Development and multicentre validation of an agar-based screening method for echinocandin susceptibility testing of Aspergillus species

Abstract

Background: Reference antifungal susceptibility testing of echinocandins against Aspergillus spp. relies on the determination of the minimal effective concentration, which is difficult to perform, time-consuming and subjective. We developed and evaluated in a multicentre study an agar-based screening method for echinocandin susceptibility testing of Aspergillus spp.

Methods: Forty WT isolates [10 Aspergillus fumigatus species complex (SC), 10 Aspergillus flavus SC, 10 Aspergillus terreus SC and 10 Aspergillus niger SC] and 4 non-WT A. fumigatus isolates with or without known fks alterations were used. The optimal test conditions and stability over time were evaluated in preliminary studies monitoring colony growth. Twenty-microlitre aliquots of 1-2 McFarland inocula in 0.1% Tween 20 aqueous solution were added to each well and plates were incubated for 24/48 h at 35 ± 2°C. Subsequently, all isolates were tested blindly at three centres using four-well screening plates, containing anidulafungin, caspofungin, micafungin or no antifungal in each of the four wells, respectively.

Results: WT isolates produced fluffy colonies on drug-free agar wells only. The non-WT isolates produced fluffy colonies on echinocandin-containing and control agar wells. Using the echinocandin concentrations of 0.25 mg/L anidulafungin, 1 mg/L caspofungin and 0.125 mg/L micafungin, and the compact (non-fluffy) versus fluffy colony morphology endpoint, all centres successfully discriminated non-WT and WT strains even after 24 h. Among the three echinocandins, anidulafungin produced the clearest endpoints.

Conclusions: The four-well plate agar method is suitable for echinocandin susceptibility screening of Aspergillus spp. and can be used to detect echinocandin non-WT isolates.

Figure 1.

Colony morphology of an A. fumigatus non-WT strain and an A. fumigatus SC WT strain on caspofungin-containing (2 mg/L) and drug-free agar after 24 and 48 h of incubation. Note the fluffy versus compact appearance. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Colony morphology of selected echinocandin WT A. fumigatus SC and non-WT A. fumigatus isolates on agar containing increasing effective concentrations (mg/L) of each echinocandin and drug-free agar (GC) in four-well plates after 24 and 48 h of incubation at 35 ± 2°C. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

Agar-based screening method for three Aspergillus spp. in four-well plates containing increasing concentrations (mg/L) of echinocandins and drug-free agar (GC) after incubation at 35 ± 2°C for 24 h. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 4.

Production of an irregular (top left photograph) or a small (colony diameter ≤8 mm) (top right photograph) halo around A. niger SC and A. terreus SC WT isolates after incubation at 35 ± 2°C for 48 h. The morphology of colonies is clearly different from strains grown on drug-free agar with and without conidiogenesis (colony diameter ≥15 mm) (bottom photographs). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 5.

Colony morphology of three A. fumigatus non-WT isolates after 24 and 48 h of incubation in the four-well agar screening plates containing 1 mg/L caspofungin (CAS), 0.25 mg/L anidulafungin (ANI) and 0.125 mg/L micafungin (MIC) and no drug (GC). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.