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Journal of Clinical Microbiology, August 2000, p. 2949-2954, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Comparison of NCCLS and 3-(4,5-Dimethyl-2-Thiazyl)-2,5-Diphenyl-2H-Tetrazolium Bromide (MTT) Methods of In Vitro Susceptibility Testing of Filamentous Fungi and Development of a New Simplified Method

Joseph Meletiadis,1,* Jacques F. G. M. Meis,1,2 Johan W. Mouton,2 J. Peter Donnelly,3 and Paul E. Verweij1

Departments of Medical Microbiology1 and Hematology,3 University Medical Center Nijmegen, and Department of Medical Microbiology, Canisius Wilhelmina Hospital,2 Nijmegen, The Netherlands

Received 6 March 2000/Returned for modification 16 May 2000/Accepted 3 June 2000

The susceptibility of 30 clinical isolates belonging to six different species of filamentous fungi (Aspergillus fumigatus, Aspergillus flavus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium solani, and Fusarium oxysporum) was tested against six antifungal drugs (miconazole, voriconazole, itraconazole, UR9825, terbinafine, and amphotericin B) with the microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (M38-P). The MICs were compared with the MICs obtained by a colorimetric method measuring the reduction of the dye 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan by viable fungi. The levels of agreement between the two methods were 96 and 92% for MIC-0 (clear wells) and MIC-1 (75% growth reduction), respectively. The levels of agreement were always higher for Aspergillus spp. (97% ± 2.5%), followed by Scedosporium spp. (87% ± 10.3%) and Fusarium spp. (78% ± 7.8%). The NCCLS method was more reproducible than the MTT method: 98 versus 95% for MIC-0 and 97 versus 90% for MIC-1. However, the percentage of hyphal growth as determined visually by the NCCLS method showed several discrepancies when they were compared with the percentages of MTT reduction. A new simplified assay that incorporates the dye MTT with the initial inoculum and in which the fungi are incubated with the dye for 48 h or more was developed, showing comparable levels of agreement and reproducibility with the other two methods. Furthermore, the new assay was easier to perform and more sensitive than the MTT method.

* Corresponding author. Mailing address: Department of Medical Microbiology, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3613514. Fax: 31-24-3540216. E-mail: j.meletiadis{at}mmb.azn.nl.

Journal of Clinical Microbiology, August 2000, p. 2949-2954, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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