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Updated: Sep 14, 2025
Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds
Published on: February 14, 2018
Multicentre evaluation of a EUCAST-based agar-screening method for terbinafine and itraconazole susceptibility of
Joseph Meletiadis1, Maria Siopi2, Karin Meinike Jørgensen3
1Clinical Microbiology Laboratory, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece; Unit of Mycology, Statens Serum Institute, Copenhagen, Denmark.
Objectives:
Resistance in Trichophyton species has become a global public health issue. Here, a four-well agar-screening method based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) definitive document (E.Def) 10.2 method was evaluated in five centres using a panel of terbinafine wild-type (WT) and non-WT Trichophyton isolates with the recently determined tentative epidemiological cut-offs of the E.Def 11.0 method.
Methods:
Forty-two Trichophyton isolates, all WT to itraconazole, 17 molecularly characterized terbinafine non-WT and 25 WT (non-WT/WT: 11/9 T. rubrum, 5/6 T. indotineae, 1/6 T. interdigitale and 0/4 T. mentagrophytes) were tested in five centres using four-well plates containing terbinafine 0.016 mg/L and 0.125 mg/L; itraconazole 1 mg/L and drug-free agar, respectively. Plates were inoculated (25 μL, 0.5 McFarland) and incubated for 5 to 7 days at 25 to 28°C. Visual growth comparable with drug-free control, ignoring faint growth/pinpoint colonies, indicated non-WT phenotype. Sensitivity and specificity in detecting Trichophyton non-WT isolates were calculated.
Results:
Most isolates produced sufficient growth after 5 days, whereas 6 to 10 of 42 isolates required 7 days of incubation. All isolates were correctly classified as WT to itraconazole by all five centres. The sensitivity (median [range among centres]) in detecting terbinafine non-WT isolates was 94% to 100% (95% CI: 79-100%), whereas the specificity for detecting WT isolates was 100%. Sensitivity and specificity were high across different species. Among the discrepancies, one false WT was observed with a T. rubrum strong mutant in one centre. WT T. indotineae grew on terbinafine 0.016 mg/L.
Discussion:
The multicentre evaluation confirmed that the agar-screening method was sensitive and specific for detecting terbinafine non-WT Trichophyton isolates and correctly identified itraconazole WT strains.
