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.
OBJECTIVE: 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.

BACKGROUND: Azole resistance screening in A. fumigatus isolates can be routinely carried out by using azole-containing plates (E.Def 10.2 method), that requires filtering conidial suspensions prior inoculum adjustment.
OBJECTIVE: We evaluated whether skipping the filtration step of conidial suspensions negatively influences the performance of the E.Def 10.2. Patients/Methods A. fumigatus sensu stricto isolates (n=92), classified as azole-susceptible or azole-resistant according to the EUCAST microdilution E.Def 9.4 method, were studied. Azole-resistant isolates had either wild type cyp51A gene sequence (n = 3) or the TR34 -L98H (n = 26), G54R (n = 5), TR46 -Y121F-T289A (n = 1), F46Y-M172V-N248T-D255E-E427K (n = 1), F165L (n=1), or G448S (n=1) cyp51A gene substitutions. In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions were obtained by adding distilled water (Tween 20 0.1%). Subsequently, the suspensions were either filtered or left unfiltered prior to inoculum adjustment to 0.5 McFarland. Using microdilution as the gold standard, agreement, sensitivity, and specificity of the agar plates inoculated with two inoculums were assessed.
RESULTS: Agreements for the agar screening method with either unfiltered or filtered conidial suspensions were high for itraconazole (100%), voriconazole (100%), and posaconazole (97.8%). Sensitivity (100%) and specificity (98.2%) of the procedure to rule in or out resistance when unfiltered suspensions were used were also high. Isolates harbouring the TR34 -L98H, G54R, and TR46 -Y121F-T289A substitutions were detected with the modified method.
CONCLUSIONS: Unfiltered conidial suspensions does not negatively influence the performance of the E.Def 10.2 method when screening for A. fumigatus sensu stricto.