Antifungal activity of a new triazole, voriconazole (UK-109496), against clinical isolates of Aspergillus spp

Antifungal activity of a new triazole, voriconazole (UK-109496), against clinical isolates of Aspergillus spp

J Infect Chemother (2000) 6:101–103 © Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases 2000 101 NOTE Shigefumi ...

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J Infect Chemother (2000) 6:101–103

© Japanese Society of Chemotherapy and the Japanese Association for Infectious Diseases 2000 101

NOTE Shigefumi Maesaki · Jun Iwakawa · Yasuhito Higashiyama Yoshitsugu Miyazaki · Katsunori Yanagihara Kazunori Tomono · Takayoshi Tashiro · Shigeru Kohno

Antifungal activity of a new triazole, voriconazole (UK-109496), against clinical isolates of Aspergillus spp.

Received: January 5, 2000 / Accepted: April 6, 2000

Abstract Voriconazole is a new triazole antifungal agent with potent activity against yeast and molds. We investigated the in-vitro activity of voriconazole compared with that of other antifungal agents against 50 clinical isolates of Aspergillus spp., measured by the National Committee for Clinical Laboratory Standards (NCCLS) reference method described in the M27-A document, and by an alamar blue colorimetric method. Voriconazole was the most potent agent against Aspergillus fumigatus (minimum inhibitory concentration [MIC]90, 0.5 mg/l) and Aspergillus niger (MIC90, 1.0 mg/l). Voriconazole was less active (MIC90, 1.0 mg/l) against Aspergillus flavus than itraconazole (MIC90, 0.5 mg/l). Voriconazole was more active than itraconazole against Aspergillus fumigatus and Aspergillus flavus by the alamar blue indicator method for the measurement of MIC. Based on these results, voriconazole has promising activity against commonly encountered isolates of Aspergillus spp., and its clinical usefulness should be established by further studies. Key words Aspergillus· Alamar blue · Voriconazole · MIC · Antifungal agents

threatening fungal infections that commonly occurs in patients with neutropenia caused by intensive anticancer therapy. The utility of triazoles, mainly itraconazole, in the treatment of these infections is limited, and amphotericin B is still the drug of choice. Voriconazole (UK-109496) is a novel wide-spectrum triazole antifungal agent that has potent in-vitro activity against Candida spp. and Aspergillus spp.1–3 Pharmacokinetic studies have shown its good bioavailability after oral or intravenous administration, and its low toxicity. Early clinical studies have documented its promising usefulness in the treatment of oropharyngeal candidiasis and invasive aspergillosis.4 Voriconazole has also been found to be effective in experimental models of pulmonary aspergillosis and in the prevention and treatment of Aspergillus endocarditis in guinea pigs.5,6 In this study, we determined the in-vitro activity of voriconazole, compared with that of other antifungal agents, against clinical isolates of Aspergillus spp. Invitro susceptibility testing was performed using the broth microdilution version of the National Committee for Clinical Laboratory Standards (NCCLS) reference method described in the M27-A document, and a colorimetric method using alamar blue.7–9

Introduction Materials and methods The azole derivatives are the largest class of antifungal antimicrobial agents used for the treatment of fungal infections. Among them, fluconazole and itraconazole have been used with considerable success in the treatment of systemic mycoses caused by Candida spp. and Cryptococcus neoformans. Invasive aspergillosis is one of the most life-

S. Maesaki · J. Iwakawa · Y. Higashiyama · Y. Miyazaki · K. Yanagihara · K. Tomono · T. Tashiro · S. Kohno (*) Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan Tel. 181-95-849-7271; Fax 181-95-849-7285 e-mail: [email protected]

Fifty recent clinical isolates were included in this study: Aspergillus fumigatus (25 isolates), Aspergillus flavus (10 isolates), and Aspergillus niger (15 isolates). All strains were obtained from the sputum, pleural effusion, or bronchial aspirate of patients with pulmonary aspergillosis at Nagasaki University Hospital. These strains were identified by both morphological identification and enzyme electrophoretic pattern.10 All isolates were stored in a small piece of agar as deep-frozen stock until they were used in the study. Before testing, each isolate was subcultured at least twice on Sabouraud Dextrose agar slants (Becton Dickinson, Cockeysville, MD, USA) to ensure its viability and purity.

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The six antifungal agents used in this study were provided as standard powders of known potency. Voriconazole and fluconazole (FLCZ) were obtained from Pfizer Central Research (Sandwich, UK). Itraconazole (ITCZ) and ketoconazole (KTZ) were obtained from Janssen Research Foundation (Beerse, Belgium). Amphotericin B (AMPH-B) was obtained from Bristol-Myers Squibb (Tokyo, Japan) and miconazole (MCZ) was obtained from Mochida Pharmaceutical (Tokyo, Japan). All drugs were diluted in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer, and dispensed into 96-well microdilution plates. The recommendations stated in the NCCLS document M27-A were followed for the dilution of each antifungal agent. The 96well flat bottomed plates containing an aliquot of 0.1 ml in each well of appropriate drug solution (2 3 final concentration) were sealed and stored at 280°C until they were used. The final concentrations ranges of the tested drugs were: AMPH-B, 0.013 to 8 mg/l; FLCZ, 0.25 to 128 mg/l; KTZ, 0.125 to 64 mg/l; MCZ, 0.125 to 64 mg/l; ITCZ, 0.013 to 8 mg/ l; and voriconazole, 0.013 to 8 mg/l. Minimum inhibitory concentrations (MICs) were determined by a broth microdilution method, using the NCCLS guidelines. Briefly, each isolate was grown on Sabouraud Dextrose agar plates (Becton Dickinson) at 35°C for a period of 7 days. The spores were collected with 0.85% sterile saline containing 0.8% Tween 80. The cell suspensions were adjusted to a final concentration of 2 3 105 spores/ml, counted by hemocytometer. A volume of 0.1 ml of cell suspension was inoculated and the plates were incubated at 35°C for 48 h. The MIC was the lowest concentration of antifungal agent that inhibited the growth of the cells, as

detected visually. The measurement of MICs using alamar blue was performed as follows: a volume of 20 µl of alamar blue (Kanto Chemical, Tokyo, Japan) was added to the 0.1-ml cell suspension. The plates were incubated at 35°C for 48 h, and MICs were determined visually. The two quality control strains recommended in the NCCLS documente M27-A, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included each time in any test.

Results and discussion The antifungal activities of voriconazole and the other azole antifungal agents tested, determined by the NCCLS testing method, are summarized in Table 1. Voriconazole showed greater activity against Aspergillus fumigatus (MIC90, 0.5 mg/l) and Aspergillus niger (MIC90, 1.0 mg/l) than ITCZ and AMPH-B. In comparison with itraconazole, voriconazole was twofold less active against Aspergillus flavus. Voriconazole was four-fold more active than AMPH-B against Aspergillus spp. The MICs of the antifungal agents measured by the alamar blue colorimetric method are shown in Table 2. Voriconazole showed the greatest activity of the agents tested against Aspergillus fumigatus (MIC90, 0.5 mg/l) and against Aspergillus niger (MIC90 2.0 mg/l). Following the publication of standardized procedures for the testing of yeast,7 the NCCLS subcommittee on antifungal susceptibility testing focused its efforts towards the development of standard guidelines for the antifungal susceptibility testing of filamentous fungi.8 Espinel-Ingroff et al.8 and McGinnis et al.3 have documented the potent in-

Table 1. Antifungal activity of antifungal agents against clinical isolates of Aspergillus species, measured by NCCLS M27-Aa Species

No. of strain

Drug

MIC (µg/ml) Range

50%

90%

Voriconazole MCZ ITCZ KTZ FLCZ AMPH

0.125–1 2–16 0.25–4 8–32 .128 0.063–4

0.25 4 0.5 8 .128 0.25

0.5 4 2 16 .128 4

Voriconazole MCZ ITCZ KTZ FLCZ AMPH

0.125–1 2–8 0.5–4 4–32 .128 1–8

0.5 8 4 16 .128 2

1 8 4 16 .128 4

Voriconazole MCZ ITCZ KTZ FLCZ AMPH

0.125–1 1–8 0.125–0.5 2–8 .128 2–16

0.25 2 0.25 4 .128 4

1 4 0.5 8 .128 16

Table 2. Antifungal activity of antifungal agents against clinical isolates of Aspergillus species, measured by the alamar blue colorimetric method Species

A. fumigatus

A. niger

A. flavus

25

15

10

MIC, Minimum inhibitory concentration; MCZ, miconazole; ITCZ, itraconazole; KTZ, ketoconazole; FLCZ, fluconazole; AMPH, amphotericin B a National Committee for Clinical Laboratory Standards, approved standard M27-A1

No. of strain

Drug

MIC (µg/ml) Range

50%

90%

A. fumigatus

25

Voriconazole MCZ ITCZ KTZ FLCZ AMPH

0.125–8 2–16 0.125–16 8–32 .128 0.063–16

0.25 4 1 4 .128 4

0.5 4 4 4 .128 16

A. niger

15

Voriconazole MCZ ITCZ KTZ FLCZ AMPH

0.25–8 2–16 0.5–32 4–32 .128 2–16

1 8 4 16 .128 4

2 8 8 32 .128 8

A. flavus

10

Voriconazole MCZ ITCZ KTZ FLCZ AMPH

0.125–4 1–8 0.125–2 2–8 .128 2–16

0.25 2 0.25 4 .128 8

1 8 0.5 8 .128 16

MCZ, Miconazole; ITCZ, itraconazole; KTZ, ketoconazole; FLCZ, fluconazole; AMPH, amphotericin B

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vitro activity of voriconazole against Aspergillus spp. and Pseudallescheria boydii. Likewise, Radford et al.11 reported the good in-vitro activity of voriconazole against a broad array of emerging and less common mold pathogens. We have also observed that voriconazole was highly active against Aspergillus spp. These promising in-vitro data seem to corroborate the encouraging in-vivo results from early clinical trials and several experimental models.5,6 We also observe that the guidelines suggested by the NCCLS subcommittee on antifungal susceptibility testing appear to be suitable for testing the filamentous fungi Aspergillus spp. and that RPMI 1640 medium sustains the adequate growth of all isolated Aspergillus strains at 48-h incubation. The susceptibility test using alamar blue dye indicated a clear endpoint and showed a good correlation with the results obtained by NCCLS methods. This colorimetric procedure could be a suitable susceptiblility test for antifungal agents against Aspergillus spp. measured in the clinical laboratory. We have previously reported the effects of antifungal agent combinations against clinical isolates of Aspergillus fumigatus.12 In that study, we tried to estimate the effects of combinations of voriconazole and amphotericin B and other antifungal agent combinations against Aspergillus spp., measured by the susceptibility test using alamar blue. The combination of voriconazole and amphotericin B showed the highest synergistic effect against several isolates of Aspergillus spp. (data not shown). The application of this synergistic effect to the therapy of murine invasive pulmonary aspergillosis is now underway. Finally, these findings should be interpreted cautiously, as with any other in-vitro susceptibility testing results. Although preliminary pharmacokinetic data suggest that serum levels of voriconazole are expected to be higher than those of itraconazole, the clinical significance of the potency of voriconazole against Aspergillus spp. awaits the results of comparative clinical trials.

References 1. Johnson EM, Szekely A, Warnock DW. In vitro activity of voriconazole, itraconazole and amphotericin B against filamentous fungi. J Antimicrob Chemother 1998;42:741–5.

2. Cuenca-Estrella M, Rodriguez Tudela JL, Mellado E, Martinez Suarez JV, Monzon A. Comparison of the in-vitro activity of voriconazole (UK-109496), itraconazole and amphotericin B against clinical isolates of Aspergillus fumigatus. J Antimicrob Chemother 1998;42:531–3. 3. McGinnis MR, Pasarell L, Sutton DA, Fothergill AW, Cooper CJ, Rinaldi MG. In vitro evaluation of voriconazole against some clinically important fungi. Antimicrob Agents Chemother 1997;41: 1832–4. 4. Ruhnke M, Schmidt WA, Trautmann M. In vitro activities of voriconazole (UK-109496) against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients with human immunodeficiency virus infection. Antimicrob Agents Chemother 1997;41:575–7. 5. Martin MV, Yates J, Hitchcock CA. Comparison of voriconazole (UK-109496) and itraconazole in prevention and treatment of Aspergillus fumigatus endocarditis in guinea pigs. Antimicrob Agents Chemother 1997;41:13–6. 6. Murphy M, Bernard EM, Ishimaru T, Armstrong D. Activity of voriconazole (UK-109496) against clinical isolates of Aspergillus species and its effectiveness in an experimental model of invasive pulmonary aspergillosis. Antimicrob Agents Chemother 1997;41: 696–8. 7. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeast. Approved standard M27-A. Wayne, PA: NCCLS; 1997. 8. Espinel-Ingroff A, Bartlett M, Bowden R, Chin NX, Cooper CJ, Fothergill, et al. Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi. J Clin Microbiol 1997;35:139–43. 9. Jahn B, Stuben A, Bhakdi S. Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and alamar blue tests. J Clin Microbiol 1996;34:2039–41. 10. Matsuda H, Kohno S, Maesaki S, Yamada H, Koga H, Tamura M, et al. Application of ubiquinone systems and electrophoretic comparison of enzymes to identification of clinical isolates of Aspergillus fumigatus and several other species of Aspergillus. J Clin Microbiol 1992;30:1999–2005. 11. Radford SA, Johnson EM, Warnock DW. In vitro studies of activity of voriconazole (UK-109496), a new triazole antifungal agent, against emerging and less-common mold pathogens. Antimicrob Agents Chemother 1997;41:841–3. 12. Maesaki S, Kohno S, Kaku M, Koga H, Hara K. Effects of antifungal agent combinations administered simultaneously and sequentially against Aspergillus fumigatus. Antimicrob Agents Chemother 1994;38:2843–5.