Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts

Abstract

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.

Novel Drug Screening Assay for Acanthamoeba castellanii and the Anti-Amoebic Effect of Carbonic Anhydrase Inhibitors

“…Even though the superiority of different drug screening assays has not been experimentally verified, the non-nutrient Escherichia coli plate assay has been suggested to function better than the LDH release assay, trypan blue and fluorescent staining. 56 The absorbance values between control curves (0) differ from each other in the testing of different inhibitors (range 0.5−1.9 in the inhibitor assay). Many factors can influence this.…”

“…The challenge linked to SRB staining is that it only adheres to proteins in trophozoites, leading to the complete exclusion of cysts from the assay, thus limiting its ability to determine infections. Even though the superiority of different drug screening assays has not been experimentally verified, the non-nutrient Escherichia coli plate assay has been suggested to function better than the LDH release assay, trypan blue and fluorescent staining …”

“…Even though the superiority of different drug screening assays has not been experimentally verified, the non-nutrient Escherichia coli plate assay has been suggested to function better than the LDH release assay, trypan blue and fluorescent staining. 56 …”

Efficacy of Off-Label Anti-Amoebic Agents to Suppress Trophozoite Formation of Acanthamoeba spp. on Non-Nutrient Agar Escherichia Coli Plates

“…In a recent study, we reported that the survival rates of drug-treated trophozoites/cysts determined by enzymatic-and dye-based viability assays might differ substantially from those yielded with the non-nutrient agar E. coli plate assay and proposed the latter test system as the gold standard for studying the treatment efficacy of drugs against Acanthamoeba spp. isolates [7]. In that study, we also observed that the off-label anti-amoebic drugs PHMB, PD, NM, and PVPI were more effective against the A. castellanii strain 1BU than the off-label anti-amoebic drugs CH, HD, DD, and MF under in vitro conditions; however, this was without providing a quantitative evaluation of the observed findings made with the non-nutrient agar E. coli plate assay [7].…”

“…isolates [7]. In that study, we also observed that the off-label anti-amoebic drugs PHMB, PD, NM, and PVPI were more effective against the A. castellanii strain 1BU than the off-label anti-amoebic drugs CH, HD, DD, and MF under in vitro conditions; however, this was without providing a quantitative evaluation of the observed findings made with the non-nutrient agar E. coli plate assay [7]. In the study presented here, we aimed to quantify the effectiveness of the off-label anti-amoebic agents PHMB, CH, HD, PD, DD, NM, MF, and PVPI in killing cysts of the A. castellanii strains 1BU (sequence type T4), 3ST (T4), and 9GU (T4), and the A. hatchetti strain 11DS (T6), under in vitro conditions.…”