Studies of in vitro activities of voriconazole and itraconazole against Aspergillus hyphae using viability staining

The minimal fungicidal concentrations (MFCs) of voriconazole and itraconazo le for five clinical isolates each of Aspergillus terreus, Aspergillus fumi gatus, Aspergillus flavus, and Aspergillus niger were determined by a broth macrodilution method. Conidial suspensions as inocula were compared to hyp hae as inocula since the invasive form of aspergillosis is manifested by th e appearance of hyphal structures. In addition, cell viability staining wit h the dye FUN-1 was performed to assess time-dependent damage of hyphae exp osed to various concentrations of the antifungal agents. With conidial inoc ula the MFC ranges of voriconazole were 0.5 to 4 mug/ml and those of itraco nazole were 0.25 to 2 mug/ml, whereas the MFCs (2 to >16 mug/ml) with hypha l inocula were substantially higher (P < 0.01) for both itraconazole and vo riconazole. Only minor differences between the tested antifungals mere obse rved since 16 of 20 and 17 of 20 of the isolates of Aspergillus spp. tested appeared to be killed by voriconazole and itraconazole, respectively. The results of FUN-1 viability staining correlated closely to colony counts, bu t various time- and dose-dependent levels of viability of hyphae were also observed. In conclusion, our study demonstrates the importance of the type of inoculum used to test antifungals and the applicability of FUN-1 stainin g as a rapid and sensitive method for assaying the viability of hyphae.