Molecular Analysis of Dirofilaria repens Isolates from Eye-Care Patients in Greece.

Abstract

Human ocular dirofilariasis is a zoonotic disease caused by several species of filarioid helminths of the genus Dirofilaria. The aim of this study was to further re-examine five preserved specimens previously isolated from patients with ocular dirofilariasis by molecular means. Four of the examined helminths had been stored in unbuffered formaldehyde solution for more than eight years; whereas, the fifth helminth was stored in ethanol buffer for more than two years. For the four specimens stored in formaldehyde, different methods of DNA recovery and amplification were applied and investigated for their efficiency in DNA extraction and PCR amplification. However, the DNA extraction and PCR amplification were successful only for the ethanol-preserved helminth. The genetic identification of the ethanol-preserved specimen as Dirofilaria repens (D. repens) and its phylogenetic position based on the analysis of mitochondrial 12S ribosomal RNA, nuclear 18S ribosomal RNA and mitochondrial cytochrome c oxidase subunit one sequences are reported in the present paper. To our knowledge, these are the only deposited sequences related to D. repens that have been isolated in Greece. Routine laboratory diagnosis is based on phenotypic characteristics of the helminthic parasites, but more accurate diagnosis requires molecular identification. Although the specimens preserved in formalin buffers may be a potential source for the enrichment of parasite genome databases, the DNA recovery of such samples is a challenging task.