A novel method using micropig stratum corneum in vitro for the evaluation of anti-Trichophyton mentagrophytes activity.

Abstract

Antifungal susceptibility testing under conditions close to clinical status is expected to provide more helpful information than that obtained by a conventional microdilution method. For this purpose, we developed a novel method to evaluate anti-Trichophyton mentagrophytes activity of antifungal agents in vitro by using disks of micropig stratum corneum epidermis (SCE). Basal agar medium containing K2HPO4, MgSO4, CaCl2 and three kinds of antibiotics. Bifonazole (BFZ), lanoconazole (LCZ) or terbinafine (TBF) was added to the basal agar medium to give serially doubling dilutions ranging from 0.0006 to 10 microg/ml. Five-hundred-microl portions of the agar media thus prepared were solidified in wells of flat-bottomed plates. SCE disks (6 mm in diameter) were placed on surfaces of the agar medium and 10(4) conidia of T. mentagrophytes were inoculated on each SCE disk. There was very good correlation between the initial concentration of the antifungal agents added to the basal agar medium (microg/ml) and the concentration of the agents impregnated into the SCE disks (microg/g) (r2>0.99). The minimum inhibitory concentration (MIC) values of BFZ, LCZ and TBF were respectively 26-, 10- and 78-times higher than those measured by the standard microdilution method. From the correlation between the concentration of the agents in the basal medium and that in the SCE disks, the above MIC values corresponded to the concentrations in SCE disks (microg/g), 832.95 for BFZ, 1.42 for LCZ and 8.87 for TBF. This novel method of antidermatophytic susceptibility testing using SCE would be useful as an in vitro screening of proper antimycotics for topical treatment of dermatophytosis.