1 Servicio de Microbiología, Hospital Universitario de Valme, Ctra Cádiz s/n, Sevilla 41014; 3 Servicio de Bioestadística, Unidad Investigación, Hospital Universitario de Valme, Sevilla, Spain; 2 Division of Infectious Diseases, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 2329-80049, USA
Received 3 November 2003; returned 23 December 2003; revised 28 January 2004; accepted 8 February 2004
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Abstract |
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Methods: Four Rhizomucor spp. isolates were included to study the suitability of the three susceptibility testing methods to detect isolates resistant to voriconazole. The disc diffusion method performed on MuellerHinton agar (Difco) supplemented with 2% glucose and Methylene Blue (0.5 mg/L) gave zone diameters with very clear border edges.
Results: The disc diffusion method showed excellent correlation with the Etest and the NCCLS methods.
Conclusion: On the basis of our results, it appears that the disc diffusion test is a useful method for testing the activity of voriconazole against Aspergillus spp.
Keywords: antifungals, susceptibility tests
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Introduction |
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Previous studies have compared the in vitro activity of these new agents against those of established agents. Currently, there is an approved standard method (M38-A) developed by the NCCLS for evaluating the susceptibilities of filamentous fungi to antifungal agents. This method is labour-intensive and not readily applicable in routine laboratories. Agar-based methods are attractive because of their simplicity and low cost, but they are not widely used for antifungal susceptibility testing. Good correlation with the reference method has been reported for fluconazole against Candida spp.23,24 The new antifungal agent voriconazole has been tested extensively in broth-based procedures, but has not been widely evaluated using agar-based testing methods.7,10,12,15,23,2528 These previous studies were mainly carried out in Candida spp., but there are a few susceptibility studies that use agar for filamentous fungi.26 This is the first study to compare the disc diffusion method with the NCCLS reference broth microdilution M38-A and the Etest method for voriconazole susceptibility testing of Aspergillus spp.29
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Materials and methods |
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A total of 77 clinical isolates were tested. These included 26 Aspergillus fumigatus, 21 Aspergillus flavus, 10 Aspergillus terreus, nine Aspergillus niger, five Aspergillus nidulellus, four Aspergillus glaucus, two Aspergillus flavipes and four Rhizomucor spp. These isolates were recovered from clinical specimens received at Valme University Hospital in Seville (Spain) and at the Medical College of Virginia, Virginia Commonwealth University (VA, USA). Identification of each strain was performed using routine mycological techniques. The mould isolates were maintained in sterile water and were subcultured on antimicrobial agent-free potato dextrose agar to ensure viability and purity.
Stock inoculum suspensions were prepared from 7-day-old cultures grown on potato dextrose agar (Difco) following NCCLS guidelines.29 Stock suspensions were adjusted spectrophotometrically to optical densities that ranged from 0.09 to 0.11 (8082% transmittance) and contained conidia or sporangiospores and hyphal fragments. The diluted (two times) inoculum sizes ranged from 0.9 x 104 to 4.7 x 104 cfu/mL, as demonstrated by quantitative colony count on Sabouraud dextrose agar. The same inoculum was used for both broth and agar methods.
Susceptibility testing
Reference antifungal susceptibility testing was performed by the reference broth microdilution method (BMD) described by the NCCLS.29 Broth microdilution MICs were determined after 48 h of incubation at 35°C. The MIC was defined as the lowest drug concentration that resulted in complete inhibition, or MIC-0. The reference powder voriconazole was obtained from Pfizer.
The Etest method was performed as described previously with RPMI agar with 2% glucose (Izasa, España).26,30 Etest strips with voriconazole concentrations ranging from 0.002 to 32 mg/L were provided by AB Biodisk (Solna, Sweden). The plates were inoculated with the inoculum suspension and incubated at 35°C for 24 h and 48 h. Etest MICs were read where the edge of inhibition ellipse intersected the MIC scale on the Etest strip.
Disc diffusion
Testing of voriconazole was performed as described in NCCLS document M44-P for yeast.31 Voriconazole discs (1 µg; Difco, Oxoid) and MuellerHinton agar (Difco) supplemented with 2% glucose and Methylene Blue (0.5 mg/L) were used throughout this study. The plates were incubated at 35°C and read at 24 h. Zone diameters were read at the point where growth decreased abruptly.
Quality control
The following strains recommended in the NCCLS M38-A were tested each time to ensure quality control: Candida parapsilosis 22019, Candida krusei 6258 and Aspergillus flavus 204304.29
Analysis of results
In the Etest method, the trailing growth inside Etest ellipse was ignored, as recommended by the manufacturer. Etest MIC endpoints were raised to the nearest two-fold dilution value that matched the NCCLS concentration ranges to facilitate MIC comparisons by both methods. The diameters of zones around 1 µg voriconazole discs were plotted against 48 h broth microdilution and 24 h and 48 h Etest MICs.
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Results and discussion |
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Footnotes |
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References |
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