Cryptococcus gattii: in vitro susceptibility to the new antifungal albaconazole versus fluconazole and voriconazole

Transcription

1 Medical Mycology September 2005, 43, 505 /510 Cryptococcus gattii: in vitro susceptibility to the new antifungal albaconazole versus fluconazole and voriconazole YOLANDA MORERA-LÓPEZ*, JOSEP M. TORRES-RODRÍGUEZ$, TERESA JIMÉNEZ-CABELLO & TERESA BARÓ-TOMÁS$ *Clinical and Experimental Mycology Research Unit, Institut Municipal d Investigació Mèdica, Barcelona and $Autonomous University of Barcelona, Barcelona, Spain Introduction Cryptococcal disease constitutes a severe complication in HIV-infected patients under inadequate or no medication [1]. Cryptococcosis may be the first manifestation of AIDS in subjects unaware of their condition as HIV-infected people [2]. It is also a frequent ailment in patients with lymphoma, leukaemia, systemic lupus erythematosus, or liver cirrhosis [3]. In a limited number of patients, cryptococcal infection occurs in the absence of underlying disorders or immunosuppression [4], Cryptococcus gattii being the most important agent in the immunocompetent population. Received 2 March 2004; Accepted 11 November 2004 Correspondence: Josep M. Torres-Rodríguez, URMEC/IMIM, Dr. Aiguader 80, E-08003, Barcelona, Spain. Tel: / ; Fax: / ; jmtorres@imim.es Minimal inhibitory concentrations (MIC) and minimal fungicidal activity of albaconazole, voriconazole and fluconazole against 55 strains of Cryptococcus gattii, clinically or environmentally isolated in Spain and some Latin American countries, were assessed. By means of the microbroth method (National Committee for Clinical Laboratory Standards; document M27-A2), the geometric mean value for fluconazole was 5.01 mg/ml; however, MIC for 12.7% of isolates ranged from 16 to 32 mg/ml, suggesting increased resistance against fluconazole. Geometric mean values of 0.02 and 0.03 mg/ml for albaconazole and voriconazole, respectively, were found, indicating not only a higher susceptibility to these new azoles but also a better performance of albaconazole (P / 0.003). Minimal fungicidal concentrations were also very low for albaconazole and voriconazole (PB/0.001; geometric mean values of mg/ml and 0.07 mg/ml, respectively). Both azoles may be good alternatives for the treatment of C. gattii cryptococcosis. Keywords albaconazole, Cryptococcus gattii, fluconazole, in vitro susceptibility, voriconazole Studies by Franzot et al. [5] on the URA5 sequences and DNA fingerprinting patterns of Cryptococcus neoformans var. neoformans suggested significant genetic differences between serotypes A and D. Therefore they proposed the creation of a new variety called Cryptococcus neoformans var. grubii for the serotype A isolates maintaining the variety neoformans for serotype D isolates. Recently, Kwon-Chung et al. [6] proposed specie status for C. neoformans var. gattii (i.e. Cryptococcus gattii), on the basis that it formed a distinct monophyletic cluster separate from C. neoformans var. grubii and C. neoformans var. neoformans. Most cases of cryptococcosis are due to C. neoformans and particularly to serotype A (C. neoformans var. grubii) [7] but C. gattii infects both animals and humans, particularly immunocompetent subjects [8]. Disseminated and meningeal forms of cryptococcosis are still associated with a high mortality rate despite antifungal treatment [9]. First-line antifungal agents 2005 ISHAM DOI: /

2 506 Morera-López et al. are amphotericin B and 5-fluorocytosine. Dangerous side-effects and capacity of the latter drug to generate resistance make compelling arguments for the search of safer and more active antibiotics against cryptococcosis [10 /12]. Some C. neoformans strains are also resistant to fluconazole [13,14], an antifungal that, although less harmful, is not the drug of choice for the treatment of severe cryptococcal disease in AIDS patients [10]. Hence the relevance of assessing the in vitro susceptibility of C. gattii against new azole agents, such as voriconazole [15] and albaconazole (UR-9825) [16]. Currently, there is a shortage of information about in vitro antifungal activity of albaconazole, although some reports have suggested a high sensitivity of Candida albicans and related species [16] and some filamentous fungi [17] to this azole. The susceptibility of 12 strains of C. neoformans to albaconazole has been reported [18]. The objective of this study was to determine minimal inhibitory concentrations (MIC) and minimal fungicidal activity of fluconazole, voriconazole and albaconazole against clinical and environmental isolates of C. gattii. Materials and methods Isolates A total of 55 C. gattii isolates were studied, 53 belonging to serotype B, and 2 to serotype C. Clinical isolates (n/41) were recovered from cerebrospinal fluid, central nervous system, broncho-alveolar lavage, lung samples from either necropsy, sputum, or blood, in patients with meningeal or pulmonary cryptococcosis admitted to hospitals in Brazil, Colombia, Mexico, Uruguay and Peru. Four isolates were taken from HIVinfected subjects while the remaining 37 specimens were from meningitis patients lacking any previous clinical history of other ailments. Results of HIV testing were negative in 24 patients but not available in 13 cases. Spanish isolates were taken from seven goats affected by cryptococcal pneumonia. Environmental samples (n /7) were recovered from plant debris (4) (Eucalyptus spp., Terminalia catappa), and from soil locations associated with pigeons (1), bats (1), and wasp nests (1) (Table 1). Two additional C. neoformans isolates were included because of their high MIC to fluconazole ( /64 mg/ml). Identification of all isolates were reconfirmed in our laboratory, by culture in Staib s medium, sugar auxanogram (Auxacolor; Biorad, Marnes la Coquette, France), urea test and culture in CGB medium (L-canavanine-glycine-bromothymol blue) to differentiate C. gattii (formerly C. neoformans var. gattii) from C. neoformans. Serotyping was performed by agglutination with capsular polysaccharide-specific polyclonal antibody (Crypto-Check commercial kit; Iatron Laboratories, Tokyo, Japan). Reference isolates, Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) were used for MIC quality control. Isolates were stored before testing at /208C in skimmed milk until study. Prior to testing, each isolate was subcultured in Sabouraud dextrose agar plates for species confirmation and to ensure purity and optimal growth. Antifungal agents Fluconazole (batch 10949/GR/19) and voriconazole (batch , 11/2000) were provided by Pfizer Pharmaceuticals, Central Research Division (Groton, CT, USA), while albaconazole (UR 9825, batch NE-5244-B-02-00) was kindly given by J. Uriach & Cía S.A. (Palau Solità i Plegamans, Barcelona, Spain). Stock solutions of fluconazole (6.4 mg/ml in water) and either voriconazole or albaconazole (400 mg/ml in dimethyl sulfoxide) were prepared. Antifungal agents were diluted as described in the NCCLS document M27-A2 [19]. Final antifungal concentrations ranged from to 64 mg/ml for fluconazole and to 4 mg/ml for voriconazole or albaconazole. MIC determination Mean inhibitory concentrations were determined on sterile microdilution plates in RPMI 1640 medium (supplemented with 18 g/l glucose, buffered to ph 7 with mol/l MOPS buffer). Aliquots of 0.1 ml were added to each well of the microdilution tray, and the final yeast inocula were adjusted to a concentration of 0.5/2.5/10 3 c.f.u./ml. In each case, the inoculum size was confirmed by colony counting. Trays were incubated at 358C, and MIC read at 48 and 72 h for Cryptococcus spp. and at 24 h for C. parapsilosis and C. krusei. Endpoint spectrophotometrical readings were measured at 414 nm with a plate reader (Multiskan MS, Labsystems, Finland). MIC were defined as the lowest drug concentration that reduced growth in 80%, compared with antifungal-free controls. Minimal fungicidal concentrations After MIC reading, aliquots (10 ml) of each yeast strain were withdrawn from wells and cultured on fresh Sabouraud dextrose agar to check fungicidal versus

3 C. gattii and albaconazole 507 Table 1 in mg/ml Characteristics of 55 Cryptococcus gattii isolates and in vitro susceptibility to albaconazole, voriconazole and fluconazole, expressed Isolate (N8) Origin Albaconazole Voriconazole Fluconazole MIC MFC MIC MFC MIC Spanish clinical GR/48 A Goat, lung, Cáceres GR/50 A Goat, lung, Cáceres GR/52 A Goat, brain, Cáceres GR/53 A Goat, lung, Cáceres GR/56 A Goat, intestinal, Cáceres GR/59 A Goat, lung, Cáceres GR/104 C Goat, brain, Cáceres Brazilian clinical GR/39 A CSF, HIV/, Paraná GR/41 A CSF, HIV/, Paraná GR/247 C CSF, HIV/, Curitiba GR/248 C CSF, HIV/, Curitiba GR/249 C CSF, HIV/, Curitiba GR/250 C CSF, HIV/, Curitiba GR/168 C CSF, HIV/, Rio de Janeiro GR/169 C CSF, Rio de Janeiro GR/170 C CSF, HIV/, Rio de Janeiro GR/171 C CSF, HIV/, Rio de Janeiro GR/172 C CSF, HIV/, Rio de Janeiro GR/173 C CSF, HIV/, Rio de Janeiro GR/174 C CSF, HIV/, Rio de Janeiro GR/175 C CSF, HIV/, Rio de Janeiro GR/160 C CSF, Porto Alegre GR/161 C CSF, Porto Alegre GR/162 C CSF, Porto Alegre GR/163 C Sputum, Porto Alegre GR/164 C BAL, Porto Alegre GR/165 C CSF, Porto Alegre GR/166 C CSF, Porto Alegre GR/167 C Sputum, Porto Alegre GR/24 C* CSF, Porto Alegre Brazilian environmental GR/73 A Soil sites associated with bats, Rio de Janeiro GR/74 A Eucalyptus camaldulensis, Rio de Janeiro Mexican clinical GR/267 C CSF, HIV/, Distrito Federal GR/268 C CSF, HIV/, Distrito Federal GR/269 C CSF, HIV/, Distrito Federal GR/270 C CSF, HIV/, Distrito Federal GR/271 C CSF, Distrito Federal GR/257 C CSF, HIV/, Distrito Federal GR/258 C CSF, HIV/, Distrito Federal GR/259 C CSF, HIV/, Distrito Federal GR/260 C CSF, HIV/, Distrito Federal GR/261 C CSF, Distrito Federal GR/263 C CSF, HIV/, Distrito Federal GR/264 C CSF, HIV/, Distrito Federal GR/265 C CSF, HIV/, Distrito Federal GR/266 C CSF, HIV/, Distrito Federal Colombian clinical GR/343 C CSF, HIV/, Arauca GR/331 C CSF, HIV/, Antioquia Colombian environmental GR/160 A* Terminalia catappa, Cúcuta GR/161 A Plant debris, Cúcuta

4 508 Morera-López et al. Table 1 (Continued) Isolate (N8) Origin Albaconazole Voriconazole Fluconazole MIC MFC MIC MFC MIC Peru clinical GR/327 C CSF, Lima Peru environmental GR/162 A Plant debris, Lima Uruguay clinical GR/310 C CSF, AIDS patient, Montevideo Uruguay environmental GR/46A Soil associated with wasp nest, Treinta y tres GR/47 A Soil, Montevideo All the isolates were B serotype except two C*. MIC, minimal inhibitory concentration; MFC: minimal fungicidal concentration. fungistatic effect of the azoles under study. Plates were then incubated at 358C for 72 h to measure growth of fungal colonies. Minimal fungicidal concentration (MFC) was the lowest concentration of antifungal required to completely inhibit fungal growth. Statistical analysis Minimal inhibitory concentration values were expressed as the geometric mean with maximum and minimum values. The Wilcoxon rank-sum test was used for statistical analysis, statistical significance being set at PB/0.05. Data were analysed with the SPSS computer program. Results Quality control strains produced MIC values against fluconazole as expected, namely 1 mg/ml for C. parapsilosis (ATCC 22019) and 64 mg/ml for C. krusei (ATCC 6258). These species were highly sensitive to albaconazole (0.008 and mg/ml, respectively) and slightly less sensitive to voriconazole (0.5 and 1 mg/ml, respectively). Table 1 presents MIC and MFC values for the 55 C. gattii isolates studied. Two additional C. neoformans isolates, not included in Table 1, were resistant to fluconazole (MIC /64 mg/ml), though highly sensitive to albaconazole (0.008 and 0.03 mg/ml) and voriconazole (0.016 and 0.06 mg/ml). This was also true for seven lesser fluconazole-sensitive C. gattii isolates (MIC between 16 and 32 mg/ml, Table 1), all of them from clinical specimens collected in Brazil (3), Colombia (2), and Mexico (2). Their MIC ranged from to 0.5 mg/ml for albaconazole and from to mg/ml for voriconazole. Environmental specimens of C. gattii showed MIC between 0.5 and 8 mg/ml for fluconazole and B/0.03 mg/ml for the new triazoles. In isolates of animal origin, MIC ranged from 1 to 8 mg/ml for fluconazole and from to mg/ml for the other two antifungals tested. Overall, the activity of these three antifungals against C. gattii followed the sequence: albaconazole /voriconazole/fluconazole (P / 0.003; Table 2). Albaconazole and voriconazole showed high fungicidal effect against all C. gattii isolates, at very low concentrations. Voriconazole MFC ranged from to 0.5 mg/ml, with the exception of one Brazilian clinical isolate whose MFC reached 1 mg/ml. Albaconazole MFC were 5/0.5 mg/ml. Discussion Cryptococcus neoformans is rarely resistant to azoles [20], although the clinical course of cryptococcosis in patients on fluconazole treatment is less favourable with high MIC [21]. Two clinical fluconazole-resistant C. neoformans isolates with MIC /64 mg/ml were included in our study in order to determine their behaviour against albaconazole and voriconazole. As both isolates were highly sensitive to either triazole, the Table 2 Geometric mean, range, minimal inhibitory concentrations (MIC) 50% and 90%, and minimal fungicidal concentrations (MFC) of albaconazole, voriconazole and fluconazole, expressed in mg/ml Antifungal agent Geometric mean Range MIC 50% MIC 90% MIC Albaconazole to Voriconazole to Fluconazole to MFC Albaconazole to Voriconazole to

5 C. gattii and albaconazole 509 possible use of these drugs in fluconazole-resistant cases of cryptococcosis might be considered. Although breakpoint values for susceptibility have not yet been determined for voriconazole or albaconazole, values reported for itraconazole in Candida spp. were taken for comparison, as suggested by others [22]. Under these criteria, none of the C. gattii strains are considered resistant to the new triazoles. In a recent study, albaconazole MIC and MFC values generated by the macrobroth method in 12 C. neoformans isolates (two of them of the formerly gattii variety) were reported [18]. In it, lower MIC and higher MFC values were obtained. The possibility of cross-resistance against fluconazole and albaconazole was also suggested. These results agree with those of Hong & Yu [14], who report MIC values of 1/2 mg/ml for itraconazole and voriconazole in fluconazoleresistant strains. Similar results were reported by Pfaller et al. [23] in North American and African clinical isolates, with MIC between 16 and 64 mg/ml for fluconazole. There are a few reports on the susceptibility patterns of Cryptococcus serotypes, pointing to a lesser susceptibility of serotype D to azoles [20], and serotype B to sordarin derivatives (GM ) [24]. Our C. gattii samples, mostly serotype B, were susceptible to the new triazoles, even those two clinical Brazilian and Mexican strains partially resistant to fluconazole (MIC 32 mg/ ml). Albaconazole and voriconazole also displayed a potent fungicidal activity at 5/0.5 mg/ml. The in vitro activity of voriconazole against C. neoformans has been assessed in different studies [25], being at least ten times higher than fluconazole [26]. Other triazoles, such as ravuconazole (BMS ) or posaconazole, are also active in vitro against C. neoformans [27,28]. Albaconazole or UR9825 [29] is a member of a new generation of triazole antifungals with higher in vitro activity than fluconazole and itraconazole, and similar or higher than voriconazole against Candida isolates [18]. Albaconazole is also active against several species of filamentous opportunistic fungi [17], reason for which its high in vitro fungicidal activity (MFC values) may prove relevant to the treatment of potentially life-threatening mycoses, such as cryptococcosis, usually refractory to treatment with established or new antifungals, some of them, like caspofungin, not active against Cryptococcus [30]. In voriconazole-treated patients with invasive fungal infections, CSF drug concentrations ranged from 0.08 to 3.9 mg/ml, with a ratio to plasma concentration from 0.22 to 1.0 [31]. Sorbera et al. [32] reported that albaconazole concentration in CSF of rabbits with experimental cryptococcosis was not higher than 15% that of plasma levels. These data suggest the potential use of both azoles in cryptococcosis. Some available data suggest cross-resistance between voriconazole and posaconazole [33]. This does not seem to be the case in our study, as no cross resistance between fluconazole and the other two triazoles was observed in the large number of C. gattii isolates tested. It has been suggested that resistance to fluconazole and voriconazole may be inherent and that subsets of C. neoformans isolated from patients with recurrent cryptococcosis may be selected from clonal populations highly resistant to fluconazole and moderately resistant to voriconazole [34]. The high susceptibility of C. gattii to voriconazole and albaconazole may decrease as both antifungals become more widely used as drugs of choice in clinical practice. So far, the former is of limited use because of its recent introduction into the market, while the latter is yet to be introduced in medical practice. It is to be expected that widespread use of both antifungals will increase the frequency of detection of more resistant isolates. Therefore, systematic prospective studies on in vitro susceptibility of C. neoformans and C. gattii are warranted. Acknowledgements This study was supported by grant PI from Fondo de Investigaciones Sanitarias (FIS), Spanish Ministry of Health. We thank Joan Vila, MsC for statistical analysis, and Drs B. Wanke (Fundación Oswaldo Cruz, Rio de Janeiro, Brazil), E. Castañeda (Instituto Nacional de Salud, Bogotá, Colombia), R. López-Martínez (Facultad de Medicina, Universidad Autónoma de México, Mexico), E. Gezuele (Instituto de Higiene, Universidad de la República, Montevideo, Uruguay), and B. Bustamante (Instituto Von Humbold, Lima, Peru) for providing the isolates used in this study, and Professor Donal W. R. Mackenzie and Ana Espinel-Ingroff for the critical review of the manuscript. References 1 Dromer F, Mathoulin-Pelissier S, Fontanet A, et al. Epidemiology of HIV-associated cryptococcosis in France (1985/2001): comparison on the pre- and post-haart eras. AIDS 2004; 18: 555/ Hajjeh RA, Brandt ME, Pinner RW. Emergence of cryptococcal disease: epidemiological perspectives 100 years after its discovery. Epidemiology Rev 1995; 17: 303/ Flagg SD, Chang YJ, Masuell CP, et al. Myositis resulting from disseminated cryptococcosis in a patient with hepatitis C cirrhosis. Clin Infect Dis 2001; 32: 1104/ Dromer F, Mathoulin S, Dupont B, et al. French Cryptococcus Study Group. Epidemiology of cryptococcosis in France: a 9-year survey (1985/1993). Clin Infect Dis 1996; 23: 82/90.

6 510 Morera-López et al. 5 Franzot SP, Salkin IF, Casadevall A. Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates. J Clin Microbiol 1999; 37: 838/ Kwon-Chung KJ, Boekhout T, Fell JW, et al. Proposal to conserve the name Cryptococcus gattii against C. hondurianus and C. bacillisporus (Basidiomycota, Hymenomycetes, Tremenomycetidae). Taxon 2002; 51: 804/ Baró T, Torres-Rodríguez JM, Morera Y, et al. Serotyping of Cryptococcus neoformans isolates from clinical and environmental sources in Spain. J Clin Microbiol 1999; 37: 1170/ Speed B, Dunt D. Clinical and host differences between infections with the two varieties of Cryptococcus neoformans. Clin Infect Dis 1995; 21: 28/34. 9 Pappas PG, Perfect JR, Cloud GA, et al. Cryptococcosis in human immunodeficiency virus negative patients in the era of effective azole therapy. Clin Infect Dis 2001; 33: 690/ Saag MS, Graybill RJ, Larsen RA, et al. Practice guidelines for the management of cryptococcal disease, Infectious Society of America. Clin Infect Dis 2000; 30: 710/ Kantarcioglu A, Yücel A. A flucytosine-resistant Cryptococcus neoformans (serotype D) strain isolated in Turkey from cutaneous lesions. Med Mycol 2002; 40: 519/ Lozano-Chiu M, Paetznick VL, Ghanoum MA, et al. Detection of resistance to Amphotericin B among Cryptococcus neoformans clinical isolates: performances of three different media assessed by using E-test and National Committee for Clinical Laboratory Standards M27-A methodologies. J Clin Microbiol 1998; 36: 2817/ Paugam A, Dupouy-Camet J, Blanche P, et al. Increased fluconazole resistance of Cryptococcus neoformans isolated for a patient with AIDS and recurrent meningitis. Clin Infect Dis 1994; 19: 975/ Hong N, Yu CY. In vitro comparative efficacy of voriconazole and itraconazole against fluconazole susceptible and resistant Cryptococcus neoformans isolates. Antimicrob Agents Chemother 1998; 42: 471/ Donnelly JP, De Pauw BE. Voriconazole / a new therapeutic agent with an extended spectrum of antifungal activity. Clin Microbiol Infect 2004; 10(Suppl. 1): 107/ Ramos G, Cuenca-Estrella M, Monzón A, et al. In vitro comparative activity of UR/9825, itraconazole and fluconazole against clinical isolates of Candida spp. J Antimicrob Chemother 1999; 44: 283/ Capilla J, Ortoneda M, Pastor FJ, et al. In vitro antifungal activity of the new triazole UR-9825 against clinically important filamentous fungi. Antimicrob Agents Chemother 2001; 45: 2635/ Miller JL, Schell WA, Wills EA, et al. In vitro and in vivo efficacies of the new triazole albaconazole against Cryptococcus neoformans. Antimicrob Agents Chemother 2004; 48: 384/ National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing for yeasts: Approved Standard M27-A2, 2nd edn. Wayne, PA: National Committee for Clinical Laboratory Standards, Baró T, Torres-Rodríguez JM, Alía C, et al. Relationship between in vitro activity of four antifungal drugs and the serotypes of Cryptococcus neoformans. J Mycol Med 2001; 11: 185/ Aller AI, Martín-Mazuelos E, Lozano F, et al. Correlation of fluconazole MICs with clinical outcome in cryptococcal infections. Antimicrob Agents Chemother 2000; 44: 1544/ Nguyen MH, Yu CY. In vitro comparative efficacy of voriconazole and itraconazole against fluconazole-susceptible and -resistant Cryptococcus neoformans isolates. Antimicrob Agents Chemother 1998; 42: 471/ Pfaller MA, Zhang J, Messer SA, et al. In vitro activities of voriconazole, fluconazole and itraconazole against 566 clinical isolates of Cryptococcus neoformans from United States and Africa. Antimicrob Agents Chemother 1999; 43: 169/ Torres-Rodríguez JM, Morera Y, Baro T, et al. In vitro susceptibility of Cryptococcus neoformans serotypes to GM derivative of the sordarin class. Mycoses 2002; 45: 1/4. 25 Espinel-Ingroff A, Boyle K, Sheehan DJ. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature. Mycopathologia 2001; 150: 101/ Brummer E, Kamei K, Miyaji M. Anticryptococcal activity of voriconazole against Cryptococcus neoformans var. gattii vs var. neoformans: comparison with fluconazole and effect of human serum. Mycopathologia 1998; 142: 3/7. 27 Yamazumi T, Pfaller MA, Messer SA, et al. In vitro activities of ravuconazole (BMS ) against 541 clinical isolates of Cryptococcus neoformans. Antimicrob Agents Chemother 2000; 44: 2883/ Perfect JR, Cox GM, Dodge RK, et al. In vitro and in vivo efficacies of the azole SCH5692 against Cryptococcus neoformans. Antimicrob Agents Chemother 1996; 40: 1910/ Bartroli J, Turmo E, Algueró M,et al. New azole antifungals. 3. Synthesis and antifungal activity of 3-substituted 4(3H)-quinazolinones. J Med Chem 1998; 41: 1869/ Krishnarao TV, Galgiani JN. Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans. Antimicrob Agents Chemother 1997; 41: 1957/ Lutsar I, Roffey S, Troke P. Voriconazole concentrations in the cerebrospinal fluid and brain tissue guinea pigs and immunocompromised patients. Clin Infect Dis 2003; 37: 28/ Sorbera LA, Bartroli J, Castañer J. Albaconazole antifungal. Drugs of the Future 2003; 28: 529/ Yildiran ST, Fothergill AW, Sutton DA, et al. In vitro susceptibilities of cerebrospinal fluid isolates of Cryptococcus neoformans collected during a ten-year period against fluconazole, voriconazole and posaconazole (SCH56592). Mycoses 2002; 45: 378/ Mondon P, Petter R, Amalfitano G, et al. Heteroresistance to fluconazole and voriconazole in Cryptococcus neoformans. Antimicrob Agents Chemother 1999; 43: 1856/1861.