A comparative study of fungicidal activities of voriconazole and amphotericin B against hyphae of Aspergillus fumigatus

Objectives: To study the in vitro fungicidal activity of voriconazole against hyphae of Aspergillus fumigatus and compare the results with those obtained for the known fungicidal drug amphotericin B. Methods: A. fumigatus mycelia were grown on Sabouraud dextrose agar and in peptone yeast extract glucose broth until the cultures reached a mid-logarithmic growth phase. The fungicidal activities of voriconazole and amphotericin B against actively growing hyphae of A. fumigatus were examined by a kill-curve experiment and a fungal cell viability test. For the kill-curve study, the drug-treated hyphae were washed, homogenized and resuspended in 1 mL of sterile water, diluted 10-1000 fold and allquots of 0.1 mL were spread on Sabouraud dextrose agar and allowed to grow for 48 h at 35 degrees C. The cfu were determined and plotted against drug concentrations for each time of exposure to obtain the kill curve. The viability of drug-treated A. fumigatus hyphae was determined by their ability to reduce tetrazolium compound 3-(4,5-dimethyl-2-thiazolyi)-2,5-diphenyl-2H-tetrazolium bromide. Results: Exposure of A. fumigatus hyphae to several concentrations (1-16mg/L) of voriconazole or amphotericin B for various time intervals killed the hyphae in a time- and drug concentration-dependent manner. Voriconazole at 1 mg/L killed > 95% of the hyphae grown on Sabouraud dextrose agar after 48 h of exposure, whereas amphotericin B at the same concentration killed similar to 70% of the hyphae after exposure for the same duration. Approximately 99% killing of hyphae grown in peptone yeast extract glucose broth was obtained for voriconazole at 1 mg/L after 48 h of exposure, whereas amphotericin IS at 1 mg/L yielded similar to 82% killing after 48 h. The fungal cell viability test by tetrazolium reduction assay showed that mycelia exposed to >= 1 mg/L (Sabouraud dextrose agar blocks) and >= 2mg/L (broth cultures) of voriconazole for 48h completely failed to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. At low concentrations (1 -2 mg/L) amphotericin B had no detectable effect on 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazollum bromide reduction by drug-treated mycelia, whereas mycelia treated with 16mg/L for 48h showed similar to 50% inhibition of 3-(4,5-dimethyl-2-thiazolyi)2,5-diphenyl-2H-tetrazolium bromide reduction compared with the control. Conclusions: Voriconazole possesses excellent fungicidal activity against actively growing hyphae of A. fumigatus. A comparison of results with those obtained for the known fungicidal drug amphotericin B shows that, in peptone yeast extract glucose broth, voriconazole has superior fungicidal activity against A. fumigatus hyphae compared with that of amphotericin B.