Antifungal activity of the echinocandin anidulafungin (VER002, LY-303366) against yeast pathogens: a comparative study with M27-A microdilution method

María Pilar Arévalo1,*, Alfonso-Javier Carrillo-Muñoz2, Javier Salgado1,3, Delia Cardenes1, Sonia Brió2, Guillermo Quindós4 and Ana Espinel-Ingroff5

1 Cátedra de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad de la Laguna, 38071 La Laguna, Tenerife; 2 Departamento de Microbiología, ACIA, Barcelona; 3 Hospital Ntra. Sra. de Candelaria, Tenerife; 4 Departamento de Immunología, Microbiología y Parasitología, Facultad de Medicina, Universidad del País Vasco, Bilbao, Spain; 5 Medical College of Virginia, Campus Virginia Commonwealth University, Richmond, VA, USA

Received 18 December 2001; returned 4 June 2002; revised 15 July 2002; accepted 28 September 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
This study further evaluated the in vitro activity of anidulafungin (VER002, Versicor Inc.) (LY303366) against 460 clinical yeast isolates. MICs of anidulafungin, fluconazole and itraconazole were determined by following the NCCLS M27-A guidelines. Minimum fungicidal concentrations (MFCs) of anidulafungin were determined for 230 isolates of Candida spp. The activity of anidulafungin in vitro was significantly superior (P < 0.05) to those of itraconazole and fluconazole against Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei, but anidulafungin was less active for Candida famata and Candida parapsilosis. The differences were not significant for the other species evaluated.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The emergence of opportunistic fungal pathogens, the increased variety of mycoses, the recovery of clinical isolates resistant to conventional antifungal agents and the adverse side effects of amphotericin B and azoles have promoted the development of new antifungal agents (triazoles and echinocandins) as well as of lipid formulations of amphotericin B and nystatin.1,2

The parallel increasing interest in antifungal susceptibility tests also reflects the need for standardized conditions to obtain data in vitro for the prediction of the susceptibility or resistance of clinical isolates to antifungal agents. In the 1990s, the NCCLS developed a reference method for the susceptibility of Candida species and Cryptococcus neoformans.3

Although Candida albicans is more often associated with serious fungal infections than other fungi, other Candida species and yeast-like organisms have emerged as aetiological agents of serious fungal infections.1 Amphotericin B and its lipid formulations, the echinocandin caspofungin and the azole derivatives fluconazole, itraconazole and voriconazole, are the only available agents for the management of severe fungal infections. However, their use is limited by either their narrow efficacy or safety problems. Although amphotericin B has been commonly used for the treatment of serious, invasive fungal infections, resistance to this agent has been documented,4 especially in immunocompromised patients infected with emerging yeast or mould pathogens.5,6 The emerging fungal pathogens are usually less susceptible to azole compounds and the management of such infections could be problematic.

Anidulafungin (VER002, Versicor Inc., CA, USA) (LY-303366) is a semi-synthetic antifungal agent with a structure related to cilofungin (LY-101216). Its structure corresponds to a cyclic lipopeptide, which inhibits ß-1,3-glucan biosynthesis by affecting the glucan-synthase enzyme. The result is fungal cell damage and death. Both the spectrum of action and the toxicity profile are superior to those of cilofungin, being highly active in vitro against fluconazole-resistant and -susceptible C. albicans, Aspergillus spp. and the cyst form of Pneumocystis carinii; it has no activity against C. neoformans.7 Kill-curves of anidulafungin are similar to those produced by amphotericin B for C. albicans, Candida glabrata and Candida krusei.8 The activity in vivo of this echinocandin has been good in animal models (per os) of systemic candidiasis and in experimental pneumonia infections due to P. carinii, as well as in infections produced by Aspergillus fumigatus (intravenous).5,9 Ongoing Phase II clinical trials will focus on the safety and efficacy of the intravenous treatment with anidulafungin for patients suffering invasive candidiasis. The present study evaluated the fungistatic (MICs) antifungal activities in vitro of anidulafungin, fluconazole and itraconazole for Candida spp. The fungicidal activity (MFCs) of anidulafungin was evaluated for 230 of the 460 isolates included in the study.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Antifungals

Anidulafungin, as the standard powder LY-303366, was kindly provided by Lilly Pharmaceuticals (Indiana, IN, USA), fluconazole by Pfizer (Sandwich, Kent, UK) and itraconazole by Janssen Research Foundation (Beerse, Belgium) as standard powders. Stock solutions of anidulafungin and itraconazole (1600 mg/L) were prepared in 100% dimethyl sulphoxide (DMSO) (Merck, Darmstad, Germany) and fluconazole stock solutions were prepared in sterile distilled water. Additive two-fold drug dilutions were prepared in the corresponding solvents (100% strength) followed by further dilutions in standard RPMI 1640 broth to twice the strength required for the final concentrations of 0.003–16 mg/L (anidulafungin, itraconazole) and 0.125–64 mg/L (fluconazole).

Strains

A total of 460 clinical yeast isolates were evaluated: C. albicans (n = 317), Candida dubliniensis (n = 38), Candida tropicalis (n = 27), C. glabrata (n = 25), C. krusei (n = 18), Candida lusitaniae (n = 15), Candida famata (n = 10) and Candida parapsilosis (n = 10). The NCCLS quality control (QC) Candida parapsilosis ATCC 22019 and C. krusei ATCC 6358 strains3 were included as controls each time a set of isolates was tested. The 460 isolates were recovered from blood, vaginal, urine and oropharyngeal clinical specimens; 210 from patients in the USA and 250 from Spain. These isolates were maintained at room temperature in sterile distilled water and subcultured before testing.

Inoculum preparation

Yeast inoculum suspensions were obtained by taking five colonies (>1 mm diameter) from 24-h-old cultures grown on Sabouraud dextrose agar. The colonies were suspended in 5 mL of sterile saline (0.85% NaCl). The inoculum suspensions were shaken for 15 s and the inoculum density was adjusted to the turbidity of a 0.5 McFarland Standard (equivalent to 1–5 x 106 cfu/mL) with sterile saline. The suspensions were diluted 1:1000 in RPMI 1640 to give a final inoculum suspension equivalent to 0.5–2.5 x 103 cfu/mL.

Microdilution tests

The broth microdilution assay was carried out following the standard conditions described by the NCCLS3 with sterile, 96-well, round-bottomed plates (Corning, New York, NY, USA) and standard RPMI 1640 buffered to pH 7 with MOPS as the test medium. On the day of the test, each microdilution well containing 100 µL of the corresponding two-fold drug dilution was inoculated with 100 µL of the two-fold-diluted cell suspension. Growth and sterility controls were included for each isolate–drug combination.

Incubation and scoring of MIC wells

The microdilution trays were incubated at 35°C and MICs were determined visually after 48 h of incubation, using an inverted mirror. The growth in each MIC well was compared with the growth in the control (antifungal-free) well. MICs of anidulafungin were the lowest drug concentration wells that were optically clear (without any visible growth/turbidity). MICs of fluconazole and itraconazole were the lowest drug concentration wells that showed a prominent reduction (approximately >=50%) in growth/turbidity.3

MFCs of anidulafungin

MFCs of anidulafungin were also determined for 215 C. albicans and five isolates each of C. krusei, C. glabrata and C. tropicalis recovered from a single clinical trial in the USA from oropharyngeal infections. Briefly, 20 µL aliquots were subcultured from each well that showed complete inhibition (optically clear) and from the growth control onto Sabouraud dextrose agar plates. The plates were incubated at 35°C until growth was seen in the growth control subculture (usually 48 h). The MFC was the lowest drug concentration that resulted in either no growth or fewer than three colonies.

Data analysis

The MIC50 and MIC90 values were calculated as the concentrations of antifungal agent that were able to inhibit 50% and 90% of the isolates, respectively. Geometric mean MICs were obtained to facilitate comparisons of the activities of the three drugs. The results were processed statistically using SPSS PC+ version 9.0 for IBM PC. The criterion for statistical significance was P < 0.05.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Efforts made to standardize methods for antifungal susceptibility testing have resulted in the development of broth macro- and microdilution standard methods by the NCCLS.3 This methodology has been applied to the evaluation of antifungal activities of investigational agents, including anidulafungin,7 which is undergoing clinical evaluations (Phase II) before FDA approval. Because of that, we further investigated the antifungal activity of anidulafungin against the 460 yeasts by following the standard conditions for broth microdilution testing described in the NCCLS document. Table 1 summarizes the data for the three agents in vitro for the 460 clinical isolates evaluated in the present study. The overall mean MIC of anidulafungin was 1.01 mg/L, which indicated a higher fungistatic antifungal activity in vitro of this echinocandin than those of itraconazole (1.97 mg/L) and fluconazole (19.4 mg/L); these differences were statistically significant (P < 0.05). The antifungal activity in vitro of anidulafungin was superior to that of itraconazole and fluconazole for C. albicans, C. tropicalis, C. glabrata and C. krusei (Table 1). But, anidulafungin was less active against C. famata and C. parapsilosis. The differences were not significant for C. dubliniensis and C. lusitaniae. Our results are in agreement with those reported by other investigators.7,9,10 As previously demonstrated,7 anidulafungin had good fungicidal activity against C. albicans, MFC range 0.03–0.5 mg/L (MFC50 0.12 mg/L and MFC90 0.5 mg/L). For other species, the MFC ranges were: C. krusei 0.06–0.5 mg/L, C. glabrata 0.2–2.0 mg/L and C. tropicalis 0.2–0.5 mg/L. MFCs were usually the same as MICs or only one to two dilutions higher.


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Table 1.  In vitro susceptibilities (mg/L) of 460 clinical yeast isolates to anidulafungin, fluconazole and itraconazole
 
In addition, our data demonstrated that anidulafungin had a potent activity in vitro against both C. krusei and C. glabrata (Table 1); these two species are either inherently resistant to fluconazole (C. krusei) or have lower susceptibility (C. glabrata) to this triazole than other Candida species. The MICs of anidulafungin are relatively high for C. parapsilosis when they are compared with those of other Candida species, but the MIC range was broad for this species. The lower susceptibility of C. parapsilosis to both echinocandins, caspofungin and anidulafungin, has been reported previously.9

In summary, anidulafungin has potential as an active antifungal agent for the treatment of fungal infections caused by most Candida species, including those species that have low susceptibility or are resistant to fluconazole. The relevance of these data in vitro has to be validated in clinical trials. However, the clinical response can be affected by the inadequate concentration of the antifungal agent at the site of the infection, by impaired host defence mechanisms or by other host–fungus and antifungal agent interactions.


    Acknowledgements
 
This study was partially supported by Lilly Pharmaceuticals.


    Footnotes
 
* Corresponding author. Tel: +34-922-319-376; Fax: +34-922-319-378; E-mail: mpareval{at}ull.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Sandven, P. (2000). Epidemiology of candidemia. Revista Iberoamericana de Micología 17, 73–81.

2 . Denning, D. W. (1997). Echinocandins and pneumocandins, a new antifungal class with a novel mode of action. Journal of Antimicrobial Chemotherapy 40, 611–4.[Free Full Text]

3 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast: Approved Standard M27-A. NCCLS, Wayne, PA, USA.

4 . Conly, J., Rennie, R., Johnson, J., Farah, S. & Hellman, L. (1992). Disseminated candidiasis due to amphotericin B-resistant Candida albicans. Journal of Infectious Diseases 165, 761–4.[ISI][Medline]

5 . Oakley, K. L., Moore, C. B. & Denning, D. W. (1998). In vitro activity of the echinocandin antifungal agent LY 303366 in comparison with itraconazole and amphotericin B against Aspergillus spp. Antimicrobial Agents and Chemotherapy 42, 2726–30.[Abstract/Free Full Text]

6 . Powderly, W. G., Kobayashi, G. S., Herzig, G. P. & Medoff, G. (1988). Amphotericin B-resistant yeast infection in severely immunocompromised patients. American Journal of Medicine 84, 826–32.[ISI][Medline]

7 . Espinel-Ingroff, A. (1998). Comparison of in vitro activities of the new triazole SCH56592 and the echinocandin MK-0991 (L-743,872) and LY303366 against opportunistic filamentous and dimorphic fungi and yeasts. Journal of Clinical Microbiology 36, 2950–6.[Abstract/Free Full Text]

8 . Karlowsky, J. A., Harding, G. A., Zelnitsky, S. A., Hoban, D. J., Kabani, A., Bello, T. Y. et al. (1997). In vitro kill curves of a new semisynthetic echinocandin, LY 303366, against fluconazole sensitive and resistant Candida species. Antimicrobial Agents and Chemotherapy 41, 2576–8.[Abstract]

9 . Zhanel, G. G., Karlowsky, J. A., Harding, G. A., Valko, T. V., Zelenitsky, S. A., Friesen, M. et al. (1997). In vitro activity of a new echinocandin, LY-303366, against systemic isolates of Candida species, Cryptococcus neoformans, Blastomyces dermatitidis, and Aspergillus species. Antimicrobial Agents and Chemotherapy 41, 863–5.[Abstract]

10 . Chavez, M., Bernal, S., Valverde, A., Gutierrez, M. J., Quindós, G. & Martin-Mazuelos, E. (1999). In-vitro activity of voriconazole (UK-109,496), LY303366 and other antifungal agents against oral Candida spp. isolates from HIV-infected patients. Journal of Antimicrobial Chemotherapy 44, 697–700.[Abstract/Free Full Text]