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


METHODS
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.


CONCLUSIONS
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.
Morcillo
N
N
Reference Laboratory of Buenos Aires Tuberculosis Control Program, Vicente Lopez, Buenos Aires, Argentina. nora_morcillo@fullzero.com.ar
Di Giulio
B
B
Testani
B
B
Pontino
M
M
Chirico
C
C
Dolmann
A
A
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D004395:Dye Dilution Technique; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D013778:Tetrazolium Salts; D013844:Thiazoles; D017186:Titrimetry / Q000379:methods; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
D000900:Anti-Bacterial Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue