Purpose

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.

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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希腊眼科患者分离株的 Dirofilaria repens 分子分析。

目的

人类眼部恶丝虫病是一种人畜共患病，由恶丝虫属的几种丝状蠕虫引起。本研究的目的是进一步重新检查以前通过分子手段从眼部纤丝虫病患者中分离出来的五个保存标本。

方法

4 只被检查的蠕虫在无缓冲的甲醛溶液中保存了 8 年以上；而第五个蠕虫在乙醇缓冲液中储存了两年多。对于储存在甲醛中的四个样本，应用了不同的 DNA 回收和扩增方法，并研究了它们在 DNA 提取和 PCR 扩增中的效率。然而，DNA 提取和 PCR 扩增仅对乙醇保存的蠕虫是成功的。

结果

本文报道了基于线粒体12S核糖体RNA、核18S核糖体RNA和线粒体细胞色素c氧化酶亚基一序列分析的乙醇保存标本为白芍(D. repens)的遗传鉴定及其系统发育位置。据我们所知，这些是与D. repens相关的唯一已在希腊分离的保藏序列。

结论

常规实验室诊断基于蠕虫寄生虫的表型特征，但更准确的诊断需要分子鉴定。尽管保存在福尔马林缓冲液中的标本可能是丰富寄生虫基因组数据库的潜在来源，但此类样品的 DNA 回收是一项具有挑战性的任务。