Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Tipificación molecular de Mycobacterium avium subsp. paratuberculosis en lecherías de Antioquia, Colombia

RESUMEN Objetivo. Determinar la diversidad molecular de Mycobacterium avium subsp. paratuberculosis (MAP) en muestras ambientales de hatos lecheros colombianos. Materiales y métodos. Las muestras ambientales de 25 hatos lecheros positivos a MAP por IS900-qPCR se cultivaron por duplicado en medio de yema de huevo de Herrold con micobactina J para obtener aislamientos. Las colonias sospechosas fueron confirmadas para MAP por IS900-qPCR. El ADN positivo se subtipó utilizando técnicas de unidades micobacterialess repetitivas intercaladas - número variable de repeticiones en tándem (MIRU-VNTR) y técnicas de repeticiones de multilocus de secuencia corta (MLSSR) para analizar las diferencias genéticas entre los aislamientos. Resultados. El subtipado reveló dos genotipos diferentes por MIRU-VNTR (INMV 2 e INMV 36). La técnica de MLSSR se realizó para aumentar el poder discriminatorio de lo obtenido por MIRU-VNTR, pero no se observaron diferencias entre los aislamientos recuperados. Conclusiones. El presente estudio representa un enfoque importante para el conocimiento del estatus epidemiológico de MAP en la población de estudio.

ABSTRACT Objective. To determine Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in environmental samples from Colombian dairy herds. Materials and methods. Environmental samples from 25 IS900-qPCR MAP-positive dairy herds were cultured by duplicate in Herrold's egg yolk medium with mycobactin J to obtain isolates. Suspicious colonies were confirmed by MAP-IS900-qPCR. Positive DNA was sub-typed using mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) and multilocus short sequence repeats (MLSSR) techniques to analyze the genetic differences between the isolates. Results. Sub-typing revealed two different genotypes by MIRU-VNTR (INMV 2 and INMV 36). MLSSR technique was carried out to increase the discriminatory power from what was obtained by MIRU-VNTR, but no differences were observed among the recovered isolates. Conclusions. The present study represents an important approach to the knowledge on MAP epidemiological status in the study population.

Uso de la técnica de vntr-miru para el estudio de transmisión intrahospitalaria de tuberculosis en el Hospital Universitario de Caracas, 2010-2011 / Use the vntr-miru technique for the study of nosocomial transmission of tuberculosis at the Hospital Universitario de Caracas, 2010-2011

La tuberculosis es una enfermedad infecciosa, causada por especies del complejo Mycobacterium tuberculosis. Es una enfermedad de fácil transmisión, lo que puede generar un problema en los sitios de cuidado de salud al incrementarse el riesgo de infección entre las personas que se encuentran hospitalizadas, personal médico y visitante. El objetivo de este estudio fue tipificar mediante VNTR-MIRU 35 aislados de M. tuberculosis, obtenidos de pacientes recluidos en el Hospital Universitario de Caracas (HUC) durante 2010­2011, para establecer si existe estrecha relación genética entre los aislados y detectar si se presentó transmisión intrahospitalaria. En la tipificación por VNTR se observaron 29 patrones genéticos únicos (82,9%) y 3 clústeres, de los cuales sólo uno estuvo conformado por patrones genéticos de aislados provenientes de pacientes con estrecha relación epidemiológica (superposición de períodos de hospitalización). Mediante el análisis comparativo de los patrones genéticos con los depositados en la base de datos de VNTR-plus utilizando el método de Neighbor-joining, se pudo observar que los aislados se agrupaban con patrones LAM (n=25), Haarlem (n=3) y S (n=7). La conformación de un clúster de dos aislados de pacientes con un vínculo epidemiológico sugiere la posible transmisión nosocomial de la TB en el HUC

Tuberculosis (TB) is an infectious disease, caused by species from the Mycobacterium tuberculosis complex (MTBC). It is an easily transmitted disease that it can increase the risk of infection among people who are in hospital, medical staff and visitors The purpose of this study was to molecular typing by VNTR-MIRU 35 isolates of Mycobacterium tuberculosis (M.tb) from hospitalized patients in the "Hospital Universitario de Caracas" during 2010-2011 to establish whether there is a close genetic relationship between isolates and to detect whether the nosocomial transmission was presented. In the analysis of VNTR typing, 29 unique genetic patterns (82,9%) and 3 clusters were observed, of which only one consisted by genetic patterns of isolates from patient with close epidemiologic relation (overlapping of periods of hospitalization). With the comparative analysis of genetic patterns with VNTR-plus database using Neighbor-joining method it was possible to observe the clustering of the isolates with LAM (n=25), Haarlem (n=3), S (n=7). The forming of one cluster with two isolates from patients with epidemiological link suggests a possible nosocomial transmission of TB in HUC

A study on pre-XDR & XDR tuberculosis & their prevalent genotypes in clinical isolates of Mycobacterium tuberculosis in north India.

Background & objectives: Pre-extensively drug resistant (pre-XDR) and extensively drug resistant tuberculosis (XDR-TB) have been areas of growing concern, and are posing threat to global efforts of TB control. The present study was planned to study the presence of pre-XDR and XDR Mycobacterium tuberculosis and their genotypes in clinical isolates obtained from previously treated cases of pulmonary TB. Methods: A total of 219 isolates obtained from previously treated cases of pulmonary TB were subjected to first-line (streptomycin, isoniazid, rifampicin and ethambutol) and second-line (ofloxacin, kanamycin, capreomycin and amikacin) drug susceptibility testing on solid Lowenstein-Jensen medium by proportion method. Genotyping was done for pre-XDR and XDR-TB isolates using 12 loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR). Results: Multi-drug resistance was observed in 39.7 per cent (87/219) isolates. Pre-XDR and XDR M. tuberculosis isolates amongst 87 multi-drug resistant (MDR) TB isolates were 43 (49.4%) and 10 (11.4%), respectively. Two most dominant genotypes among pre-XDR and XDR M. tuberculosis isolates were Beijing and Delhi/CAS types. Interpretation & conclusions: Resistance to second-line anti-tubercular drugs should be routinely assessed in areas endemic for TB. Similar genotype patterns were seen in pre-XDR and XDR-TB isolates. Beijing and Delhi/CAS were predominant genotypes.

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.

Molecular profiling of drug resistant isolates ofMycobacterium tuberculosis in the state of Santa Catarina, southern Brazil

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoBin RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

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.

Análisis genotípico de aislamientos de Mycobacterium tuberculosis en La Habana en 2009 / Genotyping of mycobacterium tuberculosis isolates in Havana, 2009

INTRODUCCIÓN: la búsqueda de alternativas al polimorfismo de la longitud de los fragmentos de restricción (RFLP, siglas en inglés) con la sonda IS 6110 en la genotipificación de Mycobacterium tuberculosis ha propiciado el desarrollo de la tipificación con número variable de repeticiones en tándem de unidades repetitivas interespaciadas de micobacterias (MIRU-VNTR, siglas en inglés). OBJETIVO: evaluar la diseminación de genotipos de M. tuberculosis en La Habana en 2009. MÉTODOS: se estudiaron 80 aislamientos procedentes de unidades de salud durante 2009 y se caracterizaron por tipificación MIRU-VNTR-15. Los genotipos se expresaron como códigos numéricos según el número de copias de cada MIRU-VNTR amplificado, y se analizaron con la herramienta bioinformática en línea MIRU-VNTR plus. Se utilizó MIRU-VNTR-24 como tipificación secundaria en los aislamientos agrupados por MIRU-VNTR-15. RESULTADOS: con MIRU-VNTR-15 se definieron 41 genotipos diferentes; entre ellos, 33 únicos (41,25 por ciento), y ocho que agruparon a 47 aislamientos (58,75 por ciento). La tipificación MIRU-VNTR-24 logró diferenciar sólo el 5 por ciento de éstos, disminuyendo el porcentaje de agrupamiento a 53,75 por ciento. CONCLUSIONES: el elevado agrupamiento encontrado sugirió transmisión reciente, lo que pudo tener influencia en la incidencia de tuberculosis en La Habana en 2009(AU)

INTRODUCTION: the search for alternatives to restriction fragment length polymorphism (RFLP) with the IS6110probe for the genotyping of Mycobacterium tuberculosis has paved the way for the development of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing (MIRU-VNTR). OBJECTIVE: evaluate the spread of M. tuberculosis genotypes in Havana in 2009. METHODS: eighty isolates obtained from healthcare centers during 2009 were examined and characterized by 15-loci MIRU-VNTR typing. The genotypes were expressed as numerical codes according to the copy number of each amplified MIRU-VNTR, and they were analyzed with the online bioinformatic tool MIRU-VNTR plus. 24-loci MIRU-VNTR was used for secondary typing of isolates grouped by 15-loci MIRU-VNTR. RESULTS: forty-one different genotypes were defined with 15-loci MIRU-VNTR. Of these, 33 were single (41.25 percent), whereas 8 clustered 47 isolates (58.75 percent). Only 5 percent of the latter could be differentiated by 24-loci MIRU-VNTR, lowering the percentage of clustering to 53.75 percent. CONCLUSIONS: the high clustering values revealed by the study suggest that transmission was recent. This may have had an influence on the incidence of tuberculosis in Havana in 2009(AU)

Presencia del genotipo Beijing entre cepas del complejo Mycobacterium tuberculosis en dos centros de salud de la Región Metropolitana-Chile / Presence of Bejing genotype among Mycobacterium tuberculosis strains in two centres of the Region Metropolitana of Chile

Background: Genotyping of Mycobacterium tuberculosis complex (cMtb) allows us to know geographically predominant lineages. Some lineages spread more rapidly and are associated with multidrug resistance, particularly Beijing, which has been reported in Latin America (Peru). There is little information about this topic in Chile and there are no reports of the presence of the Beijing genotype. Aim: To determine the most prevalent lineages in the Metropolitan Region of Chile with emphasis on the search for Beijing in two health centers. Methods: Two complementary molecular methods were used: spoligotyping, based on the variations of the direct repeat regions in the genome of cMtb and MIRU-VNTR, based in the variable number of tandem repeats of mycobacterial interspersed repetitive units, and subsequent analysis in international databases. A designed lineage was assigned to 37 of the 43 strains studied (86%); 6 isolates could not be assigned to any genotype. LAM and T genotype were the most frequent (39.5 and 32.5%, respectively) followed by Haarlem (7.0%), Beijing (4.7%) and X (2.3%). Conclusion: We describe for the first time the presence of the Beijing genotype in Chile. cMtb molecular surveillance should be implemented in our country in order to know the dynamics of its transmission.

Introducción: La genotipificación del complejo Mycobacterium tuberculosis (cMtbc) permite conocer los genotipos geográficamente predominantes. Algunos genotipos se diseminan con mayor rapidez y se asocian a multi-resistencia, tal como Beijing, reportado en América Latina en Perú. Existe poca información al respecto en Chile, sin reportes de la presencia de Beijing. Objetivo: Conocer los genotipos prevalentes en dos centros de salud de la Región Metropolitana de Chile con énfasis en la búsqueda de Beijing. Métodos: Se utilizaron dos métodos moleculares complementarios basados en la variación de las regiones de repeticiones directa en el genoma de M. tuberculosis (espoligotipificación) y número variable de repeticiones en tandem de las unidades repetitivas de interespaciadores micobacterianos (MIRU-VNTRs) y posterior análisis en bases de datos internacionales. Resultados: Se asignó un genotipo conocido a 37 de las 43 cepas estudiadas (86%), mientras que en 14% no se asignó alguno. Los genotipos LAM y T fueron los más frecuentes (39,5 y 32,5%, respectivamente), seguidos por Haarlem (7,0%), Beijing (4,7%) y X (2,3%). Conclusión: Se describe por primera vez en Chile la presencia del genotipo Beijing en cepas de cMtb. Es necesario realizar una vigilancia epidemiológica molecular en el cMtb para conocer la dinámica de la transmisión en nuestro país.

The genotyping of ofloxacin-resistant mycobacterium tuberculosis with MIRU-VNTR in Hangzhou city / 预防医学

Objective To evaluate the application of MIRU - VNTR analysis in genotyping of the ofloxacin - resistant mycobacterium tuberculosis in Hangzhou. Methods From April 2010 to June 2012,the clinical isolates of mycobacterium tuberculosis were collected from designated TB hospitals in Hangzhou for drug sensitivity test. The ofloxacin -resistant mycobacterium tuberculosis strains were typed with RD105 deletion test and MIRU-VNTR typing method respectively,and the results from both methods were compared. Results A total of 52 ofloxacin -resistant mycobacterium tuberculosis strains were collected. Using RD105 deletion test,43 strains(82. 69%)belonged to mycobacterium tuberculosis Beijing family. Using a 12-locus MIRU-VNTR typing,52 distinct MIRU-VNTR patterns were detected in the 52 ofloxacin-resistant strains. The Hunter-Gaston discriminatory index(HGI)was 0. 999. Except MIRU40 and ETR-F,the other 10 MIRU-VNTR loci showed moderate or high discriminatory power for both Beijing genotype isolates and non -Beijing genotype isolates. Conclusion The selected 10 MIRU-VNTR loci has a higher discriminatory power and are appropriate for the ofloxacin-resistant mycobacterium tuberculosis genotyping.

Mycobacterium tuberculosis belonging to family LAM and sublineage RD Rio: common strains in Southern Brazil for over 10 years

A sublineage of Mycobacterium tuberculosis called RD Rio was described in 2007. Although only recently described, this strain may have been present previously in the population, and its identification in clinical isolates will elucidate bacterial transmission dynamics and host-pathogen interactions. This study evaluated the clonal diversity of the RD Rio sublineage in clinical isolates from Rio Grande-RS obtained between 1998 and 2001. Among the 45 samples analyzed by the MIRU-VNTR method, there were six clusters with two samples each and 33 orphan strains with unique pattern. The strains were distributed across several different lineages including LAM (34.04%), X (14.89%), Haarlem (12.77%), UgandaI (10.64%), S (4.26%), NEW-1 (2.13%) and Cameroon (2.13%); 14.89% of the strains matched to multiple lineages. RD Rio strains were present in 28.9% of the samples and 81.25% of the identified strains belonged to the LAM family. The high clonal diversity observed in this study is a constant feature in this region. The RD Rio sublineage has been in Rio Grande-RS since 1998. The continued monitoring of RD Rio in clinical isolates will enhance the understanding of its epidemiological significance.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Mycobacterial Interspersed Repetitive Unit typing in Mycobacterium tuberculosis isolates from Sichuan Province in China.

Background & objectives: Emergence and spread of drug resistant Mycobacterium tuberculosis is a serious threat to tuberculosis (TB) control programme. Therefore, the objective of this study was to genotype drug-resistant M. tuberculosis strains isolated from patients in Sichuan, China, using Mycobacterial Interspersed Repetitive Units (MIRU) for epidemiological analysis. Methods: Drug-resistance testing of M. tuberculosis isolates from pulmonary TB patients was confirmed by proportion method. Twelve MIRU loci were analyzed on 80 drug-resistant and 9 susceptible isolates by polymerase chain reaction and agarose gel electrophoresis. Hunter-Gaston discriminatory index (HGI) values were determined for each 12 MIRU loci for the evaluation of their discrimination power. Results: Among 12 MIRU loci examined, polymorphic bands could be generated on 11 loci. Sixty five isolates had distinct MIRU patterns, while other 24 belonged to 8 clusters and resistant to at least one anti-TB drug tested. The association between the MIRU patterns and the mutation patterns of drug-resistance relevant target genes was not significant among the drug-resistant isolates. Interpretation & conclusions: The results showed that with a satisfactory discrimination power exhibited, the 12 loci based MIRU typing could be a valuable tool for epidemiological studies in M. tuberculosis isolates from Sichuan.

Clinical data and molecular analysis of Mycobacterium tuberculosi isolates from drug-resistant tuberculosis patients in Goiás, Brazil

Drug resistance is one of the major concerns regarding tuberculosis (TB) infection worldwide because it hampers control of the disease. Understanding the underlying mechanisms responsible for drug resistance development is of the highest importance. To investigate clinical data from drug-resistant TB patients at the Tropical Diseases Hospital, Goiás (GO), Brazil and to evaluate the molecular basis of rifampin (R) and isoniazid (H) resistance in Mycobacterium tuberculosis. Drug susceptibility testing was performed on 124 isolates from 100 patients and 24 isolates displayed resistance to R and/or H. Molecular analysis of drug resistance was performed by partial sequencing of the rpoB and katGgenes and analysis of the inhA promoter region. Similarity analysis of isolates was performed by 15 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. The molecular basis of drug resistance among the 24 isolates from 16 patients was confirmed in 18 isolates. Different susceptibility profiles among the isolates from the same individual were observed in five patients; using MIRU-VNTR, we have shown that those isolates were not genetically identical, with differences in one to three loci within the 15 analysed loci. Drug-resistant TB in GO is caused by M. tuberculosis strains with mutations in previously described sites of known genes and some patients harbour a mixed phenotype infection as a consequence of a single infective event; however, further and broader investigations are needed to support our findings.

Genotyping of Mycobacterium tuberculosis isolates from alow-endemic setting in northwestern state of Paraná in Southern Brazil

The purpose of this study was to provide information about the genetic diversity and prevalent genotype of Mycobacterium tuberculosis in a low-endemic setting in northwestern state of Paraná in Southern Brazil. We employed spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) techniques to genotype M. tuberculosisisolates from patients with pulmonary tuberculosis (TB). The 93 isolates analyzed by spoligotyping were divided into 36 different patterns, 30 of which were described in the SITVIT database. Latin American and Mediterranean, Haarlem and T families were responsible for 26.9 percent, 17.2 percent and 11.8 percent of TB cases, respectively. From the 84 isolates analyzed by MIRU-VNTR, 58 shared a unique pattern and the remaining 26 belonged to nine clusters. The MIRU loci 40, 23, 10 and 16 were the most discriminatory. A combination of MIRU-VNTR and spoligotyping resulted in 85.7 percent discriminatory power (Hunter-Gaston index = 0.995). Thus, combining spoligotyping and MIRU-VNTR typing proved to be most useful for epidemiological study in this low-endemic setting in Southern Brazil. The current study demonstrated that there is significant diversity in circulating strains in the city of Maringá and the surrounding regions, with no single genotype of M. tuberculosispredominating.

Strain Typing of Mycobacterium tuberculosis Isolates from Korea by Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats / 대한진단검사의학회지

BACKGROUND: Mycobacterium tuberculosis is one of the most clinically significant infectious agents. Especially during mass outbreaks, accurate identification and monitoring are required. The proportion of Beijing family members is very high among infecting strains, and spoligotyping is not suitable for strain typing. Therefore, we studied the homogeneity of isolates using the mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method and identified its utility for carrying out molecular epidemiologic analysis. METHODS: Eighty-one clinical M. tuberculosis isolates that had previously been analyzed by spoligotyping were used in this study. We used the 12 standard MIRU loci and further four exact tandem repeat (ETR) loci (ETR-A, -B, -C, and -F). Four strains each of randomly selected Beijing and Beijing-like families were subjected to IS6110- restriction fragment length polymorphism analysis. RESULTS: All 81 samples showed amplification products of all VNTR loci, and all of them showed differences in at least one locus. The calculation of the Hunter-Gaston diversity index (HGDI) for MIRU-VNTR gave the value of 0.965. Discriminatory index in the six loci (MIRU-10, -16, -26, -31, -39, and ETR-F) were found to be highly discriminated (HGDI >0.6). Beijing and Beijing-like family isolates were discriminated into different MIRU-VNTR types. CONCLUSIONS: MIRU-VNTR analysis by using well-selected loci can be useful in discriminating the clinical M. tuberculosis isolates in areas where the Beijing family is predominant.