Abstract
BackgroundGenotyping 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 FindingsWe 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/SignificanceStandard 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.
Highlights
Despite the existence of effective antituberculosis drugs for the last 60 years, tuberculosis (TB) continues to be a major threat worldwide
M. tuberculosis isolates were obtained from clinically confirmed TB patients, presenting pulmonary or extrapulmonary forms, diagnosed from 2000 to 2003 and reported to community health centers located in Rio de Janeiro, Porto Alegre, and Belem, the three Brazilian cities included in the study
Consistent with previous results obtained in Europe [12,13,15,16], only a minority of these MIRUVNTR clusters was split by the addition of secondary spoligotyping, resulting in a total of 321 distinct patterns, reducing the number of clusters to 28 and increasing the number of unique isolates to 293
Summary
Despite the existence of effective antituberculosis drugs for the last 60 years, tuberculosis (TB) continues to be a major threat worldwide. With 87,000 new TB cases reported in 2009, Brazil ranks 19th among these countries. Molecular typing of Mycobacterium tuberculosis is a powerful adjunct to TB control, e.g. to monitor the disease transmission, to detect or confirm outbreaks and laboratory error/cross-contamination, to distinguish endogenous reactivation from exogenous reinfection and to identify the clonal spread of successful clones, including multi-drug-resistant ones [3]. Its application in high TBburden countries has been hampered hitherto by constrained resources, technical limitations of standard IS6110 fingerprinting [5], and, in general, by the dominance of geographically specific, genetically homogeneous strain lineages 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
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