María Antonia Lezcano

Systematic Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates from Spain

ABSTRACT We used spoligotyping and restriction fragment length polymorphism (RFLP) of the IS6110-insertion sequence to study the molecular epidemiology of multidrug-resistant (MDR) tuberculosis in Spain. We analyzed 180 Mycobacterium tuberculosis complex isolates collected between January 1998 and December 2000. Consecutive isolates from the same patients (n = 23) always had identical genotypes, meaning that no cases of reinfection occurred. A total of 105 isolates (58.3%) had unique RFLP patterns, whereas 75 isolates (41.7%) were in 20 different RFLP clusters. Characterization of the katG and rpoB genes showed that 14 strains included in the RFLP clusters did not actually cluster. Only 33.8% of the strains isolated were suggestive of MDR transmission, a frequency lower than that for susceptible strains in Spain (46.6%). We found that the Beijing/W genotype, which is prevalent worldwide, was significantly associated with immigrants. The 22 isolates in the largest cluster corresponded to the Mycobacterium bovis strain responsible for two nosocomial MDR outbreaks in Spain.

Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela

BackgroundMolecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).ResultsClinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised.Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively.Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.ConclusionThis study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.

Resistencia de Mycobacterium tuberculosis en Zaragoza (1993-1997) y factores asociados

Fundamento Conocer la frecuencia de resistencias de Mycobacterium tuberculosis Zaragozay estudiar los factores asociados. Pacientes y metodo Estudio transversal de la sensibilidad de Mycobacterium tuberculosis el Hospital Universitario Miguel Servet (Zaragoza) entre 1993 y 1997; el metodo utilizado fue el de las proporciones. Se realizo un analisis estadistico con pruebas convencionales utilizando un nivel de significacion de p Resultados Se estudiaron 428 pacientes con cultivo positivo frente a Mycobacterium tuberculosis : 136 (31,8%) VIH positivos, 121 (28,3%) VIH negativos y en 171 (39,9%) no constaba la serologia. Las resistencias al menos a un farmaco se observaron en 47 pacientes (10,9%). En 22 fueron primarias (5,9%) y en 25 pacientes fueron adquiridas (42,4%). La resistencia primaria observada en los VIH positivos fue del 9,2%, mientras que en los VIH negativos fue del 7,5%; la resistencia adquirida tambien fue mayor en los pacientes infectados por el VIH, 51,8% frente al 42,8% de los seronegativos. Cuando se comparo la aparicion de resistencias entre ambos colectivos, no se observaron diferencias estadisticamente significativas. En 20 pacientes aparecieron resistencias multiples (4,7%), y 10 (2,3%) lo eran al menos a isoniacida y rifampicina. Los factores asociados con la aparicion de resistencia adquirida fueron: habito enolico ( odds ratio [OR] = 2,65; intervalo de confianza [IC] del 95%, 1,24–5,65), usuarios de drogas por via parenteral (OR = 2,33; IC del 95%, 1,05–5,17), episodios previos de tuberculosis (OR = 109,40; IC del 95%, 15,02–796,43) y ser transeunte (OR = 3,75; IC del 95%, 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 Espana. No se han observado diferencias significativas entre seropositivos y seronegativos para el VIH. Los factores asociados significativamente con la aparicion de resistencia adquirida fueron: habito enolico, adiccion a drogas por via parenteral, episodios previos de tuberculosis y ser transeunte.

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.

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

BackgroundDifferent 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.ResultsThe 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 gyrA 95. In addition, SNPs in kat G463, mgtC182, Ag85C103 and RDRio deletion were detected.ConclusionsThis 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.

Multidrug-Resistant Mycobacterium tuberculosis Strain from Equatorial Guinea Detected in Spain

To the Editor: Eleven years of molecular epidemiologic data allowed the Spanish Multidrug-resistant Tuberculosis (MDR TB) Surveillance Network to identify a specific MDR Mycobacterium tuberculosis strain that had been imported into Spain from Equatorial Guinea (1). Our study brings to light the potential dissemination of this strain (named MDR-TBEG) in Equatorial Guinea, a country where little is known about the extent and features of TB or MDR TB. It also highlights that MDR strains can spread across continents, and thus MDR TB’s emergence in any country becomes a global problem. Ten MDR M. tuberculosis isolates obtained from 10 patients from Equatorial Guinea were detected in Spain during 2000 through 2008. Evidence of clonality was found within the 10 isolates because all exhibited identical genetic profiles defined by different molecular epidemiology methods (2,3) and mutations involved in drug resistance (Figure). Notably, none of the remaining 504 MDR isolates in the Spanish database matched SIT177, a spoligotype belonging to the Latin American–Mediterranean 9 (LAM9) subfamily (4). Figure Genetic profile of the multidrug-resistant tuberculosis Equatorial Guinea (MDR-TBEG) strain. RFLP, restriction fragment length polymorphism; SIT, spoligotype international type; LAM, Latin American-Mediterranean; MIRU-VNTR, mycobacterial interspersed ... The data routinely collected for all cases of MDR TB have been previously described (1). All 10 patients in the study were from Equatorial Guinea, a small African country on the Gulf of Guinea with a population of ≈500,000, an MDR TB rate >2.0% (5) of all combined (new and previously treated) TB cases, and an estimated adult HIV prevalence rate of 3.2% (www.who.int/globalatlas/predefinedReports/EFS2008/full/EFS2008_GQ.pdf). The MDR TB isolates were collected within a 9-year period (Technical Appendix): 1 in 2000, 2 in 2001, 3 in 2003, 1 in 2004, 2 in 2007, and 1 in 2008. According to their hospitals of origin, the patients were geographically dispersed in 6 different Spanish cities. We found that the interval between the patients’ arrival in Spain to the initiation of anti-TB treatment was <3 months in 6 patients, 3 of whom were clinically ill at the time of arrival. Seven patients were adult men, 2 were adult women, and 1 was an 8-year-old girl. The patients’ mean age was 30 years (range 8–54 years). Three patients were seropositive and 4 were seronegative for HIV infection (the HIV status of 3 patients was unknown). Data on prior anti-TB treatment was available for 7 case-patients, of whom only 1 had a history of antecedent TB chemotherapy. Altogether, 3 patients died before completing treatment, including 2 patients affected by miliary TB, 1 of whom was HIV-coinfected. The third patient who died was a student without a known history of immunosuppression or previous TB who had lived for 2 years in Spain. We could not establish any epidemiologic links between these patients during their stay in Spain. Analysis of drug resistance genes showed that all isolates harbored the inhA promoter mutation –15C→T (6). Alterations in the inhA gene were previously reported in 80% of the isoniazid-resistant isolates from Equatorial Guinea (5). Notably, a double mutation in the rpoB gene affecting codons 531 (Ser531Leu) and 561 (Ile561Val) was detected in the 10 MDR isolates. The presence of this uncommon mutation, Ile561Val, outside the rifampin resistance–determining region supports the hypothesis that the MDR isolates are clonal in origin. Furthermore, we demonstrated the absence of Ile561Val mutation in 3 drug-susceptible M. tuberculosis strains with an SIT177-LAM 9 spoligotype pattern, which ruled out a relationship between this spoligotype and the Ile561Val mutation. Further analysis with phylogenetic markers assigned MDR-TBEG to the principal genetic group 2, the Euro-American lineage of M. tuberculosis and its West African sublineage, on the basis of polymorphisms in codons katG463 and gyrA95, the 7-bp pks15/1 deletion, and RD174 (7,8), respectively. The analysis of the RDRio deletion confirmed that the strain belongs to the major RDRio sublineage of the LAM M. tuberculosis spoligotype family (9). This sublineage is a major cause of TB in Rio de Janeiro (Brazil) but has disseminated globally. Additional information on the geographic distribution of SIT177-LAM 9 was obtained from the updated International Spoligotyping Database (SITVIT2) of the Institut Pasteur de Guadeloupe. SITVIT2 (consulted on 23 July 2008) contained 57 isolates belonging to SIT177. Almost 50% (n = 28) came from Brazil, and 14% from Africa (Morocco, n = 6; Senegal, n = 2). The remaining isolates with known countries of origin (n = 9) were distributed in other unrelated countries. These data indicate that this particular spoligotype pattern is widely distributed. We identified 1 MDR strain of M. tuberculosis RDRio sublineage isolated in Spain from Equatorial Guinean patients. Although the transmission of MDR-TBEG in Spain could not be conclusively ruled out, the fact that MDR TB developed in most patients within 3 months after their arrival, as well as the spatiotemporal distribution of the MDR TB cases and its clonal origin, strongly suggest that MDR-TBEG was imported into Spain and that active transmission of this particular clone could be occurring in Equatorial Guinea. However, additional molecular and epidemiologic studies should be conducted in this sub-Saharan country to ascertain its role in recent transmission of MDR TB. Greater international efforts should be made to provide appropriate tools to resource-limited areas for fighting against MDR TB and preventing development of extensively drug-resistant TB.

Global Study of IS6110 in a Successful Mycobacterium tuberculosis Strain: Clues for Deciphering Its Behavior and for Its Rapid Detection

ABSTRACT 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.