Genetic diversity among Acanthamoeba sp. isolated from clinically suspected cases of Amoebic keratitis patients attending a tertiary care hospital and proteomic differntiation between the trophozoite and cyst stage of Acanthamoeba

Abstract

Purpose: To detect the genotypes of Acanthamoeba spp. isolates from patients of clinically suspected amoebic keratitis and proteomic characterisation of trophozoite and cyst stages

Methods: Corneal scrapings from clinically suspected amoebic keratitis(AK) patients (107) attending our tertiary care hospital during last 2 years were subjected to culture on 2% non- nutrient agar plates overlaid with E.coli. DNA isolation followed by PCR assay was performed for amplification of DF3 region (280bp) of 18S rRNA gene of Acanthamoeba spp.. PCR products were sequenced and aligned using software CLUSTAL W. Phylogenetic tree reconstructions was performed with phylogenetic computer program MEGA4 using neighbour-joining method as well as UPGMA anakysis. The cultured trophozoites were encysted in laboratory conditions and the whole cell lysates of both the stages were subjected to SDS-PAGE and two dimensional gel electrophoresis.

Results: Acanthamoeba spp. was isolated from 18 of the 107 (16.82%)suspected cases of Amoebic Keratitis (AK). From the phylogenetic analysis of the nucleotide sequences, it was evident that 16 isolates clustered in one group, belonging to Genotype T4 . Two isolates formed an unique cluster . SDS-PAGE results shows clear differences between trophozoite and cyst stages. Two proteins of 35KDa and 80KDa were more expressed in cyst stages compared to trophozoites.

Conclusions: Most of the Acanthamoeba eye infections were due to T4 genotype. Using the present method, 2 of the isolates did not cluster with any known genotype. Proteomic differences between trophozoite and cyst stages could be deciphered

Keywords: 402 Acanthamoeba • 479 cornea: clinical science • 533 gene/expression