Significant Impact of Growth Medium on Itraconazole Susceptibility in Azole-Resistant Versus Wild-Type Trichophyton indotineae, rubrum, and quinckeanum Isolates

Azole resistance in dermatophytes, particularly Trichophyton indotineae , has become a growing global concern. Current antifungal susceptibility testing protocols (EUCAST, CLSI) have limitations in reproducibility and sensitivity. This study aimed to evaluate how medium composition, incubation temperature, and spore concentration influence itraconazole susceptibility testing across various dermatophyte species. Thirty-eight clinical isolates representing Trichophyton , Microsporum , and Epidermophyton species were tested using a microplate laser nephelometry system (MLN). IC 50 values for itraconazole were determined in three different media (Sabouraud glucose (SG), RPMI-based (RG), and RG supplemented with casein (RGC)) at 28 °C and 34 °C. Effects of spore concentration on growth dynamics and lag phase were also analyzed. SG medium provided clear phenotypic separation between resistant and sensitive isolates. In contrast, RG and RGC showed overlapping IC 50 values. Lower spore concentrations revealed underlying growth differences, which were masked at higher inoculum levels. Temperature and media composition significantly affected IC 50 outcomes. Genotypic analysis confirmed resistance-associated Erg11B point mutations and genomic amplifications in T. indotineae , particularly in combination with Erg1 mutations, forming distinct subpopulations. SG medium combined with reduced spore concentrations offered improved differentiation of resistant versus sensitive strains. These findings support the development of more accurate susceptibility testing protocols and highlight the need to establish species-specific ECOFF values for dermatophytes.