An Original Use of a Bioluminescence Assay to Test the In Vitro Efficacy of Polihexanide in the Eradication of Acanthamoeba Cysts.

Abstract

Acanthamoeba keratitis is a rare ocular disease caused by Acanthamoeba spp. The current Acanthamoeba keratitis treatments consist of unlicensed drugs, most commonly polihexanide (PHMB), a biguanide derivative, either as monotherapy or combined with a diamidine. The main purpose of this study was to develop an in vitro bioluminescence assay able to differentiate the antiamoebic activity of PHMB concentrations (0.02%, 0.04%, 0.06%, and 0.08%). Another objective of this study was to evaluate the antiamoebic activity of equal PHMB concentrations at different molecular weights (MWs). Adenosine triphosphate (ATP) bioluminescence assay applicability was established by a linear correlation between amoebae number and relative light unit emitted from cysts. The protocol consisted in a series of sequential tests, the first of which compared relative light unit emission and the hemocytometric method after cysts exposure to the different PHMB concentrations. In this assay, dilutions of the initial PHMB concentrations were used as working solutions. Usually, only 5% or less of the instilled dose into the eye is distributed through the cornea; thus, the antiamoebic activity of initial PHMB concentrations 1/10 and 1/100 diluted can reasonably be considered as representative of their undiluted dose administered in vivo. There was no significant difference between the 2 methods. The ATP bioluminescence assay ranked the antiamoebic activity of the working PHMB solutions and showed that the viability of the cysts was equally reduced at fixed PHMB concentration in a wide range of molecular weight. The ATP bioluminescence assay showed to be a simple and rapid way for characterizing the in vitro antiamoebic activity of working PHMB solutions at different concentrations.