| Summary: | Azole resistance in <i>Aspergillus fumigatus</i> has increasingly been reported worldwide. Its major mechanism of resistance is mediated by mutations in <i>cyp51A</i>. The objective of this study was to test the antifungal susceptibilities of <i>A. fumigatus</i> isolates from Chang Gung Memorial Hospital (CGMH), the largest tertiary referral hospital in Taiwan, and to investigate <i>cyp51A</i> mutations in azole-resistant strains. <i>A. fumigatus</i> isolates preserved in the Research Laboratory of Medical Mycology of CGMH from 2015 to 2021 were used. Antifungal susceptibility testing was performed using the YeastOne<sup>TM</sup> method. Isolates with high minimal inhibitory concentrations (MICs) against antifungals were further tested using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. Mutations in the <i>cyp51A</i> in azole-resistant strains were detected by Sanger sequencing. The overall prevalence of azole-resistant isolates was 1.77% (two out of 113 isolates). The two azole-resistant strains had tandem repeats (TR) in the promoter region and mutations in the <i>cyp51A</i> gene (TR<sub>34</sub>/L98H and TR<sub>34</sub>/L98H/S297T/F495I). One strain showed intermediate susceptibility to voriconazole, and its Cyp51A protein had five amino acid substitutions (F46Y/M172V/N248T/D255E/E427K). TR<sub>34</sub>/L98H and TR<sub>34</sub>/L98H/S297T/F495I are the most prevalent <i>cyp51A</i> mutations in Taiwan, mediating azole resistance based on current publications and our results. YeastOne<sup>TM</sup> was validated as a rapid tool for the antifungal susceptibility test; however, further confirmation by CLSI should be considered when MIC values of voriconazole, posaconazole, and amphotericin B are close to the clinical breakpoints or ecological cutoff values.
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