Single-Center Evaluation of an Agar-Based Screening for Azole Resistance in Aspergillus fumigatus by Using VIPcheck

Abstract

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,...

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