[Evaluation of discriminatory power of molecular epidemiology techniques in Mycobacterium tuberculosis Venezuelan isolates]. / Evaluación del poder discriminatorio de técnicas de epidemiología molecular en aislados Venezolanos de Mycobacterium tuberculosis.

The techniques of spoligotyping and mycobacterial interspersed repetitive unit and variable-number tandem repeat typing with 24 loci (MIRU-VNTR-24), have been used to study the molecular epidemiology of tuberculosis. The aim of this study was: to evaluate the discriminative power of MIRU-VNTR 24 loci alone and in association with spoligotyping in clinical isolates of M tuberculosis in Venezuela; the allelic diversity of the 24 loci; and the discriminative power for the combination of 24 and 15 loci, 12 traditional loci (12t), those with higher allelic diversity and a new combination named 12inv. We analyzed one set of 104 strains of different lineages and a second set of 431 strains belonging to the Latin-America and Mediterranean lineage (LAM) that is predominant in Venezuela. The determination of allelic diversity showed that 4052, 2163b, 424 y 2996 are highly discriminative. Clustering rates of MIRU-VNTR 24 loci, spoligotyping and MIRU-VNTR combined with spoligotyping for 104 isolates were 18.27%, 71.15% and 14.4%, respectively, whereas with the 431 LAM strains the values were 43.2 %, 95.8% and 37.4%. MIRU-VNTR combinations of 15, 12inv and 4 loci were more discriminatory than 12t. Clustering rates for MIRU-VNTR 15 and 12inv loci coupled with spoligotyping in the 104 isolated was 21% and 23%, while for LAM strains was 52% and 46% respectively. The number of different genetics patterns for 15 and 12inv loci were similar. In conclusion, we propose the use of a small number of informative loci MIRU-VNTR coupled to spoligotyping to investigate the transmission of tuberculosis in Venezuela.

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains. RESULTS: To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal. CONCLUSION: Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

Mycobacterium tuberculosis population structure and molecular epidemiological analysis in Sucre municipality, Miranda state, Venezuela.

Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Venezuela that consistently has more cases of tuberculosis than other municipalities in the country. To identify the neighborhoods in the municipality with the highest prevalence of tuberculosis, and determine whether the Mycobacterium tuberculosis strain distribution in this municipality is different from that previously found in the western part of Caracas and the rest of Venezuela, we collected data on all tuberculosis cases in the municipality diagnosed in 2005-6. We performed two separate molecular epidemiological studies, spoligotyping 44 strains in a first study, and spoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolates in the second. With spoligotyping, the most common patterns were Shared International Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), a distribution similar to other parts of Venezuela, except that SIT42 and SIT20 were more common. MIRU-VNTR 15 showed that six of seven SIT17 strains examined belonged to a large cluster previously found circulating in Venezuela, but all of the SIT42 strains were related to a cluster centered in the neighborhoods of Unión and Maca, with a MIRU-VNTR pattern not previously seen in Venezuela. It appears that a large percentage of the tuberculosis in the Sucre municipality is caused by the active transmission of two strain families centered within distinct neighborhoods, one reflecting communication with the rest of the country, and the other suggesting the insular, isolated nature of some sectors.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: providing guidelines for Quality Assurance when working on membranes.

BACKGROUND: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. METHODS: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. RESULTS: Seven out of the 11 laboratories (63%), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). CONCLUSIONS: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.

Most LAM Mycobacterium tuberculosis strains in Venezuela, but not SIT605, belong to the RDRio subfamily.

Tuberculosis is a global public health problem that is resurgent in Venezuela, with 13 thousand estimated new cases in 2018. Strains of the Mycobacterium tuberculosis RDRio, subfamily belong to the Latín American Mediterranean (LAM) family and are a major cause of TB in Rio de Janeiro, Brazil. LAM strains predominate in Venezuela, where spoligotype SIT605 is common, but surprisingly not found elsewhere. We sought to assess the presence of RDRio strains in tuberculosis patients in different regions of Venezuela and determine whether SIT605 also belongs to the RDRio subfamily. Using spoligotyping and MIRU-VNTR 24 loci, we identified 86 clinical LAM and SIT605 isolates from the Venezuelan capital Caracas and several Venezuelan states. Region of difference deletion loci RD174 and RDRio, and also IS1561 were used to identify strains of the RDRio subfamily, while IS6110 at position 932,204 and the Ag85C103 polymorphism were used to validate SIT 605 as a LAM family strain. We found that 69.8% of the isolates were RDRío, including 94.3% of strains isolated in Caracas, 17.9% isolated in the state of Carabobo, the two strains analyzed from Delta Amacuro, and one each from Sucre, Apure and Aragua states. RDRio was in 100% of: SIT17 (LAM 2); SIT20 (LAM 1); SITs 93, 1694, 1696, 960, 1367 (LAM 5); and SITs 216 (LAM 9); but only 75% of SIT42 (LAM 9) strains. Thus, most of the LAM strains in Venezuela belong to the RDRío subfamily. SIT 605 strains, although LAM, are not in the RDRío subfamily.

Mycobacterium tuberculosis ecology in Venezuela: epidemiologic correlates of common spoligotypes and a large clonal cluster defined by MIRU-VNTR-24.

BACKGROUND: Tuberculosis remains an endemic public health problem, but the ecology of the TB strains prevalent, and their transmission, can vary by country and by region. We sought to investigate the prevalence of Mycobacterium tuberculosis strains in different regions of Venezuela. A previous study identified the most prevalent strains in Venezuela but did not show geographical distribution nor identify clonal genotypes. To better understand local strain ecology, we used spoligotyping to analyze 1298 M. tuberculosis strains isolated in Venezuela from 1997 to 2006, predominantly from two large urban centers and two geographically distinct indigenous areas, and then studied a subgroup with MIRU-VNTR 24 loci. RESULTS: The distribution of spoligotype families is similar to that previously reported for Venezuela and other South American countries: LAM 53%, T 10%, Haarlem 5%, S 1.9%, X 1.2%, Beijing 0.4%, and EAI 0.2%. The six most common shared types (SIT's 17, 93, 605, 42, 53, 20) accounted for 49% of the isolates and were the most common in almost all regions, but only a minority were clustered by MIRU-VNTR 24. One exception was the third most frequent overall, SIT 605, which is the most common spoligotype in the state of Carabobo but infrequent in other regions. MIRU-VNTR homogeneity suggests it is a clonal group of strains and was named the "Carabobo" genotype. Epidemiologic comparisons showed that patients with SIT 17 were younger and more likely to have had specimens positive for Acid Fast Bacilli on microscopy, and patients with SIT 53 were older and more commonly smear negative. Female TB patients tended to be younger than male patients. Patients from the high incidence, indigenous population in Delta Amacuro state were younger and had a nearly equal male:female distribution. CONCLUSION: Six SIT's cause nearly half of the cases of tuberculosis in Venezuela and dominate in nearly all regions. Strains with SIT 17, the most common pattern overall may be more actively transmitted and SIT 53 strains may be less virulent and associated with reactivation of past infections in older patients. In contrast to other common spoligotypes, strains with SIT 605 form a clonal group centered in the state of Carabobo.

Turkish and Japanese Mycobacterium tuberculosis sublineages share a remote common ancestor.

Two geographically distant M. tuberculosis sublineages, Tur from Turkey and T3-Osaka from Japan, exhibit partially identical genotypic signatures (identical 12-loci MIRU-VNTR profiles, distinct spoligotyping patterns). We investigated T3-Osaka and Tur sublineages characteristics and potential genetic relatedness, first using MIRU-VNTR locus analysis on 21 and 25 samples of each sublineage respectively, and second comparing Whole Genome Sequences of 8 new samples to public data from 45 samples uncovering human tuberculosis diversity. We then tried to date their Most Recent Common Ancestor (MRCA) using three calibrations of SNP accumulation rate (long-term=0.03SNP/genome/year, derived from a tuberculosis ancestor of around 70,000years old; intermediate=0.2SNP/genome/year derived from a Peruvian mummy; short-term=0.5SNP/genome/year). To disentangle between these scenarios, we confronted the corresponding divergence times with major human history events and knowledge on human genetic divergence. We identified relatively high intrasublineage diversity for both T3-Osaka and Tur. We definitively proved their monophyly; the corresponding super-sublineage (referred to as "T3-Osa-Tur") shares a common ancestor with T3-Ethiopia and Ural sublineages but is only remotely related to other Euro-American sublineages such as X, LAM, Haarlem and S. The evolutionary scenario based on long-term evolution rate being valid until T3-Osa-Tur MRCA was not supported by Japanese fossil data. The evolutionary scenario relying on short-term evolution rate since T3-Osa-Tur MRCA was contradicted by human history and potential traces of past epidemics. T3-Osaka and Tur sublineages were found likely to have diverged between 800y and 2000years ago, potentially at the time of Mongol Empire. Altogether, this study definitively proves a strong genetic link between Turkish and Japanese tuberculosis. It provides a first hypothesis for calibrating TB Euro-American lineage molecular clock; additional studies are needed to reliably date events corresponding to intermediate depths in tuberculosis phylogeny.

High-Throughput CRISPR Typing of Mycobacterium tuberculosis Complex and Salmonella enterica Serotype Typhimurium.

Spoligotyping was developed almost 18 years ago and still remains a popular first-lane genotyping technique to identify and subtype Mycobacterium tuberculosis complex (MTC) clinical isolates at a phylogeographic level. For other pathogens, such as Salmonella enterica, recent studies suggest that specifically designed spoligotyping techniques could be interesting for public health purposes. Spoligotyping was in its original format a reverse line-blot hybridization method using capture probes designed on "spacers" and attached to a membrane's surface and a PCR product obtained from clustered regularly interspaced short palindromic repeats (CRISPRs). Cowan et al. and Fabre et al. were the first to propose a high-throughput Spoligotyping method based on microbeads for MTC and S. enterica serotype Typhimurium, respectively. The main advantages of the high-throughput Spoligotyping techniques we describe here are their low cost, their robustness, and the existence (at least for MTC) of very large databases that allow comparisons between spoligotypes from anywhere.

Combined species identification, genotyping, and drug resistance detection of Mycobacterium tuberculosis cultures by MLPA on a bead-based array.

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Evaluación del poder discriminatorio de técnicas de epidemiología molecular en aislados Venezolanos de Mycobacterium tuberculosis / Evaluation of discriminatory power of molecular epidemiology techniques in Mycobacterium tuberculosis Venezuelan isolates

La técnica de espoligotipaje y el método de unidades repetitivas micobacterianas interdiseminadas y número variable de repetidos tándem (MIRU-VNTR) 24 loci se emplean para estudiar la epidemiología molecular de tuberculosis. En el presente trabajo evaluamos la discriminación del método MIRU-VNTR 24 loci solo y en asociación con espoligotipaje en aislados clínicos de M. tuberculosis en Venezuela, la diversidad alélica de los 24 loci, y el poder discriminativo para la combinación de 24 y 15 loci, 12 loci tradicionales (12t), aquellos con más alta diversidad alélica y una nueva combinación que denominamos 12 inv. Se estudiaron 104 cepas de diferentes linajes y 431 cepas de la familia Latin-America y Mediterráneos (LAM). Los loci 4052, 2163b, 424 y 2996 presentaron la más alta diversidad alélica. Las tasas de agrupamiento de MIRU-VNTR 24 loci, espoligotipaje y MIRU-VNTR combinado con espoligotipaje para 104 aislados fueron de 18,27%, 71,15% y 14,4%, respectivamente, mientras que para cepas LAM fue de 43,2%, 95,8% y 37,4%. Las combinaciones de 15, 12inv y 4 loci MIRU-VNTR mas discriminativos, fueron más discriminatorios que 12t. Las tasas de agrupamiento para 15 y 12inv MIRU VNTR acoplados a espoligotipaje en los 104 aislados fueron de 21% y 23%, mientras que para cepas LAM fue de 52% y 46% respectivamente. El número de patrones diferentes fue similar para 12inv y 15 loci. Se propone el uso de un número reducido de loci MIRU-VNTR informativos acoplado a espoligotipaje para estudios de la transmisión de la tuberculosis en Venezuela.

The techniques of spoligotyping and mycobacterial interspersed repetitive unit and variable-number tandem repeat typing with 24 loci (MIRU-VNTR-24), have been used to study the molecular epidemiology of tuberculosis. The aim of this study was: to evaluate the discriminative power of MIRU-VNTR 24 loci alone and in association with spoligotyping in clinical isolates of M. tuberculosis in Venezuela; the allelic diversity of the 24 loci; and the discriminative power for the combination of 24 and 15 loci, 12 traditional loci (12t), those with higher allelic diversity and a new combination named 12inv. We analyzed one set of 104 strains of different lineages and a second set of 431 strains belonging to the Latin-America and Mediterranean lineage (LAM) that is predominant in Venezuela. The determination of allelic diversity showed that 4052, 2163b, 424 y 2996 are highly discriminative. Clustering rates of MIRU-VNTR 24 loci, spoligotyping and MIRU-VNTR combined with spoligotyping for 104 isolates were 18.27%, 71.15% and 14.4%, respectively, whereas with the 431 LAM strains the values were 43.2 %, 95.8% and 37.4%. MIRU-VNTR combinations of 15, 12inv and 4 loci were more discriminatory than 12t. Clustering rates for MIRU-VNTR 15 and 12inv loci coupled with spoligotyping in the 104 isolated was 21% and 23%, while for LAM strains was 52% and 46% respectively. The number of different genetics patterns for 15 and 12inv loci were similar. In conclusion, we propose the use of a small number of informative loci MIRU-VNTR coupled to spoligotyping to investigate the transmission of tuberculosis in Venezuela.

Mycobacterium tuberculosis complex CRISPR genotyping: improving efficiency, throughput and discriminative power of 'spoligotyping' with new spacers and a microbead-based hybridization assay.

The aims of the present study were to implement a microbead-based 'spoligotyping' technique and to evaluate improvements by the addition of a panel of 25 extra spacers that we expected to provide an increased resolution on principal genetic group 1 (PGG 1) strains. We confirmed the high sensitivity and reproducibility of the classical technique using the 43 spacer panel and we obtained perfect agreement between the membrane-based and the microbead-based techniques. We further demonstrated an increase in the discriminative power of an extended 68 spacer format for differentiation of PGG 1 clinical isolates, in particular for the East African-Indian clade. Finally, we define a limited yet highly informative reduced 10 spacer panel set which could offer a more cost-effective option for implementation in resource-limited countries and that could decrease the need for additional VNTR (variable number of tandem repeats) genotyping work in molecular epidemiological studies. We also present an economic analysis comparing membrane-based and microbead-based techniques.

Resolving lineage assignation on Mycobacterium tuberculosis clinical isolates classified by spoligotyping with a new high-throughput 3R SNPs based method.

We developed a new multiplexed-PCR assay to accurately classify Mycobacterium tuberculosis complex (MTC) isolates at the sublineage level by single nucleotide polymorphisms (SNPs). This method relies on 7 SNPs located in different genes of the MTC strains (recC, rec0, recR, ligB, ligC, alkA, and mgtC). Most of these genes are involved in replication, repair and recombination (3R) functions of M. tuberculosis strains, four of the mutations are synonymous, and thus neutral. Genes were chosen as a first empirical approach to assess the congruence between spoligotyping-based phylogeographical classification and SNP typing. This scheme efficiently classifies most of MTC phylogeographical groups: (1) confirming and identifying new sublineage-specific SNPs, (2) unraveling phylogenetical relationships between spoligotyping-defined MTC sublineages, (3) appropriately assigning sublineages to some spoligotypes and reassigning sublineages to other mis-labeled spoligotype signatures. This study opens the way to a more meaningful taxonomic, evolutionary and epidemiological classification. It also allows evaluation of spoligotype-signature significance towards a more comprehensive understanding of the evolutionary mechanisms of the clustered regularly interspaced short palindromic repeat (CRISPR) locus in MTC.

Mycobacterium tuberculosis population structure and molecular epidemiological analysis in Sucre municipality, Miranda state, Venezuela / Estructura poblacional y análisis epidemiológico molecular de Mycobacterium tuberculosis en el municipio Sucre, estado Miranda, Venezuela

Sucre municipality is a large, densely populated marginal area in the eastern part of Caracas, Venezuela that consistently has more cases of tuberculosis than other municipalities in the country. To identify the neighborhoods in the municipality with the highest prevalence of tuberculosis, and determine whether the Mycobacterium tuberculosis strain distribution in this municipality is different from that previously found in the western part of Caracas and the rest of Venezuela, we collected data on all tuberculosis cases in the municipality diagnosed in 2005-6. We performed two separate molecular epidemiological studies, spoligotyping 44 strains in a first study, and spoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolates in the second. With spoligotyping, the most common patterns were Shared International Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), a distribution similar to other parts of Venezuela, except that SIT42 and SIT20 were more common. MIRU-VNTR 15 showed that six of seven SIT17 strains examined belonged to a large cluster previously found circulating in Venezuela, but all of the SIT42 strains were related to a cluster centered in the neighborhoods of Unión and Maca, with a MIRU-VNTR pattern not previously seen in Venezuela. It appears that a large percentage of the tuberculosis in the Sucre municipality is caused by the active transmission of two strain families centered within distinct neighborhoods, one reflecting communication with the rest of the country, and the other suggesting the insular, isolated nature of some sectors.

El municipio Sucre es un área densamente poblada del este de Caracas, Venezuela, con más casos de tuberculosis que otros municipios del país. Para establecer las áreas en el municipio Sucre con la mas alta prevalencia de tuberculosis y determinar sí la distribución de cepas de Mycobacterium tuberculosis es diferente de las encontradas previamente en el Oeste de Caracas y el resto de Venezuela, se recolectaron los datos de todos los casos diagnosticados de tuberculosis en el municipio en el 2005-6. Además, se aplicaron dos estudios de epidemiología molecular, el primero con 44 aislados en 2006 y el segundo con 131 aislados del 2006 al 2011, todos caracterizados por spoligotyping. Fue aplicada la técnica MIRU VNTR15 sobre 21 aislados agrupados. Con spoligotyping, los patrones encontrados fueron SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), presentando una distribución similar en otras partes de Venezuela, con la diferencia de que el SIT42 y el SIT20 fueron comunes en el municipio. MIRU VNTR15 mostró que seis de las siete cepas SIT17 pertenecían a un gran grupo encontrado previamente en Venezuela, mientras las cepas SIT42, estaban relacionados a un grupo concentrado en los Barrios Unión y Maca, con un patrón MIRU VNTR no visto previamente en Venezuela. Los resultados indicarían que un gran porcentaje de tuberculosis en el municipio Sucre es causada por transmisión activa de dos familias, una reflejando comunicación con el resto del país, y otra sugiriendo que es un aislado propio de algunos Barrios del municipio.