Genotyping Acanthamoeba Keratitis (AK) Infections: Cytochrome Oxidase I (COI) Barcoding and Phylogenetic Analysis

Abstract

Purpose: : Acanthamoeba is responsible for a rare but devastating eye infection in which the protist adheres to the cornea and invades the stroma. Although Acanthamoeba is found ubiquitously in nature, infections are rare, are only associated with certain genotypes of Acanthamoeba, and are often misdiagnosed as viral or bacterial infections. Successful prognosis in AK is associated with early diagnosis and treatment, but standard methods to identify Acanthamoeba require specialized equipment and expertise. A simple genotyping method using cytochrome oxidase I (COI) barcoding is examined for its ability to identify specific genotypes of Acanthamoeba.

Methods: : Acanthamoeba were cultured using standard protocols, and genomic DNA was extracted. The mitochondrial COI gene was amplified via PCR using universal primers, and sequenced by automated fluorescent sequencing. Genetic similarity of 40 isolates, including environmental and keratitis isolates, was determined by comparison of the COI sequences.

Results: : Phylogenetic relationships determined from partial COI gene sequences for 40 Acanthamoeba isolates were compared to those produced using the nuclear 18S ribosomal RNA gene (rDNA), the standard normally used to identify Acanthamoeba genotypes. COI barcode analysis produces genotypic relationships analogous to the 18S rDNA, and easily identifies genotypic subgroups of Acanthamoeba.

Conclusions: : Barcoding Acanthamoeba isolates using COI produces results highly consistent with 18S rDNA genotype analysis, and offers an accurate, rapid, and inexpensive method to identify Acanthamoeba isolates causing keratitis infections.

Keywords: Acanthamoeba