Detection and differentiation of pathogenic Acanthamoeba
Acanthamoeba are free-living and opportunistic protozoa that are ubiquitous in nature. Human infection due to Acanthamoeba can involve the brain, skin, lung and eyes. However, eye infection, Acanthamoeba keratitis, is the most common infection. Acanthamoeba keratitis is a sight threatening corneal disease producing a progressive, blinding infection of the corneal surface. Several species of Acanthamoeba have been identified as human pathogens, however, current laboratory techniques for identification and speciation of Acanthamoeba are time consuming and laborious. The purpose of this study was to develop and improve current methods for the detection and differentiation of Acanthamoeha spp. Initially a number of isolates and species were obtained and their pathogenicity determined using animal model studies and cytopathic effect assays. Based on these data Acanthamoeba were classified in pathogenic and non-pathogenic groups. Using this information a variety of techniques were developed to differentiate these groups. Simple plating assays were used to differentiate pathogenic Acanthamoeba. We successfully differentiated pathogenic and non-pathogenic Acanthamoeba on the basis of physical characteristics such as growth at 37°C and ability to grow in the presence of 1 M mannitol. Morphological characteristics were observed using scanning electron microscopy. Although these were useful, not all Acanthamoeba tested could be differentiated. Due to the increased analytical sensitivity and and specificity, PCR based assays were used to identify and speciate Acanthamoeba spp. A new DNA extraction method was developed which can be directly used with the given specimen without the prior need of cultivation of parasites. This method together with Acanthamoeba specific primers makes an ideal assay for the detection of Acanthamoeba. Using this assay we were able to identify Acanthamoeba up to 5 cells. Ribosomal DNA sequencing was performed to aid in differentiation of pathogenic and non-pathogenic Acanthamoeba. Acanthamoeba isoates from different sources (environmental and eye infections) were clustered into distinct genotypes according to their rONA sequence. All tested pathogenic Acanthamoeba fell into one group indicating that virulence may be associated with specific cluster of cladistic groups of Acanthamoeba. Owing to simplicity and popularity of immunoassays as diagnostic methods we isolated antibody fragments from a phage display antibody library which can be potentially used for the clinical diagnosis of Acanthamoeba spp. These antibody fragments were tested against a range of different organisms and cell types for their specificities. In a recent study by Cao et al. (1998), a correlation between proteases and rabbit primary corneal epithelial cell damage has been shownbut the study is limited as only one isolate of Acanthamoeba was used. In our study we have shown clear differentiation between pathogenic and non-pathogenic species of Acanthamoeba on the basis of extracellular protease activity. In addition we have also observed a contact-dependent protease when whole pathogenic Acanthamoeba were added to primary corneal epithelial cell monolayers. These data will help in furthering our understanding of the basic mechanisms of Acanthamoeba pathogenicity and thus to prevent the development of infection.