Authors

Discussion

Introduction: Acanthamoeba is a genus of free living but opportunistic amoebae usually found in soil, fresh water and many other habitats. Acanthamoeba exist in two morphologically forms; a motile reproductive trophozoites and a dormant yet viable cyst. The most common type of infections caused by this organism is a keratitis that up until recently was associated with poor hygiene by contact lens users. In the UK there are currently no licenced anti-amoeba drugs for the treatment of this infection. Such infections are currently managed using biocides such as polyhexanide (polyhexamethylene biguanide), chlorhexidine and propamidine isetionate; however failure to kill the cyst forms have been implicated in resistance and reinfection. Development of drugs for the treatment of this infection requires use of appropriate assay methods that can be used for both morphological forms and can be adapted for high throughput screening. In this work we have shown that and ATP viability assay is a sensitive, reproducible assay that can be used for both cysts and trophozoites forms

Method: In this study an ATP- based antimicrobial susceptibility assay for Acanthamoeba cells was developed by optimising cell lysis and assay conditions. For both life cycle forms the assays were similar in that the compounds were incubated with cells, however after incubation, trophozoites were lysed using conventional detergent lysis methods and for cysts, the cyst wall was enzymatically lysed with lysozyme (lysis was confirmed using  TEM). After lysis ATP was measured using the luciferase+ luciferin assay. For our assay development two biocidal compounds were used as our test compounds; Povidone-iodine (PvP-1) and Menadione

Results: Results shows a concentration dependent inhibition of Acanthamoeba cells in both the cysts and the trophozoites, but with different MIC values. We were able to extrapolate the minimal inhibitory concentration for PVP by taking RLU values where linearity of our plot stops. These MIC values have RLU similar to control wells were no drug was added. From our data and as supported by previous work using PVP on Acanthamoeba cysts and trophozoites the minimal inhibitory concentration for the cysts is about 1 % (w/v), and about 0.06 % (w/v) for the trophozoites. Menadione showed inhibition of Acanthamoeba cyst and trophozoites at   0.06mM concentration and inhibition was concentration dependent. The data generated by this assay has shown remarkable reproducibility and the results obtained are similar to those reported by other researchers.

Conclusion: From our results, this optimised ATP bioluminescence assay showed a good correlation with the conventional plate microscopy count method and the resazurin assay. Both of these methods however have limitations, growth based microscopy show high levels of variation and the resazurin assay does not work for cyst forms and requires large numbers of trophozoites. This method can be engineered into a HTS assay and would be an invaluable tool in the search for new anti-amoebic drugs