Azole resistance screening in Aspergillus fumigatus sensu stricto using the azole-containing agar method (EUCAST E.Def 10.2): conidial suspension filtration and inoculum adjustment before inoculum preparation may not be needed

Abstract

Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing agar plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculum preparation are time-consuming. We evaluated whether skipping the filtration and inoculum adjustment steps negatively influenced the performance of the E.Def 10.2 procedure. A. fumigatus sensu stricto isolates (n = 98), previously classified as azole susceptible or azole resistant (E.Def 9.4 method), were studied. Azole-resistant isolates had either the wild-type cyp51A gene sequence (n = 1) or the following cyp51A gene substitutions: TR₃₄-L98H (n = 41), G54R (n = 5), TR₄₆-Y121F-T289A (n = 1), or G448S (n = 1). In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions obtained by adding distilled water (Tween 20 0.1%) were either filtered and the inocula adjusted to 0.5 McFarland or left unfiltered and unadjusted. Agreements between the agar screening methods using inocula prepared by each procedure were high for itraconazole (99%), voriconazole (100%), and posaconazole (94.9%). Sensitivity and specificity (considering the susceptibility category as per the microdilution E.Def 9.4 method as the gold standard) of E.Def 10.2 were 100% to rule in or rule out resistance when unfiltered and unadjusted suspensions were used; the resistance phenotype of isolates harboring the TR₃₄-L98H, G54R, or TR₄₆-Y121F-T289A substitutions was correctly detected. Unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto.

Importance: Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculation of plates are time-consuming. We, here, showed that unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto.

Keywords: Aspergillus fumigatus; E.Def 10.2; EUCAST; azole resistance; azole-containing plates; detection.

Fig 1

A conidial suspension of isolate 6118 was prepared in distilled water and then serially diluted to 1/10,000. The figure shows the aspect of each dilution of the unfiltered-unadjusted inoculum.