| Abstract: | Objective: This study aimed to compare the YeastOne and reference CLSI M38-A2 microdilution methods for antifungal susceptibility testing against common Aspergillus species, including TR34/L98H A. fumigatus. Methods: A total of 100 isolates were evaluated, including clinical isolates of 31A. fumigatus (5 TR34/L98H isolates), 23 A. flavus,13 A. terreus, and 10 A. niger, and 23 environmental A. fumigatus isolates (19 TR34/L98H isolates). Aspergillus species was identified by morphology and ITS and calmodulin sequencing. For M38-A 2 testing, each well of the microdilution trays containing 100 μL of the 2x drug concentrations (amphotericin B, itraconazole, posaconazole, and voriconazole, respectively [all Sigma-Aldrich]) was inoculated with 100μL of the 2x conidial inoculum suspension. The final volume and inoculum density in each well was 200 μl and 0.4 × 104 to 5 × 104 CFU/ml, and the final drug concentrations were 0.015 to 8 mg/L. Microdilution trays were incubated at 35◦C and MICs at 48 hours were read as the lowest concentration that prevent any discernible growth. For YeastOne testing, Sensititre YeastOne panels (Trek Diagnostic) containing serial twofold dilution of amphotericin B, itraconazole, posaconazole, and voriconazole (0.008 to 16 mg/L) were used, and each well of the panels was rehydrated with 100 μLofthe inoculum suspension (density 0.4 × 104 to 5 × 104 CFU/ml) and incubated at 35◦C. MICs at 48 hours were read as the lowest concentration in which the growth indicator remained blue. Results: The overall agreement (within ± 2log2 dilutions) between two methods was best for voriconazole (100%), followed by posaconazole (95%), itraconazole (92%), and amphotericin B (90%). Discrepancies were mostly due to lower posaconazole and itraconazole MICs and higher amphotericin B MICs obtained by YeastOne. Of all isolates, the geometric mean (GM) MICs of voriconazole by YeastOne and M38-A2 were nearly identical (0.732 vs. 0.758 mg/L). YeastOne GM MICs of itraconazole and posaconazole were about one two-fold dilution lower than reference GM MICs (itraconazole 0.293 vs. 0.567 mg/L; posaconazole 0.096 vs. 0.214 mg/L), whereas the YeastOne GMMIC of amphotericin B was 3.3-fold higher than the reference GMMIC (1.959 vs 0.602 mg/L). Of 24 TR34/L98H A. fumigatus isolates, itraconazole resistance could be identified by YeastOne in all isolates, and MICs of itraconazole and voriconazole, respectively, obtained by two methods correlated well in terms of GM MICs, MIC ranges, and agreement within ± 1log2 dilution (100% for both). Four A. niger isolates exhibited trailing growth with microscopically aberrant small, rounded, compact hyphal forms in itraconazole-containing wells with concentrations ranged from 0.5 to 8 mg/L, whereas the corresponding itraconazole MICs by YeastOne were all categorized as wild-type MICs (≤ 0.5 mg/L) without trailing growth microscopically. Conclusion: YeastOne can be applied in clinical laboratories for MIC determination of itraconazole, posaconazole, and voriconazole against Aspergilli and for detecting azole-resistant TR34/L98H A. fumigatus. However, YeastOne tended to result in a higher amphotericin B MIC and failed to detect trailing growth in itraconazole-resistant A. niger, and hence, MIC interpretation on both occasions should be undertaken with caution. An improvement of amphotericin B MIC testing should also be made for the YeastOne method. |
