Characterization of Free-living Amoebae Isolated from cases of Amoebic Keratitis in Sassari.

The cosmopolitan and ubiquitous free-living amoebae (FLA) are amphizoic protozoa with the ability to exist both as free-living organisms in nature and as parasites within host tissues. Numerous reports indicate that some FLA can cause infections in humans. Several species of Acanthamoeba are the causative agents of amoebic keratitis, a corneal infection mainly associated with contact lens use. AIM: Given the lack of data on the occurrence of pathologies caused by FLA in our geographical area, we have studied cases of suspected amoebic keratitis in patients admitted to the Institute of Ophthalmology, University of Sassari, Sassari, Italy, from January 2008 to January 2010. Patients with eye infections were classified on the basis of epidemiological data, clinical manifestations, the evolution of the disease, response to pharmacological treatment and recurrence of infection. Axenic cultures obtained from amoebae isolated were used to study their phenotypic and genotypic features. MATERIALS AND METHODS: Corneal scrapings from non- and soft contact lens wearers with suspected Acanthamoeba keratitis at an early stage (presence of dendritic epithelial lesions and/or diffuse punctate epitheliopathy) were plated on Petri plates, containing 1.5% agar in Page’s amoeba saline (PAS). Whenever possible, patients’ contact lens solutions and samples of the water used for domestic purposes were also analyzed. Amoebic isolates were identified morphologically and by polymerase chain reaction (PCR) analysis with primers P-FLA-F/P-FLA-R (generic for FLA), JDP1/JDP2 (specific for Acanthamoeba) and JITS-F/JITS-R (described for vahlkampfiid species). All patients were treated with PHMB 0.02% (SIFI S.p.A., Catania, Italy) and propamidine isetionate 0.1% eye-drops. RESULTS: Three genera of FLA (Acanthamoeba, Hartmannella and Vahlkampfia), were identified by microscopy examination. One patient had mixed Hartmannella/Vahlkampfia keratitis. PCR analysis with primers FLA yielded 800 bp amplicons (typical of Hartmannella) in seven cases. PCR analysis with primers JDP1/JDP2 yielded 450 bp amplicons (specific for Acanthamoeba T4 genotype) in 1 case and 550 bp amplicons in 2 cases (Acanthamoeba non T4). Both these PCRs not provided amplification products for Vahlkampfia isolates. Works are in progress to identificate they by amplification of ITS and 5.8S rDNA sequences using primers JTS-F/JTS-R. All patients soft contact lens wearers had amoebic contamination of their care solutions. It is cause for great concern that we found contamination with Acanthamoeba in some unopened 10-ml bottles of disinfecting solutions containing polyquaternium-1 (result confirmed by the Istituto Superiore di Sanità, Rome, Italy). Treatment with PHMB 0.02% and propamidine isetionate 0.1% was always successful. Some patients non contact lens wearers had amoebic contamination of their domestic water or frequented swimming pools. CONCLUSIONS: Our results provided evidence that the ability to cause corneal disease may not be restricted to Acanthamoeba genus. Early diagnosis and proper anti-amoebic treatment are crucial to yield a cure. The causes of this cluster of amoebic keratitis in soft contact lens wearers is still under investigation. Preliminary data suggest exposure to contaminated tap water in some cases and contamination of contact lens solutions in others. The comparative evaluation of the pathogenicity of these isolates on human corneal and congiuntival cell lines are in progress. This study will be useful to acquire epidemiological data and promote suitable prophylaxis intervention with regards to these emerging pathogens in our geographic area.