Antifungal susceptibility testing is an essential tool for guiding therapy, although EUCAST and CLSI reference methods are often available only in specialized centers. We studied the performance of an agar-based screening method for the detection of azole resistance in Aspergillus fumigatus cultures. The VIPcheck consists of four wells containing voriconazole, itraconazole, posaconazole, or a growth control. Ninety-six A. fumigatus isolates were used. Thirty-three isolates harbored a known resistance mechanism: TR 34 /L98H (11 isolates), TR 46 /Y121F/T289A (6 isolates), TR 53 (2 isolates), and 14 isolates with other cyp51A gene point mutations. Eighteen resistant isolates had no cyp51A-mediated azole resistance. Forty-five isolates had a wild-type (WT) azole phenotype. Four technicians and two inexperienced interns, blinded to the genotype/phenotype, read the plates visually after 24 h and 48 h and documented minimal growth, uninhibited growth, and no growth. The performance was compared to the EUCAST method. After 24 h of incubation, the mean sensitivity and specificity were 0.54 and 1.00, respectively, with uninhibited growth as the threshold. After 48 h of incubation, the performance mean sensitivity and specificity were 0.98 and 0.93, respectively, with minimal growth. The performance was not affected by observer experience in mycology. The interclass correlation coefficient was 0.87 after 24 h and 0.85 after 48 h. VIPcheck enabled the selection of azole-resistant A. fumigatus colonies, with a mean sensitivity and specificity of 0.98 and 0.93, respectively. Uninhibited growth on any azole-containing well after 24 h and minimal growth after 48 h were indicative of resistance. These results indicate that the VIPcheck is an easy-to-use tool for azole resistance screening and the selection of colonies that require MIC testing.KEYWORDS VIPcheck, broth microdilution, antifungal resistance, susceptibility, azole resistance, antifungal susceptibility testing A zole resistance is an emerging problem in Aspergillus fumigatus (1), with increasing evidence that patients with azole-resistant aspergillosis fail azole therapy (2-5). Two routes of resistance selection have been signified, through patient therapy and through the exposure of A. fumigatus to azole fungicides in the environment. There are important differences between these routes of resistance selection, including patient risk factors and fungal resistance mechanisms. Resistance mechanisms that are associated with the environmental route include TR 34 /L98H and TR 46 /Y121F/T289A (6-8).Surveillance studies in The Netherlands show that of the clinical isolates that are azole resistant, between 83% and 95% harbor mutations associated with the environmental route, while approximately 15% exhibit an azole-resistant phenotype without known resistance mutations (9). As patients inhale these airborne azole-resistant spores, a resistant infection may occur in any Aspergillus disease and in patients who have never been treated with medical triazoles (3, 7, 10,...

The in vitro susceptibilities of 21 Aspergillus isolates were tested against three antifungal agents in RPMI 1640 and yeast nitrogen base at pH 5.0 and 7.0 by a broth microdilution format of the NCCLS method. The MICs of amphotericin B and itraconazole were higher, while those of flucytosine were lower, at pH 5.0 than at pH 7.0. The poor correlation between in vitro results and clinical outcome could be due to a difference in pH between the in vitro susceptibility test and at the site of infection.In vitro susceptibility testing of filamentous fungi has become increasingly important. A good standard in vitro susceptibility test must give reproducible results, predict the resistance of molds, and correlate with clinical outcome (5).In 1998 the National Committee for Clinical Laboratory Standards (NCCLS) proposed a standard method for the determination of the in vitro antifungal susceptibility of conidium-forming filamentous fungi. This document is now approved (3). Although the standardized NCCLS method has been found to give better inter-and intracenter reproducibility, in vitro antifungal susceptibility testing of filamentous fungi is still faced with several problems such as the correlation of in vitro results with clinical outcome.Clinical outcome may be affected by various factors related to the host, the drugs, the fungus, and their interactions (9). These factors are not taken into account in the in vitro tests, and it may be for this reason that the prediction of antifungal efficacy or failure from in vitro susceptibility tests remains difficult. One of the host-related-factors is the pH at the site of infection. In the human body the pH is carefully regulated at 7.4, but it may be lower at the site of infection due to necrosis (7), the production of organic acids by fungi (6), or lysosome activity of granulocytes and macrophages (1).The aim of this study was to investigate the effect of pH on the in vitro activities of three different antifungal agents against 21 Aspergillus isolates in two different media.Twenty-one clinical Aspergillus isolates were tested: five itraconazole (ITZ)-susceptible and five ITZ-resistant Aspergillus fumigatus isolates, five A. flavus isolates, and six A. terreus isolates. Isolates had been frozen in glycerol broth at Ϫ80°C; they were revived by subculturing twice on Sabouraud glucose agar tubes supplemented with 0.5% chloramphenicol and were incubated for 5 to 7 days at 35°C. All isolates were tested in duplicate on different days.Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258 were used for quality control. Amphotericin B (AMB; BristolMyers Squibb, Woerden, The Netherlands), ITZ (Janssen Pharmaceutica B.V., Tilburg, The Netherlands), and flucytosine (5FC; ICN Pharmaceuticals, Zoetermeer, The Netherlands) were obtained as powders. AMB and ITZ were dissolved in dimethyl sulfoxide, and 5FC was dissolved in distilled water. The final concentrations of the antifungal agents at pH 5.0 ranged from 256 to 0.25 g/ml for AMB, from 16 to 0.016 g/ml for ITZ, and from 1,024 to 0...