Development and multicentre evaluation of a screening method for echinocandin susceptibility testing of Aspergillus spp.
J.Meletiadis1,2, M.Siopi1, L.Kanioura2, K.Meinike Jørgensen4,  J.W.Mouton2, M.C.Arendrup4,5,6

1Attikon University General Hospital, Athens, Greece, 2Erasmus MC, Rotterdam, The Netherlands, 4Statens Serum Institut, Copenhagen, Denmark, 5 Rigshospitalet, Copenhagen, Denmark 6University of Copenhagen, Copenhagen, Denmark


Objectives:
Antifungal susceptibility testing of Aspergillus spp. against echinocandins relies on the microscopic determination of the minimal effective concentration (MEC) with broth microdilution method which is difficult-to-perform, time consuming and subjective even for trained staff. We therefore developed and evaluated in a mulicentre study the performance of an echninocandin screening agar method for susceptibility testing of Aspergillus spp.
Methods:
A total of 40 Aspergillus wild-type isolates (10 A. fumigatus, 10 A. flavus, 10 A. terreus and 10 A. niger) and 4 non wild-type A. fumigatus isolates with high MEC with fks alterations (AFS678P SSI-1794-homo, AFS678P SSI-765-hetero, DPLMD24053) or without (DPLDS55985) were used. MECs were determined with EUCAST E.Def 9.3. All isolates were tested blindly at three centres using 4-well screening plates, containing either caspofungin, micafungin, anidulafungin or a drug free agar. The optimal concentrations of each echinocandin were evaluated in preliminary studies using 0.001-4 mg/L concentrations and morphologic endpoints (fluffy vs. compact appearance, aerial mycelia around the colony, diameter of colony). Stability of 4-well plates was assessed with QC isolates C. krusei ATCC6258, C. parapsilosis ATCC22019, A. fumigatus ATCC MYA-3626 up to 3 months. 20 μl of 1-2 McFarland inocula in 0.1% Tween20 were added to each well and plates were incubated for 48 h at 37oC. Formation of fluffy colonies similar to the drug free control was recorded for each well after 24h and 48h. 
Results:
The median (range) MECs (mg/L) were 0.5 (0.125-1) of caspofungin, 0.03 (0.002-0.12) of micafungin and 0.015 (0.001-0.12) of anidulafungin for the 40 susceptible Aspergillus isolates and >16(2->16) for the resistant isolates (the MEC against DPLMD24053 was 2-4) for all three echinocandins. The optimal concentrations that prevented susceptible Aspergillus isolates to form fluffy colonies similar to the ones observed in drug free media and by resistant A. fumigatus isolates were 1 mg/L of caspofungin, 0.25 mg/L of anidulafungin and 0.12 mg/L of micafungin. The 4-well plates were stable up to 3 months with the same colonial appearance of Aspergillus isolates. All centres successfully detected resistant isolates adopting fluffy colonies after 24h at echinocandin-containing agar as endpoint criteria (Figure). None of the susceptible isolates produced such colonies after 24 h. Some susceptible isolates (8/40 for centre 1, 3/40 for centre 2, 0/40 for centre 3) produced a small (colony diameter <8 cm) or irregular halos around the colonies after 48 h but the morphology were clearly different from the colonies on drug free control agars (colony diameter >11 cm).
Conclusion:
The 4-well plate agar method is suitable for antifungal susceptibility screening of Aspergillus isolates and could be combined with the azole-screening method in order to detect azole- and echinocandin-resistant isolates.

4 / Development and multicentre evaluation of a screening method for echinocandin susceptibility testing of Aspergillus spp.