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.