Azole-Resistant Aspergillus fumigatus Clinical Isolate Screening in Azole-Containing Agar Plates (EUCAST E.Def 10.1): Low Impact of Plastic Trays Used and Poor Performance in Cryptic Species

Abstract

Azole-containing agar is used in routine Aspergillus fumigatus azole resistance screening. We evaluated the impact of the type of plastic used to prepare in-house agar plates on the procedure's performance against A. fumigatus sensu stricto and cryptic species. A. fumigatus sensu stricto (n = 91) and cryptic species (n = 52) were classified as susceptible or resistant (EUCAST E.Def 9.3.2; clinical breakpoints v10). In-house azole-containing agar plates were prepared following EUCAST E.Def 10.1 on three types of multidish plates. We assessed the sensitivity, specificity, and agreement values of the agar plates to screen for azole resistance. Overall, sensitivity and specificity values of the agar screening method were 100% and 93.3%, respectively. The type of tray used did not affect these values. All isolates harboring TR₃₄-L98H substitutions were classified as resistant to itraconazole and voriconazole by the agar method; however, false susceptibility (very major error) to posaconazole was not uncommon and happened in isolates with posaconazole MICs of 0.25 mg/liter. Isolates harboring G54R and TR₄₆-Y121F-T289A substitutions were correctly classified by the agar method as itraconazole/posaconazole resistant and voriconazole resistant, respectively. False resistance (major error) occurred in isolates showing tiny fungal growth. Finally, agreements between both procedures against cryptic species were much lower. Azole-containing agar plates are a convenient and reliable tool to screen for resistance in A. fumigatus sensu stricto; the type of plastic tray used minimally affects the method. On the contrary, the performance against cryptic species is rather poor.

Keywords: Aspergillus fumigatus; EUCAST E.Def 10.1; agar plates; azoles; resistance.

FIG 1

Number of Aspergillus fumigatus sensu stricto isolates falling in each MIC value according to the E.Def 9.3.2 method with (bars above the x axis) or without (bars below the x axis) fungal growth on agar plates containing itraconazole (a to c), voriconazole (d to f), and posaconazole (g to i). Susceptible isolates are depicted in green, and resistant isolates are depicted in other colors depending on the cyp51A gene mutation. Samples were obtained from isolates inoculated on Nunc tissue-treated plates (a, d, and g), Nunc nontissue-treated plates (b, e, and h), and Labclinics tissue-treated plates (c, f, and i).

FIG 2

Example of major errors (false resistance) detected for itraconazole-susceptible (a; isolate code 6142) and voriconazole-susceptible (c; isolate code 6430) Aspergillus fumigatus sensu stricto isolates and for an itraconazole-susceptible Neosartorya udagawae isolate (b; isolate code 6158). Fungal growth was observed for all isolates on agar wells with a scoring of 0.5 or 1. Wells in the plates contained itraconazole (4 mg/liter; well 1), voriconazole (2 mg/liter; well 2), posaconazole (0.5 mg/liter; well 3), and no azole (growth control; well 4). The A. fumigatus sensu stricto isolate 6430 harbored the G54R mutation. Images of the isolates inoculated on Labclinics tissue-treated trays were obtained.

FIG 3

Number of cryptic species isolates falling in each MIC value according to the E.Def 9.3.2 method and showing fungal growth with scorings of 0, 0.5, 1, 2, or 3 on agar plates containing itraconazole (a to c), voriconazole (d to f), and posaconazole (g to i). Isolates with or without fungal growth on the agar plates are represented as bars above and below the x axis, respectively. Scores obtained from Nunc tissue-treated plates (a, d, and g), Nunc nontissue-treated plates (b, e, and h), and Labclinics tissue-treated plates (c, f, and i) are shown.

FIG 4

Growth pattern and score of azole-resistant A. fumigatus sensu stricto isolates harboring the following cyp51A gene substitutions: F165L (a), TR₄₆-Y121F-T289A (b), G54R (c), and TR₃₄-L98H (d). Wells in the plates contained itraconazole (4 mg/liter; well 1), voriconazole (2 mg/liter; well 2), posaconazole (0.5 mg/liter; well 3), and no azole (growth control; well 4).