Technique for Determining the Viability of Acanthamoeba Cysts Treated with a Cysticidal Agent Based on Membrane Integrity

This study presents a straightforward and reliable method for determining the viability of Acanthamoeba cysts. A standard method for determining Acanthamoeba cyst viability in an in vitro cytotoxicity analysis is required to ensure that the double-walled and sturdy cysts are affected by the substance tested. In this study, a new approach was used to determine the cysticidal potential of redox Cleland’s reagent, dithiothreitol (DTT), against Acanthamoeba cysts. This approach constitutes a significant breakthrough, as the cyst form of Acanthamoeba is known for its high resistance to various chemicals and drugs used to treat infections of the central nervous system and eyes caused by Acanthamoeba. Cyst viability was evaluated based on the intensity of the cyst population under fluorescence produced by propidium iodide (PI) dye and measured using an enzyme-linked immunosorbent assay (ELISA) reader at an absorbance of 636 nm. The results were validated using high-content screening (HCS). For analysis, an individual cell was imaged and examined for phenotypic changes in the Acanthamoeba cyst at the cyst population level. Fluorescence intensity of the cysts in each well in a 96-well plate was measured using Image J software. HCS is an automated technique that uses fluorescence microscopy to produce quantitative data.

Dithiothreitol; Fluorescence intensity; High content screening; Keratitis; Propidium iodide

    Acanthamoebae are opportunistic protozoan parasites distributed in diverse environments, such as air, soil, freshwater, seawater, tap water, bottled mineral water, laboratory distilled water wash bottles, chlorinated swimming pools and sewage. Fish, reptiles, birds and mammals and are known to be one of the most ubiquitous organisms’ host (Khan and Paget, 2002). Although Acanthamoebae exist primarily as free-living amoebae, they can infect the eye, brain and skin and can spread haemotogenously to the central nervous system (CNS) and various organs. Acanthamoeba spp. are dangerous ocular pathogens, particularly in wearers of contact lenses, as shown in a study that found 95% of reported cases involved contact lens wearers (Kilvington et al., 2004). Mazur et al. (1995) reported that the cysts of Acanthamoeba can survive in vitro for more than 20 years and under many unfavorable conditions, such as desiccation and starvation. Aksozek et al. (2002) reported that Acanthamoeba are resistant to a variety of chemicals, such as disinfectants and antimicrobials, and physical agents, including heat, freezing and UV radiation. The sturdy double-walled cyst consists of cellulose and proteinaceous elements, and a standard positive control with which to ensure the cysticidal effect of chemicals or agents on the Acanthamoeba cyst is necessary. A number of antimicrobials have shown efficacy against amoeba in vitro, but there is no evidence that those same drugs will be effective clinically (Schuster and Visvesvara, 2004). In addition, Acanthamoeba cysts are more resistant than trophic amoebae, which are sensitive to the antimicrobials used in treating eye infections caused by Acanthamoeba. 

In the previous research, the fluorescence properties of samarium (Sm) and europium (Eu) have been reported by Usman et al. (2018) and Kusrini et al. (2014), respectively. Preparation of chitosan nanoparticles (CHN NPs) using potassium persulfate has also been reported by Kusrini et al. (2015). Furthermore, biocompatible chitin-encapsulated cadmium sulfide quantum dots (CdS@CTN) that synthesized using the colloidal chemistry method has also been reported by Lim et al. (2021). This CdS@CTN was also screened as antibacterial agent (Lim et al., 2021). This CdS@CTN compound is potential drug carriers and useful in biology, biomedical, fluorescent labelling, and diagnostic applications (Lim et al., 2021). Previously studies can be useful as references for preparation of complexes, nanoparticles and quantum dots.

In the cyst stage, Acanthamoeba is hard to eradicate, and its standard viability quantifications remain problematic. Cyst viability determination is necessary to establish the effectiveness of any substance on Acanthamoeba in the cyst stage. The potential of these substances as drugs to fight the diseases caused by Acanthamoeba at the cyst stage can be determined by the method presented in this study. In previous work by Lazuana et al. (2019) described the application of of trypan blue dye and a haemocytometer to evaluate the number of cysts affected by the cellulase enzyme. Assessing cell viability based on cell wall integrity and discriminating through staining with propidium iodide (PI) intensity in vitro is preferably because it allows for the reading of absorbance changes without bias. Absorbance detection using an enzyme-linked immunosorbent assay (ELISA) reader and HCS can be combined as an effective and simple method of determining the cytotoxicity of treatments for Acanthamoeba cysts. The data from this study show a reduction in Acanthamoeba cyst viability after treatment with DTT (also called Cleland’s reagent) when analyzed using HCS and fluorescence intensity. To our knowledge, this is the first published study on Acanthamoeba cyst cytotoxicity induced by DTT in which viability was assessed by the amount of leakage from the endo- and ecto-cyst walls caused by DTT. This study can be useful as a reference for future research on Acanthamoeba cyst viability. These data will benefit the protozoologist, pharmacologist, cell biologist and microbiologist interested in studying the mechanism of cysticidal agents on Acanthamoeba cysts. The method described in this report can facilitate the study of Acanthamoeba cyst viability, as the mechanism of cysticidal activities is currently difficult to determine. The efficacy of this method was supported in this study by using it on images of increasing intensity and with increasing concentrations of the death agent.

        There have been many studies on toxicity to the trophozoite of Acanthamoeba, but studies on toxicity to the cyst are limited because a standard method for measuring mortality at the cyst stage was lacking. Acanthamoeba cyst viability determination based on membrane integrity, fluorescence intensity and absorbance is a simple, accurate and quick method for assessing Acanthamoeba cyst viability. It is hoped that this method will inspire future toxicity studies involving Acanthamoeba cysts that will identify potent substances to kill Acanthamoeba at the cystic stage.

    This study was funded by UMT TAPE-RG 55115 (Talent and Publication Enhancement Research Grant). The facilities were supported by the Institute of Marine Biotechnology, Universiti Malaysia Terengganu.

Aksozek, A., McClellan, K., Howard, K., Niederkorn, J.Y., Alizadeh, H., 2002. Resistance of Acanthamoeba castellanii Cysts to Physical, Chemical, and Radiological Conditions. Journal of Parasitology, Volume 88(3), pp. 621–623

Anwar, A., Khan, N.A., Siddiqui, R., 2018. Combating Acanthamoeba spp. cysts: What are the Options? Parasites & Vectors, Volume 11(1), pp. 1–6

Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., Traganos, F., 1997. Cytometry in Cell Necrobiology: Analysis of Apoptosis and Accidental Cell Death (Necrosis). Cytometry: The Journal of the International Society for Analytical Cytology, Volume 27(1), pp. 1–20

Hashim, F., Amin, N.M., 2017, February. Insights into the Prominent Effect of Mahanimbine on Acanthamoeba Castellanii: Cell Profiling Analysis Based on Microscopy Techniques. In: AIP Conference Proceedings, AIP Publishing LLC, Volume 1817, No. 1, p. 030002

Khan, N.A., Paget, T.A., 2002. Molecular Tools for Speciation and Epidemiological Studies of Acanthamoeba. Current Microbiology, Volume 44(6), pp. 444–449

Kilvington, S., Gray, T., Dart, J., Morlet, N., Beeching, J.R., Frazer, D.G., Matheson, M., 2004. Acanthamoeba keratitis: The Role of Domestic Tap Water Contamination in the United Kingdom. Investigative Ophthalmology & Visual Science, Volume 45(1), pp. 165–169

Kusrini, E., Tristantini, D., Slamet, Setianingrum, V.M., Yulizar, Y., 2014. Fluorescence Properties of Microcomposites Europium Triethylene Glycol Picrate Complex Doped in Polymer. International Journal of Technology, Volume 5(1), pp. 70–78

Kusrini, E., Shiong, N.S., Harahap, Y., Yulizar, Y., Dianursanti, Arbianti, R., Pudjiastuti, A.R., 2015. Effects of Monocarboxylic Acids and Potassium Persulfate on Preparation of Chitosan Nanoparticles. International Journal of Technology, Volume 6(1), pp. 11–21

Kusrini, E., Hashim, F., Saleh, M.I., Adnan, R., Usman, A., Zakaria, I.N., Prihandini, W.W., Putra, N., Prasetyanto, E.A., 2020. Monoclinic Cerium (III) Picrate Tetraethylene Glycol Complex: Design, Synthesis and Biological Evaluation as Anti-Amoebic Activity Against Acanthamoeba sp. Journal of Materials Science, Volume 55(23), pp. 9795–9811

Kusrini, E., Hashim, F., Gunawan, C., Mann, R., Azmi, W.N.N.W.N., Amin, N.M., 2018. Anti-amoebic Activity of Acyclic and Cyclic-Samarium Complexes on Acanthamoeba. Parasitology Research, Volume 117(5), pp. 1409–1417

Kusrini, E., Hashim, F., Azmi, W.N.N.W.N., Amin, N.M., Estuningtyas, A., 2016. A Novel Antiamoebic Agent Against Acanthamoeba Sp. — A Causative Agent for Eye Keratitis Infection. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Volume 153, pp. 714–721

Lazuana, T., Astuty, H., Sari, I. P. 2019. Effect of Cellulase Enzyme Treatment on Cyst Wall Degradation of Acanthamoeba sp. Journal of Parasitology Research. Volume 2019, pp. 1–16

Lim, M.J., Shahri, N.N.M., Taha, H., Mahadi, A.H., Kusrini, E., Lim, J.W., Usman, A., 2021. Biocompatible Chitin-encapsulated CdS Quantum Dots: Fabrication and Antibacterial Screening. Carbohydrate Polymers, Volume 260, https://doi.org/10.1016/j.carbpol.2021.117806

Mazur, T., Hada?, E., Iwanicka, I., 1995. The Duration of the Cyst Stage and the Viability and Virulence of Acanthamoeba Isolates. Tropical Medicine and Parasitology: Official Organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ), Volume 46(2), pp. 106–108

Nakisah, M.A., Muryany, M.I., Fatimah, H., Fadilah, R.N., Zalilawati, M.R., Khamsah, S., Habsah, M., 2012. Anti-amoebic Properties of a Malaysian Marine Sponge Aaptos sp. on Acanthamoeba castellanii. World Journal of Microbiology and Biotechnology, Volume 28(3), pp. 1237–1244

Riedy, M.C., Muirhead, K.A., Jensen, C.P., Stewart, C.C., 1991. Use of a Photolabeling Technique to Identify Nonviable Cells in Fixed Homologous or Heterologous Cell Populations. Cytometry: The Journal of the International Society for Analytical Cytology, Volume 12(2), pp. 133–139

Schuster, F.L., Visvesvara, G.S., 2004. Free-Living Amoebae as Opportunistic and Non-Opportunistic Pathogens of Humans and Animals. International Journal for Parasitology, Volume 34(9), pp. 1001–1027

Usman, A., Kusrini, E., Widiantoro, A.B., Hardiya, E., Abdullah, N.A., Yulizar, Y., 2018. Fabrication of Chitosan Nanoparticles Containing Samarium Ion Potentially Applicable for Fluorescence Detection and Energy Transfer. International Journal of Technology, Volume 9(6), pp. 1112–1120

Tihonov, M.M., Kim, V.V., Noskov, B.A., 2016. Impact of a Reducing Agent on the Dynamic Surface Properties of Lysozyme Solutions. Journal of Oleo Science, Volume 65(5), pp. 413–418