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Advantages of Using Mitochondrial 16S rDNA Sequences to Classify Clinical Isolates of Acanthamoeba

¹From the Department of Molecular Genetics, The Ohio State University, Columbus, Ohio; ²L. V. Prasad Eye Institute, Hyderabad, India; the ³Centre for Cellular and Molecular Biology, Hyderabad, India; and the ⁴Parasitology Research Program, University of Witwatersrand, Johannesburg, South Africa.

PURPOSE. This work was intended to test the classification of Acanthamoeba into genotypes based on nuclear ribosomal RNA gene (18S rDNA, Rns) sequences. Nearly all Acanthamoeba keratitis (AK) isolates are genotype RnsT4. This marked phylogenetic localization is presumably either due to an innate potential for pathogenicity or to a peculiarity of the gene sequences used. To differentiate between these possibilities, relationships among isolates have been reexamined, using a second gene.

METHODS. Phylogenetic relationships among isolates of Acanthamoeba were studied, using sequences of the mitochondrial small subunit ribosomal RNA gene (16S rDNA; rns). Genotypes based on complete sequences of approximately 1540 bp were determined for 68 strains, by using multiple phylogenetic analyses.

RESULTS. Each strain’s mitochondria contained a single intron-free rns sequence (allele). The 68 strains had 35 different sequences. Twenty-eight strains had unique sequences, and 40 strains each shared one of the seven remaining sequences. Eleven mitochondrial rns genotypes corresponding to 11 of 12 previously described nuclear Rns genotypes were identified. Genotype rnsT4 was subdivided into eight distinct clades, with seven including Acanthamoeba keratitis (AK) isolates.

CONCLUSIONS. The phylogenetic clustering of AK isolates was confirmed and thus is not specific to the nuclear gene. Rns and rns sequences are both suitable for genotyping of Acanthamoeba. However, the mitochondrial sequences are shorter and more consistent in length, have a higher percentage of alignable bases for sequence comparisons, and have none of the complications caused by multiple alleles or introns, which are occasionally found in Rns. In addition, the more common occurrence of strains with identical rns sequences simplifies identification and clustering of isolates.

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