Abstract

Acanthamoeba keratitis is a devastating eye disease that requires prompt diagnosis and treatment. Currently, there are no targeted antimicrobial drugs that can be used in the United Kingdom to treat Acanthamoeba infections. There are treatment options available but require the prolonged application of low concentrations of biocides. These biocides cause significant damage to host tissue (cornea) and are frequently the major contributor to the loss of visual acuity seen with this type of infection. In addition to this, Acanthamoeba trophozoites can undergo cell transformation into a cyst form as a response to stress (including biocide treatment); cysts are less sensitive to biocides. The development of new anti-amoebal drugs has been hampered by the limited interest in this rare infection by big pharma and the lack of a robust in-vitro susceptibility assay that can be used to assay trophozoites as well as cysts.
A novel approach that may be useful as a treatment modality for Acanthamoeba trophozoites and cysts is the use of antimicrobial photodynamic therapy (APDT). This approach combines light and a photo-activated drug to generate reactive oxygen species that cause cell death. Although this approach often causes host cell toxicity, these drugs can be targeted through chemical modification, the use of antibodies and via the use of precise light targeting.

The aim of the present study was to evaluate the use of photodynamic therapy for the treatment of all morphological forms of Acanthamoeba. The objectives are
• To asses existing assay methodologies for susceptibility testing and validate their adaptability to assay for drug activity against all morphological forms of Acanthamoeba.
• To identify useful photosensitisers for Acanthamoeba photodynamic therapy.
We were able to develop a novel optimised ATP assay that used traditional detergent-based cell lysis for trophozoites. Cysts could not be lysed using this or similar approaches thus we introduced enzymatic (lysozyme) lysis of cysts. The assay was validated using a range of clinically relevant biocides and drugs that have known in vitro activity against Acanthamoeba.
Results showed that photodynamic therapy using LED light (85 j/cm2) and cationic porphyrins (10µM), was able to inactivate more than 80% of both cysts and trophozoites. These results suggest that APDT is a promising approach for the treatment of Acanthamoeba infections in the eye and that it may be possible to enhance the anti-amoebic activity of porphyrins via chemical modification. We also suggest that improved targeting of these drugs could be achieved using Acanthamoeba specific peptides conjugated to porphyrins targeting photodynamic therapy.