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. 2014;58(3):1523-8.
doi: 10.1128/AAC.02254-13. Epub 2013 Dec 23.

In Vitro Efficacy of Corifungin Against Acanthamoeba Castellanii Trophozoites and Cysts

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In Vitro Efficacy of Corifungin Against Acanthamoeba Castellanii Trophozoites and Cysts

Anjan Debnath et al. Antimicrob Agents Chemother. .
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Abstract

Painful blinding keratitis and fatal granulomatous amebic encephalitis are caused by the free-living amebae Acanthamoeba spp. Several prescription eye medications are used to treat Acanthamoeba keratitis, but the infection can be difficult to control because of recurrence of infection. For the treatment of encephalitis, no single drug was found useful, and in spite of the use of a combination of multiple drugs, the mortality rate remains high. Therefore, efficient, novel drugs are urgently needed for the treatment of amebic keratitis and granulomatous amebic encephalitis. In this study, we identified corifungin, a water-soluble polyene macrolide, as amebicidal. In vitro, it was effective against both the trophozoites and the cysts. Transmission electron microscopy of Acanthamoeba castellanii incubated with corifungin showed the presence of swollen mitochondria, electron-dense granules, degeneration of cytoplasm architecture, and loss of nuclear chromatin structure. These changes were followed by lysis of amebae. Corifungin also induced the encystment process of A. castellanii. There were alterations in the cyst cell wall followed by lysis of the cysts. Corifungin is a promising therapeutic option for keratitis and granulomatous amebic encephalitis.

Figures

FIG 1
FIG 1
Effect of corifungin on A. castellanii growth. Trophozoites were incubated with 25, 100, and 200 μM corifungin for 24, 48, 72, 96, and 120 h, and viable trophozoites were calculated as the percentage of trophozoites compared with the means of controls (as 100%). Values plotted are means and standard deviations for three independent experiments.
FIG 2
FIG 2
A. castellanii viability determination by trypan blue exclusion method. Trophozoites were incubated with 200 μM corifungin for different time periods. (A) Amebae with fresh medium only. (B) Amebae incubated with corifungin for 24 h. (C) Amebae incubated with corifungin for 48 h. (D) Amebae incubated with corifungin for 72 h. (E) Amebae incubated with corifungin for 96 h. (F) Amebae incubated with corifungin for 120 h. Cells stained blue were considered nonviable (arrowheads). Magnification, ×60.
FIG 3
FIG 3
Light microscopy of epoxy-embedded semithin sections, stained with toluidine blue, of A. castellanii trophozoites incubated in the presence and absence of corifungin. Trophozoites were incubated with 200 μM corifungin for different time periods. (A) Amebae (arrows) with fresh medium only. (B) Amebae incubated with 200 μM corifungin for 24 h. Arrows indicate trophozoites, and the arrowhead indicates a cyst. (C) Amebae incubated with 200 μM corifungin for 48 h. The damage in the trophozoites (arrows) is evident, and there was an increase in the number of the cysts (arrowheads). (D) Amebae incubated with 200 μM corifungin for 72 h. More damage is seen in the trophozoites (arrows), and damage is also found in the cysts (arrowheads). (E and F) Amebae incubated with 200 μM corifungin for 96 to 120 h. Cell remains of trophozoites (arrows) and more damage in the cysts (arrowheads) are seen. Magnification, ×60.
FIG 4
FIG 4
Transmission electron microscopy of A. castellanii trophozoites incubated with 200 μM corifungin for different time periods. (A) Trophozoites treated with only fresh medium. (B) Trophozoites treated with corifungin for 24 h. The alteration of the plasma membrane is evident (arrowheads), and electron-dense granules (eg) appeared in the degenerated cytoplasm. (C) Trophozoites treated with corifungin for 48 h. Mitochondria (arrows) showed damage, and glycogen (*) was present. (D) Trophozoites treated with corifungin for 72 h. Mitochondria (arrows) are dilated, and the nucleus (N) shows alterations. (E) Trophozoites treated with corifungin for 96 h. The edematous mitochondria (arrows) and damaged nucleus (N) are evident. The inset image is the higher magnification of mitochondria. (F) Trophozoites treated with corifungin for 120 h. The ameba shows vacuolization, the electron-dense granules (eg) increase in number, and cell membrane damage is evident (arrowheads). Bars, 2 μm.
FIG 5
FIG 5
Transmission electron microscopy of A. castellanii cyst formation after incubation of trophozoites with 200 μM corifungin for 120 h. Encystment of the ameba is evident. Cell wall (arrowheads) is present. Inside the cyst, the trophozoite (arrow) presents signs of damage (*). Electron-dense granules also appear (eg). In many cysts, loss of the inner wall and cell wall disruption are observed (arrowheads). Bars, 2 μm.

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