ACCUMULATION OF AMPHOTERICIN-B IN HUMAN MACROPHAGES ENHANCES ACTIVITYAGAINST ASPERGILLUS-FUMIGATUS CONIDIA - QUANTIFICATION OF CONIDIAL KILL AT THE SINGLE-CELL LEVEL

A cytofluorometric assay that allowed assessment of damage to phagocyt osed Aspergillus fumigatus conidia at the single-cell level was develo ped. After ingestion by monocyte-derived macrophages (MDMs), conidia w ere reisolated by treatment of the cells with streptolysin O, a pore-F orming toxin with lytic properties on mammalian cells but not on fungi . The counts obtained by staining of damaged conidia with propidium io dide and quantification by cytofluorometry correlated with colony coun ts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damage Aspergill us conidia in vitro. The killing rate 12 h after phagocytosis was foun d to be only 10 to 15%. However, intracellular loading of the phagocyt es with amphotericin B (AmB) dose dependently enhanced the anticonidia l activity. Preincubation of macrophages with only 1 mu g of AmB per m i resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the r apid quantification of anticonidial activity against A. fumigatus in v itro. Intracellular accumulation elf AmB may represent an important fa ctor underlying the efficacy of this antifungal drug in the prophylaxi s and treatment of Aspergillus infections.