Genetic Diversity Of Mycobacterium Tuberculosis Complex Research Articles

Molecular diversity of Mycobacterium tuberculosis isolates in Treatment-experienced Patients from Andhra Pradesh, India.

To get a comprehensive idea about the transmission and epidemiology of TB globally and locally, the use of molecular typing methods has become imperative not only for understanding genetic diversity but also the population structure of Mycobacterium tuberculosis complex (MTBC). We aimed to investigate the drug resistance pattern and genetic diversity of MTBC among previously treated patients with sputum smear-positive pulmonary tuberculosis in a South Indian population. 104 patients with sputum smear positivity and who had previously undergone treatment were selected. Drug susceptibility testing, Spoligotyping, MIRU-VNTR, and SNP typing were performed. Mono-resistance to isoniazid 16 (15.38%) was the highest among all drugs. Out of 104 isolates, 24 (23%) isolates were classified as MDR strains. The distributions of most common lineages were: EAI3-Ind-20 (19.23%), EAI5-13 (12.50%), Beijing-12 (11.54%), CAS1-Delhi- 9 (8.65%), and 7 (6.73%) each of T-H37rv, Unknown and Orphan types. MIRU-VNTR-based analysis revealed that there are two major groups: CAS1-Delhi and Beijing groups. Out of 104 isolates, 82 belonged to well-defined lineages and 6 clusters, and the remaining 22 were singletons. SNP analysis showed no mutations associated with five sets of genes in 33 strains. The occurrence of 11.54% Beijing strains in South India is an important finding. High frequency of Isoniazid mono resistance noticed. Spoligotyping along with MIRU-VNTR and SNP typing is the best approach to the identification of strain lineages. No mutation with Antigen85C gene represents, can be used for vaccine candidates.

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity.

The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains. The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection. We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4=1.2, L4=1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs. Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

The within-host evolution of antimicrobial resistance in Mycobacterium tuberculosis.

ABSTRACTTuberculosis (TB) has been responsible for the greatest number of human deaths due to an infectious disease in general, and due to antimicrobial resistance (AMR) in particular. The etiological agents of human TB are a closely-related group of human-adapted bacteria that belong to the Mycobacterium tuberculosis complex (MTBC). Understanding how MTBC populations evolve within-host may allow for improved TB treatment and control strategies. In this review, we highlight recent works that have shed light on how AMR evolves in MTBC populations within individual patients. We discuss the role of heteroresistance in AMR evolution, and review the bacterial, patient and environmental factors that likely modulate the magnitude of heteroresistance within-host. We further highlight recent works on the dynamics of MTBC genetic diversity within-host, and discuss how spatial substructures in patients’ lungs, spatiotemporal heterogeneity in antimicrobial concentrations and phenotypic drug tolerance likely modulates the dynamics of MTBC genetic diversity in patients during treatment. We note the general characteristics that are shared between how the MTBC and other bacterial pathogens evolve in humans, and highlight the characteristics unique to the MTBC.

Molecular epidemiology of clinical Mycobacterium tuberculosis complex isolates in South Omo, Southern Ethiopia

BackgroundTuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC). Mapping the genetic diversity of MTBC in high TB burden country like Ethiopia is important to understand principles of the disease transmission and to strengthen the regional TB control program. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis complex (MTBC) isolates circulating in the South Omo, southern Ethiopia.MethodsMTBC isolates (N = 156) were genetically analyzed using spacer oligotyping (spoligotyping) and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Major lineages and lineages were identified using MTBC databases. Logistic regression was used to correlate patient characteristics with strain clustering.ResultsThe study identified Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), Lineage_7/Aethiops vertus, Mycobacterium bovis and Mycobacterium africanum major lineages in proportions of 67.3% (105/156), 22.4% (35/156), 6.4% (10/156), 1.9% (3/156), 1.3% (2/156) and 0.6% (1/156), respectively. Lineages identified were Delhi/CAS 23.9% (37/155), Ethiopia_2 20.6% (32/155), Haarlem 14.2% (22/155), URAL 14.2%(22/155), Ethiopia_3 8.4% (13/155), TUR 6.5% (10/155), Lineage_7/Aethiops vertus 1.9% (3/155), Bovis 1.3% (2/155), LAM 1.3% (2/155), EAI 0.6% (1/155), X 0.6% (1/155) and Ethiopia H37Rv-like strain 0.6% (1/155). Of the genotyped isolates 5.8% (9/155) remained unassigned. The recent transmission index (RTI) was 3.9%. Orphan strains compared to shared types (AOR: 0.09, 95% CI: 0.04–0.25) were associated with reduced odds of clustering. The dominant TB lineage in pastoral areas was EAI and in non-pastoral areas was EA.ConclusionThe epidemiological data, highly diverse MTBC strains and a low RTI in South Omo, provide information contributing to the TB Control Program of the country.

Molecular Analysis of Mycobacterium tuberculosis Isolated in the North Central Zone of Nigeria

Tuberculosis (TB) incidence in Nigeria is high, with a significant burden of TB/Human Immunodeficiency Virus (HIV). Genotyping and drug susceptibility of Mycobacterium tuberculosis Complex (MTBC) are important in order to improve the control of the disease. This study sought to determine drug susceptibility and genetic diversity of MTBC in the country. The sputum samples of 202 patients [133 (65.8%) males/69 (34.2%) females] were collected in the North Central zone of Nigeria and cultured using Lowenstein–Jensen medium. Immunochromatography for the primary identification and Drug Susceptibility Testing (DST) by proportion method, as well as IS6110 typing, regions of difference 1, 4, 9, 12, 702, and 711, and spoligotyping were carried out on the isolates. Following the DST on 202 isolates, 51 (25.2%) showed resistance to at least one drug. Multidrug resistance was observed in 29/202 (14.4%) cases. HIV positivity [37/202 (18.3%) patients] was associated with rifampicin 9/37 (24.3%) resistance (p = 0.012) as well as gender (p = 0.009). Of the 202 isolates, 150 (74.3%) were identified as the Cameroon sublineage, followed by the UgandaI, Haarlem, and West Africa 1 with 18 (8.9%), 10 (5%), and 6 (3%), respectively. The LAM10_CAM was the most prevalent genetic family [128/202 (63.4%)], with the shared international type 61 [111 (55%) isolates] the largest cluster. Gender (p = 0.038) and age (p = 0.015) had significant associations with the LAM10_CAM family but neither with HIV (p = 0.479) nor drug resistance. Rifampicin resistance in TB/HIV coinfected patient is a major concern in the study area. The Mycobacterium africanum lineage showed a marked decrease, and the need to educate females most at risk of TB/HIV coinfection is advocated.

Clinical isolates of the modern Mycobacterium tuberculosis lineage 4 evade host defense in human macrophages through eluding IL-1\u03b2-induced autophagy

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected over 1.7 billion people worldwide and causes 1.4 million deaths annually. Recently, genome sequence analysis has allowed the reconstruction of Mycobacterium tuberculosis complex (MTBC) evolution, with the identification of seven phylogeographic lineages: four referred to as evolutionarily “ancient”, and three “modern”. The MTBC strains belonging to “modern” lineages appear to show enhanced virulence that may have warranted improved transmission in humans over ancient lineages through molecular mechanisms that remain to be fully characterized. To evaluate the impact of MTBC genetic diversity on the innate immune response, we analyzed intracellular bacterial replication, inflammatory cytokine levels, and autophagy response in human primary macrophages infected with MTBC clinical isolates belonging to the ancient lineages 1 and 5, and the modern lineage 4. We show that, when compared to ancient lineage 1 and 5, MTBC strains belonging to modern lineage 4 show a higher rate of replication, associated to a significant production of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and induction of a functional autophagy process. Interestingly, we found that the increased autophagic flux observed in macrophages infected with modern MTBC is due to an autocrine activity of the proinflammatory cytokine IL-1β, since autophagosome maturation is blocked by an interleukin-1 receptor antagonist. Unexpectedly, IL-1β-induced autophagy is not disadvantageous for the survival of modern Mtb strains, which reside within Rab5-positive phagosomal vesicles and avoid autophagosome engulfment. Altogether, these results suggest that autophagy triggered by inflammatory cytokines is compatible with a high rate of intracellular bacilli replication and may therefore contribute to the increased pathogenicity of the modern MTBC lineages.

Immunological consequences of strain variation within the Mycobacterium tuberculosis complex.

In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies.

Mapping of Mycobacterium tuberculosis Complex Genetic Diversity Profiles in Tanzania and Other African Countries.

The aim of this study was to assess and characterize Mycobacterium tuberculosis complex (MTBC) genotypic diversity in Tanzania, as well as in neighbouring East and other several African countries. We used spoligotyping to identify a total of 293 M. tuberculosis clinical isolates (one isolate per patient) collected in the Bunda, Dar es Salaam, Ngorongoro and Serengeti areas in Tanzania. The results were compared with results in the SITVIT2 international database of the Pasteur Institute of Guadeloupe. Genotyping and phylogeographical analyses highlighted the predominance of the CAS, T, EAI, and LAM MTBC lineages in Tanzania. The three most frequent Spoligotype International Types (SITs) were: SIT21/CAS1-Kili (n = 76; 25.94%), SIT59/LAM11-ZWE (n = 22; 7.51%), and SIT126/EAI5 tentatively reclassified as EAI3-TZA (n = 18; 6.14%). Furthermore, three SITs were newly created in this study (SIT4056/EAI5 n = 2, SIT4057/T1 n = 1, and SIT4058/EAI5 n = 1). We noted that the East-African-Indian (EAI) lineage was more predominant in Bunda, the Manu lineage was more common among strains isolated in Ngorongoro, and the Central-Asian (CAS) lineage was more predominant in Dar es Salaam (p-value<0.0001). No statistically significant differences were noted when comparing HIV status of patients vs. major lineages (p-value = 0.103). However, when grouping lineages as Principal Genetic Groups (PGG), we noticed that PGG2/3 group (Haarlem, LAM, S, T, and X) was more associated with HIV-positive patients as compared to PGG1 group (Beijing, CAS, EAI, and Manu) (p-value = 0.03). This study provided mapping of MTBC genetic diversity in Tanzania (containing information on isolates from different cities) and neighbouring East African and other several African countries highlighting differences as regards to MTBC genotypic distribution between Tanzania and other African countries. This work also allowed underlining of spoligotyping patterns tentatively grouped within the newly designated EAI3-TZA lineage (remarkable by absence of spacers 2 and 3, and represented by SIT126) which seems to be specific to Tanzania. However, further genotyping information would be needed to confirm this specificity.

Combined Genotypic, Phylogenetic, and Epidemiologic Analyses of Mycobacterium tuberculosis Genetic Diversity in the Rhône Alpes Region, France.

BackgroundThe present work relates to identification and a deep molecular characterization of circulating Mycobacterium tuberculosis complex (MTBC) strains in the Rhône-Alpes region, France from 2000 to 2010. It aimed to provide with a first snapshot of MTBC genetic diversity in conjunction with bacterial drug resistance, type of disease and available demographic and epidemiologic characteristics over an eleven-year period, in the south-east of France.MethodsMycobacterium tuberculosis complex (MTBC) strains isolated in the Rhône-Alpes region, France (n = 2257, 1 isolate per patient) between 2000 and 2010 were analyzed by spoligotyping. MIRU-VNTR typing was applied on n = 1698 strains (with full results available for 974 strains). The data obtained were compared with the SITVIT2 database, followed by detailed genotyping, phylogenetic, and epidemiologic analyses in correlation with anonymized data on available demographic, and epidemiologic characteristics, and location of disease (pulmonary or extrapulmonary TB).ResultsThe most predominant spoligotyping clusters were SIT53/T1 (n = 346, 15.3%) > SIT50/H3 (n = 166, 7.35%) > SIT42/LAM9 (n = 125, 5.5%) > SIT1/Beijing (n = 72, 3.2%) > SIT47/H1 (n = 71, 3.1%). Evolutionary-recent strains belonging to the Principal Genetic Group (PGG) 2/3, or Euro-American lineages (T, LAM, Haarlem, X, S) were predominant and represented 1768 or 78.33% of all isolates. For strains having drug resistance information (n = 1119), any drug resistance accounted for 14.83% cases vs. 1.52% for multidrug resistance (MDR); and was significantly more associated with age group 21–40 years (p-value<0.001). Extra-pulmonary TB was more common among female patients while pulmonary TB predominated among men (p-value<0.001; OR = 2.16 95%CI [1.69; 2.77]). Also, BOV and CAS lineages were significantly well represented in patients affected by extra-pulmonary TB (p-value<0.001). The origin was known for 927/2257 patients: 376 (40.6%) being French-born vs. 551 (59.4%) Foreign-born. French patients were significantly older (mean age: 58.42 yrs 95%CI [56.04; 60.80]) than Foreign-born patients (mean age: 42.38 yrs. 95%CI [40.75; 44.0]).ConclusionThe study underlined the importance of imported TB cases on the genetic diversity and epidemiologic characteristics of circulating MTBC strains in Rhône-Alpes region, France over a large time-period. It helps better understand intricate relationships between certain lineages and geographic origin of the patients, and pinpoints genotypic and phylogenetic specificities of prevailing MTBC strains. Lastly, it also demonstrated a slow decline in isolation of M. africanum lineage in this region between 2000 and 2010.

Mycobacterium africanum Is Associated with Patient Ethnicity in Ghana

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.

Multi-drug resistant Mycobacterium tuberculosis complex genetic diversity and clues on recent transmission in Punjab, Pakistan

Multi-Drug Resistant Tuberculosis (MDR-TB), i.e. bacilli resistant to rifampicin (RIF) and isoniazid (INH), is a major Public Health concern in Pakistan according to WHO estimates (3.5% and 32% of new and retreated cases, respectively). Previous Pakistanis reports identified a correlation between being MDR and belonging to Beijing or EAI lineages in one study, and belonging to “H4”-Ural Euro-American sublineage in another study. In addition, MDR-TB transmission was suspected in Karachi.We tested MDR characteristics on a Punjab sample of 278 clinical isolates (without selection for Multi-Drug Resistance) including new and retreated cases collected from 2008 to 2012. All samples were characterized by a new, microbead-based method named “TB-SPRINT” (molecular diagnostic including spoligotype identification, and genetic resistance determinants to first-line anti-TB drugs RIF and INH). Isolates from 2011 to 2012 (n=100) were further analyzed using 24-loci MIRU-VNTR.We detected 8.7% MDR isolates (CI95%=[5.0; 12.5]), mainly among CAS lineage that predominates in this central-East region of Pakistan. Out of 20 MDR-TB cases, 12 different TB-SPRINT profiles were identified, limiting the suspicion of MDR-TB transmission. 24 MIRU-VNTR confirmed the unrelatedness of isolates with different TB-SPRINT profiles and discriminated 3 isolates with identical TB-SPRINT profiles.In conclusion, our study did not confirm any of the correlations between Multi-Drug Resistance and lineage or sublineage in Punjab, Pakistan. MDR-TB isolates were diverse indicating that transmission is not pervasive. TB-SPRINT proved useful as a first step for detecting MDR-TB likely transmission events, before more extensive genotyping such as 15 or 24 MIRU-VNTR and thorough epidemiological investigation.

Four year longitudinal study of Mycobacterium tuberculosis complex isolates in a region of North-Eastern Italy

Recent reports have suggested a change of Mycobacterium tuberculosis complex genetic diversity in Western Europe due to an increasing proportion of imported cases of tuberculosis (TB). This study analyzed a total of 705 M. tuberculosis strains isolated from 2006 to 2009 in Veneto, a North-Eastern Italian region, to see the impact of foreign-born cases vs. Italian patients on prevailing TB epidemiology. Strains were genotyped using spoligotyping followed by comparison with international genotyping database SITVIT2. Six spoligotyping clusters with suspected phylogeographical specificity for imported cases, were typed by 15-loci MIRUs for a finer characterization. Overall, 410 (58.16%) strains were isolated from foreign-born patients, while 295 (41.84%) were isolated from Italian patients. Older patients (>70years, i.e., 46.4% of cases) predominated among Italians while younger age groups prevailed among foreign-born patients. Our results suggest that despite a high proportion of reactivation of latent TB infection in elderly Italian-born patients, active TB transmission between foreign-born and Italian patients may be ongoing, and argue in favor of an increased TB surveillance among immigrants to combat TB epidemic in Italy.