Aplicación de métodos moleculares para la identificación de las especies del complejo Mycobacterium tuberculosis / Differentiation of species within the Mycobacterium tuberculosis complex by molecular techniques

Introducción En el complejo Mycobacterium tuberculosis se engloban las especies M. tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-Calmette y Guérin, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii y Mycobacterium canettii. Estas especies son las causantes de la tuberculosis en humanos y animales. Tradicionalmente la identificación de estas especies se ha basado en el estudio de métodos fenotípicos. No obstante, en los últimos años se han desarrollado numerosas técnicas moleculares. El objetivo de este trabajo es la evaluación de cada una de éstas para crear un esquema de identificación rápido y sencillo. Material y métodos Mediante el esquema propuesto se analizaron 251 cepas escogidas al azar entre las estudiadas en el año 2004 y se analizaron 797 cepas recibidas en el Laboratorio de Referencia de Micobacterias entre los años 2005 y 2007. La caracterización fenotípica de 4.183 cepas aisladas en este período se realizó mediante el estudio morfológico de la colonia, el aspecto del cultivo, la reducción de nitratos, la producción de niacina y el crecimiento en presencia de (..) (AU)

Introduction The Mycobacterium tuberculosis complex includes the following species: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-BCG, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii, and Mycobacterium canettii. These species cause tuberculosis in humans and animals. Identification of mycobacterial strains has classically been performed by phenotype study. Over the last years, laboratories have developed several molecular techniques to differentiate between these species. The aim of this study is to evaluate these methods and develop a simple, fast, identification scheme. Material and methods We analyzed 251 strains randomly obtained from the strains studied in 2004, and 797 strains received by the Reference Laboratory between 2005 and 2007. Phenotype characterization of 4183 strains isolated during that period was done by studying the colony morphology, characteristics in culture, nitrate (..) (AU)

[Differentiation of species within the Mycobacterium tuberculosis complex by molecular techniques]. / Aplicación de métodos moleculares para la identificación de las especies del complejo Mycobacterium tuberculosis.

INTRODUCTION: The Mycobacterium tuberculosis complex includes the following species: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-BCG, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii, and Mycobacterium canettii. These species cause tuberculosis in humans and animals. Identification of mycobacterial strains has classically been performed by phenotype study. Over the last years, laboratories have developed several molecular techniques to differentiate between these species. The aim of this study is to evaluate these methods and develop a simple, fast, identification scheme. MATERIAL AND METHODS: We analyzed 251 strains randomly obtained from the strains studied in 2004, and 797 strains received by the Reference Laboratory between 2005 and 2007. Phenotype characterization of 4183 strains isolated during that period was done by studying the colony morphology, characteristics in culture, nitrate reduction, niacin accumulation, and growth in the presence of thiophen-2-carboxylic acid hydrazide 10 microg/mL and pyrazinamide 50 microg/mL. The molecular identification scheme designed was as follows: 1) gyrB PCR-RFLP with RsaI, TaqI or SacII and hsp65 RFLP/PCR with HhaI., and 2) multiplex-PCR to determine the presence/absence of the RD9 and RD1 regions. RESULTS: The results showed 100% agreement between phenotype study and the molecular scheme. DISCUSSION: This molecular identification scheme is a simple and fast method, with 100% sensitivity and specificity, that can be implemented in most clinical laboratories at a low cost.

[Phenotypic and genotypic characteristics of Mycobacterium kansasii strains isolated in Spain (2000-2003)]. / Caracterización fenotípica y genotípica de cepas de Mycobacterium kansasii aisladas en España (2000-2003).

INTRODUCTION: Mycobacterium kansasii is an opportunistic pathogen that mainly causes pulmonary infections. This species accounted for 9.7% of Mycobacteria other than tuberculosis complex identified in the reference laboratory of the Spanish Centro Nacional de Microbiologia during the period of 2000-2003. METHODS: In this study we analyzed the phenotypic and genotypic characteristics of 298 M. kansasii strains isolated over this 4-year period. The phenotypic characteristics were determined by conventional methods: biochemical testing, culture and morphological study. Genotypic characteristics were studied using PCR restriction fragment analysis of a fragment of the hsp65 gene and digestion with BstEII and HaeIII, according to the method of Telenti. RESULTS: Among the total of tested strains, 57.4% had the typical phenotypic characteristics described for M. kansasii. The rest had atypical patterns that we grouped into 17 biotypes. Strains belonging to six of the seven described genotypes were identified, with 86.6% of the strains falling into genotype I. CONCLUSION: Analysis of the phenotypic characteristics of M. kansasii showed a higher discrimination index for intraspecific differentiation than genotypic methods. Nevertheless, the high variability of phenotypic characteristics, some of which were very specific for the species (e.g., photochromogenicity), could complicate their identification. Hence both conventional and molecular methods should be used to accurately identify the atypical isolates.

Caracterización fenotípica y genotípica de cepas de Mycobacterium kansasii aisladas en España (2000-2003) / Phenotypic and genotypic characteristics of Mycobacterium kansasii strains isolated in Spain (2000-2003)

Introducción. Mycobacterium kansasii se define como un patógeno oportunista que causa mayoritariamente enfermedad pulmonar. Esta especie representa el 9,7% de las micobacterias no tuberculosas identificadas en el Centro Nacional de Microbiología durante los años 2000-2003. Métodos. En el presente estudio se analizan las características fenotípicas y genotípicas de 298 cepas identificadas como M. kansasii durante este período de tiempo. La caracterización fenotípica se realizó mediante métodos convencionales: características morfológicas, de crecimiento y pruebas bioquímicas. La caracterización genotípica se realizó mediante amplificación de un fragmento del gen hsp65 y posterior digestión con las enzimas BstEII y HaeIII según el método descrito por Telenti. Resultados. El 57,4% de las cepas estudiadas poseían las características fenotípicas propias de M. kansasii, pero en el resto se obtuvieron otros resultados que nos permitieron definir 17 biotipos. Se identificaron cepas pertenecientes a 6 de los 7 genotipos descritos, el 86,6% de las cepas se agrupaban en el genotipo I. Conclusión. Al analizar las características fenotípicas, se obtiene una mayor discriminación para la diferenciación intraespecífica de M. kansasii que al aplicar métodos genotípicos, pero debido al gran número de cepas con características fenotípicas atípicas, algunas de ellas muy específicas para la especie como la fotocromogenicidad, se aconseja la utilización de métodos convencionales y la aplicación de métodos moleculares para la correcta identificación de la especie (AU)

Introduction. Mycobacterium kansasii is an opportunistic pathogen that mainly causes pulmonary infections. This species accounted for 9.7% of Mycobacteria other than tuberculosis complex identified in the reference laboratory of the Spanish Centro Nacional de Microbiología during the period of 2000-2003. Methods. In this study we analyzed the phenotypic and genotypic characteristics of 298 M. kansasii strains isolated over this 4-year period. The phenotypic characteristics were determined by conventional methods: biochemical testing, culture and morphological study. Genotypic characteristics were studied using PCR restriction fragment analysis of a fragment of the hsp65 gene and digestion with BstEII and HaeIII, according to the method of Telenti. Results. Among the total of tested strains, 57.4% had the typical phenotypic characteristics described for M. kansasii. The rest had atypical patterns that we grouped into 17 biotypes. Strains belonging to six of the seven described genotypes were identified, with 86.6% of the strains falling into genotype I. Conclusion. Analysis of the phenotypic characteristics of M. kansasii showed a higher discrimination index for intraspecific differentiation than genotypic methods. Nevertheless, the high variability of phenotypic characteristics, some of which were very specific for the species (e.g., photochromogenicity), could complicate their identification. Hence both conventional and molecular methods should be used to accurately identify the atypical isolates (AU)