Mycobacterium "habana" TMC 5135 as a vaccine candidate against tuberculosis: In vitro studies.

Background: Tuberculosis (TB) is one of the biggest problems of global health, at present. Bacillus-Calmette-Guérin is the only vaccine available against this disease. It protects only against the severe forms of TB in the childhood, which is a challenge in the search of new vaccine candidates. Taking into account the protective history of Mycobacterium "habana" against experimental TB, we proposed to provide the elements that support the use of M. "habana" TMC 5135 as a vaccine candidate against TB by infection studies in murine macrophages cell cultures. Methods: The production of microbicidal compounds dependent on oxygen metabolism as nitric oxide and hydrogen peroxide by murine peritoneal macrophages was detected. The invasive and toxigenic capacity of M. "habana" to infect this cell type was also evaluated through the quantification of intracellular alive bacillus and 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, respectively. Results: The results suggest that M. "habana" TMC 5135 is able to persist into peritoneal macrophages, to resist the effectors mechanisms of respiratory burst, and to keep the viability of the target cell. The demonstration of these effector mechanisms and the survival capacity of M. "habana" in this niche are relevant aspects of this research assuring the continuity of this candidate to next phases of preclinical development. Conclusion: The present investigation contributes to the characterization of the infection by this mycobacteria in its main target cells of innate immunity and it suggest future investigations to evaluate the activation of effector mechanisms of the innate immunity against this candidate.

Antituberculosis Drug Resistance in Pulmonary Isolates of Mycobacterium tuberculosis, Cuba 2012-2014.

INTRODUCTION Systematic surveillance of antituberculosis drug resistance allows identification of multidrug-resistant and extensively drug-resistant isolates of Mycobacterium tuberculosis. Surveillance studies of antituberculosis drug resistance systematically conducted in Cuba for over 15 years have revealed low circulation of multidrug-resistant tuberculosis, under 1% in new cases. OBJECTIVE Characterize antituberculosis drug resistance in isolates of M. tuberculosis recovered from patients with pulmonary tuberculosis in Cuba in 2012-2014. METHODS The nitrate reductase assay was used to test 997 isolates of M. tuberculosis for sensitivity to isoniazid and rifampicin. Isolates identified as multidrug resistant were tested for sensitivity to isoniazid, rifampicin, streptomycin, ethambutol, ofloxacin, amikacin, kanamycin and capreomycin by the proportion method, as well as genetic resistance mutations in rpoB, katG, inhA, gyrA, rrs and embB genes, using GenoType MTBDRplus and MTBDRsl commercial kits. RESULTS Some 95.6% of isolates from new cases and 89.6% of isolates from previously treated patients were sensitive to isoniazid and rifampicin. Multidrug resistance was found in 0.8% of new and 5.2% of previously treated patients, a statistically significant difference. One extensively drug-resistant isolate was detected among previously treated cases. All isolates examined with the molecular method had mutations in the rpoB gene, which is associated with resistance to rifampicin; only seven showed mutations in the katG gene and one in the inhA gene associated with isoniazid resistance. In one isolate, we found mutations in both gyrA and rrs genes, which are associated with resistance to fluoroquinolones and second-line injectable drugs and therefore, extensive resistance. CONCLUSIONS Results corroborate the low frequency of multidrug-resistant and extensively resistant M. tuberculosis strains in Cuba and highlight the need for continuous improvement of surveillance of antituberculosis drug resistance in Cuba. KEYWORDS Mycobacterium tuberculosis, multidrug resistance, extensively drug-resistant tuberculosis, Cuba.

[Surveillance of antituberculosis-drug resistance in Cuba, 2010-2011]. / Vigilancia de la resistencia a los medicamentos antituberculosos en Cuba, 2010-2011.

INTRODUCTION: Antituberculosis-drug resistance surveillance is very important to identify multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis isolates. OBJECTIVE: To determine the prevalence of resistance in M. tuberculosis strains isolated between 2010 and 2011, and to demonstrate the laboratory performance in the external quality control of drug susceptibility testing. MATERIALS AND METHODS: A prospective longitudinal study was carried out to determine antituberculosis-drug resistance in 657 M. tuberculosis isolates obtained throughout the country. The nitrate reductase assay was used to detect resistance to isoniazid and rifampin. The proportion method was performed to confirm resistance to these drugs and to further investigate in multidrug-resistant isolates their susceptibility to streptomycin, ethambutol, ofloxacin, kanamycin and capreomycin. Additionally, as part of external quality control, susceptibility was evaluated in two M. tuberculosis strain panels. RESULTS: In 95.69% of the isolates recovered from new tuberculosis cases, and in 72.64 % of isolates from previously treated patients we found susceptibility to isoniazid and rifampicin; multidrug resistance was 1,03 and 10.38%, respectively. We found two extensively resistant isolates. Except for ethambutol and capreomycin, the efficiency of all other drugs was 100% in the external quality control. CONCLUSION: The study confirmed the low prevalence of M. tuberculosis multidrug-resistant isolates in Cuba. This result was confirmed by the external quality control of drug susceptibility testing.

Vigilancia de la resistencia a los medicamentos antituberculosos en Cuba, 2010-2011 / Surveillance of antituberculosis-drug resistance in Cuba, 2010-2011

Introducción. La vigilancia de la resistencia a medicamentos antituberculosos permite alertar sobre el hallazgo de aislamientos de Mycobacterium tuberculosis multirresistentes y extremadamente resistentes . Objetivo. Determinar los patrones de resistencia de los aislamientos de M. tuberculosis recuperados en Cuba entre los años 2010 y 2011 y demostrar el desempeño del Laboratorio Nacional de Referencia en la ejecución de las pruebas de sensibilidad. Materiales y métodos. Se realizó un estudio prospectivo longitudinal en el que se incluyeron 657 aislamientos de M. tuberculosis recibidos de todo el país. Se empleó el método de la nitrato reductasa para detectar resistencia a isoniacida y rifampicina, y el método de las proporciones para corroborar la resistencia a dichos medicamentos e investigar la sensibilidad a estreptomicina, etambutol, ofloxacina, kanamicina y capreomicina en aislamientos multirresistentes. Como parte del control de calidad externo de las pruebas de sensibilidad, se evaluaron dos paneles de cepas de M. tuberculosis . Resultados. En 95,69 % de los aislamientos recuperados de casos nuevos de tuberculosis y en 72,64 % de los recuperados de casos previamente tratados, se encontró sensibilidad a isoniacida y rifampicina, siendo la multirresistencia de 1,03 y 10,38 %, respectivamente. Se encontraron dos aislamientos extremadamente resistentes. Con la excepción del etambutol y la capreomicina, para todos los medicamentos la eficiencia fue de 100% en el control de calidad externo. Conclusiones. Se confirmó la baja prevalencia de aislamientos de M. tuberculosis multirresistentes en Cuba, resultado avalado por el excelente desempeño demostrado en el control de calidad externo de las pruebas de sensibilidad.

Introduction: Antituberculosis-drug resistance surveillance is very important to identify multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis isolates. Objective: To determine the prevalence of resistance in M. tuberculosis strains isolated between 2010 and 2011, and to demonstrate the laboratory performance in the external quality control of drug susceptibility testing. Materials and methods: A prospective longitudinal study was carried out to determine antituberculosis-drug resistance in 657 M. tuberculosis isolates obtained throughout the country. The nitrate reductase assay was used to detect resistance to isoniazid and rifampin. The proportion method was performed to confirm resistance to these drugs and to further investigate in multidrug-resistant isolates their susceptibility to streptomycin, ethambutol, ofloxacin, kanamycin and capreomycin. Additionally, as part of external quality control, susceptibility was evaluated in two M. tuberculosis strain panels. Results: In 95.69% of the isolates recovered from new tuberculosis cases, and in 72.64 % of isolates from previously treated patients we found susceptibility to isoniazid and rifampicin; multidrug resistance was 1,03 and 10.38%, respectively. We found two extensively resistant isolates. Except for ethambutol and capreomycin, the efficiency of all other drugs was 100% in the external quality control. Conclusion: The study confirmed the low prevalence of M. tuberculosis multidrug-resistant isolates in Cuba. This result was confirmed by the external quality control of drug susceptibility testing.

Vigilancia de la resistencia a los fármacos antituberculosos en Cuba, 2000–2009 / Surveillance of resistance to antitubercular drugs in Cuba, 2000–2009

Objetivo. Determinar la prevalencia de la resistencia a los fármacos antituberculosos en Cuba en el decenio 2000–2009. Métodos. Se realizó un estudio prospectivo longitudinal. El universo de trabajo estuvo constituido por un total de 2 285 aislamientos de Mycobacterium tuberculosis obtenidos de todo el país en el período comprendido entre el 1 de enero de 2000 y el 31 de diciembre de 2009. Se empleó el método de las proporciones en medio Löwenstein-Jensen con los fármacos de primera línea: isoniazida, estreptomicina, etambutol y rifampicina. Resultados. La resistencia entre los casos nuevos y los pacientes con antecedente de tratamiento previo fue de 8,5% y 37,0%, respectivamente; para estas mismas categorías de caso, la multirresistencia fue de 0,4% y 8,8%, respectivamente. Conclusiones. El presente estudio muestra baja prevalencia de cepas multirresistentes en Cuba. Estos resultados reflejan los avances logrados por el programa nacional de control, que trabaja en la actualidad hacia la eliminación de la tuberculosis como problema de salud pública en el país.

Objective. Determine the prevalence of resistance to antitubercular drugs in Cuba in the 2000–2009 decade. Methods. A prospective longitudinal study was conducted. The sample group consisted of 2 285 Mycobacterium tuberculosis isolates obtained from throughout the country in the period from 1 January 2000 to 31 December 2009. The proportion method was used in Löwenstein-Jensen media with the first-line drugs: isoniazid, streptomycin, ethambutol, and rifampicin. Results. In the new cases and patients with a history of previous treatment, resistance was 8.5% and 37.0%, respectively. In these case categories, multidrug resistance was 0.4% and 8.8%, respectively. Conclusions. This study shows low prevalence of multidrug-resistant strains in Cuba. The results reflect the progress made by the national control program, which is currently working on the elimination of tuberculosis as a public health problem in the country.

[Surveillance of resistance to antitubercular drugs in Cuba, 2000-2009]. / Vigilancia de la resistencia a los fármacos antituberculosos en Cuba, 2000-2009.

OBJECTIVE: Determine the prevalence of resistance to antitubercular drugs in Cuba in the 2000-2009 decade. METHODS: A prospective longitudinal study was conducted. The sample group consisted of 2,285 Mycobacterium tuberculosis isolates obtained from throughout the country in the period from 1 January 2000 to 31 December 2009. The proportion method was used in Löwenstein-Jensen media with the first-line drugs: isoniazid, streptomycin, ethambutol, and rifampicin. RESULTS: In the new cases and patients with a history of previous treatment, resistance was 8.5% and 37.0%, respectively. In these case categories, multidrug resistance was 0.4% and 8.8%, respectively. CONCLUSIONS: This study shows low prevalence of multidrug-resistant strains in Cuba. The results reflect the progress made by the national control program, which is currently working on the elimination of tuberculosis as a public health problem in the country.

Evaluation of colorimetric methods using nicotinamide for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis.

The direct detection of pyrazinamide resistance in Mycobacterium tuberculosis is sufficiently difficult that many laboratories do not attempt it. Most pyrazinamide resistance is caused by mutations that inactivate the pyrazinamidase enzyme needed to convert the prodrug pyrazinamide to its active form. We evaluated two newer and simpler methods to assess pyrazinamidase activity, the nitrate reductase and malachite green microtube assays, using nicotinamide in place of pyrazinamide. A total of 102 strains were tested by these methods and the results compared with those obtained by the classic Wayne assay. Mutations in the pncA gene were identified by sequencing the pncA genes from all isolates in which pyrazinamide resistance was detected by any of the three methods. Both the nitrate reductase and malachite green microtube assays showed sensitivities of 93.75% and specificities of 97.67%. Mutations in the pncA gene were found in 14 of 16 strains that were pyrazinamide resistant and in 1 of 4 strains that were sensitive by the Wayne assay. Both of these simple methods, used with nicotinamide, are promising and inexpensive alternatives for the rapid detection of pyrazinamide resistance in limited-resource countries.

Diagnóstico de mycobacterium tuberculosis en pacientes sintomáticos incluyendo los infectados con el síndrome de inmunodeficiencia adquirida (SIDA) / Diagnosis of mycobacterium tuberculosis in symptomatic patients including those infected with the acquired immunodeficiency syndrome (AIDS)

Con la aparición del Virus de Inmunodeficiencia Humana (VIH), la tuberculosis ha tenido un significativo aumento, se ha planteado que esta coinfección es denominada como el "dúo mortal": uno acelera el avance de la otra y viceversa, por lo que cada día su temprano diagnóstico se hace más necesario. El objetivo de nuestro trabajo fue analizar, en el periodo de enero 2000 a diciembre 2006, la positividad en el diagnóstico bacteriológico de Mycobacterium tuberculosis en pacientes sintomáticos, incluyendo los pacientes VIH+/SIDA tratados en nuestra Institución Hospitalaria. Las muestras estudiadas fueron procesadas por las técnicas establecidas para el diagnóstico convencional; examen directo, examen de cultivo, e identificación. Mycobacterium tuberculosis solamente fue aislado de un total de 8151 muestras analizadas, en 205 muestras para un 2.5 por ciento de positividad; conjuntamente a las cepas identificadas se les realizó estudios de resistencia y/o sensibilidad por el método de Canetti, donde no se encontró cepas resistentes a ninguna de las drogas antibacilares empleadas, por lo que afirmamos que estudios como éste son imprescindibles para la vigilancia epidemiológica de esta enfermedad. Los resultados obtenidos muestran que la asociación VIH/SIDA/TB en nuestro país ha evolucionado en forma lenta, el incremento no es comparable con los reportados en la literatura internacional, demostrando la eficiencia de los Programas Nacionales de Control establecidos para estas dos enfermedades.

Nitrate reductase assay for detection of drug resistance in Mycobacterium tuberculosis: simple and inexpensive method for low-resource laboratories.

The nitrate reductase assay (NRA) was used as an alternative method for detection of resistance to the first-line antituberculous drugs isoniazid, rifampicin, ethambutol and streptomycin. A total of 320 strains of Mycobacterium tuberculosis were studied and the results compared with the proportion method (PM) on Löwenstein-Jensen medium. The mean time to obtain results was 10 days and the overall agreement between the NRA and PM was 98.8 %. The NRA was easy to perform and represents a useful tool for rapid and accurate determination of drug-resistant M. tuberculosis strains in low-resource countries.

Evaluation of phage assay for rapid phenotypic detection of rifampicin resistance in Mycobacterium tuberculosis.

BACKGROUND: Conventional methods for susceptibility testing require several months before results can be reported. However, rapid methods to determine drug susceptibility have been developed recently. Phage assay have been reported as a rapid useful tools for antimicrobial susceptibility testing. The aim of this study was to apply the Phage assay for rapid detection of resistance on Mycobacterium tuberculosis strains in Cuba. METHODS: Phage D29 assay was performed on 102 M. tuberculosis strains to detect rifampicin resistance. The results were compared with the proportion method (gold standard) to evaluate the sensitivity and specificity of Phage assay. RESULTS: Phage assay results were available in 2 days whereas Proportion Methods results were obtain in 42 days. A total of 44 strains were detected as rifampicin resistant by both methods. However, one strains deemed resistant by Proportion Methods was susceptible by Phage assay. The sensitivity and specificity of Phage assay were 97.8 % and 100% respectively. CONCLUSION: Phage assay provides rapid and reliable results for susceptibility testing; it's easy to perform, requires no specialized equipment and is applicable to drug susceptibility testing in low income countries where tuberculosis is a major public health problem.

Drug-resistant tuberculosis in Cuba. Results of the three global projects.

SETTING: The spread of multidrug-resistant tuberculosis (MDR TB) in the world remains a major public health problem. Surveillance of anti-TB drug resistance is therefore an essential tool for monitoring the effectiveness of TB control program and, through policy development, for improving national and global TB control. OBJECTIVE: To determine the prevalence of anti-TB drug resistance in Cuba during the execution of the three global projects. DESIGN: Drug-resistance was determined using the proportion method in 1528 Mycobacterium tuberculosis strains to first line anti-TB drugs. RESULTS: Resistance in new cases was 8.3%, 4.6% and 5.0%; MDR was 0.7%, 0% and 0.3% in the first (1997), second (2000) and third (2004) global projects, respectively. In new cases, none showed resistance to the four drugs. There were among previously treated cases statistically significant decreases in the prevalence of resistance to at least one drug when comparing the results obtained in the three global projects. CONCLUSIONS: The contributed data through Cuba demonstrated that our country is relatively free of MDR strains, reflecting the good National Control Program and the possibility of TB elimination in Cuba.

Comparative evaluation of the nitrate reduction assay, the MTT test, and the resazurin microtitre assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis.

OBJECTIVES: To evaluate the performance of three rapid low-cost methods for the detection of resistance to first-line drugs in Mycobacterium tuberculosis. METHODS: One hundred M. tuberculosis clinical isolates were tested by the nitrate reductase assay (NRA), the MTT test and the resazurin microtitre assay (REMA), and the results compared with those obtained with the gold standard proportion method (PM) on Lowenstein Jensen medium. RESULTS: The results using the three methods showed a good sensitivity and specificity between 94% and 100% for the detection of rifampicin and isoniazid resistance. Specificity for ethambutol and streptomycin using MTT and resazurin was low (58-89%). In contrast, NRA showed a good agreement for all first-line drugs tested. CONCLUSIONS: This study shows a high level of agreement of these three low-cost methods compared with the PM for rapid detection of rifampicin and isoniazid resistance. However, more standardization is needed for ethambutol and streptomycin using the MTT test and resazurin microtitre assay. The nitrate reductase assay might represent an inexpensive procedure for rapid detection of resistance to first-line drugs in low-resource countries.

[Surveillance of resistance of Mycobacterium tuberculosis to anti-TB drugs in Cuba, 1995-1998]. / Vigilancia de la resistencia de Mycobacterium tuberculosis a las drogas antituberculosas en Cuba, 1995-1998.

Drug-resistance surveillance by means of culture and drug susceptibility test (DST) allows the estimation of the regional and global magnitude of multidrug-resistance (MDR). Two reports on anti-tuberculosis drug resistance by the WHO/IUATLD showed that drug-resistance TB varied widely across regions. This work determined the prevalence of drug resistance among new cases and among previously treated cases of tuberculosis in Cuba during the period 1995-1998. Drug resistance was determined using the proportion method in 1379 Mycobacterium tuberculosis strains to first line antituberculosis drugs (isoniazid, streptomycin, ethambutol and rifampicin). The resistance in new cases was 8.3% and 6.5%; MDR was 0.7% and 0% in the first and second study respectively. The contributed data through Cuba demonstrated that our country is relatively free of MDR strains, recognizing it to world level the good tuberculosis-control programs and the success of the application in our country of the directly-observed-treatment strategy from the year 1971.

Vigilancia de la resistencia de Mycobacterium tuberculosis a las drogas antituberculosas en Cuba, 1995-1998

L a vigilancia de la resistencia a fármacos a través del cultivo y de las pruebas de susceptibilidad in vitro permite conocer la magnitud regional y mundial de la resistencia en tuberculosis. En el presente trabajo se determinó la prevalencia de la resistencia a fármacos antituberculosos en Cuba, durante el periodo 1995-1998 en casos nuevos y en aquéllos que han recibido tratamiento previo. Los resultados incluidos en este estudio forman parte de los dos proyectos mundiales organizados por la OMS/UICTER. La resistencia a los medicamentos se evaluó usando el método de las proporciones en 1.379 cepas de Mycobacterium tuberculosis a los fármacos de primera línea (isoniacida, rifampicina, estreptomicina y etambutol). La resistencia en casos nuevos fue del 8,3% y 6,5% y la resistencia múltiple a fármacos ( multidrug-resistance, MDR) fue del 0,7% y 0% en el primer y segundo estudio, respectivamente. Estos resultados permitieron demostrar la escasa circulación de cepas MDR en Cuba; se reconoció a nivel mundial el buen funcionamiento del Programa Nacional de Control y el éxito de la aplicación en nuestro país de la estrategia del tratamiento estrictamente supervisado desde 1971.

Surveillance of resistance of Mycobacterium tuberculosis to anti-TB drugs in Cuba, 1995-1998 Drug-resistance surveillance by means of culture and drug susceptibility test (DST) allows the estimation of the regional and global magnitude of multidrug-resistance (MDR). Two reports on anti-tuberculosis drug resistance by the WHO/IUATLD showed that drug-resistance TB varied widely across regions. Thecurrent study determined the prevalence of drug resistance among new cases and among previously treated cases of tuberculosis in Cuba during the period 1995-1998. Using the proportion method, drug resistance was determined in 1,379 Mycobacterium tuberculosis strains to four front-line anti- tuberculosis drugs (isoniazid, streptomycin, ethambutol and rifampicin). In two separate studies, the overall resistance in new cases was 8.3% and 6.5%, and the MDR was 0.7% and 0%, respectively. The Cuba data demonstrated that Colombia, in comparison, is relatively free of MDR strains, indicating effective tuberculosis control programs in Colombia and the success of the direct observation-treatment strategy that has been in continuous use since 1971.

Detección de plásmidos en cepas de Mycobacterium fortuitum / Detection of plasmids in stubs of mycobacterium fortiutum

La presencia de plásmidos ha sido demostrada en algunas especies de micobacterias como: Mycobacterium fortuitum, Mycobacterium avium, Mycobacterium intracellulare y Mycobacterium scrofulaceum(1,2). A estos plásmidos se les ha atribuido algunas características importantes, como, son: resistencia a antibióticos(3) y a metales pesados (cobre y mercurio) (4), y además se piensa que tengan un posible rol en la virulencia de las cepas que los posean(5)

Aplicación del enzimoinmunoensayo para el diagnóstico de la tuberculosis / Serodiagnosis of tuberculosis by enzyme immunoassay

En el presente trabajo se utilizó un EIE para la detección de anticuerpos IgG circulantes anti-PPD. El antígeno PPD(Derivado Proteico Purificado) fue elaborado en el IPK a partir de Mycobacterium tuberculosis cepa H37Rv. Se estudió un total de 220 sueros: Setenta sueros de pacientes sintomáticos respiratorios, de los cuales 50 pertenecen a pacientes con diagnóstico clínico, radiológico y bacteriológico de tuberculosis pulmonar (41 resultaron positivos al EIE, para una sensibilidad del 82 por ciento). Al analizar los 20 sueros restantes pertenecientes a pacientes con otras patologías no tuberculosas, 4 resultaron positivos (2 correspondían a sueros de pacientes con neoplasia de pulmón y 2 sueros de pacientes con histoplasmosis

Diagnóstico serológico de tuberculosis pulmonar por la técnica contrainmunoelectroforesis (CIE) / Serodiagnosis of tuberculosis by counterimmunoelectrophoresis (CIE)

Se estudiaron por la técnica Contrainmunoelectroforesis (CIE) 3 grupos de sueros, los cuales incluyeron 56 pacientes con tuberculosis pulmonar, 50 con otras patologías pulmonares y 75 de individuos supuestamente sanos. Se utilizaron en dicha técnica 2 extractos antigénicos celulares crudos de las cepas Mycobacterium tuberculosis H37Ra y Mycobacterium bovis BCG elaborados según Rojas - Espinoza y Quesada - Pascual; se discute la utilidad de la CIE para el diagnóstico serológico de la tuberculosis reportando una sensibilidad de 80,35 por ciento y una especificidad de 93,33 por ciento