We aimed to compare LDH release assay, trypan blue and fluorescent stainings, and
non-nutrient Escherichia coli plate assay in determining treatment efficacy of antiamoebic
agents against Acanthamoeba castellanii trophozoites/cysts, in vitro. 1BU trophozoites/cysts
were challenged with 0.02% polyhexamethylene biguanid (PHMB), 0.1% propamidine isethionate
(PD), and 0.0065% miltefosine (MF). Efficacies of the drugs were determined by LDH
release and trypan blue assays, by Hoechst 33343, calcein-AM, and ethidium homodimer-1
fluorescent dyes, and by a non-nutrient agar E. coli plate assay. All three antiamoebic
agents induced a significant LDH release from trophozoites, compared to controls (p
< 0.0001). Fluorescent-dye staining in untreated 1BU trophozoites/cysts was negligible,
but using antiamoebic agents, there was 59.3%-100% trypan blue, 100% Hoechst 33342,
0%-75.3% calcein-AM, and 100% ethidium homodimer-1 positivity. On E. coli plates,
in controls and MF-treated 1BU trophozoites/cysts, new trophozoites appeared within
24 h, encystment occurred after 5 weeks. In PHMB- and PD-treated 1BU throphozoites/cysts,
irregularly shaped, smaller trophozoites appeared after 72 h, which failed to form
new cysts within 5 weeks. None of the enzymatic- and dye-based viability assays tested
here generated survival rates for trophozoites/cysts that were comparable with those
yielded with the non-nutrient agar E. coli plate assay, suggesting that the culture-based
assay is the best method to study the treatment efficacy of drugs against Acanthamoeba.
