Evaluation of a colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for rapid detection of rifampicin resistance in Mycobacterium tuberculosis

Abstract

Objectives: To standardise the colorimetric assay based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) for the rapid detection of rifampicin-resistant Mycobacterium tuberculosis in clinical practice and to evaluate the assay on a collection of 92 clinical isolates.

Design: The Bactec method was used as the reference method. Rifampicin was used for the susceptibility testing in the Bactec method at a concentration of 2 microg/ml. The MTT assay was performed in tubes containing 3 ml Dubos broth; the assay is based on the principle that live cells convert the yellow tetrazolium salt into a blue formazan. A final concentration of 2 microg/ml rifampicin was used in the assay. Optical density (OD) values at 570 nm were recorded on the third and sixth day. A strain was defined as susceptible when the relative optical density unit (RODU) (i.e., OD of rifampicin containing tube/OD of undiluted control) was < or = 0.2, and when the OD value of the rifampicin-containing tube on the sixth day was lower than the OD value on the third day. A strain was defined as resistant when the RODU was more than 0.5, and when there was an increase in OD value in the rifampicin-containing tube on the sixth day. The tubes were also read visually.

Results and conclusion: The result obtained by the MTT assay perfectly matched the result obtained by the Bactec method. The MTT assay was also interpretable by the naked eye. This simple, inexpensive assay could be used as a rapid screening method for identification of rifampicin-resistant strains in low-income countries.