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.