Journal of Antimicrobial Chemotherapy (2001) 48, 713-715
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Effect of the growth medium on the in vitro antifungal activity of micafungin (FK-463) against clinical isolates of Candida dubliniensis
a Department of Paediatrics, b Institute for Molecular Biology of Infection, and c Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Strasse 2, D-97080 Würzburg, Germany
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Abstract |
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Micafungin (FK-463), a member of the new candin family of antifungal agents, was highly active against clinical isolates of Candida albicans and Candida dubliniensis. The in vitro activity of micafungin suggested that it was more potent than fluconazole, flucytosine, amphotericin B or voriconazole against C. albicans, and comparable or moderately less effective against C. dubliniensis isolates when high-resolution medium (HR) was used. Lower MICs of micafungin were recorded when RPMI 2% or AM3 2% media were used, indicating an influence of the growth medium on the MIC.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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As a member of the new candin family of antifungal agents, FK-463, micafungin, has demonstrated fungicidal activity against a number of Candida spp. including azole- and polyene-resistant isolates.1,2 However, for Candida tropicalis and Candida guilliermondii the minimal fungicidal concentrations (MFCs) were >64 mg/L.2 Micafungin inhibits Aspergillus spp. and prolongs survival in murine models of aspergillosis, but the antifungal activity against moulds appears to be fungistatic rather than fungicidal. The in vitro blood:plasma concentration of micafungin in humans is in the range 0.82–0.85 mg/L over a micafungin concentration of 0.1–10 mg/L. Micafungin is currently under investigation in Phase III trials for the treatment of systemic Candida and Aspergillus infections as well as for empirical use.
The recently described yeast Candida dubliniensis has been isolated from different anatomical sites from HIV-infected and non-HIV-infected individuals.3,4 As yet there are no data evaluating the in vitro susceptibilities to micafungin of clinical isolates of Candida albicans in comparison with C. dubliniensis with respect to different growth media.
In the present study, we investigated the in vitro susceptibilities of chlamydospore-positive clinical Candida isolates to one member of the new candin family of antifungal agents, micafungin, in comparison with fluconazole, flucytosine, amphotericin B and voriconazole. We investigated the semisynthetic high-resolution (HR) medium supplemented with glucose and asparagine. In addition, RPMI with 2% dextrose and antibiotic medium 3 (AM3) with 2% dextrose were assessed for their effect on the activity of micafungin.
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Materials and methods |
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One hundred and one chlamydospore-positive yeast isolates were recovered from patients suffering from oropharyngeal candidosis between May and December 1998.4 Unequivocal classification of the strains to the species level as either C. albicans or C. dubliniensis was achieved by a novel PCR test and confirmed by sequencing the V3 region of the 25S rDNA. Twenty-one isolates from 14 HIV-infected patients were identified as C. dubliniensis, and 80 isolates were identified as C. albicans.4
Broth microdilution testing was performed with minor modifications to the NCCLS M27-A reference method. Dimethyl sulphoxide (Merck, Darmstadt, Germany) was used to solubilize amphotericin B (Sigma–Aldrich, Taufkirchen, Germany) and voriconazole (Pfizer, Sandwich, UK), while fluconazole (Pfizer) was solubilized in methanol and distilled water, flucytosine (ICN, Frankfurt, Germany) in 0.9% NaCl, and micafungin (Fujisawa, Japan) in methanol and 0.9% NaCl. Sterile 96-well microtitration plates were prepared with two-fold serial dilutions of the antifungal agents. The susceptibility testing medium was HR antifungal assay medium (Oxoid, Wesel, Germany) for fluconazole, flucytosine, amphotericin B, voriconazole and micafungin; in addition micafungin was tested with RPMI 2% and AM3 2% medium for the purpose of media comparison. The final concentrations of the antifungal agents were 0.125–128 mg/L of fluconazole, 0.03–32 mg/L of flucytosine, 0.03–16 mg/L of amphotericin B and voriconazole, and 0.007–8 mg/L of micafungin. The C. albicans and C. dubliniensis inoculum size ranged from 0.5 x 103 to 2.5 x 103 cfu/mL. In each case, the inoculum size was verified by colony counting. The 96-well plates were incubated at 37°C in air. The MICs were recorded at 48 h. The MIC endpoints were determined visually and spectrophotometrically in order to avoid subjective interpretation. The MICs of fluconazole, flucytosine and voriconazole were read as the lowest concentration at which a prominent decrease in turbidity (c. 50% inhibition) relative to the growth control was observed. The MICs of amphotericin B and micafungin were read as the lowest concentration at which no visible growth was observed.5
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Results |
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The in vitro susceptibility testing of all 101 isolates revealed that the C. dubliniensis isolates were more susceptible to fluconazole, flucytosine, amphotericin B and voriconazole than the C. albicans isolates tested (Table 1
). While for micafungin the MICs for C. dubliniensis were moderately higher than for the C. albicans isolates tested when HR antifungal assay medium or RPMI 2% medium was used, they were in the same range when AM3 2% medium was used (Table 2). Micafungin was highly active against all clinical C. albicans and C. dubliniensis isolates tested. The in vitro potency of micafungin was greater than that of fluconazole, flucytosine, amphotericin B or voriconazole against C. albicans, and comparable to or moderately lower against C. dubliniensis isolates when HR medium was used (Table 2). The MICs of micafungin suggested greater susceptibility when RPMI 2% or AM3 2% media were used, indicating an influence of the different growth media on the MIC (Table 2).
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Discussion |
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In a recent study, another member of the candin family, caspofungin (MK-0991) was shown to be highly active against 71 clinical isolates of C. dubliniensis with a MIC range 0.03–0.5 mg/L.6 An influence of the culture medium has been shown for HR medium on the in vitro susceptibility of selected opportunistic fungi to fluconazole.7 A distinct media effect has been reported for caspofungin and amphotericin B: the MIC90 values for caspofungin and amphotericin B were at least four-fold lower for a number of Candida spp. when tested in AM3 supplemented with 2% dextrose in comparison with RPMI.5 In time–kill studies with LY-303366 (VER-002, V-Echinocandin) MICs for Candida spp. were found to be 10 to 100 times lower in AM3 compared with RPMI 1640 buffered with MOPS.8 In addition it has been reported recently that human serum acts synergically with caspofungin to enhance its inhibitory activity against Aspergillus fumigatus.9 In contrast micafungin MIC activity was decreased 32-fold against C. albicans when tested in the presence of 4% human serum albumin.2 While different formulations of RPMI 1640 medium provide a suitable substrate for producing reproducible results on MIC testing with fluconazole, this issue needs to be readdressed for the candins.10
In conclusion, micafungin, a member of the new class of candin antifungal agents, is highly active against clinical isolates of C. dubliniensis. The in vitro potency of micafungin was generally greater than that of the other antifungal agents tested against C. albicans, and was comparable to or moderately lower than the other antifungal agents against C. dubliniensis isolates when HR medium was used. The lower MICs of micafungin obtained in RPMI 2% and AM3 2% medium indicate an influence on the MIC by the different media. Careful evaluation of ongoing in vivo studies with the candin antifungal agents will determine which growth medium provides the best correlation between in vitro and in vivo findings.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We thank Andrea Staudigel and Stefanie Müksch for expert technical assistance. This work was supported by a grant from Fujisawa Pharm, Co., Ltd, Japan. We thank Pfizer and Fujisawa for supplying the antifungal powders. F.-M.C.M. was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF). O.K. is supported by a student fellowship from the Studienstiftung des Deutschen Volkes. Work in the laboratory of F.M. is supported by the Deutsche Forschungsgemeinschaft.
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Notes |
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* Corresponding author. Tel: +49-931-201-3728; Fax: +49-931-201-3720; E-mail: fmmueller{at}mail.uni-wuerzburg.de
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References |
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1 . Ikeda, F., Wakai, Y., Matsumoto, S., Maki, K., Watabe, E., Tawara, S. et al. (2000). Efficacy of FK463, a new lipopeptide antifungal agent, in mouse models of disseminated candidiasis and aspergillosis. Antimicrobial Agents and Chemotherapy 44, 614–8.
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4 . Kurzai, O., Korting, H. C., Harmsen, D., Bartsch, W., Molitor, M., Frosch, M. et al. (2000). Molecular and phenotypic identification of the yeast pathogen Candida dubliniensis. Journal of Molecular Medicine 78, 521–9.[Web of Science][Medline]
5 . Hicks, P. S., Dorso, K., Gerckens, L., Lynch, L., Sinclair, P., Suber, D. et al. (2000). Comparative in vitro susceptibility of clinical trial isolates to the glucan synthesis inhibitor caspofungin. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy. Abstract 193, p. 351. American Society for Microbiology, Washington, DC.
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Received 11 April 2001; returned 19 July 2001; revised 27 July 2001; accepted 17 August 2001
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