a Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK; b Infectious Diseases Departments, Janssen Research Foundation, B-2340 Beerse, Belgium
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Abstract |
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Introduction |
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For some years we have included hydroxy-itraconazole in routine antifungal susceptibility test panels with clinical isolates of fungal pathogens. We now report the activities of both compounds against a large group of fungal isolates.
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Materials and methods |
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Microplates were incubated at 37°C for 48 h (Candida and Saccharomyces spp.) or at 30°C until control wells were visibly turbid (other fungi). For all plates, growth turbidity was measured at 405 nm, background absorbance was substracted from the readings and the OD for each culture in the presence of each agent was expressed as a percentage of control absorbance. Plates in which control ODs were less than 0.15 were excluded from analysis. MIC was determined as IC50, i.e. the lowest concentration of test compound that reduced absorbance below 50% of control. For yeasts, this end point gave the best agreement with MIC values determined by the NCCLS macrodilution reference method.4,5 The IC50 values for itraconazole and hydroxy-itraconazole were extracted retrospectively from a computer database created over a 5 year period.
To investigate susceptibility differences between itraconazole and hydroxy-itraconazole for five bioassay marker yeasts,6 MICs with the two agents were repeated in five separate replicate tests. For each isolate, the averages of the percentage control turbidity data from the replicate tests were used to construct a doseresponse curve for determination of IC50.
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Results |
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The Table summarizes the differentials in antifungal activity measured between itraconazole and hydroxyitraconazole. For most isolates the MICs determined for the two agents were the same within a range of three test dilutions of the compound (a maximum difference of two dilutions). From the data generated for the 144 isolates tested in replicate, 1518% of results would be expected by chance to show MIC differences exceeding three or more dilutions owing to experimental variability. The only fungi for which more than 18% of the isolates showed differences of this magnitude between MICs of itraconazole and hydroxy-itraconazole were Candida glabrata, Trichophyton mentagrophytes and Trichophyton rubrum. For the other fungi the large MIC differences between the two azole antifungals occurred rarely, and within the bounds of experimental variation.
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In five replicate tests with two bioassay strains of C. kefyr (SA and ATCC 46764) the itraconazole IC50 was 0.016 mg/L and the hydroxy-itraconazole IC50 value was 0.032 mg/L. In replicate tests with three Candida albicans bioassay strains (NCPF 3241, 3153A and RV 1488) the IC50 values for itraconazole and hydroxy-itraconazole were all 0.008 or 0.016 mg/L.
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Discussion |
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In bioassays designed to measure itraconazole and hydroxy-itraconazole concentrations in clinical samples, laboratories use various marker yeast strains in the absence of a standardized method.6 Since a bioassay result reflects the total antifungal activity of itraconazole + hydroxyitraconazole in a fluid sample, it may be thought more useful than a precise quantification of itraconazole alone by a more sensitive procedure such as HPLC. However, bioassays for a sample containing a mixture of the two azoles usually overestimate the true concentrations of itraconazole + hydroxy-itraconazole with itraconazole as reference standard and underestimate them with hydroxy-itraconazole as the standard.6,7 This discrepancy might arise from susceptibility differences to the two azoles in the marker strains, differences in agar diffusion rates of the two azoles or both factors.2,6,7 A previous study found that C. kefyr SA was two-fold less susceptible to itraconazole than to hydroxy-itraconazole,2 whereas the present study showed a two-fold difference in the opposite direction. Differences of only two dilutions fall well within the range of normal replicate variability in this study and in the ranges for quality control yeast isolates recommended by the NCCLS;5 they are therefore insufficient to permit conclusions concerning differential susceptibility of these isolates to the two azole antifungal agents. The low precision of bioassay, its relative insensitivity and the dependence of results on the choice of reference compound make bioassay at best only a semi-quantitative indicator of effective antifungal levels in an itraconazole-treated patient. Since our results show the compounds to be equipotent, HPLC measurements of both itraconazole and hydroxy-itraconazole concentrations could be reliably reported as the sum of the two concentrations to indicate bioactive levels.
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Acknowledgments |
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Notes |
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References |
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2 . Hostetler, J. S., Heykants, J., Clemons, K. V., Woestenborghs, R., Hanson, L. H. & Stevens, D. A. (1993). Discrepancies in bioassay and chromatography determinations explained by metabolism of itraconazole to hydroxyitraconazole: studies of interpatient variations in concentrations. Antimicrobial Agents and Chemotherapy 37, 22247.[Abstract]
3 . Mikami, Y., Sakamoto, T., Yazawa, K., Gonoi, T., Ueno, Y. & Hasegawa, S. (1994). Comparison of in vitro antifungal activity of itraconazole and hydroxy-itraconazole by colorimetric MTT assay. Mycoses 37, 2733.[ISI][Medline]
4 . Odds, F. C., Vranckx, L. & Woestenborghs, F. (1995). Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation. Antimicrobial Agents and Chemotherapy 39, 205160.[Abstract]
5 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of YeastsApproved Standard M27-A. NCCLS, Wayne, PA.
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Odds, F. C., Dupont, B., Rinaldi, M. G., Stevens, D. A., Warnock, D. W. & Woestenborghs, R. (1999). Bioassays for itraconazole blood levels: an interlaboratory collaborative study. Journal of Antimicrobial Chemotherapy 43, 7237.
7 . Law, D., Moore, C. B. & Denning, D. W. (1994). Bioassay for serum itraconazole concentrations using hydroxyitraconazole standards. Antimicrobial Agents and Chemotherapy 38, 15616.[Abstract]
Received 9 July 1999; returned 30 September 1999; revised 2 November 1999; accepted 9 November 1999