65% of human TB in Ghana confirms our previous report from Ghana [13], and is in agreement with findings from neighbouring countries. In particular, the Cameroon sub-lineage was previously found to cause 40% of human TB in Cameroon [21], 45% in Nigeria [22] and 33% in Chad [23]. The reasons for the success of this sub-lineage in this region of Africa are unclear but could be due to a founder effect and/or particularly high fitness in the corresponding patient populations. Similarly, other successful sub-lineages of Lineage 4 have been observed in other regions of Africa, including Uganda [24] and Zimbabwe [25].
We found that in Ghana, M. africanum still accounts for 17.1% of all human TB, which is similar to the prevalence we reported several years ago [13]. This is in contrast to a study in Cameroon [21] that indicated a sharp decrease in TB caused by M. africanum during the last decades. A potential explanation for the decline of M. africanum in some West African countries includes possible out-competition by M. tuberculosis, as M. africanum has been associated with reduced virulence in animal models [26]–[27], and a longer latency and a slower rate of progression to active disease in humans [28]. Of note, our finding that smoking was associated with infection by M. africanum as opposed to M. tuberculosis sensu stricto is consistent with the notion that M. africanum might be less virulent in immunocompetent hosts [7]. This notion is also supported by a previous study in the Gambia reporting a significant association between M. africanum West Africa II and HIV co-infection [29]. However, no such association was found between M. africanum West Africa I and II in Ghana [30]. Because information on HIV status was not available for the present study, we could not explore this question here. Taken together, there is a need for further investigation to ascertain why M. africanum is declining in some regions of West Africa, but not in Ghana, and whether this phenomenon can be attributed to differences in virulence and/or other factors.
One reason for why the prevalence of M. africanum might be more stable in Ghana than in some other countries is that this bacterial lineage might be particularly well adapted to (some) human populations in Ghana. Our finding that M. africanum was independently associated with Ewe ethnicity supports this possibility. Moreover, this association was largely driven by Lineage 5, and not the result of a single outbreak as the spoligotyping patterns among M. africanum isolates from Ewe patients were diverse (Table 5). From available data, we know that M. africanum, in particular Lineage 5 is prevalent in countries around the Gulf of Guinea [13], [31], and particularly frequent in Benin and Ghana [13], [32], two countries with large Ewe populations [33]. The Ewe speaking ethnic group traditionally forms part of the Gbe language family which includes the Fons of Benin, the Aja of Togo and the Phla-phera of western Nigeria [33], [34]. Although the Ewe, Fons, Aja and phla-phera are different dialects of the same Gbe language family, members of theses individual groups are interrelated [33], [34]. Together they constitute the indigenous inhabitants of coastal West Africa.
Table 5. Spoligotyping profiles of M. africanum isolates from patients of Ewe ethnicity.
Species | SNP | RD726 | RD711 | RD702 | Spoligotyping profile | Sub-lineagea | SIT | No | % |
Mafric | L5 | ND | Del | Undel | ▪▪▪▪▪▪▪□□□□□▪▪▪▪□□□□□□□□▪▪□□□□□▪▪▪▪▪□□□▪▪▪▪ | WA I | 2 | 8.8 | |
Mafric | L5 | ND | Del | Undel | ▪▪▪▪▪▪▪□□□□□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 438 | 5 | 21.7 |
Mafric | L5 | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□▪▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 331 | 7 | 30.4 |
Mafric | L5 | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□▪▪□▪▪▪▪▪□□▪□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | Orphan | 1 | 4.3 |
Mafric | L5 | ND | Del | Undel | ▪□▪▪▪▪▪▪□□□□□□▪▪▪▪▪▪▪□□□□▪▪▪▪▪▪▪▪▪▪▪□□□▪▪▪▪ | WA I | 319 | 2 | 8.8 |
Mafric | L5 | ND | Del | Undel | ▪▪▪▪▪▪▪▪□□□□□▪▪▪▪▪▪▪▪□□□□□▪□□□□□□□□□□□□□▪▪▪ | WA I | 1592 | 1 | 4.3 |
Mafric | L6 | ND | Undel | Del | ▪▪▪▪▪▪□□□▪▪▪▪▪□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪ | WA 2 | 324 | 2 | 8.8 |
Mafric | L6 | ND | Undel | Del | ▪▪▪▪▪▪□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪ | WA 2 | 181 | 1 | 4.3 |
Mafric | L6 | ND | Undel | Del | ▪□▪▪▪▪□□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪ | WA 2 | 318 | 1 | 4.3 |
Mafric | L6 | ND | Undel | Del | ▪▪▪▪▪▪▪▪▪□▪▪▪□□▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪▪□▪▪▪▪ | WA 2 | Orphan | 1 | 4.3 |
Sub-lineage as defined by the MIRU-VNTRplus database, Undel = not deleted, Del = deleted, ND = Not done.
Associations between particular MTBC lineages and human ethnicities have been observed before. For example, in San Francisco, Lineage 1, 2 and 4 were strongly associated with Filipino, Chinese, and “white” ethnicities, respectively [11]. More recently, Hui ethnicity was found to be associated with the Beijing family of MTBC in China [35]. While social “cohesion” is likely to restrict intermingling between individuals belonging to different ethnic groups and thus transmission of MTBC between these groups, biological factors could also play a role in the association between different MTBC genotypes and human populations. Self-defined ethnicity has been shown to be a reliable proxy for human ancestry [36], and human genetic diversity has been linked to an increased or reduced susceptibility to TB [37]. Importantly, recent studies indicate that human genetic susceptibility to TB is further influenced by the MTBC genotype [10]. In particular, studies have reported human genetic polymorphisms that influence the susceptibility to TB caused by M. africanum but not M. tuberculosis sensu stricto or vice versa [38]. For example, a study performed in Ghana reported a human polymorphism in 5-lipoxygenase (ALOX5) associated with increased TB risk [39]. Stratification by MTBC lineage revealed that this association was mainly driven by M. africanum indicating that this human polymorphism increases the risk of TB in a MTBC lineage-specific matter. ALOX5 is involved in the synthesis of leukotrienes and lipoxins, which are important mediators of the inflammatory response [39]. Conversely, a human polymorphism reported recently in the Mannose Binding Lectin (MBL) was associated with protection against TB caused by M. africanum but not M. tuberculosis sensu stricto [40]. Moreover, this latter study also found that M. africanum bound human recombinant MBL more efficiently, perhaps leading to an improved uptake of M. africanum by macrophages and selection of deficient MBL variants among human populations exposed to M. africanum [40].
Our study has several limitations. First, data on HIV co-infection was not available. This might have influenced our results on the patient characteristics associated with M. africanum. Secondly, this study was not population-based as patients were recruited only at three government hospitals. Hence, some degree of selection bias cannot be excluded.
In conclusion, our study provides novel insights into the interaction between environmental, host and pathogen variability in human TB. In particular, the observed association between M. africanum and Ewe patient ethnicity suggests a possible explanation for the geographical restriction of M. africanum to parts of West Africa. Our findings also highlight the need to consider this variability in the development of new tools and strategies to control TB.
Supporting Information
Acknowledgments
We express our gratitude to all laboratory staff and study participants of the various health facilities for their time and cooperation during the study period.
Data Availability
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding Statement
This study was supported by the Leverhulme-Royal Society Africa Award (grant AA080019 to DYM and SG), the National Tuberculosis Program Ghana, and the Swiss National Science Foundation (PP00P3_150750). AAP was supported by the “Amt für Ausbildungsbeiträge”, Canton of Basel, Switzerland and the government of Ghana. Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
References
- 1.World Health Organization (2014) Global Tuberculosis Report, Geneva: World Health Organization.
- 2. Brosch R, Gordon SV, Marmiesse M, Brodin P, Buchrieser C, et al. (2002) A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A 99: 3684–3689. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Firdessa R, Berg S, Hailu E, Schelling E, Gumi B, et al. (2013) Mycobacterial lineages causing pulmonary and extrapulmonary tuberculosis, Ethiopia. Emerg Infect Dis 19: 460–463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Gagneux S, Small PM (2007) Global phylogeography of Mycobacterium tuberculosis and implications for tuberculosis product development. Lancet Infect Dis 7: 328–37. [DOI] [PubMed] [Google Scholar]
- 5. de Jong BC, Antonio M, Gagneux S (2010) Mycobacterium africanum—Review of an important cause of human tuberculosis in West Africa. PLoS Negl Trop Dis 4: e744. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Achtman M (2008) Evolution, population structure, and phylogeography of genetically monomorphic bacterial pathogens. An Rev Microbiol 62: 53–70. [DOI] [PubMed] [Google Scholar]
- 7. Coscolla M, Gagneux S (2010) Does M. tuberculosis genomic diversity explain disease diversity? Drug Discov Today Dis Mech 7: e43–e59. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Nicol MP, Wilkinson Robert J (2008) The clinical consequences of strain diversity in Mycobacterium tuberculosis Trans R Soc Trop Med Hyg. 102: 955–965. [DOI] [PubMed] [Google Scholar]
- 9. Comas I, Coscolla M, Luo T, Borrell S, Holt KE, et al. (2013) Out-of-Africa migration and neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat Genet 45: 1176–1182. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Gagneux S (2012) Host–pathogen coevolution in human tuberculosis Phil Trans R Soc B. 367: 850–859. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Gagneux S, DeRiemer K, Van T, Kato-Maeda M, de Jong BC, et al. (2006) Variable host-pathogen compatibility in Mycobacterium tuberculosis . Proc Natl Acad Sci USA 103: 2869–2873. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Fenner L, Egger M, Bodmer T, Furrer H, Ballif M, et al. (2013) HIV infection disrupts the sympatric host–pathogen relationship in human tuberculosis. PLoS Genet 9: e1003318. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Yeboah-Manu D, Asante-Poku A, Bodmer T, Stucki D, Koram K, et al. (2011) Genotypic diversity and drug susceptibility patterns among M. tuberculosis complex isolates from South-Western Ghana. PLoS ONE 6: e21906. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Government of Ghana (2010) Ghana Demographic and Health Survey, Final Report.
- 15. Yeboah-Manu D, Bodmer T, Mensah-Quainoo E, Owusu S, Ofori-Adjei D (2004) Evaluation of decontamination methods and growth media for primary isolation of Mycobacterium ulcerans from surgical specimens. J Clin Microbiol 42: 5875–5876. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Yeboah-Manu D, Yates MD (2001) Stuart Mark (2001) Wilson Application of a simple multiplex polymerase chain reaction to aid in the routine work of the mycobacterium reference laboratory. J Clin Microbiol 39: 4166–4168. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Stucki D, Malla B, Hostettler S, Huna T, Feldmann J, et al. (2012) Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages. PLoS ONE 7: e41253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. (1997) Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35: 907–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Demay C, Liens B, Burguiere T, Hill V, Couvin D, et al. (2012) SITVITWEB–a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology. Infect Genet Evol 12: 755–766. [DOI] [PubMed] [Google Scholar]
- 20. Weniger T, Krawczyk J, Supply P, Niemann S, Harmsen D (2010) MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria. Nucleic Acids Res 38: W326–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Niobe-Eyangoh SN, Kuaban C, Sorlin P, Cunin P, Thonnon J, et al. (2003) Genetic biodiversity of Mycobacterium tuberculosis complex strains from patients with pulmonary tuberculosis in Cameroon. J Clin Microbiol 41: 2547–2553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Lawson L, Zhang J, Gomgnimbou MK, Abdurrahman ST, Le Moullec S, et al. (2012) A molecular epidemiological and genetic diversity study of tuberculosis in Ibadan, Nnewi and Abuja, Nigeria. PLoS ONE 7: e38409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Diguimbaye C, Hilty M, Ngandolo R, Mahamat HH, Pfyffer GE, Baggi F, et al. (2006) Molecular characterization and drug resistance testing of Mycobacterium tuberculosis isolates from Chad. J Clin Microbiol 44: 1575–1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Wampande EM, Mupere E, Debanne SM, Asiimwe BB, Nsereko M, et al. (2013) Long-term dominance of Mycobacterium tuberculosis Uganda family in peri-urban Kampala-Uganda is not associated with cavitary disease. BMC Infectious Diseases 13: 484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Easterbrook PJ, Gibson A, Murad S, Lamprecht D, Ives N, et al. (2004) High rates of clustering of strains causing tuberculosis in Harare, Zimbabwe: a molecular epidemiological study. J Clin Microbiol 42: 4536–4544. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Castets M, Sarrat H (1969) Experimental study of the virulence of Mycobacterium africanum (preliminary note). Bull Soc Med Afr Noire Lang Fr 14: 693–696. [PubMed] [Google Scholar]
- 27. Bold TD, Davis DC, Penberthy KK, Cox LM, Ernst JD, et al. (2012) Impaired fitness of Mycobacterium africanum despite secretion of ESAT-6. J Infect Dis 205: 984–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. de Jong BC, Hill PC, Aiken A, Awine T, Antonio M, et al. (2008) Progression to active tuberculosis, but not transmission, varies by Mycobacterium tuberculosis lineage in the Gambia. J Infect Dis 198: 1037–1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. de Jong BC, Hill PC, Brookes RH, Otu JK, Peterson KL, et al. (2005) Mycobacterium africanum: a new opportunistic pathogen in HIV infection? AIDS 19: 1714–1715. [DOI] [PubMed] [Google Scholar]
- 30. Meyer CG, Scarisbrick G, Niemann S, Browne EN, Chinbuah MA, et al. (2008) Pulmonary tuberculosis: virulence of Mycobacterium africanum and relevance in HIV co-infection. Tuberculosis (Edinb) 88: 482–489. [DOI] [PubMed] [Google Scholar]
- 31. Gehre F, Antonio M, Faïhun F, Odoun M, Uwizeye C, et al. (2013) The First phylogeographic population structure and analysis of transmission dynamics of M. africanum West African 1— combining molecular data from Benin, Nigeria and Sierra Leone. PLoS ONE 8: e77000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Affolabi D, Anyo G, Faihun F, Sanoussi N, Shamputa IC, et al. (2009) First molecular epidemiological study of tuberculosis in Benin. Int J Tuberc Lung Dis 13: 317–322. [PubMed] [Google Scholar]
- 33.The Ewe People. en.wikipedia.org/wiki/Ewe_people. Accessed on July 02, 2014.
- 34. Kofi Anyidoho (2003) The back without which there is no front. Africa Today 50: 3–18. [Google Scholar]
- 35. Pang Y, Song Y, Xia H, Zhou Y, et al. (2012) Risk factors and clinical phenotypes of Beijing genotype strains in tuberculosis patients in China. BMC Infectious Disease 12: 354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36. Rosenberg NA, Pritchard JK, Weber JL, Cann HM, Kidd KK, et al. (2002) Genetic structure of human populations. Science 298: 2381. [DOI] [PubMed] [Google Scholar]
- 37. Abel L, El-Baghdadi J, Bousfiha AA, Casanova J-L, Schurr E (2014) Human genetics of tuberculosis: a long and winding road. Phil Trans R Soc B 369: 20130428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Intemann CD, Thye T, Niemann S, Browne ENL, Chinbuah MA, et al. (2009) Autophagy gene variant IRGM2261T contributes to protection from tuberculosis caused by Mycobacterium tuberculosis but not by M. africanum strains. PLoS Pathog 5: e1000577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Herb F, Thye T, Niemann S, Browne ENL, Chinbuah MA, et al. (2008) ALOX5 variants associated with susceptibility to human pulmonary tuberculosis. Hum Mol Genet 17: 1052–60. [DOI] [PubMed] [Google Scholar]
- 40. Thye T, Niemann S, Walter K, Homolka S, Intemann CD, et al. (2011) Variant G57E of Mannose Binding Lectin associated with protection against tuberculosis caused by Mycobacterium africanum but not by M. tuberculosis . PLoS ONE 6: e20908. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.