The forest behind the tree: phylogenetic exploration of a dominant Mycobacterium tuberculosis strain lineage from a high tuberculosis burden country.

BACKGROUND: Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. METHODOLOGY/PRINCIPAL FINDINGS: We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. CONCLUSIONS/SIGNIFICANCE: Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay.

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.