In-vitro activity of five antifungal agents against uncommon clinical isolates of Candida spp.

Francesco Barchiesia,*, Anna Maria Tortoranob, Luigi Falconi Di Francescoa, Massimo Cogliatib, Giorgio Scalisea and Maria Anna Vivianib

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A broth microdilution method and an agar dilution method were used for testing fluconazole, itraconazole, ketoconazole, flucytosine and amphotericin B against 98 clinical isolates belonging to seven species of Candida. The approximate rank order of fluconazole MICs was Candida lusitaniae {approx} Candida kefyr< Candida famata{approx}Candida guilliermondii< Candida pelliculosa {approx} C. lipolytica {approx} Candida inconspicua. Candida lypoliticaand C. pelliculosa were the species least susceptible to itraconazole and ketoconazole. Flucytosine MICs revealed the highest prevalence of resistant strains among C. lipolyticaand C. lusitaniae. All isolates were susceptible to amphotericin B.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The risk of opportunistic infections is greately increased in patients who are severely immunocompromised. Although Candida albicansis the organism most often associated with serious fungal infections, other Candidaspp. have emerged as clinically important pathogens associated with opportunistic infections. 1,2

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A panel of 98 yeast isolates belonging to seven species of Candidawas used. They represented strains from two culture collections: Istituto di Igiene e Medicina Preventiva, Universitàdi Milano, and Istituto di Malattie Infettive e Medicina Pubblica, Università di Ancona, Italy. They included 16 isolates of Candida kefyr (Kluyveromyces marxianus), 15 isolates each of Candida famata (Debariomyces hansenii), Candida inconspicua, Candida lusitaniae (Clavispora lusitaniae) and Candida pelliculosa (Pichia anomala), 13 isolates of Candida guilliermondii (Pichia guilliermondii/ohmeri) and nine isolates of Candida lipolytica (Yarrowia lipolytica). The isolates were recovered from the gastrointestinal, respiratory or urinary tracts, from blood or from other sterile body fluids. Each strain represented a unique isolate from a patient managed in the two university medical centres. Yeast isolates were identified at species level by conventional morphological and biochemical methods (API 20 C AUX or API 32 ID; bio Mérieux, Marcy l' Etoile, France). C. inconspicua isolates were identified by API 20 C AUX or API 32 ID, production of pseudohyphae, growth at 42°C and glucose fermentation.

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


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluconazole MICs obtained by broth dilution revealed a wide range (<=0.125 to >64 mg/L) (Table I). Candida inconspicuashowed the highest MIC50 (16 mg/L) and C. kefyrthe lowest (0.25 mg/L). Itraconazole and ketoconazole MIC ranges were narrower than that for fluconazole: <=0.03 to 0.5 mg/L and <=0.03 to 1.0 mg/L, respectively. The highest MIC50 of itraconazole was observed for C. pelliculosa, C. guilliermondii and C. lipolytica (all 0.125 mg/L), while the lowest MIC50 was observed for C. lusitaniae (<=0.03 mg/L). The highest MIC50 of ketoconazole was observed for C. lipolytica (0.5 mg/L), and the lowest for C. kefyrand C. lusitaniae (both <=0.03 mg/L). Flucytosine MICs revealed a wide range (<=0.125 to >64 mg/L). Candida lipolyticaand C. lusitaniaeshowed the highest MIC50s: 8.0 mg/L and 4.0 mg/L, respectively. Amphotericin B MICs determined in antibiotic medium 3 yielded a slightly wider range than those determined in RPMI 1640 (data not shown) with C. lusitaniaeand C. lipolytica showing the highest MIC50s: 0.125 mg/L and 0.25 mg/L, respectively.


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Table I. In-vitro activity of five antifungal agents against 98 clinical isolates of Candida spp.
 
All three azoles tested in agar dilution produced higher MICs than those tested in broth dilution, with the highest difference for fluconazole (approximately two dilutions; Table I). Itraconazole and ketoconazole MICs tested in agar dilution were approximately one dilution higher than those tested in broth dilution. No difference was found between flucytosine MICs produced by the two methods. Amphotericin B MICs tested in agar dilution were approximately two dilutions lower than those tested in broth dilution, regardless of the medium used for testing. Overall, the agreements between broth and agar dilution MICs within two dilutions were always >80%, ranging from 83% to 100%.

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{approx}C. kefyr< C. famata{approx}C. guilliermondii< C. pelliculosa{approx} C. lipolytica{approx} 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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Table II. Prevalence of isolates with reduced susceptibility to azoles and to flucytosine in seven species of Candida
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To investigate the drug susceptibility of uncommon species of Candida, two different methods were used: a microformat adaptation of the NCCLS broth macrodilution procedure and an agar dilution method which is widely employed in European countries. Both methods were able to demonstrate several important features of drug susceptibilities among the seven species of Candidaconsidered.

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.


    Acknowledgments
 
This work was supported in part by a grant from Istituto Superiore di Sanità, Rome, Italy (I AIDS project) and by a grant from IRCCS Ospedale Maggiore di Milano, Milano, Italy.


    Notes
 
* Correspondence address. Istituto di Malattie Infettive e Medicina Pubblica, Università degli Studi di Ancona, Ospedale Umberto I, Largo Cappelli 1, 60121 Ancona, Italy. Tel: +39-71-5963467; Fax: +39-71-5963468; E-mail: cmalinf{at}popcsi.unian.it Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Morrison, V. A., Haake, R. J. & Weisdorf, D. J. (1993). The spectrum of non-Candida fungal infections following bone marrow transplantation. Medicine 72, 78–89.[ISI][Medline]

2 . Nguyen, M. H., Peacock, J. E., Morris, A. J., Tanner, D. C., Nguyen, M. L., Snydman, D. R. et al. (1996). The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. American Journal of Medicine 100,617 –23.

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.

4 . Drouhet, E., Dupont, B., Improvvisi, L., Viviani, M. A. & Tortorano, A. M. (1986). Disc agar diffusion and microplate automatized techniques for in vitro evaulation of antifungal agents on yeasts and sporulated pathogenic fungi. In In Vitro and In Vivo Evaluation of Antifungal Agents (Iwata, K. & Vanden Bossche, H., Eds), pp. 31–49. Elsevier Science, Amsterdam.

5 . Tortorano, A. M., Viviani, M. A., Barchiesi, F., Arzeni, D., Rigoni, A. L., Cogliati, M. et al. (1998). Comparison of three methods for testing azole susceptibilities of Candida albicans strains isolated sequentially from oral cavities of AIDS patients. Journal of Clinical Microbiology 36, 1578–83.[Abstract/Free Full Text]

6 . Rex, J. H., Cooper, C. R., Merz, W. G., Galgiani, J. N. & Anaissie, E. J. (1995). Detection of amphotericin B-resistant Candida isolates in a broth-based system. Antimicrobial Agents and Chemotherapy39 , 906–9.[Abstract]

7 . Bland, J. M. & Altman, D. G. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. Lancet i, 307–11.

8 . Barchiesi, F., Hollis, R. J., McGough, D. A., Scalise, G., Rinaldi, M. G. & Pfaller, M. A. (1995). DNA subtypes and fluconazole susceptibilities of Candida albicans isolates from the oral cavities of patients with AIDS. Clinical Infectious Diseases 20, 634–40.[ISI][Medline]

9 . Viviani, M. A. (1995). Flucytosine—what is its future? Journal of Antimicrobial Chemotherapy 35, 241–4.[ISI][Medline]

Received 11 May 1998; returned 2 July 1998; revised 12 August 1998; accepted 15 September 1998