Physiological tolerance of acanthamoeba genotype T4 isolated from contact lens paraphernalia / Khairul Ameera Kamaruddin

Amoeba from the genera Acanthamoeba is known as a free-living opportunistic protozoan that consumes on other environmental micro-organisms. These ubiquitous pathogen has the ability to survive in very harsh environmental circumstances and have been isolated from different sources including contact lens paraphernalia. Genotype T4 had been acknowledged as the most virulent genotype associated with Acanthamoeba keratitis. However, genotyping alone does not provide enough indication of the pathogenicity of an isolate. For this reason, the characterization of Acanthamoeba is magnified by the assessment of physiological properties. Physiological tolerance assays comprising osmo- and thermotolerance were performed in order to investigate the pathogenic potential of Acanthamoeba genotype T4 isolated from contact lens paraphernalia. Overall, a total of 14 contact lens paraphernalia that had been confirmed with Acanthamoeba genotype T4 from a previous study were used in this study. Each sample was repeated in duplicate. A clinical strain of Acanthamoeba castellanii (ATCC 50492-T4) was used as positive control for both assays. In osmo-tolerance assay, amoeba cysts from 14 positive contact lens samples were directly inoculated (approximately 10³ cysts) onto a labeled non-nutrient agar plate supplemented with 0.5 M and 1 M mannitol, lawn with 2 mL of E. coli suspension. Meanwhile, for the thermotolerance assay was performed by incubating the cultivated Acanthamoeba into 37⁰C and 42⁰C. The number of trophozoites or cysts for each sample were observed (20 mm away from the center of each plate) and counted on the 7th day of incubation in five microscope fields under X40 objectives lens of a light microscope. Demographic and clinical details were collected using pre-tested questionnaire. Of the 14 contact lens samples, all sample isolates (100%) were able to show positive growth at 30⁰C with 0.5 M mannitol. However, only nine (64%) of them were able to grow at a higher osmolarity concentration, 1 M mannitol at 30⁰C. Meanwhile, for thermo-tolerance assay, only 13 (93%) of the isolates capable to develop at 37⁰C, while, nine (64%) of them have the ability to grow up to 42⁰C. In addition, all of the subjects in this study noted with variation progressive symptoms of the infections that uphold the perceptions for Acanthamoeba keratitis. In conclusion, there is a clear need for more detailed knowledge about the distribution of Acanthamoeba genotype T4 in different environments and their direct and indirect virulence factors. Thus, the determination of in-vivo pathogenicity of the Acanthamoeba isolates should be conducted in the future.