Dataset Information

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.

ABSTRACT: There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for phylogenetic studies and strain classification. By contrast, single nucleotide polymorphisms (SNP) are ideal markers for classifying MTBC into phylogenetic lineages, as they exhibit very low degrees of homoplasy. In this study, we developed two complementary SNP-based genotyping methods to classify strains into the six main human-associated lineages of MTBC, the "Beijing" sublineage, and the clade comprising Mycobacterium bovis and Mycobacterium caprae. Phylogenetically informative SNPs were obtained from 22 MTBC whole-genome sequences. The first assay, referred to as MOL-PCR, is a ligation-dependent PCR with signal detection by fluorescent microspheres and a Luminex flow cytometer, which simultaneously interrogates eight SNPs. The second assay is based on six individual TaqMan real-time PCR assays for singleplex SNP-typing. We compared MOL-PCR and TaqMan results in two panels of clinical MTBC isolates. Both methods agreed fully when assigning 36 well-characterized strains into the main phylogenetic lineages. The sensitivity in allele-calling was 98.6% and 98.8% for MOL-PCR and TaqMan, respectively. Typing of an additional panel of 78 unknown clinical isolates revealed 99.2% and 100% sensitivity in allele-calling, respectively, and 100% agreement in lineage assignment between both methods. While MOL-PCR and TaqMan are both highly sensitive and specific, MOL-PCR is ideal for classification of isolates with no previous information, whereas TaqMan is faster for confirmation. Furthermore, both methods are rapid, flexible and comparably inexpensive.

SUBMITTER: Stucki D

PROVIDER: S-EPMC3401130 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.

Stucki David D Malla Bijaya B Hostettler Simon S Huna Thembela T Feldmann Julia J Yeboah-Manu Dorothy D Borrell Sonia S Fenner Lukas L Comas Iñaki I Coscollà Mireia M Gagneux Sebastien S

PloS one 20120720 7

There is increasing evidence that strain variation in Mycobacterium tuberculosis complex (MTBC) might influence the outcome of tuberculosis infection and disease. To assess genotype-phenotype associations, phylogenetically robust molecular markers and appropriate genotyping tools are required. Most current genotyping methods for MTBC are based on mobile or repetitive DNA elements. Because these elements are prone to convergent evolution, the corresponding genotyping techniques are suboptimal for ...[more]

PMID: 22911768

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Project description:INTRODUCTION:Recurrent tuberculosis (TB) is one of the main challenges in TB control. Genotyping based on Mycobacterial Interspersed Repetitive Units-Variable Tandem Repeats (MIRU-VNTR) has been widely used to differentiate between relapse and reinfection, which are the two main causes of recurrent TB. There is a lack of data regarding the causes of TB recurrence in Georgia, and while differentiating between relapse and reinfection plays a key role in defining appropriate interventions, the required genotyping methodologies have not been implemented. The objective of this study was to implement MIRU-VNTR genotyping at the National Center for Tuberculosis and Lung Diseases (NCTBLD) and differentiate between relapse and reinfection in multidrug resistant (MDR-) TB patients from Tbilisi, Georgia. METHODS:Recurrent MDR tuberculosis cases from 2014-2016 diagnosed at NCTLD were included in the study when bacterial samples from both episodes were available. Genotyping based on the MIRU-VNTR 24 loci was implemented and used for differentiating between relapse and reinfection. Paired samples showing the same MIRU-VNTR pattern or one locus difference were classified as relapse, while two and more loci differences were treated as reinfection. Exact logistic regression was used to identify predictors of recurrence. RESULTS:Thirty two MDR-TB patients (64 samples) were included and MIRU-VNTR 24 typing was performed on the corresponding paired samples. Of the 32 patients, 25 (83.3%) were identified as relapse while 5 (16.7%) were due to re-infection. Patients with a history of incarceration were significantly associated with TB reinfection (p< 0.05). CONCLUSION:Recurrent TB in MDR patients in Georgia are mainly caused by relapse, raising concerns on the efficacy of the TB control program. An association between incarceration and reinfection likely reflects high levels of ongoing TB transmission in prisons, indicating the need for better TB infection control measures in these settings. Our results add to the rationale for implementing genotypic surveillance of TB more broadly to support TB control in Georgia.

Project description:BackgroundGenotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis.Methods and findingsIn M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing < 8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts < or = 42 cycles in internal transcribed spacer-real time PCR.ConclusionsThese results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst).

Dataset Information

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.

Publications

Two new rapid SNP-typing methods for classifying Mycobacterium tuberculosis complex into the main phylogenetic lineages.

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