Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km. 2, 28220 Majadahonda (Madrid), Spain
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Abstract |
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Introduction |
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New drugs are needed to improve the treatment of fungal infections. The fungal cell wall is a potential target for new antifungal agents. Echinocandins are cyclic hexapeptides that disrupt cell wall glucan formation.1,2 The mechanism of action is thought to involve non-competitive inhibition of (1,3)-ß-D-glucan synthase.2 LY303366 is a semi-synthetic echinocandin B derivative possessing in vitro activity against Candida species and filamentous fungi.15 However, this echinocandin is not active in vitro against Cryptococcus neoformans or Blastomyces dermatitidis; the lack of activity may arise because of the greater proportion of (1,3)--D-glucan linkages in cell wall polymers of these fungi.3 In vivo, this compound has shown efficacy in rabbit models of pulmonary aspergillosis and disseminated candidosis, displaying good bioavailability and low toxicity.6
Previous investigations have demonstrated the potent antifungal activity of LY303366 against Candida spp., and azole-resistant isolates appear not to be cross-resistant to this compound. However, the number of clinical isolates resistant to antifungal agents included in previous studies has been limited.1,35 We have evaluated the in vitro activity of LY303306 against 156 clinical isolates of Candida spp. with decreased susceptibility to fluconazole (MIC 16 mg/L), and compared the results with those obtained with amphotericin B and itraconazole.
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Material and methods |
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A broth microdilution test was performed according to the NCCLS reference method,7 with minor modifications (RPMI2% glucose).8 LY303366 (Lilly S.A., Madrid, Spain), amphotericin B (Bristol Myers Squibb, Madrid, Spain), itraconazole (Janssen Farmaceútica, Madrid, Spain) and fluconazole (Pfizer, Madrid, Spain) were obtained as standard powders. Sterile flat-bottomed microtitration plates were prepared with 100 µL aliquots of the antifungal agent in each well and were inoculated with 100 µL aliquots of an inoculum preparation to give a final concentration of 0.52.5 x 105 cfu/mL. Spectrophotometric readings were performed with a Labsystems IEMS Reader MF (Labsystems, Barcelona, Spain) at 540 nm. MICs of LY303366 and amphotericin B were defined as the lowest concentration resulting in 80% inhibition of growth compared with drug-free control. MICs of fluconazole and itraconazole were defined as 50% inhibition.
The NCCLS has proposed that strains showing a fluconazole MIC of 1632 mg/L should be defined as susceptible dependent on dose.7 We have proposed 16 mg/L as the resistance breakpoint in strains causing oropharyngeal candidosis in AIDS patients as this has shown correlation in vivo.9 In this study, we therefore defined 16 mg/L as the breakpoint for decreased susceptibility to fluconazole.
Data are reported as ranges and the concentration of each antifungal agent required to inhibit 50% (MIC50) and 90% (MIC90) of the isolates tested. The correlation between MICs obtained with the different antifungal agents was determined by Pearson's r coefficient.
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Results |
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The Table summarizes the distribution of the MICs of itraconazole, amphotericin B and LY303366 for 156 fluconazole-resistant isolates. For 49 strains, the MIC of itraconazole was
1 mg/L. The MICs of itraconazole correlated well with the MICs of fluconazole (Pearson's r coefficient 0.56; P < 0.01). As expected for compounds with different mechanisms of action the MICs of amphotericin B and LY303366 did not correlate with those of fluconazole (Pearson's r 0.065 and 0.033, P = 0.358 and 0.657, respectively).
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Discussion |
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Our results show that susceptibility testing performed according to NCCLS guidelines with minor modifications (higher inoculum and spectrophotometric reading) is also an acceptable means of evaluating the in vitro activity of this new antifungal agent. Furthermore, in rabbits receiving 1 mg/kg of LY303366 as a single iv bolus, the concentration (Cmax) of this antifungal agent in serum was 3.56 ± 0.644 mg/L;6 these pharmacokinetic data warrant further studies.
In summary, it appears that LY303366 is more potent in vitro against fluconazole-resistant clinical isolates of C. albicans, C. glabrata, C. krusei and C. tropicalis than either itraconazole or amphotericin B. This echinocandin is least active against fluconazole-resistant isolates of C. parapsilosis and C. guilliermondii. The excellent in vitro activity against azole-resistant Candida spp. strains may have important implications for the treatment of infections by these yeasts. LY303366 is a promising new antifungal agent that merits more comprehensive clinical studies to determine the correlation between these data and the clinical outcome.
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Acknowledgments |
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Notes |
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References |
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2 . Pfaller, M. A., Marco, F., Messer S. A. & Jones, R. N. (1998). In vitro activity of two echinocandin derivatives, LY303366 and MK-0991 (L-743 792), against clinical isolates of Aspergillus, Fusarium, Rhizopus, and other filamentous fungi. Diagnostic Microbiology and Infectious Diseases 30, 2515.[ISI][Medline]
3 . Zhanel, G. G., Karlowsky, J. A., Harding, G. A., Balko, T. V., Zelenitsky, S. A., Friesen, M. et al. (1997). In vitro activity of a new semisynthetic echinocandin, LY303366 against systemic isolates of Candida species, Cryptococcus neoformans, Blastomyces dermatitidis, and Aspergillus species. Antimicrobial Agents and Chemotherapy 41, 8635.[Abstract]
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Espinel-Ingroff, A. (1998). Comparison of in vitro activities of the new triazole SCH56592 and the echinocandins MK-0991 (L-743872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts. Journal of Clinical Microbiology 36, 29506.
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Chavez, M., Bernal, S., Valverde, A., Gutierrez, M. J., Quindós, G. & Mazuelos, E. M. (1999). In vitro activity of voriconazole (UK-109496), LY303366 and other antifungal agents against oral Candida spp. isolates from HIV-infected patients. Journal of Antimicrobial Chemotherapy 44, 697700.
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Petraitiene, R., Petraitis, V., Groll, A. H., Candelario, M., Sein, T., Bell, A. et al. (1999). Antifungal activity of LY303366, a novel echinocandin B, in experimental disseminated candidiasis in rabbits. Antimicrobial Agents and Chemotherapy 43, 214855.
7 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. NCCLS, Villanova, PA.
8 . Rodríguez-Tudela, J. L. & Martínez-Suárez, J. V. (1994). Improved medium for fluconazole susceptibility testing of Candida albicans. Antimicrobial Agents and Chemotherapy 38, 458.[Abstract]
9 . Rodríguez-Tudela, J. L., Martínez-Suárez, J. V., Dronda, F., Laguna, F., Chaves, F. & Valencia, E. (1995). Correlation of in-vitro susceptibility test results with clinical response: a study of azole therapy in AIDS patients. Journal of Antimicrobial Chemotherapy 35, 793804.[Abstract]
Received 4 February 2000; returned 25 April 2000; revised 12 May 2000; accepted 23 May 2000