Assessing the Incubation Time in Antifungal Susceptibility Testing Using the Agar-Based Disk Diffusion Method

Background: The use of agar-based disk diffusion (ABDD) for susceptibility testing of fungal pathogens is well-established. However, the incubation time may affect the result. Therefore, this study aims to determine the optimal incubation period for assessing the inhibition zone against Trichophyton spp., one of the most common pathogens among patients with dermatophytosis. Methods: This in-vitro study involved 129 cryopreserved strains of Trichophyton spp., including T. rubrum complex (67 strains) and T. mentagrophytes complex (62 strains). The strains were recovered in Sabouraud’s dextrose agar, and conidia were harvested after 5 to 7 days of culture. The ABDD method was performed in Müller-Hinton agar with five antifungals: itraconazole (ITC), griseofulvin (GRI), clotrimazole (CLO), miconazole (MCI), and voriconazole (VOR). The inhibition diameter zone (IZD) was measured consecutively on days 3, 5, and 7 by the caliper. Data analysis was performed by SPSS 25. Results: The mean IZD of all antifungals on T. rubrum was highest on day 5. The results were similar in T. mentagrophytes except VOR presented the highest IZD on day 3. We noticed significant differences in the IZD of ITC, CLO, MCI, and VOR for T. rubrum. In T. mentagrophytes, only VOR showed a marked difference between periods. Conclusion: The study’s findings recommend the optimal incubation time in dermatophyte susceptibility testing. Specifically, the incubation time should be 5 days for ITC, GRI, CLO, and MCI for both T. rubrum and T. mentagrophytes. Regarding VOR, it should be 5 days for T. rubrum and 3 days for T. mentagrophytes.