Genetic diversity of the Mycobacterium tuberculosis Beijing family in Brazil and Mozambique and relation with infectivity and induction of necrosis in THP-1 cells.

INTRODUCTION: The success of the Mycobacterium tuberculosis Beijing (MtbB) lineage in different geographical regions has been attributed to high transmission, increased virulence, drug resistance and rapid adaptation to the host. In some countries of secondary MtbB dispersion like South Africa and Peru, rising prevalence of the Beijing strains is registered. However, in neighboring countries to affected regions such as Mozambique and Brazil, respectively, the prevalence of these strains is still low and this could be due to biological particularities of the circulating MtbB strains and/or differentiated host susceptibility. OBJECTIVE: To characterize genetically and phenotypically MtbB strains isolated in Brazil (n = 8) and Mozambique (n = 17). METHODS: This is a descriptive study of genotypes of the MtbB isolates, determined by spoligotyping, MIRU-VNTR typing, analysis of the IS6110 copy number in the NTF region and screening for mutations in mutT2, mutT4, rpoB, katG and pks 15/1 genes. Virulence-associated properties of the studied isolates were verified in the in vitro model of infection of human THP-1 cells. RESULTS: The genotypes defined by the 24VNTRs were distinct for all isolates included in this study and presented an HGDI of 0.997. The VNTR patterns with seven copies of MIRU26 and seven copies of QUB26, representative for the previously described MtbB genotype B0, dominant in Russia, were detected in 38.5% of the studied isolates. In addition, all isolates presented RD105 deletion and a 7 bp insertion in pks15/1 gene. Almost all tested strains belonged to the RD181 sublineage, with the exception of two strains from Mozambique of RD150 sublineage. Combined analysis of the NTF region integrity and mutations in mutT genes showed that 62.5% and 47% of isolates obtained in Brazil and Mozambique, respectively, were of the ancestral genotype. The virulence index of the ancient isolates, evaluated in the THP-1 cells, was significantly lower than that of the modern genotype group. CONCLUSIONS: These data demonstrate genotype particularities of the Beijing strains isolated in Brazil and Mozambique, two countries of low prevalence of the MtbB lineage in local Mtb populations. In contrast to the neighboring countries with high prevalence of the MtbB strains of modern sublineage, significant proportions of the isolates obtained in Brazil and Mozambique were presented by the strains of the ancient sublineage. Our data suggest that lower virulence of the ancient strains, compared with the modern strains, could be involved in the slow spread of the MtbB strains in some regions.

Maintenance of Mycobacterium tuberculosis on glass beads.

The intent of this study was to estimate the shelf life of Mycobacterium tuberculosis strains, and to observe the loss of viability in some of them from year to year. From 2000 to 2004, 10,015 cultures of M. tuberculosis were preserved by freezing on glass beads at -70 degrees C. With the expectation that the loss of viability might be around 5-10%/yr of storage, 730 strains were analyzed in order to establish the prevalence of recovery within a 5% margin of error. This study shows that 94% of the strains preserved at -70 degrees C on glass beads could be recovered within 30 days. The recovery rates for drug-susceptible and drug-resistant strains showed no significant differences. The growth rates and the number of strains that showed abundant growth before the 30th day of incubation represent important features, since the subculture of a strain preserved for future use ought to quickly produce abundant growth in order to avoid misinterpretation of the tests. Our experience indicates that storage of M. tuberculosis on glass beads at -70 degrees C is a suitable procedure for an active culture collection in a public health laboratory like ours, where maintenance of M. tuberculosis cultures is a complementary activity and must be quick, practical, effective, and economical.

Mycobacterium kansasii: antibiotic susceptibility and PCR-restriction analysis of clinical isolates.

Mycobacterium kansasii is the second most common cause of non-tuberculosis mycobacterial diseases in Sao Paulo, Brazil. An important component of the management of infections caused by this organism is antibiotic susceptibility testing. The objective of this study was to determine the drug susceptibility profiles and genotypes of clinical isolates of M. kansasii obtained from patients with or without an infection that met the American Thoracic Society's case definition criteria of M. kansasii disease. One hundred and sixty-nine clinical isolates of M. kansasii collected between 1993 and 1998 in Sao Paulo, Brazil, were tested consecutively. The isolates were genotyped by PCR restriction-enzyme pattern analysis (PRA). Most of the M. kansasii strains were susceptible to isoniazid, streptomycin, rifabutin, rifampicin, clarithromycin, ethionamide, amikacin, clofazimine and cycloserine, and resistant to ethambutol, ciprofloxacin and doxycycline. Of 169 isolates, 167 belonged to the type I PRA genotype and one each belonged to type II and III genotypes. There was no correlation between PRA subtype and M. kansasii disease according to the American Thoracic Society case definition. Clinical trials may be needed to better correlate MIC values with treatment outcomes to identify appropriate parameters for drug-resistance testing of M. kansasii.

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean.

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.

Distribución de patrones PRA en aislamientos clínicos del complejo Mycobacterium avium procedentes de España y Suramérica

La infección por el complejo Mycobacterium avium (MAC) es la infección sistémica más frecuente en la fase terminal del SIDA. Las sondas de ADN disponibles en el mercado para la identificación de micobacterias son muy precisas pero extremadamente costosas. Por eso, la mayoría de los laboratorios clínicos de Latinoamérica aún tipifican micobacterias mediante pruebas fenotípicas que son lentas, laboriosas y poco precisas. En este trabajo se aplicó el análisis del polimorfismo de los fragmentos de restricción del gen hsp65 (PRA) a la identificación de MAC en 163 aislamientos clínicos procedentes de España y Suramérica. El genotipo PRA predominante en cada país fue: M. avium tipo I en Argentina (23/42, 55%) y Brasil (48/72, 67%), M. avium tipo II en España (18/26, 69%) y M. avium tipo III en Colombia (10/23, 43%). Este último genotipo, que aún no fue descrito fuera del continente americano, resultó muy infrecuente en los otros tres países del estudio. Se discuten ventajas e inconvenientes de la aplicación del PRA al diagnóstico micobacteriológico.

Distribution of PRA patterns of clinical isolates of the Mycobacterium avium complex from Spain and South America Mycobacterium avium complex (MAC) infections are the most frequent systemic infections associated with advanced AIDS. DNA probes for accurate identification of mycobacteria are available but are very expensive in many Latin American settings. Consequently, most Latin American diagnostic laboratories employ inaccurate and outdated tests for mycobacteria identification. Therefore, PCR restriction analysis (PRA) of the hsp65 gene was evaluated for the identification of 163 MAC human isolates originated from Spain and South America. The predominant PRA type in each country was: M. avium type I in Argentina (23/42, 55%) and Brazil (48/72, 67%), M. avium type II in Spain (18/26, 69%) and M. avium type III in Colombia (10/ 23, 43%). The Colombia frequency is noteworthy, since the PRA type III was quite infrequent in the other three countries. Furthermore, its presence has not been reported outside the Americas. The advantages and disadvantages of PRA in diagnostic mycobacteriology are discussed.