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Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients.

ABSTRACT: BACKGROUND: Tuberculosis remains a major public health problem. Clinical tuberculosis manifests often as pulmonary and occasionally as extra-pulmonary tuberculosis. The emergence of drug resistant tubercle bacilli and its association with HIV is a formidable challenge to curb the spread of tuberculosis. There have been concerted efforts by whole genome sequencing and bioinformatics analysis to identify genomic patterns and to establish a relationship between the genotype of the organism and clinical manifestation of tuberculosis. Extra-pulmonary TB constitutes 15-20 percent of the total clinical cases of tuberculosis reported among immunocompetent patients, whereas among HIV patients the incidence is more than 50 percent. Genomic analysis of M. tuberculosis isolates from extra pulmonary patients has not been explored. RESULTS: The genomic DNA of 5 extra-pulmonary clinical isolates of M. tuberculosis derived from cerebrospinal fluid, lymph node fine needle aspirates (FNAC) / biopsies, were sequenced. Next generation sequencing approach (NGS) was employed to identify Single Nucleotide Variations (SNVs) and computational methods used to predict their consequence on functional genes. Analysis of distribution of SNVs led to the finding that there are mixed genotypes in patient isolates and that many SNVs are likely to influence either gene function or their expression. Phylogenetic relationship between the isolates correlated with the origin of the isolates. In addition, insertion sites of IS elements were identified and their distribution revealed a variation in number and position of the element in the 5 extra-pulmonary isolates compared to the reference M. tuberculosis H37Rv strain. CONCLUSIONS: The results suggest that NGS sequencing is able to identify small variations in genomes of M. tuberculosis isolates including changes in IS element insertion sites. Moreover, variations in isolates of M. tuberculosis from non-pulmonary sites were documented. The analysis of our results indicates genomic heterogeneity in the clinical isolates.

SUBMITTER: Das S

PROVIDER: S-EPMC3699378 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients.

Das Sarbashis S Roychowdhury Tanmoy T Kumar Parameet P Kumar Anil A Kalra Priya P Singh Jitendra J Singh Sarman S Prasad H K HK Bhattacharya Alok A

BMC genomics 20130617

<h4>Background</h4>Tuberculosis remains a major public health problem. Clinical tuberculosis manifests often as pulmonary and occasionally as extra-pulmonary tuberculosis. The emergence of drug resistant tubercle bacilli and its association with HIV is a formidable challenge to curb the spread of tuberculosis. There have been concerted efforts by whole genome sequencing and bioinformatics analysis to identify genomic patterns and to establish a relationship between the genotype of the organism a ...[more]

PMID: 23773324

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Project description:BackgroundPhylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world.Principal findingsA total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1).ConclusionsThis study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage.

Project description:BackgroundPeru holds the fourth highest burden of tuberculosis in the Americas. Despite an apparently well-functioning DOTS control program, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase. To worsen this situation, cases of extensively drug resistance tuberculosis (XDR-TB) have been detected. Little information exists about the genetic diversity of drug-susceptible vs. MDR-TB and XDR-TB.MethodsCryopreserved samples of XDR strains from 2007 to 2009 (second semester), were identified and collected. Starting from 227 frozen samples, a total of 142 XDR-TB strains of Mycobacterium tuberculosis complex (MTBC; 1 isolate per patient) were retained for this study. Each strain DNA was analyzed by spoligotyping and the 15-loci Mycobacterial Interspersed Repetitive Unit (MIRU-15).ResultsAmong the 142 isolates analyzed, only 2 samples (1.41%) could not be matched to any lineage. The most prevalent sublineage was Haarlem (43.66%), followed by T (27.46%), LAM (16.2%), Beijing (9.15%), and X clade (1.41%). Spoligotype analysis identified clustering for 128/142 (90.1%) isolates vs. 49/142 (34.5%) with MIRUs. Of the samples, 90.85% belonged to retreated patients. The drug resistant profile demonstrated that 62.67% showed resistance to injectable drugs capreomycin (CAP) and kanamycin (KAN) vs. 15.5% to CAP alone and 21.8% to KAN alone. The SIT219/T1 and SIT50/H3 were the most prevalent patterns in our study. The spoligoforest analysis showed that SIT53/T1 was at the origin of many of the T lineage strains as well as a big proportion of Haarlem lineage strains (SIT50/H3, followed by SIT47/H1, SIT49/H3, and SIT2375/H1), as opposed to the SIT1/Beijing strains that did not appear to evolve into minor Beijing sublineages among the XDR-TB strains.ConclusionIn contrast with other Latin-American countries where LAM sublineage is the most predominant, we found the Haarlem to be the most common followed by T sublineage among the XDR-TB strains.

Project description:Conventionally, facultative intracellular pathogen, Mycobacterium tuberculosis, the tuberculosis (TB) causing bacilli in human is cleared by cell-mediated immunity (CMI) with CD4(+) T cells playing instrumental role in protective immunity, while antibody-mediated immunity (AMI) is considered non-protective. This longstanding convention has been challenged with recent evidences of increased susceptibility of hosts with compromised AMI and monoclonal antibodies conferring passive protection against TB and other intracellular pathogens. Therefore, novel approaches toward vaccine development include strategies aiming at induction of humoral response along with CMI. This necessitates the identification of mycobacterial proteins with properties of immunomodulation and strong immunogenicity. In this study, we determined the immunogenic potential of M. tuberculosis Zinc metalloprotease-1 (Zmp1), a secretory protein essential for intracellular survival and pathogenesis of M. tuberculosis. We observed that Zmp1 was secreted by in vitro grown M. tuberculosis under granuloma-like stress conditions (acidic, oxidative, iron deficiency, and nutrient deprivation) and generated Th2 cytokine microenvironment upon exogenous treatment of peripheral blood mononulear cells PBMCs with recombinant Zmp1 (rZmp1). This was supported by recording specific and robust humoral response in TB patients in a cohort of 295. The anti-Zmp1 titers were significantly higher in TB patients (n = 121) as against healthy control (n = 62), household contacts (n = 89) and non-specific infection controls (n = 23). A significant observation of the study is the presence of equally high titers of anti-Zmp1 antibodies in a range of patients with high bacilli load (sputum bacilli load of 300+ per mL) to paucibacillary smear-negative pulmonary tuberculosis (PTB) cases. This clearly indicated the potential of Zmp1 to evoke an effective humoral response independent of mycobacterial load. Such mycobacterial proteins can be explored as antigen candidates for prime-boost vaccination strategies or extrapolated as markers for disease detection and progression.

Project description:BackgroundPatients with multidrug-resistant (MDR) tuberculosis may have phenotypic heterogeneity in results of drug-susceptibility tests (DSTs). However, the impact of this on clinical outcomes among patients treated for MDR tuberculosis is unknown.MethodsPhenotypic DST heterogeneity was defined as presence of at least 1 Mycobacterium tuberculosis isolate susceptible to rifampicin and isoniazid recovered <3 months after MDR tuberculosis treatment initiation from a patient with previous documented tuberculosis due to M. tuberculosis resistant to at least rifampicin and isoniazid. The primary outcome was defined as good (ie, cure or treatment completion) or poor (ie, treatment failure, treatment default, or death). A secondary outcome was time to culture conversion. Cox proportional hazard models were used to determine the association between phenotypic DST heterogeneity and outcomes.ResultsPhenotypic DST heterogeneity was identified in 33 of 475 patients (7%) with MDR tuberculosis. Poor outcome occurred in 126 patients (28%). Overall, patients with MDR tuberculosis who had phenotypic DST heterogeneity were at greater risk of poor outcome than those with MDR tuberculosis but no phenotypic DST heterogeneity (adjusted hazard ratio [aHR], 2.1; 95% confidence interval [CI], 1.2-3.6). Among HIV-infected patients with MDR tuberculosis, the adjusted hazard for a poor outcome for those with phenotypic DST heterogeneity was 2.4 (95% CI, 1.3-4.2) times that for those without phenotypic DST heterogeneity, whereas among HIV-negative patients with MDR tuberculosis, the adjusted hazard for those with phenotypic DST heterogeneity was 1.5 (95% CI, .5-4.3) times that for those without phenotypic DST heterogeneity. HIV-infected patients with MDR tuberculosis with phenotypic DST heterogeneity also had a longer time to culture conversion than with HIV-infected patients with MDR tuberculosis without phenotypic DST heterogeneity (aHR, 2.9; 95% CI, 1.4-6.0).ConclusionsPhenotypic DST heterogeneity among persons with HIV infection who are being treated for MDR tuberculosis is associated with poor outcomes and longer times to culture conversion.

Project description:Genome sequencing plays a key role in understanding the genetic diversity of Mycobacterium tuberculosis (M.tb). The genotype-specific character of M. tb contributes to tuberculosis severity and emergence of drug resistance. Strains of M. tb complex can be classified into seven lineages. The Nonthaburi (NB) genotype, belonging to the Indo-Oceanic lineage (lineage 1), has a unique spoligotype and IS6110-RFLP pattern but has not previously undergone a detailed whole genome analysis. In addition, there is not much information available on the whole genome analysis of M. tb isolates from tuberculous meningitis (TBM) patients in public databases. Isolates CSF3053, 46-5069 and 43-13838 of NB genotype were obtained from the cerebrospinal fluids of TBM Thai patients in Siriraj Hospital, Bangkok. The whole genomes were subjected to high throughput sequencing. The sequence data of each isolate were assembled into draft genome. The sequences were also aligned to reference genome, to determine genomic variations. Single nucleotide polymorphisms (SNPs) were obtained and grouped according to the functions of the genes containing them. They were compared with SNPs from 1,601 genomes, representing the seven lineages of M. tb complex, to determine the uniqueness of NB genotype. Susceptibility to first-line, second-line and other antituberculosis drugs were determined and related to the SNPs previously reported in drug-resistant related genes. The assembled genomes have an average size of 4,364,461 bp, 4,154 genes, 48 RNAs and 64 pseudogenes. A 500 base pairs deletion, which includes ppe50, was found in all isolates. RD239, specific for members of Indo Oceanic lineage, and RD147c were identified. A total of 2,202 SNPs were common to the isolates and used to classify the NB strains as members of sublineage 1.2.1. Compared with 1,601 genomes from the seven lineages of M. tb complex, mutation G2342203C was found novel to the isolates in this study. Three mutations (T28910C, C1180580T and C152178T) were found only in Thai NB isolates, including isolates from previous study. Although drug susceptibility tests indicated pan-susceptibility, non-synonymous SNPs previously reported to be associated with resistance to anti-tuberculous drugs; isoniazid, ethambutol, and ethionamide were identified in all the isolates. Non-synonymous SNPs were found in virulence genes such as the genes playing roles in apoptosis inhibition and phagosome arrest. We also report polymorphisms in essential genes, efflux pumps associated genes and genes with known epitopes. The analysis of the TBM isolates and the availability of the variations obtained will provide additional resources for global comparison of isolates from pulmonary tuberculosis and TBM. It will also contribute to the richness of genomic databases towards the prediction of antibiotic resistance, level of virulence and of origin of infection.

Dataset Information

Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients.

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Genetic heterogeneity revealed by sequence analysis of Mycobacterium tuberculosis isolates from extra-pulmonary tuberculosis patients.

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