[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.

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 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.

The diagnosis of two cases of cutaneous ulcer caused by infection with Mycobacterium haemophilum: direct identification in a clinical sample by polymerase chain reaction-restriction endonuclease analysis.

BACKGROUND: Mycobacterium haemophilum was first recovered from subcutaneous lesions of a patient with Hodgkin's disease. Because of its special growth requirements (it grows at 30-32 degrees C and requires iron-supplemented medium), the organism cannot be isolated using routine culture techniques for other mycobacteria. Only a few developed countries have reported infection with this mycobacterium. We report the first two cases diagnosed in Venezuela. METHODS: The diagnosis of the first case was established using polymerase chain reaction (PCR)-restriction endonuclease analysis of the gene encoding the 65-kDa heat shock protein (hsp65) for the direct identification of M. haemophilum in a clinical specimen in which bacilli were observed on acid-fast smear, but growth was not detected by standard culture procedures. RESULTS: After recognizing this bacterium as a possible cause of infection in our setting, clinical samples of cutaneous lesions were routinely cultured on blood agar at 30 degrees C for at least 6 weeks, which resulted in the diagnosis of the second case. CONCLUSIONS: Dermatologists should consider this bacterium in immunocompromised patients with cutaneous ulcerating lesions. Material from the lesions can be screened for mycobacteria using an acid-fast stain and, if acid-fast bacilli are seen, PCR analysis of mycobacterial hsp65 can be an effective tool for early diagnosis. Appropriate culture methods are required for bacteriologic confirmation of infection with M. haemophilum.

Mycobacterium bovis cultured from commercially pasteurized cows' milk: laboratory cross-contamination.

The ability of Mycobacterium paratuberculosis to survive the commercial pasteurization process of raw milk remains controversial. In a study undertaken in Venezuela to culture M. paratuberculosis from commercially pasteurized cows' milk, 83-200 ml containers of milk were processed and cultured on Herrold's egg yolk slants. No M. paratuberculosis was cultured but a total of six colonies of Mycobacterium bovis were isolated from one container each from two different milk providers. Because laboratory cross-contamination was suspected, the laboratory records were reviewed and spoligotyping was carried out on the isolated individual colonies. On the day before these milk specimens were processed, the biological safety cabinet had been used for the isolation of M. bovis from lymph nodes from infected cattle. Spoligotyping showed that that the colonies isolated from the milk all had the same pattern as the strains isolated from the lymph nodes that were processed the previous day. As far as we know, this is the first report of cross-contamination in a veterinary mycobacterial laboratory. False-positive cultures in the mycobacterial laboratory are not rare. In this setting M. bovis was isolated because it is the most common manipulated organism in this laboratory. We believe that reports on the isolation of M. paratuberculosis from commercially pasteurized milk should exclude cross-contamination before reporting, especially when this organism is routinely isolated from animal material in the same lab.