A first assessment of the genetic diversity of Mycobacterium tuberculosis complex in Cambodia

<p>Abstract</p> <p>Background</p> <p>Cambodia is among the 22 high-burden TB countries, and has one of the highest rates of TB in South-East Asia. This study aimed to describe the genetic diversity among clinical <it>Mycobacterium tuberculosis </it>complex (MTC) isolates collected in Cambodia and to relate these findings to genetic diversity data from neighboring countries.</p> <p>Methods</p> <p>We characterized by 24 VNTR loci genotyping and spoligotyping 105 <it>Mycobacterium tuberculosis </it>clinical isolates collected between 2007 and 2008 in the region of Phnom-Penh, Cambodia, enriched in multidrug-resistant (MDR) isolates (n = 33).</p> <p>Results</p> <p>Classical spoligotyping confirmed that the East-African Indian (EAI) lineage is highly prevalent in this area (60%-68% respectively in whole sample and among non-MDR isolates). Beijing lineage is also largely represented (30% in whole sample, 21% among non-MDR isolates, OR = 4.51, CI_95%[1.77, 11.51]) whereas CAS lineage was absent. The 24 loci MIRU-VNTR typing scheme distinguished 90 patterns with only 13 multi-isolates clusters covering 28 isolates. The clustering of EAI strains could be achieved with only 8 VNTR combined with spoligotyping, which could serve as a performing, easy and cheap genotyping standard for this family. Extended spoligotyping suggested relatedness of some unclassified "T1 ancestors" or "Manu" isolates with modern strains and provided finer resolution.</p> <p>Conclusions</p> <p>The genetic diversity of MTC in Cambodia is driven by the EAI and the Beijing families. We validate the usefulness of the extended spoligotyping format in combination with 8 VNTR for EAI isolates in this region.</p

A Model-Based Bayesian Estimation of the Rate of Evolution of VNTR Loci in Mycobacterium tuberculosis

Variable numbers of tandem repeats (VNTR) typing is widely used for studying the bacterial cause of tuberculosis. Knowledge of the rate of mutation of VNTR loci facilitates the study of the evolution and epidemiology of Mycobacterium tuberculosis. Previous studies have applied population genetic models to estimate the mutation rate, leading to estimates varying widely from around to per locus per year. Resolving this issue using more detailed models and statistical methods would lead to improved inference in the molecular epidemiology of tuberculosis. Here, we use a model-based approach that incorporates two alternative forms of a stepwise mutation process for VNTR evolution within an epidemiological model of disease transmission. Using this model in a Bayesian framework we estimate the mutation rate of VNTR in M. tuberculosis from four published data sets of VNTR profiles from Albania, Iran, Morocco and Venezuela. In the first variant, the mutation rate increases linearly with respect to repeat numbers (linear model); in the second, the mutation rate is constant across repeat numbers (constant model). We find that under the constant model, the mean mutation rate per locus is (95% CI: ,)and under the linear model, the mean mutation rate per locus per repeat unit is (95% CI: ,). These new estimates represent a high rate of mutation at VNTR loci compared to previous estimates. To compare the two models we use posterior predictive checks to ascertain which of the two models is better able to reproduce the observed data. From this procedure we find that the linear model performs better than the constant model. The general framework we use allows the possibility of extending the analysis to more complex models in the future

Investigation on Mycobacterium tuberculosis Diversity in China and the Origin of the Beijing Clade

Contains fulltext : 124314.pdf (publisher's version ) (Open Access

Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing.

Contains fulltext : 125549.pdf (publisher's version ) (Open Access)BACKGROUND: In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. METHODS: Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. RESULTS: Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994)). 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994) were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5%) revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. CONCLUSIONS: Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination

Automated High-Throughput Genotyping for Study of Global Epidemiology of Mycobacterium tuberculosis Based on Mycobacterial Interspersed Repetitive Units

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607–2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen