In vitro susceptibility of clinical isolates of Zygomycota to amphotericin B, flucytosine, itraconazole and voriconazole

A. Gómez-López, M. Cuenca-Estrella,*, A. Monzón and J. L. Rodriguez-Tudela

Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, 28220 Majadahonda, Madrid, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The in vitro susceptibility of 29 clinical isolates of fungi belonging to the division Zygomycota was reviewed. A broth microdilution test was performed by following the NCCLS reference method, with minor modifications. The species of Zygomycota tested were resistant in vitro to flucytosine, itraconazole and voriconazole. Amphotericin B showed the best activity against most of the strains evaluated.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Zygomycosis is the currently accepted designation of the disease processes caused by the members of the division Zygomycota. Fungi in this group are common worldwide and have been isolated from decaying vegetation, soil, house dust and air. In addition, these fungi are usually isolated as contaminants in the laboratory and all are opportunistic in human cases associated with immunosuppression.1 The vast majority of the human pathogenic Zygomycota belong to the order Mucorales. Absidia corymbifera, Apophysomyces elegans, Cokeromyces recur-vatus, Cunninghamella bertholletiae, Mucor circinelloides, Mucor ramosissimus, Rhizopus oryzae, Rhizopus micro-sporus, Rhizopus pusillus, Saksenaea vasiformis and Syn-cephalastrum racemosum are the most frequent species causing human infections. These organisms can produce rhinocerebral, pulmonary, gastrointestinal, cutaneous or disseminated infections in predisposed individuals.2 The different clinical forms are often associated with particular underlying conditions and are potentially lethal. The term zygomycosis also includes diseases caused by another group of Zygomycota, those belonging to the order Ento-mophthorales. This last type of zygomycosis usually afflicts patients considered physiologically and immunologically normal.

Human zygomycosis occurs as a sporadic infection in compromised hosts. However, its rapid progressive and potentially fatal course makes necessary an effective anti-fungal chemotherapy. It has been reported that Mucorales are susceptible in vitro to amphotericin B, but therapy with this agent has produced variable results, since it is dependent upon the organism and the immune status of the patient.1,2 Thus, new therapeutic strategies are clearly needed. Mucorales are generally resistant to azoles, with MICs that exceed 16 mg/L, and to other agents such as flucytosine and naftifine, and new agents such as the echinocandins.2

The purpose of the present study was to examine the activity of four antifungal agents in vitro against a variety of clinical isolates of Mucorales, following a standardized methodology.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Fungi

A collection of 29 clinical isolates was included, compris-ing: A. corymbifera four, C. bertholletiae one, M. circinel-loides one, M. ramosissimus three, R. microsporus five, R. oryzae 14 and S. vasiformis one. All strains were recovered during a period of 5 years (1995–1999) from 15 Spanish hospitals. Each clinical isolate represented a unique isolate from a patient. Paecilomyces variotii ATCC 22319 and Aspergillus fumigatus ATCC 9197 were used as reference strains for quality control and to monitor the reproducibility of susceptibility tests.

Antifungal agents

Antifungal agents used were amphotericin B (Sigma– Aldrich Química, Madrid, Spain), flucytosine (Sigma–Ald-rich Química), itraconazole (Janssen Pharmaceutica, Madrid, Spain) and voriconazole (Pfizer Ltd, Sandwich, UK). They were obtained as standard powders and stock solutions were prepared in 100% dimethyl sulphoxide (DMSO; Sigma–Aldrich Química), except for flucytosine, which was dissolved in sterile distilled water. Solutions were stored at -70°C for each drug.

Test medium

The susceptibility testing medium was RPMI 1640 with l-glutamine buffered to pH 7 with 0.165 M morpholine-propanesulphonic acid (MOPS) and 10 M NaOH (Oxoid, Madrid, Spain), and supplemented with glucose 18 g/L (RPMI–2% glucose). This medium was prepared as a double strength solution.

Antifungal susceptibility testing

A broth microdilution test was performed by following the NCCLS reference method,3 with minor modifications.4 Initial solutions of amphotericin B, itraconazole and voriconazole were diluted in DMSO and the stock solution of flucytosine with RPMI–2% glucose.3 The final concentra-tions ranged from 16 to 0.03 mg/L for amphotericin B, from 128 to 0.25 mg/L for flucytosine, from 8 to 0.015 mg/L for itraconazole and from 64 to 0.12 mg/L for voriconazole. Sterile plastic microtitration plates with 96 flat-bottom wells each were employed. These plates contained two-fold serial dilutions of the antifungal drugs and two drug-free medium wells for sterility and growth controls.

Inoculum suspensions were obtained by rubbing the surface of agar cultures with a loop after the addition of sterile, distilled water (10 mL/slant) and transferring the material to sterile tubes with sterile distilled water. A starting inoculum of 1 x 106 to 5 x 106 cfu/mL was obtained by adjusting the density of suspensions between 0.15 and 0.17 units of absorbance or 68–70% of transmittance at 530 nm using a spectrophotometer. The suspension was then diluted 1:10 with sterile water to get a final working inoculum of 1–5 x 105 cfu/mL. The trays were inoculated with 0.100 mL into each well. The plates were incubated at 35°C for 24, 48 and 72 h in a humid atmosphere. Visual readings were performed with the help of a mirror.

Endpoint determination

MICs were defined as the lowest concentration of the anti-fungal agent that completely inhibited fungal growth.

Statistical analysis

Statistical analysis was done with Statistical Package for the Social Sciences (SPSS, v. 10.0.) (SPSS S.L, Madrid, Spain).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All fungi produced detectable growth after 48–72 h of in-cubation. The MICs of the four agents for the control organisms were consistent within three two-fold dilutions. The TableGo summarizes the MIC results for the 29 isolates tested. A wide range of MICs was noted for all agents across the species tested, with the exception of flucytosine, which was inactive against all except S. vasiformis. Ampho-tericin B was the most active drug against the majority of fungi studied, with MICs ranging from 0.12 to >16 mg/L. One isolate of A. corymbifera was not inhibited by amphotericin B (MIC > 16 mg/L) or itraconazole (MIC > 8 mg/L). It was isolated from a wound exudate of a patient with a chronic venous leg ulcer. In general, MICs of azole compounds were significantly higher than those of ampho-tericin B, except for one isolate of C. bertholletiae where the MIC of amphotericin B (4 mg/L) was higher than that of itraconazole (2 mg/L). Voriconazole did not show more in vitro activity against Zygomycota than itraconazole.


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Table. In vitro susceptibilities of Zygomycota (n = 29) included in the study
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our data show that the antifungal tests often showed elevated MICs for this group of Mucorales. However, the results of in vitro susceptibility testing were species dependent and even isolate dependent. Amphotericin B was active against most of the isolates studied, but a wide range of MICs was observed, indicating resistance in some, particularly Cunninghamella spp. As several reports have indicated, these species are resistant to amphotericin B in vitro.5,6

The MICs of itraconazole were elevated against most of the fungi studied, indicating that this antifungal agent is likely to be ineffective in the treatment of this kind of infec-tion. Voriconazole is a new triazole that is active against A. fumigatus and other species of moulds.7 Several previous works have reported that some species of Zygomycota were resistant to this agent.8–10 In our study, overall, MICs of voriconazole were significantly higher than those of amphotericin B and even than those of itraconazole. Our data show that all species of Zygomycota tested are resistant to voriconazole in vitro.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by grant 99/198 from the Fondo de Investigaciones Sanitarias and by grant 99/1199 of the Institute de Salud Carlos III. A.G.-L. is a Fellow of the Fondo de Investigaciones Sanitarias (grant 99/198).


    Notes
 
* Corresponding author. Tel: +34-91-5097961; Fax: +34-91-5097966; E-mail: mcuenca-estrella{at}isciii.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Ribes, J. A., Vanover-Sams, C. L. & Baker, D. J. (2000). Zygo-mycetes in human disease. Clinical Microbiology Reviews 13, 236–301.[Abstract/Free Full Text]

2 . Otcenasek, M. & Buchta, V. (1994). In vitro susceptibility to 9 anti-fungal agents of 14 strains of Zygomycetes isolated from clinical specimens. Mycopathology 128, 135–7.

3 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Test-ing of Conidium-Forming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PA.

4 . Rodriguez-Tudela, J. L. & Martínez-Suárez, J. V. (1994). Improved medium for fluconazole susceptibility testing of Candida albicans. Antimicrobial Agents and Chemotherapy 38, 45–8.[Abstract]

5 . Rotowa, N. A., Shadomy, H. J. & Shadomy, S. (1990). In vitro activities of polyene and imidazole antifungal agents against unusual opportunistic fungal pathogens. Mycoses 33, 203–11.[ISI][Medline]

6 . Zeilender, S., Drenning, D., Glauser, F. L. & Berchard, D. (1990). Fatal Cunninghamella bertholletiae infection in an immunocompetent patient. Chest 97, 1482–3.[Abstract]

7 . Cuenca-Estrella, M., Rodriguez-Tudela, J. L., Mellado, E., Martínez-Suárez, J. V. & Monzón, A. (1998). Comparison of the in-vitro activity of voriconazole (UK-109,496), itraconazole and ampho-tericin B against clinical isolates of Aspergillus fumigatus. Journal of Antimicrobial Chemotherapy 42, 531–3.[Abstract]

8 . Wildfeuer, A., Seidl, H. P., Paule, I. & Haberreiter, A. (1998). In vitro evaluation of voriconazole against clinical isolates of yeast, moulds and dermatophytes in comparison with itraconazole, ketoco-nazole, amphotericin B and griseofulvin. Mycoses 41, 309–19.[ISI][Medline]

9 . Kappe, R. (1999). Antifungal activity of a new azole UK-109, 496 (voriconazole). Mycoses 42, Suppl. 2, 83–6.[ISI][Medline]

10 . Espinel-Ingroff, A., Boyle, K. & Sheehan, D. J. (2001). In vitro antifungal activities of voriconazole and reference agents as deter-mined by NCCLS methods: review of the literature. Mycopathologia 150, 101–15.[ISI][Medline]

Received 10 April 2001; returned 9 August 2001; revised 28 August 2001; accepted 30 August 2001