María Soledad Jiménez

New Multiplex PCR for Rapid Detection of Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates

ABSTRACT In this study, we describe a multiplex PCR to detect a AGC→ACC (serine to threonine) mutation in the katG gene and a −15 C-to-T substitution (inhAC−15T) at the 5′ end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.

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.

Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders.

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.

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.

Orquiepididimitis por Mycobacterium africanum

blicado estudios que documentan un incremento de las infecciones óseas de origen comunitario en la población pediátrica causadas por S. aureus productor de LPV2-3. Las presentaciones clínicas más frecuentes de las infecciones osteoarticulares por S. aureus en la infancia son la osteomielitis aguda hematógena que suele afectar a las metáfisis de los huesos largos y la artritis séptica que afecta principalmente a las grandes articulaciones de miembros inferiores7. En ambos casos, el compromiso plurifocal, particularmente de tres o más sitios, es excepcional. Además, la evolución clínica suele ser excelente con un diagnóstico precoz y una pauta antibiótica adecuada7. Sin embargo, las infecciones osteoarticulares por S. aureus productor de LPV revisten especial gravedad dado que se asocian a afectación multifocal, peor evolución clínica y mayor número de complicaciones. Dos estudios realizados en niños por el mismo grupo de investigadores en Texas ilustran claramente la mayor virulencia de estas cepas. En el primero de ellos se comparó prospectivamente la evolución de 26 pacientes con osteomielitis aguda por cepas de S. aureus LPV negativas (todas SASM) con 59 pacientes con cepas LPV positivas (3 SASM y 56 SARM)2. Los pacientes con osteomielitis aguda por S. aureus productor de LPV presentaron significativamente mayor afectación multifocal y elevación de los reactantes de fase aguda (VSG y PCR), mayor porcentaje de hemocultivos positivos e ingresos en la unidad de cuidados intensivos y mayor proporción de complicaciones como piomiositis, abscesos subperiósticos e intraóseos. En el segundo se evidenció que de 9 cepas aisladas de niños diagnosticados de osteomielitis aguda por S. aureus (7 SARM) complicada con piomiositis y trombosis venosa profunda adyacente al foco infeccioso, 7 eran productoras de LPV8. Aunque en esta comunicación describimos el primer caso de infección osteoarticular multifocal por SASM productor de LPV en España, un estudio realizado recientemente en el sur de la Comunidad de Madrid6 detectó la presencia, por primera vez en nuestro país, de 7 aislamientos comunitarios resistentes a meticilina productores de la exotoxina LPV en pacientes pediátricos, 4 de ellos asociados a infecciones de piel y partes blandas, 2 a otitis y 1 a piomiositis bacteriémica. Otra característica destacable de estos aislamientos fue que 3 cepas del mismo genotipo procedían de pacientes de origen ecuatoriano. La emergencia y diseminación de cepas comunitarias de S. aureus productoras de LPV, resistentes o sensibles a meticilina, constituye un hecho preocupante que puede condicionar complicaciones en el tratamiento y empeorar la evolución y el pronóstico de las infecciones estafilocócicas. De ahí la importancia de futuros estudios epidemiológicos que aporten información sobre la prevalencia y las características genéticas y fenotípicas de estas cepas en nuestro país.

Mycobacterium mageritense meningitis in an immunocompetent patient with an intrathecal catheter

Mycobacterium mageritense is a non-pigmented rapidly growing microorganism identified as a new species in the year 1997 in Madrid, Spain.1 Since 2002, there are several published papers of clinical infections caused by M. mageritense (e.g., sinusitis, pneumonia, skin and soft tissue infections, and catheterrelated bacteraemia).2–6 Different antimicrobials (doxicicline, ciprofloxacine, amykacine, imipenem, linezolid and trimethoprim/sulfamethoxazole) have been used for the treatment of these infections. We report here our experience with an immunecompetent patient diagnosed of M. mageritense meningitis likely associated to an intrathecal catheter. In July 2008, a 39-year-old woman was admitted at her reference hospital because of fever. She was carrier of an intrathecal catheter (with a reservoir for epidural analgesia); also, she was taking several psychotropic drugs because of posttraumatic back pain and left radiculopathy (following a car crash). There was a purulent discharge in the reservoir area. Therefore, both the epidural catheter and the reservoir were removed. Microbial cultures (three set of blood, the exudates and the catheter tip) were all negative. An empirical combination of intravenous (IV) vancomycin and gentamicin was prescribed during 14 days. There was total resolution of fever and the patient was discharged from her reference hospital. Twenty days later (August 2008), she developed a new fever (38.5 ◦C), headache and somnolence, and the back pain increased significantly. She was remitted to our hospital. The cerebrospinal fluid (CSF) study showed a count of 246 leukocyte/ L (60% of mononuclear’s cells), 33 mg/dL of glucose, 155 mg/dL of protein, and the adenosinedeaminase (ADA) was 29 U/L (normal range: <9 U/L). CSF usual stains, bacterial cultures and LCR serology (VDRL, Brucella sp., Listeria monocytogenes, Coxiella burnetii, Leptospira sp., and Borrelia sp.) were all negative. A Mantoux test was negative. Chest X-ray was within normal limits. Empirical treatment with rifampin, isoniazid, pyrazinamide and ethambutol in standard doses was prescribed. Twelve days after, the patient developed a sudden facio-braquio-crural hemiplegy. NMR study showed several ischaemic infarcts (at the protuberance and right intern capsule). The first CSF culture in Löwenstein medium (August 2008) was positive for a mycobacterium. The isolate was sent to the National Reference Laboratory for Mycobacteria and identified by phenotypic methods as colonies non-pigmented after 3 days of incubation at 22, 30, 37 and 42 ◦C, negative for Tween Hydrolysis and heat-stable catalase test, positive for arylsulfatase activity at 3 days, and for nitrate reductase. Furthermore, it was identified as M. mageritense by PCR-RFLP of hsp65 gene.8 The strain showed three fragments (240, 130 and 85 bp) by BstEII restriction enzyme digestion and three fragments (145, 120 and 60 bp) by HaeIII restriction enzyme digestion. In addition, the identification was confirmed by sequencing of 16S rRNA gene. Susceptibility testing was made by the proportions method (on Agar 7H10) that showed resistance to isoniazide, streptomycin, ethambutol, rifampicin, P.A.S., kanamycin, cycloserine and ethionamide. Pirazinamide susceptibility on MGIT 960 was made following the manufacturer recommendations. The susceptibility to other drugs (amikacin, norfloxacin, ofloxacin, ciprofloxacin, imipenen, linezolid, trimetil-sulfametoxazol, capreomycin, doxiciclin, claritromycin, amoxicillin-clavulanic and tobramycin) was made by E-TEST (on Mueller-Hinton agar). Table 1 Mycobacterium mageritense infections.

Mycobacterium kumamotonense, another Member of the Mycobacterium terrae Complex Unusually Carrying Two Copies of the Ribosomal RNA Operon

The novel slow-grower Mycobacterium kumamotonense was previously misidentified as Mycobacterium tuberculosis complex using commercial probes. Similarly to other slow-growers that cross-react with the tubercle bacilli using commercial probes, M. kumamotonense is carrying two copies of the ribosomal RNA operon (rrn) per genome. Analysis of the corresponding rrn regions allowed the identification of sequences putatively involved in that cross-reactivity.