Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor.

Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.

Impaired fitness of Mycobacterium tuberculosis resistant isolates in a cell culture model of murine macrophages.

OBJECTIVES: We analysed the ability of Mycobacterium tuberculosis clinical isolates to penetrate and grow inside murine macrophages as a surrogate of fitness. METHODS: Thirty-five drug-resistant and 10 drug-susceptible M. tuberculosis isolates were studied in a murine macrophage model from the J774.2 cell line in a 6 day protocol, performing semi-quantitative counts in Middlebrook 7H11 medium. The mycobacterial penetration index (MPI) after infection and the mycobacterial growth ratio (MGR) inside the macrophages were determined to evaluate the fitness of isolates. RESULTS: Isolates with the katG S315T mutation and multidrug-resistant (MDR) isolates had a significantly lower MGR compared with drug-susceptible isolates. The MPI of the isolates with the katG S315T mutation showed a significant decrease compared with the MPI of those without this mutation. A trend to significantly lower values was also observed on comparing the MPI of the MDR isolates with that of the drug-susceptible isolates and the isolates resistant to isoniazid. CONCLUSIONS: The isoniazid-resistant and MDR isolates with mutations in the katG gene showed decreased multiplication inside murine macrophages, suggesting a lower fitness of M. tuberculosis with these resistance patterns.

Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis clinical isolates in the area of Barcelona.

OBJECTIVES: To determine the proportion and type of mutations in Mycobacterium tuberculosis isolates resistant to streptomycin, and their relationship with the level of resistance and with the epidemiological molecular pattern of the isolates. METHODS: Sixty-nine streptomycin-resistant isolates from a M. tuberculosis strain collection (1995-2005) from Barcelona were studied. The MIC of streptomycin for each isolate was determined using the proportions method with Middlebrook 7H11 medium. The entire rpsL gene and two specific fragments of the rrs gene (the 530 loop and the 912 region) were sequenced. IS6110-restriction fragment length polymorphism and spoligotyping were performed in each isolate. RESULTS: Twenty-six (26/69, 37.7%) streptomycin-resistant isolates presented a mutation in either the rpsL gene and/or the rrs530 loop, with no mutation in the rrs912 region. Seventeen (24.6%) isolates showed rpsL mutations (codons 43 and 88) associated with high MIC levels. Nine (13.0%) isolates had alterations in the rrs gene (A513T, A513C and C516T). Nineteen isolates (19/64, 29.7%) were classified into seven clusters (containing 2-5 isolates per cluster). Nineteen different spoligotype patterns were found. All the LAM3 spoligotype isolates (10/67, 14.9%) were associated with a C491T change in the rrs gene, being also observed in all LAM3 streptomycin-susceptible isolates. CONCLUSIONS: Mutations in the rpsL and rrs genes were detected in 37.7% of streptomycin-resistant M. tuberculosis isolates. High-level resistance was associated with mutations in the rpsL gene, whereas wild-type isolates showed low MIC levels. The presence of the C491T substitution in the rrs gene in streptomycin-susceptible and -resistant isolates demonstrates that this change is an epidemiological marker associated with LAM3 sublineage.

Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain.

The aim of this study was to analyze the factors associated with conventional contact tracing (CCT) and molecular epidemiology (ME) methods in assessing tuberculosis (TB) transmission, comparing the populations studied and the epidemiological links established by both methods. Data were obtained from TB case and CCT registries, and ME was performed using IS6110-based restriction fragment length polymorphism (RFLP) analysis and mycobacterial interspersed repetitive unit 12 (MIRU12) typing as a secondary typing method. During two years (2003 and 2004), 892 cases of TB were reported, of which 687 (77%) were confirmed by culture. RFLP analysis was performed with 463 (67.4%) of the 687 isolated strains, and MIRU12 types in 75 strains were evaluated; 280 strains (60.5%) had a unique RFLP pattern, and 183 (39.5%) shared patterns, grouping into 65 clusters. CCT of 613 (68.7%) of 892 cases detected 44 clusters involving 101 patients. The results of both CCT and ME methods yielded 96 clusters involving 255 patients. The household link was the one most frequently identified by CCT (corresponding to 80.7% of the cases clustered by this method), whereas nonhousehold and unknown links were associated with 94.1% of the strains clustered by ME. When both methods were used in 351 cases (39.3%), they showed the same results in 214 cases (61%). Of the remainder, 106 (30.2%) were clustered only by ME, 19 (5.5%) were clustered only by CCT, and 12 (3.4%) were clustered by both methods but into different clusters. Patients with factors potentially associated with social problems were less frequently studied by CCT (P = 0.002), whereas patients of <15 years of age, most with negative cultures, were less frequently studied by ME (P = 0.005). Significant differences in the populations studied by ME versus CCT were observed, possibly explaining the scarce correlation found between the results of these methods. Moreover, ME allowed the detection of nonhousehold contact relationships, whereas CCT was more useful for tracing transmission chains involving patients of <15 years of age. In conclusion, the two methods are complementary, suggesting the need to improve the methodology of contact study protocols.

Worldwide emergence of extensively drug-resistant tuberculosis.

Mycobacterium tuberculosis strains that are resistant to an increasing number of second-line drugs used to treat multidrug-resistant tuberculosis (MDR TB) are becoming a threat to public health worldwide. We surveyed the Network of Supranational Reference Laboratories for M. tuberculosis isolates that were resistant to second-line anti-TB drugs during 2000-2004. We defined extensively drug-resistant TB (XDR TB) as MDR TB with further resistance to > or = 3 of the 6 classes of second-line drugs. Of 23 eligible laboratories, 14 (61%) contributed data on 17,690 isolates, which reflected drug susceptibility results from 48 countries. Of 3,520 (19.9%) MDR TB isolates, 347 (9.9%) met criteria for XDR TB. Further investigation of population-based trends and expanded efforts to prevent drug resistance and effectively treat patients with MDR TB are crucial for protection of public health and control of TB.

Multicenter laboratory evaluation of the MB/BacT Mycobacterium detection system and the BACTEC MGIT 960 system in comparison with the BACTEC 460TB system for susceptibility testing of Mycobacterium tuberculosis.

In this multicenter study, the reliability of two nonradiometric, fully automated systems, the MB/BacT and BACTEC MGIT 960 systems, for testing the susceptibilities of 82 Mycobacterium tuberculosis strains to isoniazid, rifampin, ethambutol, and streptomycin was evaluated in comparison with the radiometric BACTEC 460TB system. The arbitration of discrepant results was done by the reanalysis of the strain, the determination of the MIC, and the molecular characterization of some resistance determinants. The overall level of agreement with BACTEC 460TB results was 96% with the MB/BacT test and 97.2% with the BACTEC MGIT 960 system. With both methods, the level of agreement with BACTEC 460TB results was 96.3% for isoniazid, 98.8% for rifampin, and 98.8% for ethambutol. The level of agreement for streptomycin was 90.2% with MB/BacT and 97.5% with BACTEC MGIT 960. Overall, there were 11 very major errors and 2 major errors with the MB/BacT method and 5 very major errors and 2 major errors with the BACTEC MGIT 960 system. In general, the MB/BacT and BACTEC MGIT 960 systems showed good performance for susceptibility testing with first-line antituberculosis drugs.

[Tuberculous meningitis: a comparative study in relation to concurrent human immunodeficiency virus infection]. / Meningitis tuberculosa: estudio comparativo en relación con la coexistencia de infección por el virus de la inmunodeficiencia humana.

INTRODUCTION: This study assesses the epidemiological and clinical data, as well as therapy and evolution in a recent series of patients with tuberculous meningitis (MT). A comparative study was conducted between adult MT patients with and without concurrent human immunodeficiency virus (HIV) infection. METHODS: From 1987 to 2000, 75 episodes of MT were diagnosed, 39 of them (52%) in patients with prior HIV infection. A comparative study was performed of variables related to the presence or absence of HIV and MT coinfection. RESULTS: MT was more frequent in HIV patients (6.4% versus 1.2%, p < 0.01). CD41 lymphocyte value in HIV patients was 52 +/- 66 cells/mm3. There were no significant differences in clinical manifestations or cerebrospinal fluid biochemical alterations between the two groups. Extrameningeal TB was more frequent in patients with HIV coinfection than those without (61.5% vs. 36.1%, p = 0.03). Radiological alterations on cranial studies were more frequent in HIV-infected patients. Treatment with four antituberculosis drugs was also more frequent in HIV-infected patients (61.5% vs. 13.9%, p = 0.01). There were no differences in adverse effects between the groups. Overall mortality (20.5% vs. 22.51%) and neurological sequelae (7.7% vs. 5.6%) were also similar. CONCLUSIONS: Half of our MT patients were coinfected with HIV. Their clinical, microbiological and evolutionary characteristics were comparable to those of patients without HIV infection. These results indicate that the diagnostic and therapeutic strategies applied in MT patients with or without HIV coinfection can be similar.

Meningitis tuberculosa: estudio comparativo en relación con la coexistencia de infección por el virus de la inmunodeficiencia humana / Tuberculous meningitis: a comparative study in relation to concurrent human immunodeficiency virus infection

Introducción. El objetivo de este estudio fue evaluar los datos epidemiológicos, clínicos, terapéuticos y evolutivos de una serie reciente de meningitis tuberculosa (MT) y efectuar un estudio comparativo entre los pacientes adultos con y sin infección por el virus de la inmunodeficiencia humana (VIH). Métodos. Desde 1987 a 2000 se diagnosticaron 75 episodios de MT, 39 de ellos (52%) en pacientes infectados por el VIH. Se realizó un estudio comparativo de las diferentes variables en relación a la coinfección. Resultados. La MT fue más frecuente en los pacientes infectados por el VIH (6,4% frente a 1,2%; p < 0,01). Los linfocitos CD41 en los pacientes infectados por el VIH fueron de 66 +/- 52 cél./ml. No se detectaron diferencias significativas en las manifestaciones clínicas y en las alteraciones bioquímicas del líquido cefalorraquídeo entre ambos grupos. La tuberculosis extrameníngea fue más frecuente en casos de coinfección por el VIH (61,5% frente a 36,1%; p = 0,03). Las alteraciones radiológicas fueron más frecuentes en los pacientes infectados por el VIH. La terapéutica con 4 fármacos antituberculosos fue más frecuente en los pacientes infectados por el VIH (61,5% frente a 13,9%; p = 0,01). Los efectos adversos no mostraron diferencias entre ambos grupos. La mortalidad global (20,5% frente a 22,51%) y las secuelas neurológicas (7,7% frente a 5,6%) fueron similares. Conclusiones. La mitad de los pacientes con MT están coinfectados por el VIH. Las características clínicas, microbiológicas y evolutivas son similares a la de los pacientes sin coinfección. La estrategia diagnóstica y terapéutica en la MT de los enfermos con o sin infección por el VIH pueden ser similares (AU)

Introduction. This study assesses the epidemiological and clinical data, as well as therapy and evolution in a recent series of patients with tuberculous meningitis (MT). A comparative study was conducted between adult MT patients with and without concurrent human immunodeficiency virus (HIV) infection. Methods. From 1987 to 2000, 75 episodes of MT were diagnosed, 39 of them (52%) in patients with prior HIV infection. A comparative study was performed of variables related to the presence or absence of HIV and MT coinfection. Results. MT was more frequent in HIV patients (6.4% versus 1.2%, p < 0.01). CD41 lymphocyte value in HIV patients was 52 +/- 66 cells/mm 3. There were no significant differences in clinical manifestations or cerebrospinal fluid biochemical alterations between the two groups. Extrameningeal TB was more frequent in patients with HIV coinfection than those without (61.5% vs. 36.1%, p = 0.03). Radiological alterations on cranial studies were more frequent in HIV-infected patients. Treatment with four antituberculosis drugs was also more frequent in HIV-infected patients (61.5% vs. 13.9%, p = 0.01). There were no differences in adverse effects between the groups. Overall mortality (20.5% vs. 22.51%) and neurological sequelae (7.7% vs. 5.6%) were also similar. Conclusions. Half of our MT patients were coinfected with HIV. Their clinical, microbiological and evolutionary characteristics were comparable to those of patients without HIV infection. These results indicate that the diagnostic and therapeutic strategies applied in MT patients with or without HIV coinfection can be similar (AU)

Infections due to the newly described species Mycobacterium parascrofulaceum.

We report on four cases of infection by the recently described species Mycobacterium parascrofulaceum. In two cases the mycobacterium was isolated from AIDS patients, while in the others it was responsible for pulmonary disease in elderly men. Our findings suggest that M. parascrofulaceum is an opportunistic pathogen, like many other nontuberculous mycobacterial species.

¿Hay que añadir un cuarto fármaco a la pauta actual de tratamiento de la tuberculosis inicial? / Is it necessary to add a fourth drug to the initial treatment of tuberculosis?

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Micobacterias ambientales en España: aislamientos en el período 1976-1996 / Non-tuberculous mycobacteria in Spain: isolations from 1976 to 1996

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Farmacorresistencia de mycobacterium tuberculosis. Estudio multicéntrico en el área de Barcelona / Drug resistance of Mycobacterium tuberculosis. Multicenter study in Barcelona, Spain

Fundamento: El objetivo fue conocer, mediante un estudio multicéntrico y con la colaboración de clínicos, microbiólogos y epidemiólogos, el nivel de resistencias de Mycobacterium tuberculosis en el área de Barcelona y su relación con datos clínicos de los pacientes para establecer posibles grupos de riesgo. Pacientes y método: Se incluyeron las cepas de M. tuberculosis aisladas de los pacientes diagnosticados bacteriológicamente desde octubre de 1995 a septiembre de 1997, revisándose su historial clínico. Se estudió la resistencia a isoniacida, rifampicina, estreptomicina, etambutol y pirazinamida mediante el sistema Bactec 460. Los factores asociados a resistencia se analizaron mediante regresión logística. Resultados: El total de pacientes fue de 1.749 (1.535 iniciales y 214 tratados anteriormente). La resistencia primaria fue 5,7 por ciento (isoniacida, 3,8 por ciento; rifampicina, 1,0 por ciento, estreptomicina, 2,1 por ciento, etambutol, 0,3 por ciento y pirazinamida, 1,0 por ciento). La resistencia adquirida alcanzó el 20,5 por ciento (isoniacidas, 17,3 por ciento, rifampicina, 9,8 por ciento, etambutol, 1,9 por ciento, estreptomicina, 4,7 por ciento y pirazinamida, 6,5 por ciento). La multirresistencia primaria fue 0,9 por ciento, y la adquirida 9,3 por ciento. Las resistencias primarias totales, a isoniacida y a pirazinamida se asociaron con inmigración y se objetivaron algunas diferencias entre laboratorios atribuibles a diferencias de población. Las resistencias adquiridas totales y a isoniacida se asociaron a más de 60 años y al sexo femenino. Conclusiones: El bajo nivel de resistencias primarias a isoniacida permite tratar los casos iniciales con 3 fármacos, salvo en determinados grupos de inmigrantes. Se recomienda realizar pruebas de sensibilidad en todas las cepas aisladas y realizar estudios de farmacorresistencia en tuberculosis basados en trabajos coordinados para evitar sesgos de población. (AU)