Medical Mycology May 2005, 43, 281 /284
Short Communication
In vitro activities of new and established triazoles against opportunistic filamentous and dimorphic fungi
Med Mycol Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/12 For personal use only.
GLORIA M. GONZA´LEZ*$, ANNETTE W. FOTHERGILL$, DEANNA A. SUTTON$, MICHAEL G. RINALDI$% & DAVID LOEBENBERG§ *Departamento de Microbiologı´a, Facultad de Medicina, Universidad Auto´noma de Nuevo Leo´n, Monterrey, Nuevo Leo´n, Me´xico, $Department of Pathology, University of Texas Health Science Center at San Antonio and %Audie L. Murphy Division, South Texas Veterans Health Care System, San Antonio, Texas, and §Schering-Plough Research Institute, Kenilworth, New Jersey, USA
The in vitro activities of three new triazoles were determined and compared to those of itraconazole and fluconazole against 306 clinical isolates of Blastomyces dermatitidis, Cladophialophora carrionii, Coccidioides immitis, Fonsecaea pedrosoi, Fusarium spp., Histoplasma capsulatum, Paecilomyces lilacinus, Pseudallescheria boydii and Sporothrix schenckii. Minimum inhibitory concentrations (MIC) were determined by a broth macrodilution method of the National Committee for Clinical Laboratory Standards M38-A procedure. Itraconazole (geometric mean MIC, 0.16 0.65 mg/ml), voriconazole (geometric mean MIC, 0.18 1.44 mg/ml), ravuconazole (geometric mean MIC, 0.18 1.09 mg/ml), and posaconazole (geometric mean MIC, 0.18 1.38 mg/ml), had relatively uniform values showing potent in vitro inhibitory activity against B. dermatitidis, C. carrionii, C. immitis, F. pedrosoi, H. capsulatum, and S. schenckii. The in vitro activity was variable with strains of P. boydii, P. lilacinus and Fusarium spp. /
/
/
/
Keywords
fluconazole, itraconazole, posaconazole, ravuconazole, voriconazole
Introduction In the last 20 years, opportunistic fungal infections have increased in frequency. Among the opportunistic mycoses, invasive diseases caused by filamentous fungi are commonly associated with high morbidity and mortality. While Aspergillus species are the most common filamentous pathogens, Pseudallescheria boydii , Paecilomyces lilacinus and Fusarium species have emerged as important human pathogens, particularly in the immunocompromised host [1 /4]. On the other hand, the number of infections
Received 16 March 2004; Accepted 5 February 2005 Correspondence: Gloria M. Gonza´lez, Facultad de Medicina, Universidad Auto´ noma de Nuevo Leo´ n, Departamento de Microbiologı´a, Madero y Dr Eduardo A. Pequen˜o s/n, Colonia Mitras Centro, Monterrey, NL, Me´xico 64460. Tel.: /5281 8329 4166; Fax: /5281 8348 5477; E-mail:
[email protected]
– 2005 ISHAM
caused by dimorphic fungi such as Coccidioides immitis, Histoplasma capsulatum and others have also increased in these patient populations [5,6]. Unfortunately, conventional antifungal therapy is frequently associated with therapeutic failures and/ or toxicity [7]. As a result of these trends, several antifungal compounds are under clinical evaluation. Among these, three new triazoles: voriconazole (VCZ), posaconazole (POS), and ravuconazole (RVZ) have potent activity against both yeast and molds [8 /14]. Although several studies of the in vitro activities of some conventional and investigational triazoles have been conducted, none of these have included head-tohead comparisons. In this study, we evaluated the in vitro activity of VCZ, POS and RVZ against a wide number of opportunistic filamentous and dimorphic fungi and compared it to those of itraconazole (ITZ), fluconazole (FCZ). DOI: 10.1080/13693780500088416
282
Gonza´lez et al.
Materials and methods
Med Mycol Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/12 For personal use only.
Microorganisms A total of 306 clinical isolates of opportunistic filamentous and dimorphic fungi were selected for this study. The strains, from very diverse clinical sources, were Blastomyces dermatitidis (6), Cladophialophora carrionii (17), Coccidioides immitis (125), Fonsecaea pedrosoi (8), Fusarium spp. (57), Histoplasma capsulatum (28), Paecilomyces lilacinus (22), Pseudallescheria boydii (28) and Sporothrix schenckii (15). The isolates were identified at the Microbiology Department, Medical School, Autonomous University of Nuevo Leon, Mexico. Each isolate was maintained as a suspension in water at room temperature until testing was performed.
Growth of inocula Fungi were grown on potato flakes agar slants prepared in-house [15] at 358C for 10/15 days in duplicate to ensure viability.
forming filamentous fungi [16]. After adequate sporulation occurred, the mycelium was overlaid with sterile distilled water, and suspensions were made by softly scraping the colonies with wooden applicators. Heavy fragments were allowed to settle, and the upper, homogeneous supernatant was transferred to sterile tubes. Inoculum suspensions of 106 c.f.u./ml were prepared by hemacytometer counts and then diluted to obtain a final organism concentration of 1 /5 /104 c.f.u./ml. The inoculum size for all tests was verified by plating 10 ml of each inoculum onto Sabouraud dextrose agar plates, incubating the plates at 358C, and counting the number of colonies. Previously prepared frozen drug samples containing 0.1 ml of each drug were allowed to thaw and inoculated with 0.9-ml volumes of the inoculum suspensions. A drug-free growth control tube was included for each isolate. Tubes were incubated at 358C. The MIC were determined when the control tubes displayed appropriate growth. The MIC was defined as the drug concentration that had turbidity corresponded to 50% inhibition of the growth control tube.
Antifungal agents FCZ and VRZ (Pfizer, New York, NY), ITZ (Janssen Pharmaceutica, Beerse, Belgium), RVZ (Bristol-Myers Squibb, Princeton, NJ), and POS (Schering-Plough Research Institute, Kenilworth, NJ) were obtained as reagent-grade powders from their respective manufacturers. Stock solutions were prepared in polyethylene glycol (ITZ, VCZ, POS), water (FCZ) and dimethyl sulfoxide (RVZ). Serial two-fold dilutions of each antifungal agent were prepared to obtain final drug concentration ranges from 0.125 to 64 mg/ml for FCZ and VCZ and from 0.015 to 8.0 mg/ml for ITZ, POS and RVZ. The drug dilutions were dispensed as 0.1-ml volumes into sterile polystyrene tubes 12 /75 mm, and stored at /208C until used.
Medium Testing was performed in RPMI-1640 with L-glutamine and morpholinepropanesulfonic acid buffer at a concentration of 165 mmol/l (Angus, Niagara Falls, NY).
Broth macrodilution method Isolates were evaluated by using National Committee for Clinical Laboratory Standards broth macrodilution approved standard reference method M38-A for broth dilution antifungal susceptibility testing of conidium-
Quality control A Paecilomyces variotii UTHSC 90-459 strain was included in all testing for quality control.
Analysis of the results The MIC ranges, geometric mean MIC and MIC of the RVZ, ITZ, FCZ, VCZ, and PCZ necessary to inhibit 50 and 90% of the isolates were calculated.
Results and discussion Most of the isolates produced sufficient growth to determine the MIC within 72 h of incubation. The exceptions were seven strains of H. capsulatum and all the strains of B. dermatitidis, which needed 5 days of the incubation. The MIC range, geometric mean MIC and MIC50 and MIC90 for RVC, ITZ, FCZ, VCZ and POS for each fungal species are summarized in Table 1. The MIC ranges for the P. variotii control strain were within the expected ranges: ITZ, 5/0.015 /0.06; FCZ, 4 /8; VCZ, 0.125 /0.25; POS, 0.015 /0.06. Overall, ITZ, VCZ, RVC and POS have a uniform in vitro activity against this group of filamentous fungi. The FCZ MIC were generally greater than the others triazoles. Our results showed that ITZ, VCZ, RVZ and POS have a potent activity against C. immitis, H. capsulatum, B. dermatitidis, C. carrionii, F. pedrosoi – 2005 ISHAM, Medical Mycology, 43, 281 /284
Triazoles against filamentous and dimorphic fungi
283
Table 1 In vitro susceptibilities of 306 isolates to ravuconazole, itraconazole, fluconazole, voriconazole and posaconazole
Med Mycol Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/12 For personal use only.
Fungi (no. of isolates)
MIC (mg/ml) Compound
Range
B. dermatitidis (6)
RVZ ITZ FCZ VRZ POS
0.125 /4 0.03 /4 4 /32 0.06 /2 0.06 /2
C. carrionii (17)
RVZ ITZ FCZ VRZ POS
0.125 /0.5 0.06 /0.5 16 /64 0.06 /0.5 0.06 /0.5
C. immitis (125)
RVZ ITZ FCZ VRZ POS
0.06 /1 0.03 /1 2 /64 0.06 /1 0.06 /1
F. pedrosoi (8)
RVZ ITZ FCZ VRZ POS
0.125 /0.5 0.06 /0.5 16 /64 0.25 /1 0.25 /1
0.22 0.19 22.62 0.35 0.32
/ / / / /
/ / / / /
Fusarium spp. (57)
RVZ ITZ FCZ VRZ POS
2 / /16 4 / /8 /64 2 /8 2 /8
NA NA NA 5.90 5.97
8 8 /64 8 8
16 /8 /64 8 8
H. capsulatum (28)
RVZ ITZ FCZ VRZ POS
0.125 /2 0.06 /1 2 /32 0.06 /2 0.03 /2
0.45 0.31 5.79 0.47 0.40
P. lilacinus (22)
RVZ ITZ FCZ VRZ POS
0.25 /2 1 / /8 16 / /64 0.5 /4 0.5 /2
1 NA NA 1.32 1
1 2 32 1 1
2 4 64 2 2
P. boydii (28)
RVZ ITZ FCZ VRZ POS
0.5 /8 0.5 /4 4 /64 0.125 /2 0.125 /2
2.21 1.56 12.19 1.13 1.05
2 2 16 2 1
4 4 32 2 2
S. schenckii (15)
RVZ ITZ FCZ VRZ POS
0.5 /4 0.25 /2 /64 1 /4 1 /4
1.09 0.65 NA 1.44 1.38
1 0.5 /64 1 1
and S. schenckii . These findings are similar and confirm other reports [12,17,18]. However, the three new triazoles and ITZ displayed a variable in vitro activity against P. boydii, P. lilacinus and Fusarium spp. and with some strains the drugs appear to lack in vitro inhibitory activity. The lack of – 2005 ISHAM, Medical Mycology, 43, 281 /284
Geometric Mean 1 0.55 8.97 0.62 0.55 0.30 0.18 26.09 0.21 0.21 0.18 0.16 9.24 0.18 0.18
50%
90%
/ / / / /
/ / / / /
0.25 0.25 16 0.25 0.25 0.125 0.125 8 0.125 0.125
0.5 0.25 4 0.5 0.5
0.5 0.5 64 0.25 0.25 0.5 0.5 32 0.5 0.5
1 1 16 1 2
4 2 /64 4 2
in vitro activity was not a surprise because of the poor activities of the available triazoles against these fungi have been published by several [19,20]. In contrast, Carrillo and Guarro [21] reported that RVZ, POS and VCZ were very active against Scedosporium apiospermum (asexual state of P. boydii ) with geometric mean
Med Mycol Downloaded from informahealthcare.com by Nyu Medical Center on 05/31/12 For personal use only.
284
Gonza´lez et al.
MIC being 0.125, 0.08 and 0.06 mg/ml, respectively. Minassian et al . [22] in their study showed that VCZ was more active against S. apiospermum (MIC /0.5 mg/ml), while RVZ and ITZ were moderately active (median MIC /2 mg/ml). For Fusarium spp., triazoles had poor activity. ITZ was the least active against Fusarium strains, generally with MIC /16 mg/ml. VCZ was moderately active (MIC 5/8 mg/ml) against many Fusarium isolates. One of the most noteworthy aspect of this in vitro study is the good activity displayed for VCZ, POS and RVZ against the strains of P. lilacinus. Until now this fungus has always been resistant to the established antifungals. We found that none of the new and established triazoles evaluated were active against all the filamentous fungi tested. Although several studies of the in vitro activities of the three new antifungal agents have been conducted, none of these have included head-tohead comparisons with conventional triazoles. On the basis of the available in vitro information described here VCZ, RVZ and POS have promising antifungal activity against some of filamentous fungi that merits further assessment of the clinical significance.
9
10
11
12
13
14
15 16
References 1 Braddley JW, Stroud TP, Salzman D, et al . Invasive mold infections in allogenic bone marrow transplant recipients. Clin Infect Dis 2001; 32: 1319 /1324. 2 Ginter G, Petutschnig, Pierer G, et al . Atypical cutaneous pseudallescheriosis refractory to antifungal agents. Mycoses 1998; 42: 507 /511. 3 Chan-Tack KM, Thio CL, Miller NS, et al . Paecilomyces lilacinus fungemia in an adult bone marrow transplant recipient. Med Mycol 1999; 37: 57 /60. 4 Patterson TF, Andriole VT, Zervos MJ, et al . The epidemiology of pseudallescheriasis complicating transplantation: nosocomial and community-acquired infection. Mycoses 1990; 33: 297 /302. 5 Fish DG, Ampel NM, Galgiani JN, et al . Coccidioidomycosis during human immunodeficiency virus infection. Medicine 1990; 69: 384 /391. 6 Johnson PC, Sarosi GA, Septimus EJ. Progressive disseminated histoplasmosis in patients with the acquired immune deficiency syndrome: a report of 12 cases and a literature review. Semin Resp Infect 1986; 1: 1 /8. 7 Gallis HA, Drew RH, Pickard WW. Amphotericin B: 30 years of clinical experience. Rev Infect Dis 1990; 12: 308 /329. 8 Hata K, Kimura J, Miki H, et al . Efficacy of ER-30346, a novel oral triazole antifungal agent, in experimental models of
17
18
19
20
21
22
aspergillosis, candidiasis, and cryptococosis. Antimicrob Agents Chemother 1996; 40: 2243 /2247. Hata K, Kimura J, Miki H, et al . In vitro and in vivo antifungal activities of ER-30346, a novel oral triazole with a broad antifungal spectrum. Antimicrob Agents Chemother 1996; 40: 2237 /2242. Galgiani JN, Lewis ML. In vitro studies of activities of the antifungal traizoles SCH 56592 and itraconazole against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts. Antimicrob Agents Chemother 1997; 41: 180 /183. Oakley KL, Moore CB, Denning DW. In vitro activity of SCH56592 and comparison with activities of amphotericin B and itraconazole against Aspergillus spp. Antimicrob Agents Chemother 1997; 41: 1124 /1126. McGinnis MR, Pasarell L, Sutton DA, et al . In vitro activity of voriconazole against selected fungi. Antimicrob Agents Chemother 1998; 36: 239 /242. McGinnis MR, Pasarell L. In vitro testing of susceptibilities of filamentous ascomycetes to voriconazole, itraconazole, and amphotericin B, with consideration of phylogenetic implications. Antimicrob Agents Chemother 1998; 36: 2353 /2355. Clemons KV, Stevens DA. Efficacy of ravuconazole in treatment of mucosal candidosis in SCID mice. Antimicrob Agents Chemother 2001; 45: 3433 /3436. Rinaldi MG. Use of potato flakes agar in clinical mycology. J Clin Microbiol 1982; 15: 1159 /1160. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Approved standard. NCCLS document M38-A. Wayne, PA: National Committee for Clinical Laboratory Standards, 2000. Chapman SW, Rogers PD, Rinaldi MG, et al . Susceptibilities of clinical and laboratory isolates of Blastomyces dermatitidis to ketoconazole, itraconazole, and fluconazole. Antimicrob Agents Chemother 1998; 42: 978 /980. Johnson EM, Szekely A, Warnock DW. In vitro activity of voriconazole, itraconazole, and amphotericin B against filamentous fungi. J Antimicorb Chemother 1998; 42: 741 /745. Arikan S, Lozano-Chiu M, Paetznick V, et al . Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species. J Clin Microbiol 1999; 37: 3946 /3951. Gonza´lez GM, Tijerina R, Najvar LK, et al . Activity of posaconazole against Pseudallescheria boydii: In vitro and in vivo assays. Antimicrob Agents Chemother 2003; 47: 1436 / 1438. Carrillo AJ, Guarro J. In vitro activities of four novel triazoles against Scedosporium spp. Antimicrob Agents Chemother 2001; 45: 2151 /2153. Minassian B, Huczko E, Washo T, et al . In vitro activity of ravuconazole against zygomycetes, Scedosporium and Fusarium isolates. Clin Microbiol Infect 2003; 9: 1250 /1252.
– 2005 ISHAM, Medical Mycology, 43, 281 /284
