a Division of Infectious Diseases, Department of Medicine, Wayne State University School of Medicine, Detroit, MI 48201; b ScheringPlough Research Institute, Kenilworth, NJ 07033, USA
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Abstract |
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Introduction |
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Materials and methods |
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The clinical isolates of A. fumigatus (n = 284), Aspergillus niger (n = 66), Aspergillus flavus (n = 31) and Aspergillus spp. (n = 43) used in this investigation were obtained from the Detroit Medical Center from January 1994 to December 1998. The majority of the isolates (>95%) were from individual patients (mainly, but not limited to, immunocompromised; one isolate per patient). The original cultures obtained on Sabouraud dextrose agar (SDA) slants were subcultured on the same medium to confirm viability and purity of the cultures. For long-term storage, conidial suspensions in 25% glycerol were kept at 80°C. Working cultures of the isolates were maintained on SDA slants at 4°C.
Amphotericin B-resistant isolates of A. fumigatus were selected in the laboratory from a susceptible clinical isolate (ATCC 208996) by UV mutagenesis as described previously.17 Spontaneous mutants of A. fumigatus that showed reduced susceptibility to itraconazole were selected in the laboratory on SDA containing itraconazole as described previously.18
Voriconazole-resistant mutants of A. fumigatus that showed reduced susceptibility to this drug were selected in the laboratory from the clinical isolate ATCC 208996 by stepwise selection on SDA containing voriconazole. Briefly, a conidial suspension of A. fumigatus ATCC 208996 was prepared as described previously19 from a 6 day old culture and the conidial density was determined by haemocytometry. Approximately 1 x 106 conidia/plate were spread on 20 peptone yeast extract glucose (PYG: peptone 1 g, yeast extract 1 g, glucose 3 g, agar 15 g/L in distilled water) agar plates containing voriconazole 0.5 mg/L. A. fumigatus colonies that grew on PYG agar plates in the presence of voriconazole 0.5 mg/L after 3 days of incubation at 35°C (F1 colonies) were collected and stored as conidial suspensions at 80°C.
In the second step of the selection process, conidial suspensions from two representatives of the F1 colonies that showed voriconazole MICs 1 mg/L were plated on PYG agar containing voriconazole 4 mg/L and the agar plates were incubated in plastic sleeves for 6 days at 35°C. Colonies that grew in the presence of voriconazole 4 mg/L were collected and stored as conidial suspensions in glycerol at 80°C. These isolates obtained after the second step of selection (F2 colonies) were used for subsequent studies.
Antifungal agents
Various antifungal agents used in this study were obtained as pure powders from the manufacturers. Itraconazole (R51 211, batch no. STAN-9304-005-1) was obtained from Janssen Pharmaceutica, Beerse, Belgium. Voriconazole (batch no. 25381-57-8) was from Pfizer Pharmaceuticals, New York, NY, USA. Amphotericin B (batch no. 20-914-29670) was obtained from Squibb Institute for Medical Research, Princeton, NJ, USA. Posaconazole (batch no. 97-56592-X-208) was obtained from ScheringPlough Research Institute, Kenilworth, NJ, USA. All the antifungal agents were dissolved in dimethylsulphoxide at a concentration of 1 mg/mL and stored as 0.25 mL aliquots at 20°C. The frozen stock was thawed at room temperature and gently vortexed several times to ensure that any remaining crystals were completely dissolved before use. Where applicable, comparable concentrations of dimethylsulphoxide were used to examine its effect on the growth of the organism.
MIC and MFC determinations
The in vitro susceptibilities of various isolates of Aspergillus spp. to antifungal agents were determined by a broth macrodilution technique as previously described,1922 except that PYG medium was used instead of RPMI 1640 for amphotericin B-resistant isolates. Briefly, fresh conidia were collected23 from various aspergillus isolates and suspended in RPMI 1640 at a density of 2 x 104 conidia/mL. Drug solutions of double the required concentration were prepared in the same medium (0.5 mL) by serial dilution in sterile 6 mL polystyrene tubes (Falcon 2054, BectonDickinson, Lincoln Park, NJ, USA) and inoculated with an equal volume (0.5 mL) of the conidial suspension. The tubes were incubated at 35°C for 48 h and scored for visible growth after vortexing the tubes gently. The MIC was defined as the lowest concentration of the drug which produced no visible growth (i.e. 100% inhibition). Each MIC determination was performed in duplicate and the experiment was repeated once. The concentrations of the antifungal agents used for the MIC studies ranged from 0.0625 to 16 mg/L. A drug-free growth control and a set of tubes with RPMI 1640 alone for monitoring contamination of the medium were used.
The minimum fungicidal concentrations (MFCs) were determined in duplicate by subculturing 0.1 mL aliquots from all MIC tubes showing no visible growth on to SDA plates. The plates were incubated at 35°C for 48 h for growth and the MFC was defined as the lowest concentration of the antifungal agent that provided 10 colonies per MIC tube (c. 99% killing).
Kill curves
The fungicidal activity of various antifungal agents against A. fumigatus ATCC 208996 conidia was determined by timekill experiments. Five millilitres of conidial suspension prepared in PYG broth (106 conidia/mL) was incubated at 35°C in the presence of various concentrations (08 mg/L) of amphotericin B, itraconazole, voriconazole and posaconazole. At various time intervals, 0.1 mL aliquots of the conidial suspension were removed and diluted appropriately to obtain 10- to 104-fold dilutions, and 0.1 mL aliquots were spread in duplicate on SDA plates. The plates were incubated at 35°C for 48 h, and the numbers of colony forming units (cfu)/mL of conidial suspension were determined.
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Results |
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As shown in Figure 2, exposure of A. fumigatus conidia to posaconazole killed the fungal cells in a time- and concentration-dependent manner. For example, posaconazole 4 mg/L killed
99% of A. fumigatus conidia within 48 h, and the fungicidal activity of this compound was slightly superior to that obtained for itraconazole and voriconazole, but inferior to amphotericin B, which at the same concentration killed >99% of A. fumigatus conidia within 4 h.
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Discussion |
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The azole-resistant isolates of A. fumigatus used in our study were selected in the laboratory from the same parental strain. These could be spontaneous mutants with identical genetic variation(s). Thus, it is possible that multiple isolates we obtained may be clones of the same isolate since they all underwent the same selection process. On the other hand, it is possible that spontaneous mutations resulting in different mechanisms of resistance would have occurred randomly and the phenotypic expression of the resistance trait resulted in their selection in the presence of azole. A detailed study of the mechanism(s) associated with the resistance in several isolates is required to understand whether they all carry the same mechanism for azole resistance.
The primary objective of this study was to compare the in vitro activity of posaconazole against various species of aspergillus, including those isolates with reduced susceptibility to other azoles and the polyenes. Members of the azole family of antifungals are generally fungistatic against pathogenic yeasts such as Candida spp. Exposure of Candida albicans cells to azoles such as fluconazole, itraconazole, voriconazole and posaconazole arrests their growth but does not kill them. On the other hand, we showed previously that itraconazole and voriconazole not only inhibit the growth of Aspergillus spp. but also kill them.23 The fungicidal activity of posaconazole further confirms our previous observation that certain members of the azole family have organism-dependent fungicidal activity against Aspergillus spp. The comparatively good in vitro activity of posaconazole against clinical and polyene- and azole-resistant isolates of aspergillus suggests that it is a promising candidate for further development.
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Acknowledgments |
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Notes |
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References |
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Received 24 September 1999; returned 29 December 1999; revised 28 February 2000; accepted 20 March 2000