Journal of Antimicrobial Chemotherapy (2000) 46, 475-477
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B
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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The in vitro activity of LY303366 was compared with those of itraconazole and amphotericin B against 156 fluconazole-resistant (MIC
16 mg/L) clinical isolates of Candida spp. An adaptation of the NCCLS reference method was employed for determination of MICs. LY303366 was more potent than either itraconazole or amphotericin B against Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis, even against isolates with itraconazole MICs 1 mg/L. LY303366 was less potent in vitro against Candida parapsilosis and Candida guilliermondii isolates. LY303366 has promising antifungal activity and warrants further investigation. |
Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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Infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. Fluconazole and itraconazole are frequently employed in the treatment of these infections.1,2 Their widespread use has been accompanied by an increase in antifungal resistance and by a noticeable shift toward non-albicans species with relative resistance to fluconazole and itraconazole. Amphotericin B has been the mainstay of treatment for severe fungal infections for several decades, but its use has been limited by a number of serious adverse effects.3
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.1–5 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.1,3–5 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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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The 156 isolates of Candida spp. were recovered from clinical samples processed at 51 different Spanish hospitals during the last 5 years. Ninety-four samples were obtained from oropharyngeal exudate, 36 from vaginal samples, 12 from blood cultures and 14 from other specimens. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included as quality control strains.
A broth microdilution test was performed according to the NCCLS reference method,7 with minor modifications (RPMI–2% 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 0.5–2.5 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 16–32 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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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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All isolates produced detectable growth after 24–48 h of incubation. The MIC values obtained for the control organisms varied by no more than one three-fold dilution and were similar in range to reference values.8
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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A broad range of MICs was observed with each antifungal agent for the various Candida spp. tested. The MICs of LY303366 were lower than those obtained with amphotericin B and itraconazole. The least susceptible isolates were Candida parapsilosis and Candida guilliermondii strains; the MIC50 results of LY303366 were 0.06 and 2 mg/L respectively.
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
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The results obtained support and extend findings reported previously. We found that LY303366 had very potent activity against fluconazole-resistant clinical isolates of a variety of Candida spp., including Candida albicans, Candida glabrata, C. krusei and Candida tropicalis. In addition, the spectrum of activity was broader than that of itraconazole. LY303366 had potent activity against species usually considered refractory to azoles such as C. krusei and C. glabrata. We found that fluconazole-resistant isolates with reduced susceptibility to itraconazole did not demonstrate reduced susceptibility to LY303366, which could be a reflection of its different mechanism of action. The MICs of LY303366 for the five isolates of C. parapsilosis and the three of C. guilliermondii were high, confirming earlier reports. However, because of the very small number of strains tested this finding requires further investigation.1,4
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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We thank Lilly S.A., Janssen Farmacéutica, Pfizer and Bristol Myers Squibb for supplying the antifungal powders. This work was supported in part by grant 96/0598 from the Fondo de Investigaciones Sanitarias. T. M. Díaz-Guerra is a Fellow of the Instituto de Salud Carlos III (grant 99/4149)
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Notes |
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* Corresponding author. Tel: +34-91-5097961; Fax: +34-91-5097966; E-mail: micologia{at}isciii.es
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References |
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1 . Pfaller, M. A., Messer, S. A. & Coffman, S. (1997). In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative, LY303366, and other antifungal agents. Antimicrobial Agents and Chemotherapy 41, 763–6.[Abstract]
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, 251–5.[Web of Science][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, 863–5.[Abstract]
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Received 4 February 2000; returned 25 April 2000; revised 12 May 2000; accepted 23 May 2000
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