Comparison of a 3-(4,5-Dimethyl-2-Thiazyl)-2,5 Diphenyl-2H-Tetrazolium Bromide (MTT) with the Proposed Standard of NCCLS Method for In Vitro Susceptibility Testing of Filamentous Fungi.



Background: A colorimetric method based on the detection of mitochondrial activity of viable cells with MTT was compared with the proposed NCCLS method for filamentous fungi. Methods: A set of 30 clinical isolates (5 of each species) of Aspergillus fumigatus, A.flavus, Scedosporium prolificans, S. apiospermum, Fusarium solani and F. oxysporum were tested for the susceptibility to six antifungal drugs: amphotericin B (AB), miconazole (MCZ), itraconazole (ITZ), voriconazole (VCZ), UR9825 (UR), terbinafine (TB) by a broth microdilution method. The plates were incubated at 37oC and read after 48 h for Aspergillus spp. and Fusarium spp. and after 72 h for Scedosporium spp. The MICs were determined by NCCLS method and by two methods incorporating MTT. In the first method (M3) MTT was added at a final concentration of 0.5mg/ml after incubation and the plates were further incubated for 3h. The formed formazan was extracted by isopropanol+5%HCl 1M and the OD of each well was measured at 550nm. In the second method (M48) the MTT was added to the inoculum at a concentration of 0.1 mg/ml and the plates were incubated with MTT. The percentage agreement between the MTT and NCCLS methods was defined as the fraction of isolates with MICs within 1 dilution step of the NCCLS method. A percentage of agreement of 80% or greater was considered to be acceptable. The possible toxic effect of MTT against the filamentous fungi was investigated by incubating the moulds with different concentrations of MTT for 48 and 72h and determining the level of inhibition of growth. Results: No toxic effect of MTT was observed at concentrations of 0.25 mg/ml MTT and lower after 48 and 72h for all isolates tested. The percentage of agreement for each method-endpoint-species combination was > 80% for all Aspergillus and Scedosporium spp., but for only 54% of Fusarium spp. Conclusion: A good correlation was observed between the MTT methods and NCCLS for Aspergillus and Scedosporium spp. And can be considered as alternative method.

abstract No: 


Full conference title: 

39th Interscience Conference on Antimicrobial Agents and Chemotherapy
    • ICAAC 39th