Development of a Visual Assay Based on Colony Diameter Measurement for Drug Susceptibility Testing of Poorly Sporulating Fungi.

Antifungal susceptibility testing is essential for guiding the clinical management of fungal infections. However, the standard method for broth dilution testing of filamentous fungi requires spore suspensions and is therefore unsuitable for poorly sporulating fungal species or strains. Some Aspergillus lentulus strains sporulate poorly, and Schizophyllum commune is a clinically relevant non-sporulating fungus for which preparing homogeneous inocula remains challenging. In this study, we developed an agar‑based visual assay to evaluate relative growth inhibition without requiring spores. Using 6‑well plates containing varying concentrations of itraconazole (ITCZ) or voriconazole (VRCZ), the colony diameters of A. lentulus and S. commune were measured after 24 hours and 48 hours, respectively, and normalized to those of the drug‑free control. A. lentulus exhibited clear growth inhibition at 0.25 µg/mL ITCZ, but further increases in concentration did not result in proportionally greater inhibition under the present assay conditions. In contrast, VRCZ markedly and dose‑dependently reduced colony diameter, resulting in only < 10% growth at 4 µg/mL. ITCZ susceptibility varied widely among S. commune strains, whereas VRCZ consistently inhibited growth at concentrations ≥ 1 µg/mL. These susceptibility patterns aligned with previously reported minimum inhibitory concentration ranges for both species. Notably, our method also accommodated slow‑growing strains and allowed extended incubation when necessary. Although not directly comparable to standardized minimum inhibitory concentration methods, our agar‑based assay provides a practical and visually interpretable means to assess drug susceptibility in A. lentulus and S. commune under the tested conditions.