Synergy of itraconazole with macrophages in killing Blastomyces dermatitidis

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Abstract
We examined in vitro interaction between the azole antifungal agents itraconazole and ketoconazole and macrophages and their activities against Blastomyces dermatitidis. Fungistatic and fungicidal concentrations for B. dermatitidis in vitro were assessed in a microculture system in which fungistasis was measured as inhibition of multiplication and fungicidal activity was measured as reduction of inoculum CFU. Resident peritoneal murine macrophages, which surround but do not phagocytize the fungus, were not fungicidal for B. dermatitidis isolates but were fungistatic for some isolates studied. Synergy was demonstrated when fungistatic concentrations (e.g., 0.01 micrograms/ml) of itraconazole, which limited growth 55% compared with that of controls, were cocultured with macrophages; this resulted in fungicidal activity (85% killing) against B. dermatitidis (ATCC 26199) in 72-h assays. This synergy could occur even if itraconazole was added after the macrophages had surrounded the fungus. Ketoconazole at fungistatic concentrations did not act synergistically with macrophages to kill B. dermatitidis. Lymph node lymphocytes could not substitute for macrophages in synergy with itraconazole to kill B. dermatitidis. When B. dermatitidis was separated by a filter from macrophages in Transwell cultures, fungicidal synergy with itraconazole was less efficient. Pretreatment of B. dermatitidis with itraconazole for 24 h did not render the fungus susceptible to killing by macrophages in the absence of itraconazole, whereas pretreatment of nonfungistatic macrophages with itraconazole rendered them fungistatic in a dose-dependent manner. Three other isolates were killed by otherwise fungistatic concentrations of itraconazole when the isolates were cocultured with macrophages. These findings indicate that one basis for the efficacy of itraconazole versus ketoconazole in treating blastomycosis could be synergy of a fungistatic concentration of itraconazole with macrophages in killing of B. dermatitidis.