Single nucleotide polymorphism (SNP) analysis used for the phylogeny of the Mycobacterium tuberculosis complex based on a pyrosequencing assay.

BACKGROUND: Different polymorphisms have been described as markers to classify the lineages of the Mycobacterium tuberculosis complex. The analysis of nine single nucleotide polymorphisms (SNPs) was used to describe seven SNPs cluster groups (SCGs). We attempted to classify those strains that could not been categorized into lineages by the genotyping methods used in the routine testing. RESULTS: The M. tuberculosis complex isolates collected in 2010 in our region were analysed. A new method based on multiplex-PCRs and pyrosequencing to analyse these SNPs was designed. For the pyrosequencing assay nine SNPs that defined the seven SCGs were selected from the literature: 1977, 74092, 105139, 232574, 311613, 913274, 2460626, 3352929 and gyrA95. In addition, SNPs in katG(463), mgtC(182), Ag85C(103) and RD(Rio) deletion were detected. CONCLUSIONS: This work has permitted to achieve a better classification of Aragonian strains into SCGs and in some cases, to assign strains to its certain lineage. Besides, the description of a new pattern shared by two isolates "SCG-6c" reinforces the interest of SNPs to follow the evolution of M. tuberculosis complex.

Global study of IS6110 in a successful Mycobacterium tuberculosis strain: clues for deciphering its behavior and for its rapid detection.

The Mycobacterium tuberculosis insertion sequence IS6110, besides being a very useful tool in molecular epidemiology, seems to have an impact on the biology of bacilli. In the present work, we mapped the 12 points of insertion of IS6110 in the genome of a successful strain named M. tuberculosis Zaragoza (which has been referred to as the MTZ strain). This strain, belonging to principal genetic group 3, caused a large unsuspected tuberculosis outbreak involving 85 patients in Zaragoza, Spain, in 2001 to 2004. The mapping of the points of insertion of IS6110 in the genome of the Zaragoza strain offers clues for a better understanding of the adaptability and virulence of M. tuberculosis. Surprisingly, the presence of one copy of IS6110 was found in Rv2286c, as was recently described for a successful Beijing sublineage. As a result of this analysis, a rapid method for detecting this particular M. tuberculosis strain has been designed.

Long-term molecular surveillance of multidrug-resistant tuberculosis in Spain.

The data presented here span 11 years (1998-2008) of monitoring of multidrug-resistant tuberculosis (MDR-TB) clustering through molecular typing techniques in Spain. The molecular and epidemiological data of 480 multidrug-resistant Mycobacterium tuberculosis complex isolates were analyzed. Thirty-one clusters involving 157 (32.7%) patients were identified. The proportion of immigrants increased substantially over the study period reaching 65% in 2008; however, the clustering rate remained stable indicating that local transmission was little influenced by imported MDR-TB. The three major clusters respond to the persistence of two autochthonous strains throughout the study period and an extensively drug-resistant (XDR) Mycobacterium bovis outbreak with only two cases was reported since 2002. Molecular and epidemiological evidence for the importation of new strains and their spread within the community was found. Immigrant-only clusters most often grouped patients infected abroad with strains belonging to rare spoligotypes. Conversely, widespread spoligotypes of the Latin-American and Mediterranean (LAM) and Haarlem families were responsible for the majority of the MDR-TB local transmission. The demonstration of clusters spanning several Spanish regions that have been ongoing throughout the study period makes it advisable to maintain a continuous molecular surveillance in order to monitor the spread of MDR-TB.

Infección cutánea esporotricoide por Mycobacterium chelonae en paciente inmunocompetente / Sporotrichoid cutaneous infection due to Mycrobacterium chelonae in a immunocompent patient

Una mujer de 58 años consultó por múltiples lesiones cutáneas en su pierna derecha en un patrón lineal ascendente (diseminación esporotricoide) hacia la rodilla. En el cultivo de la biopsia realizada se aisló Mycobacterium chelonae. No existía ninguna situación de inmunodepresión en la paciente. La anamnesis reveló la existencia de un traumatismo en esa pierna con una hamaca de playa 1 año antes (AU)

Resistencia de mycobacterium tuberculosis en Zaragoza (1993-1997) y factores asociados / Resistance of Mycobacterium tuberculosis in Zaragoza, Spain (1993-1997) and related factors

Fundamento: Conocer la frecuencia de resistencias de Mycobacterium tuberculosis en Zaragoza y estudiar los factores asociados. Pacientes y método: Estudio transversal de la sensibilidad de Mycobacterium tuberculosis en el Hospital Universitario Miguel Servet (Zaragoza) entre 1993 y 1997; el método utilizado fue el de las proporciones. Se realizó un análisis estadístico con pruebas convencionales utilizando un nivel de significación de p < 0,05. Resultados: Se estudiaron 428 pacientes con cultivo positivo frente a Mycobacterium tuberculosis: 136 (31,8 por ciento) VIH positivos, 121 (28,3 por ciento) VIH negativos y en 171 (39,9 por ciento) no constaba la serología. Las resistencias al menos a un fármaco se observaron en 47 pacientes (10,9 por ciento). En 22 fueron primarias (5,9 por ciento) y en 25 pacientes fueron adquiridas (42,4 por ciento). La resistencia primaria observada en los VIH positivos fue del 9,2 por ciento, mientras que en los VIH negativos fue del 7,5 por ciento; la resistencia adquirida también fue mayor en los pacientes infectados por el VIH, 51,8 por ciento frente al 42,8 por ciento de los seronegativos. Cuando se comparó la aparición de resistencias entre ambos colectivos, no se observaron diferencias estadísticamente significativas. En 20 pacientes aparecieron resistencias múltiples (4,7 por ciento), y 10 (2,3 por ciento) lo eran al menos a isoniacida y rifampicina. Los factores asociados con la aparición de resistencia adquirida fueron: hábito enólico (odds ratio [OR] = 2,65; intervalo de confianza [IC] del 95 por ciento, 1,24-5,65), usuarios de drogas por vía parenteral (OR = 2,33; IC del 95 por ciento, 1,05-5,17), episodios previos de tuberculosis (OR = 109,40; IC del 95 por ciento, 15,02-796,43) y ser transeúnte (OR = 3,75; IC del 95 por ciento, 1,26-11,17); cuando estudiamos la resistencia primaria, no encontramos diferencias significativas entre los diferentes factores de riesgo. Conclusiones: El porcentaje de resistencias a M. tuberculosis observado en Zaragoza es similar a otros descritos en diferentes regiones de España. No se han observado diferencias significativas entre seropositivos y seronegativos para el VIH. Los factores asociados significativamente con la aparición de resistencia adquirida fueron: hábito enólico, adicción a drogas por vía parenteral, episodios previos de tuberculosis y ser transeúnte. (AU)