a Servicio de Microbiología Clínica, Hospital Universitario de Valme, E-41014 Sevilla; b Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Universidad del País Vasco, E-48080 Bilbao, Spain
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Abstract |
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Introduction |
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In the present study, we used the broth microdilution method of the National Comittee of Clinical Laboratory Standards (NCCLS)7 to compare the in-vitro activity of voriconazole and LY303366 with the activities of four other antifungal agents, including fluconazole, itraconazole, 5-fluorocytosine (5FC) and amphotericin B against 219 Candida spp. strains isolated from the oral cavities of HIV-infected patients.
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Materials and methods |
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The NCCLS M27-A broth microdilution method7 was used. Yeasts were obtained from 24 h cultures on Sabouraud dextrose agar (SDA, Difco, MI, USA) at 35°C. The turbidities of the yeast suspensions were adjusted by a spectrophotometric method, and the suspensions were diluted 1:1000 in RPMI medium supplemented with 2% glucose, resulting in twice the test inoculum (1 x 103 to 5 x 103 cfu/mL) as demonstrated by quantitative colony counts on SDA. Voriconazole (Pfizer, Sandwich, UK), LY303366 (Eli-Lilly, Indianapolis, IN, USA), fluconazole (Pfizer), itraconazole (Janssen, Beerse, Belgium) and amphotericin B (Squibb, Barcelona, Spain) stock solutions were prepared in 100% dimethyl sulphoxide (DMSO; Sigma, USA). Additive two-fold dilutions of these agents were prepared in DMSO at final concentration, followed by further dilutions (1:50) in RPMI 1640 medium (Sigma) buffered to pH 7.0 with 0.165 M morpholine propane sulphonic acid (MOPS) buffer (Sigma). 5FC (Roche Laboratories, Basel, Switzerland) was prepared in sterile water and diluted in RPMI medium instead of DMSO. Final drug concentrations were 0.0316 mg/L for voriconazole, LY303366 and amphotericin B, 0.0632 mg/L for itraconazole and 5FC and 0.1264 mg/L for fluconazole. Microdilution trays were incubated at 35°C and examined at 24 h or 48 h or until growth was evident (heavy growth) for MIC determination. For voriconazole, fluconazole, itraconazole and 5FC the MIC was defined as the lowest concentration showing prominent growth inhibition (approximately 50%), for amphotericin B and LY303366, the MIC was defined as the lowest concentration showing 100% growth inhibition.
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Results |
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The MICs of fluconazole for 43 Candida spp. isolates were 64 mg/L (Table II).
The MIC90 of voriconazole and LY303366 for all these strains was 0.25 mg/L. C.
tropicalis showed a voriconazole MIC90 of
0.03 mg/L and an LY303366
MIC90 of 1 mg/L.
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Discussion |
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The results of the present study support the findings reported previously by other investigators.1,4,6,8,9,10 In-vitro studies have demonstrated that voriconazole and LY303366 are as active as amphotericin B and more active than fluconazole, itraconazole and 5FC against most Candida spp.
We observed that both voriconazole and LY303366 had a very potent activity (MIC90 0.25 mg/L and 0.12 mg/L, respectively) against isolates of C. krusei and C. glabrata, species usually considered resistant to azoles such as fluconazole. This excellent in-vitro activity against fluconazole-resistant Candida spp. is remarkable and it may have important implications for the management of Candida spp. infections, as voriconazole and LY303366 are available in both oral and parenteral form.3
We found that LY303366 possessed good activity (MICs 0.25 mg/L) against C.
albicans and C. tropicalis, including strains for which the fluconazole MICs were
64 mg/L. The difference in potency between LY303366 and the triazoles is emphasized by
the extremely low MICs of LY303366 (
0.25 mg/L) observed with isolates of Candida
spp. for which there are elevated fluconazole and itraconazole MICs.10 Echinocandin and pneumocandin MICs have been demostrated to correlate
well with in-vivo activity.9 Results for C. parapsilosis
are inconclusive due to the small number of isolates studied.
Voriconazole possessed good activity against C. albicans and C. tropicalis
at concentrations of 2 mg/L. However, we found, as did Rhunke et al.2 and Marco et al.,5 that
for C. albicansisolates for which fluconazole MICs were high, voriconazole MICs were
proportionally higher than those for fluconazole-susceptible C. albicans isolates (<64
mg/L), which may indicate cross-resistance. Barry & Brown1
also found a small number of C. albicans isolates with MICs higher than 4
mg/L. We observed three C. albicans isolates with MICs of
16 mg/L. Reduced
susceptibility and cross-resistance between azole antifungals are not uncommon and are probably
related to the same mechanisms of action. Whether MICs higher than 4 mg/L might reflect
resistance to this agent needs to be substantiated by clinical studies and its clinical consequences
need to be evaluated. Further assessment of the clinical significance of the relative potency of
voriconazole and LY303366 awaits the generation of human pharmacokinetic data and the
results of comparative clinical trials.The present results suggest that voriconazole and LY303366
may be potent agents in the treatment of fungal infections due to Candida spp. Clinical
trials currently in progress will determine the efficacy and safety in the treatment of human
candidosis.
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Acknowledgments |
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Notes |
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References |
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2 . Schwartz, S., Milatovic, D. & Thiel, E. (1997). Succesful treatment of cerebral aspergillosis with a novel triazole (voriconazole), in a patient with acute leukaemia. British Journal of Haematology 97, 6635.[ISI][Medline]
3
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Marco, F., Pfaller, M. A., Messer, S. & Jones, R. N.
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6 . Ruhnke, M., Schmidt-Westhausen, A. & Trautmann, M. (1997). In vitro activities of voriconazole (UK-109,496) against fluconazole susceptible and resistant Candida albicans isolates from oral cavities of patients with human immunodeficiency virus infection. Antimicrobial Agents and Chemotherapy 42, 57582.
7 . National Comittee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of YeastsApproved Standard M27-A. NCCLS, Villanova, PA.
8
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Espinel-Ingroff, A. (1998). In vitro
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and dimorphic fungi and common and emerging yeast pathogens. Journal of Clinical
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9 . Krishnarao, T. V. & Galgiani, J. N. (1997). Comparison of the in vitro activities of the echinocandine LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 41,1957 60.[Abstract]
10 . Uzun, Ö., Kocagöz, S., Çetinkaya, Y., Arikan, S. & Ünal, S. (1997). In vitro activity of a new echinocandin, LY303366, comaprred with those of amphotericin B and fuconazole against clinical yeaste isolates. Antimicrobial Agents and Chemotherapy 41, 11567.[Abstract]
Received 14 December 1998; returned 26 March 1999; revised 16 April 1999; accepted 28 April 1999