a Istituto di Malattie Infettive e Medicina Pubblica, Università degli Studi di Ancona; b Istituto di Igiene e Medicina Preventiva, Università degli Studi di Milano, IRCCS Ospedale Maggiore di Milano, Italy
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Abstract |
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Introduction |
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At present, few drugs are available for the treatment of fungal infections and little is known about the in-vitro activity of the major antifungal compounds against Candidaspp. other than C. albicans.
The present study was undertaken to determine the in-vitro activity of five antifungal agents against clinical yeast isolates belonging to seven species of Candida.
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Materials and methods |
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The antifungal agents used in this study were fluconazole and amphotericin B intravenous preparations, flucytosine (Sigma Chemical, Milano, Italy), itraconazole and ketoconazole powders (both from Janssen Pharmaceutica, Beerse, Belgium). In preliminary experiments there was no difference between the results obtained with the commercial preparation of fluconazole and amphotericin B and those obtained with pure powders (Pfizer Inc., New York, NY, USA, and Sigma, respectively). Drugs were tested at the following concentration ranges: fluconazole and flucytosine, 0.125-64 mg/L; itraconazole and ketoconazole, 0.03-4.0 mg/L; and amphotericin B, 0.015-4.0 mg/L.
Antifungal susceptibility testing was performed by either a broth microdilution method or an agar dilution method. Broth dilution was performed in RPMI 1640 (Sigma) as outlined in the NCCLS M27-A document.3 The medium used for the agar dilution method depended on the drug: azole and amphotericin B MICs were tested in phosphate-buffered casitone agar while flucytosine was tested in yeast nitrogen base with added glucose (pH 5.6).4,5 MICs, recorded after 48 h of incubation at 35°C, were defined as the lowest concentration which suppressed any visible growth.4 In addition to the standard media described above, amphotericin B MICs were tested in antibiotic medium 3 (Difco) supplemented with 2% glucose by both methods. 6
MIC ranges were determined for each species-drug combination tested. MICs for
50% and 90% of the isolates of each species tested (MIC50 and MIC90,
respectively) were determined for the yeast species with 10 isolates. Both
on-scale and off-scale results were included in the analysis. MIC results obtained by the two
methods were logarithmically transformed, deviated to avoid negative values (log-MIC) and
compared according to the method of Bland & Altman.
7 The mean of the differences between the log-MIC
obtained by broth dilution and agar dilution was calculated and used to determine the
percentage of agreement within one and two dilutions as previously described.5
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Results |
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To determine how drug susceptibility varied according to the species of Candidatested, we analysed the prevalence of isolates with reduced susceptibility to azoles and
flucytosine. Amphotericin B was not included in this analysis since, with the exception of one
strain of C. lipolytica(amphotericin B MIC > 4.0 mg/L by agar dilution;
Table I), all
the isolates were inhibited by concentrations 1.0 mg/L. Based on the recent NCCLS standard
3 and on our previous experience with C. albicansstrains,5,8 we defined
isolates with reduced susceptibility to fluconazole as those strains with MICs
8.0 mg/L, and
isolates with reduced susceptibility to itraconazole and ketoconazole as those strains with MICs
0.25 mg/L by the broth dilution method. According to the mean differences between agar
and broth dilution MICs obtained in this study, we defined isolates with reduced susceptibility to
fluconazole as those strains with MICs
32 mg/L, and isolates with reduced susceptibility to
itraconazole and ketoconazole as those strains with MICs
0.5 mg/L by the agar dilution
method. On the basis of the recommendations of the NCCLS
3 and of the equivalences between MICs obtained by both
methods, an MIC
8.0 mg/L was selected to define isolates with reduced susceptibility to
flucytosine (Table II). Overall, both methods showed a species-specific
trend of azole MICs. The
approximate rank order of fluconazole MICs was: C. lusitaniae
C. kefyr< C. famata
C. guilliermondii< C. pelliculosa
C.
lipolytica
C. inconspicua. All isolates of C. pelliculosatested against
fluconazole by agar dilution had reduced susceptibility to this triazole, while only 53%
(eight out of 15 isolates) were shown to be fluconazole-resistant by the broth dilution method. It
must be noted, however, that the other seven isolates had fluconazole MICs of 4.0 mg/L when
tested by the broth dilution method. The same phenomenon was seen even when itraconazole
and ketoconazole were tested against isolates of C. pelliculosa: only 40% of the
strains had reduced susceptibility to both azoles when tested by the broth dilution method,
compared with 93% and 87%, respectively, when tested by the agar dilution
method. Similarly, the nine isolates which were considered susceptible to itraconazole and
ketoconazole as judged by the broth dilution method all had MICs of both these drugs of 0.125
mg/L. Overall, C. lipolyticaand C. pelliculosashowed the highest prevalence of
reduced susceptibility to itraconazole and ketoconazole, while C. incospicuashowed
high prevalence of reduced susceptibility to ketoconazole. The lowest prevalence of
flucytosine-susceptible strains was seen in C. lipolyticaand C. lusitaniae (Table II).
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Discussion |
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We identified three species of Candidawith reduced susceptibility to fluconazole: C. inconspicua, C. lipolyticaand C. pelliculosa.All three of these species were cross-resistant to ketoconazole; only C. lipolyticaand C. pelliculosawere cross-resistant to itraconazole, while C. inconspicuawas itraconazole susceptible. Our in-vitro data on flucytosine showed that in two species, C. lusitaniaeand C. lipolytica, there was a high prevalence of strains with low susceptibility. Although flucytosine is rarely employed in monotherapy due to the frequent development of resistance, its usefulness in combination therapy has been repeatedly documented. 9 We found that only 50% of isolates of C. lusitaniaeand 33% of isolates of C. lipolyticawere flucytosine susceptible. These findings suggest that the use of flucytosine in the treatment of fungal infections caused by these two species of Candidashould be avoided unless their susceptibility has been proven in vitro.
Although amphotericin B MICs tested in antibiotic medium 3 yielded a slightly broader range than that observed in RPMI, we were not able to detect amphotericin B-resistant isolates. The same result was obtained with the agar dilution method. The lack of amphotericin B-resistant Candidaisolates found in this study probably reflects the fact that our isolates were randomly selected and that no efforts to test clinical isolates from patients who failed amphotericin B therapy were made.
In conclusion, our results clearly show that species vary in their drug susceptibility. Our data could be of aid in choosing the best antifungal therapy according to the organism causing the infection.
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Acknowledgments |
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Notes |
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References |
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3 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.
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Received 11 May 1998; returned 2 July 1998; revised 12 August 1998; accepted 15 September 1998