1 The JONES Group/JMI Laboratories, Inc., 345 Beaver Kreek Centre, Suite A, North Liberty, IA 52317; 2 Tufts University School of Medicine, Boston, MA, USA
Received 7 July 2004; returned 3 September 2004; revised 5 October 2004; accepted 19 October 2004
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Abstract |
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Methods: MICs of anidulafungin, amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole and voriconazole were determined using reference method (M27-A2) guidelines. The M38-A reference method was used for the filamentous fungi, including determination of minimum effective concentrations (MECs) of anidulafungin.
Results: Anidulafungin was more active when compared with fluconazole and itraconazole for Candida albicans (MIC90, 0.06 mg/L), Candida tropicalis (MIC90, 0.06 mg/L), Candida glabrata (MIC90, 0.12 mg/L), Candida krusei (MIC90, 0.06 mg/L) and Candida lusitaniae (MIC90, 1 mg/L) as well as the less-often encountered yeast species. Anidulafungin was less active against Candida parapsilosis, Candida guilliermondii and Candida famata (MIC50, 12 mg/L). Anidulafungin also exhibited excellent activity against all Aspergillus spp. (MEC90, 0.03 mg/L). Anidulafungin was also evaluated comparing two end point reading criteria and two incubation intervals. Data indicate that longer incubation periods do not significantly influence overall MIC ranges. These international surveillance results for anidulafungin confirm the activity observed in studies of smaller numbers of isolates.
Keywords: yeast , susceptibility , antifungals , echinocandins
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Introduction |
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Beginning in the early 1990s, the National Committee for Clinical Laboratory Standards (NCCLS) developed standardized consensus testing methods for determining susceptibilities of Candida species and Cryptococcus neoformans.5 This reference method5 established consistency in routine testing and aided the comparison of different classes of antifungal agents against the opportunistic yeasts. In 2003, the NCCLS approved a format for determining susceptibilities of the filamentous fungi (M38-A).6 Although work is ongoing to determine correlation between in vitro results and clinical outcome,7 both NCCLS reference methods have contributed to added clarity of available antifungal options against a broad range of opportunistic fungal pathogens. Anidulafungin is a novel semi-synthetic echinocandin antifungal agent that compromises cell wall structural integrity through non-competitive inhibition of ß-1,3-glucan biosynthesis, causing cell death. It has demonstrated excellent broad spectrum in vitro and in vivo activity against a wide variety of fungal pathogens.3,4,811 We present here, initial data from a longitudinal study comparing the activity of anidulafungin, as well as an activity comparison with amphotericin B, 5-fluorocytosine and four azole agents.
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Materials and methods |
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A total of 880 clinical yeast isolates and 68 mould isolates from 64 medical centres in North America, Latin America and Europe were tested. The isolates were collected in 2002 and 2003, and were sent to a central reference laboratory for testing. All isolates were maintained in vials of sterile distilled water at ambient room temperature until tested. The collection of yeasts included Candida albicans (n=500), C. parapsilosis (n=106), C. tropicalis (n=106), C. glabrata (n=105), C. krusei (n=23), C. lusitaniae (n=13), C. guillermondii (n=8), C. kefyr (n=5), C. lipolytica (n=5), C. pelliculosa (n=3), C. dubliniensis (n=2), C. famata (n=2), and one strain each of C. rugosa and C. pulcherrima. The collection of moulds included Aspergillus fumigatus (n=30), A. flavus (n=7), A. niger (n=6), Penicillium spp. (n=6), A. terreus (n=4), Trichosporon spp. (n=3), A. nidulans (n=2), A. versicolor (n=2), Curvularia spp. (n=2), Paecilomyces spp. (n=2), Fusarium spp. (n=2) and Rhizopus spp. (n=2).
Antifungal agents
Anidulafungin (Vicuron, Inc., King of Prussia, PA, USA), fluconazole and voriconazole (Pfizer, Inc., New York, NY, USA), itraconazole and ketoconazole (Janssen, Beerse, Belgium), and amphotericin B and 5-fluorocytosine (Sigma Chemical Co., St. Louis, MO, USA) were obtained as standard powders and stock solutions prepared according to NCCLS guidelines at TREK Diagnostics (Westlake, OH, USA).5,6 Briefly, two-fold dilution microdilution format 2x concentration was incorporated using standard RPMI 1640 broth (Sigma). Microdilution panels were shipped frozen and stored at 80°C until used. Antifungal concentrations were brought to final concentration with addition of an equal volume of inoculum. Final concentration ranges were as follows: anidulafungin (0.00816 mg/L), amphotericin B (0.01616 mg/L), 5-fluorocytosine (0.0364 mg/L), fluconazole (0.12256 mg/L), itraconazole (0.00816 mg/L), ketoconazole (0.00816 mg/L), and voriconazole (0.00816 mg/L).
Broth microdilution tests
Sterile 96-well round-bottomed panels (Corning, New York, NY, USA) with wells containing 100 µL of two-fold antifungal dilutions in RPMI 1640 growth medium buffered to pH 7.0 with 0.165 M MOPS were used for testing.
Broth microdilution testing for yeasts followed standard conditions described in the NCCLS M27-A2 reference method.5 Starting inocula were adjusted to an equivalent 0.5 McFarland turbidity standard (1.05.0 x 106 cfu/mL) and diluted 1:1000 before being dispensed into the microdilution wells (1.05.0 x 103 cfu/mL). Then 100 µL of diluted inocula were dispensed into each microdilution well containing 100 µL of the 2x antifungal agent, to give a 1x final drug concentration and an inoculum range of 0.52.5 x103 cfu/mL. Colony counts were carried out to verify the inoculum concentration.
All filamentous fungi were tested using conditions described in the NCCLS M38-A reference method.6 Isolates were grown on potato dextrose agar slants for 7 days and the conidia harvested with gentle probing. Inocula were adjusted for turbidity as described in the reference method, based on percent transmittance and species. Final inocula in the panel ranged from 0.4 to 3.3 x 104 cfu/mL. All preparations and MIC readings were carried out in a biosafety hood.
Quality control (QC) isolates C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 from the American Type Culture Collection were used as recommended (NCCLS) and all results were observed within published ranges.5,6,12
Panel incubation and determination of MICs for yeasts
All microdilution panels were incubated in enclosed, humid containers at 35°C and visualized using a reading mirror at both 24 and 48 h. Following incubation, the MIC values for anidulafungin, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole and voriconazole were read at the lowest concentration at which a significant decrease in turbidity (50%) was detected compared with the growth control. Amphotericin B MICs were determined as the lowest concentration at which no visible growth was discerned.5 In addition, a second MIC value for anidulafungin was determined as the lowest concentration at which no visible growth was detected.12
Interpretive breakpoints for susceptibility (S) to fluconazole [S at 8 mg/L; S dose-dependent (SDD) at 1632 mg/L; and resistant (R) at
64 mg/L], 5-fluorocytosine [S at
4 mg/L; intermediate (I) at 816 mg/L; and R at
32 mg/L] and itraconazole (S at
0.12 mg/L; SDD at 0.250.5 mg/L; and R
1 mg/L) were those published by Rex et al.13 and the NCCLS.9 Interpretive criteria for echinocandins, amphotericin B, ketoconazole and voriconazole have not been established. However, Stone et al.14 proposed a breakpoint of
1 mg/L for caspofungin (same class as anidulafungin) based on serum levels of >1 mg/L being achieved over the entire dosing interval.15
Panel incubation and determination of MIC and MEC values for moulds
Panels were incubated at 35°C in an enclosed container and visualized with a reading mirror at 24 and 48 h in a biosafety hood. MIC results for amphotericin B, itraconazole and voriconazole were determined as the concentration at which no visible growth was detected. MIC values for 5-fluorocytosine, fluconazole and ketoconazole were detected as the lowest concentration in which a prominent decrease in growth (50%) was visualized compared to the growth control.6 As described for the echinocandins,8 the minimum effective concentration (MEC) for anidulafungin was determined as the lowest concentration at which pronounced morphological change from filamentous growth to non-filamentous growth was observed.
Data analysis
MIC50 and MIC90 values were determined for each tested yeast or mould species and were calculated as the concentration of each antifungal agent that inhibited 50% and 90% of the isolates, respectively. MEC50 and MEC90 values were determined for anidulafungin against the filamentous fungi.
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Results and discussion |
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Acknowledgements |
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Footnotes |
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References |
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