Direct colorimetric assay for rapid detection of rifampin-resistant Mycobacterium tuberculosis

Abstract

The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was standardized for direct detection of rifampin-resistant Mycobacterium tuberculosis in sputum samples. The sensitivity and specificity of the direct MTT assay matched those of the standard indirect susceptibility assay on 7H10 medium, and interpretable results were obtained for 98.5% of the samples within 2 weeks. Traditional methods of in vitro drug susceptibility testing are time consuming and laborious. Susceptibility tests on clinical isolates require 6 to 9 weeks, and tests conducted directly on smear-positive samples take about 3 weeks (International Union Against Tuberculosis and Lung Disease, The public health service national tuberculosis reference laboratory and the national laboratory network. Minimum requirements, role and operation in a low-income country, Paris, France, 1998, and P. T. Kent and G. P. Kubica, Public health mycobacteriology. A guide for the level III laboratory, Centers for Disease Control and Prevention, Atlanta, Ga., 1985). More-rapid methods are available but are very expensive for routine use under program conditions in countries with high levels of tuberculosis endemicity.

FIG. 1.

Flow chart of MTT assay. Abbreviations: BC, bacterial control without rifampin; R, tube with 2 μg of rifampin/ml; C, control without bacteria. LJ, Löwenstein-Jensen.

FIG. 2.

Growth patterns of rifampin-susceptible (n = 37 samples in the first week, 56 in the second week, and 58 in the third week) and rifampin-resistant (n = 6 in the first week and 8 in the subsequent weeks) strains of M. tuberculosis as reflected by RODU values (means ± standard errors) [(RODU = OD₅₇₀ of rifampin-containing medium)/(OD₅₇₀ of drug-free medium)]. The difference in RODU values in each week was statistically significant (P < 0.01 [Mann-Whitney test]).