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

Shi, Lei and Stachon, Tanja and Latta, Lorenz and Elhawy, Mohamed Ibrahem and Gunaratnam, Gubesh and Orosz, Erika and Kiderlen, Albrecht F. and Seitz, Berthold and Bischoff, Markus and Szentmáry, Nóra (2020) Comparison of in vitro assays to study the effectiveness of antiparasitics against Acanthamoeba castellani trophozoites and cysts. ACTA MICROBIOLOGICA ET IMMUNOLOGICA HUNGARICA, 67 (1). pp. 23-32. ISSN 1217-8950

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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.

Item Type: Article
Uncontrolled Keywords: trypan blue assay; Acanthamoeba castellanii 1BU; LDH release assay; non-nutrient agar E. coli plate assay;
Subjects: Q Science / természettudomány > QR Microbiology / mikrobiológia
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 19 Oct 2021 06:42
Last Modified: 19 Oct 2021 06:42
URI: http://real.mtak.hu/id/eprint/132704

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