Abstract
Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 μm/0.4-0.6 μm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking.
Highlights
Acanthamoeba keratitis is a rare but serious water-borne parasitic infection of the eye caused by the ubiquitous protozoa Acanthamoeba, which can result in permanent visual impairment or blindness
Standard diagnostic methods include corneal biopsy and in vivo reflection corneal microscopy
Miltefosine has been demonstrated to be useful in the treatment of Acanthamoeba keratitis in animal models and has been granted an orphan designation for the treatment of Acanthamoeba keratitis in humans by the European Commission: Fig. 1. (a) The alkylphospholipid miltefosine used as carrier of the fluorescent dye. (b) The Acanthomoeba fluorescent marker MT-11C-BDP
Summary
Acanthamoeba keratitis is a rare but serious water-borne parasitic infection of the eye caused by the ubiquitous protozoa Acanthamoeba, which can result in permanent visual impairment or blindness. Diagnosing Acanthamoeba keratitis is a clinical challenge Initial symptoms resemble those of bacterial keratitis [1–3], the clinical management of Acanthamoeba keratitis differs substantially. The standard diagnosis for Acanthamoeba keratitis in suspect patients is made by culture and isolation of organisms from a corneal culture or detection of trophozoites and/or cysts on histopathology [5]. This requires corneal scrapping or collection of a corneal biopsy, and typically takes days to confirm diagnosis. Molecular diagnostic assays (based on Polymerase Chain Reaction Analysis) have proved to be more rapid [6–10], as well as more sensitive and specific than smear and culture, but they still require corneal scraping to obtain the samples [11], as well as trained personnel
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