[Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis]. / Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 microg/ml; streptomycin (SM), 4.00 microg/ml; ethambutol (EMB), 2.00 microg/ml and rifampin (RMP), 40.00 microg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 microg/ml; SM, 8.00-0.25 microg/ml; EMB, 32.00-1.00 microg/ml and RMP, 2.00-0.06 microg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 microg/ml, RMP, 0.50 microg/ml, SM, 4.00 microg/ml and EMB, 4.00 microg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

Evaluación de un micrométodo colorimétrico para determinar la concentración inhibitoria mínima de drogas antituberculosas frente a Mycobacterium tuberculosis / Evaluation of a colorimetric micromethod for determining the minimal inhibitory concentration of antibiotics against Mycobacterium tuberculosis

La tuberculosis multidrogorresistente (MDR), originada por aislamientos de cepas simultáneamente resistentes a isoniacida y rifampicina, es reconocida en la actualidad como un problema sanitario mundial. Nuevas técnicas que permitan detectar en forma temprana cepas MDR son necesarias para evitar su dispersión en la comunidad. En este trabajo hemos evaluado el empleo de un micrométodo colorimétrico para determinar las concentraciones inhibitorias mínimas (CIM) de las drogas de primera línea frente a dichas cepas, usando el indicador: bromuro de 3-(4,5 dimetiltiazol-2-yl)-2,5 difeniltetrazolio (M-MTT). Junto a la cepa de referencia H37Rv se procesaron 603 aislamientos clínicos, 507 provenientes de casos pulmonares (84,1%) y 96 de extrapulmonares (15,9%). Como estándar de referencia se utilizó el método de proporciones en medio de Löwenstein-Jensen (MP), con isoniacida (INH), 0,20 µg/ml; estreptomicina (SM), 4,00 µg/ml; etambutol (EMB), 2,00 µg/ml y rifampicina (RMP), 40,00 µg/ml. Los intervalos de concentraciones de las drogas empleadas en el M-MTT fueron: INH, 1,00-0,03 µg/ml; SM, 8,00-0,25 µg/ml; EMB, 32,00-1,00 µg/ml y RMP: 2,00-0,06 µg/ml. El resultado de la CIM por el M-MTT fue obtenido en un tiempo promedio de 8 días (rango total: 7 a 12 días). Los puntos de corte para cada una de las drogas, calculados mediante el análisis de la curva ROC, fueron: INH, 0,25 µg/ml; RMP, 0,50 µg/ml; SM, 4,00 µg/ml y EMB, 4,00 µg/ml. Los valores de sensibilidad y especificidad fueron 100% en el caso de RMP; y 97,8% y 99,5%, respectivamente, para INH. El análisis estadístico de los resultados permitió concluir que el M-MTT es un método seguro para la rápida detección de MDR. Por su sencillez y bajo costo, podría ser aplicado en los países en vías de desarrollo.

Multidrug-resistant tuberculosis (MDR) caused by strains resistant to both isoniazid and rifampin is now considered a serious sanitary problem worldwide. New technical tools for the early detection of these strains are urgently needed to avoid their spread within the community. We have evaluated a microplate colorimetric-based method to determine the minimal inhibitory concentration (MIC) of first-line antituberculosis drugs by using 3-(4, 5 dimethylthiazolyl 1-2 yl)-2,5 diphenyl tetrazolium bromide as a bacterial growth indicator (MTT) (M-MTT). A total of 603 clinical isolates, 507 from respiratory cases (84.1%) and 96 from non-respiratory cases (15.9%) were processed. The proportion method on a Löwenstein-Jensen medium (PM) with isoniazid (INH), 0.20 µg/ml; streptomycin (SM), 4.00 µg/ml; ethambutol (EMB), 2.00 µg/ml and rifampin (RMP), 40.00 µg/ml, was used as the gold standard. The drugs and the concentration range tested were: INH, 1.00-0.03 µg/ml; SM, 8.00-0.25 µg/ml; EMB, 32.00-1.00 µg/ml and RMP, 2.00-0.06 µg/ml. MIC results were obtained on an average of 8 days (range: 7-12). The cut-off values for each drug, calculated by the ROC curve method, were: INH, 0.25 µg/ml, RMP, 0.50µg/ml, SM, 4.00 µg/ml and EMB, 4.00 µg/ml. Sensitivity and specificity for RMP were 100 %, while for INH, they were 97.8% and 99.5% respectively. The results obtained suggested that M-MTT is a low cost and easy to set up method that could be applied to MDR clinical diagnosis in developing countries.

Multicenter study of MTT and resazurin assays for testing susceptibility to first-line anti-tuberculosis drugs.

OBJECTIVE: A multicentre evaluation was performed to assess two rapid low-cost methods, MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) and resazurin assays, for testing the susceptibility of Mycobacterium tuberculosis to the first-line anti-tuberculosis drugs rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and streptomycin (SM). METHODS: Thirty coded M. tuberculosis strains were sent to seven laboratories located in Latin America, representing six countries. Each site performed the colorimetric assays, MTT and resazurin, blind for the first-line drugs RMP, INH, EMB and SM. The minimum inhibitory concentration results obtained were compared to the conventional proportion method on Lowenstein-Jensen medium. RESULTS: After establishing the breakpoint concentrations, excellent results were obtained for RMP, INH and EMB, with levels of specificity and sensitivity of between 96% and 99%. CONCLUSION: MTT and resazurin assays are promising, accessible new alternative methods for middle- and low-resource countries that need low-cost methods to perform rapid susceptibility testing of M. tuberculosis to key anti-tuberculosis drugs.

A microplate indicator-based method for determining the susceptibility of multidrug-resistant Mycobacterium tuberculosis to antimicrobial agents.

SETTING: Reference Laboratory, Buenos Aires Province Tuberculosis Control Program, Dr Cetrangolo Hospital, Argentina. OBJECTIVE: To obtain a rapid, inexpensive method of determining minimal inhibitory concentrations (MIC) of several drugs acting on multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a colorimetric, microplate-based assay (M-MTT) was developed. DESIGN: One hundred and one clinical isolates were studied. Drugs were placed in a microtiter plate, and several two fold dilutions were made in situ. Kanamycin, capreomycin, ethionamide, para-aminosalicylic acid and clarithromycin were tested in a range concentration of 8.0-0.25 microg/ml, cycloserine 60.0-1.9 microg/ml, clofazimine 3.0-0.10 microg/ml, levofloxacin 4.0-0.13 microg/ml and rifabutin 1.0-0.13 microg/ml. General indicator 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) at 5.0 mg/ml was used for visualising cellular growth and viability. The proportion method with Middlebrook 7H11 was used as the gold standard. RESULTS: MICs by M-MTT were obtained at on average 8 days and correlated with those obtained using the proportion method. In our conditions, the total cost of MIC determination for nine drugs was 5.00 US dollars. CONCLUSION: M-MTT could be used as a simple, rapid, low-cost technology to test the susceptibility of MDR-TB strains to several second-line and alternative drugs, with the objective of orienting future treatment regimens.