Effective Detection of Azole Resistance in Aspergillus Fumigatus Sensu Stricto Using a Gradient Diffusion Plastic Strip: A Comparison of Filtered-Adjusted Vs. Unfiltered-Unadjusted Inocula.
Medical mycology（2025）
Abstract
Gradient diffusion plastic strips are utilized for azole susceptibility testing against Aspergillus fumigatus in some clinical microbiology laboratories; however, they lack proper validation. We evaluated the performance of this method for detecting azole resistance in A. fumigatus sensu stricto, and we assessed whether skipping filtration and inoculum adjustment of conidial suspensions negatively influenced its performance. A total of 98 A. fumigatus sensu stricto isolates, previously classified as azole-susceptible or resistant, were studied. Azole-resistant isolates had specific cyp51A gene mutations, including TR34-L98H ± S297T-F495I, G54R, TR46-Y121F-T289A, and G448S. Conidial suspensions were prepared following the principles of the E.Def 9.4 method or left unfiltered and unadjusted. Based on minimum inhibitory concentration (MIC) distributions obtained with the gradient diffusion plastic strip using both types of inoculum preparations, cutoff values were proposed to classify isolates as resistant: >1 mg/l for itraconazole and voriconazole and >0.25 mg/l for posaconazole. These cutoffs coincided with EUCAST breakpoints and correctly classified all except one (TR46-Y121F-T289A) isolate. High essential agreement values (95.9%-98%) were obtained, regardless of inoculum preparation. Overall, sensitivity/specificity values of the gradient diffusion plastic strip using the proposed cutoff values for itraconazole (100%/98%), voriconazole (100%/100%), and posaconazole (100%/98%) to screen for azole resistance were not impacted by the inoculum preparation method. Thus, the gradient diffusion plastic strip is a reliable method for screening azole resistance in A. fumigatus isolates using the proposed MIC cutoffs, and inoculum preparation without filtration and adjustment of conidial suspensions did not negatively influence the performance of the method.
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