Ikonomopoulos J, Kokotas S, Gazouli M, Zavras A, Stoitsiou M, Gorgoulis V G
Department of Anatomy-Physiology, Faculty of Animal Science, Agricultural University of Athens, Iera Odos 75, Botanikos, 18875 Athens, Greece.
Vet Parasitol. 2003 Apr 18;113(2):99-113. doi: 10.1016/s0304-4017(03)00061-x.
Leishmaniosis is a zoonotic, parasitic disease caused by members of the genus Leishmania. The disadvantages of the traditional methods have currently rendered the polymerase chain reaction (PCR), the most reliable alternative for the laboratory diagnosis of this disease. Several relevant protocols have been described in the past but their application is in most cases limited to research use. The latter combined with the diagnostic problems that can be caused by the genetic variability of the different Leishmania strains or the presence of PCR inhibitors, indicate that an alternative approach should be followed for the development of a standard diagnostic tool for leishmaniosis. In the present study, we have evaluated several PCR-based protocols, in order to identify a primer combination that would allow the reliable detection of Leishmania DNA from clinical material and the verification of its results, in a manner that could be applicable even for routine use. The evaluation consisted of a BLAST verification of the specificity of the previously described primers, PCR testing, and optimisation of the reaction conditions. Our assessment was completed with the comparative evaluation of the results produced by the proposed PCR assay, light microscopy, and indirect fluorescent antibody technique (IFAT), on clinical samples collected from dogs suspected of leishmaniosis. The proposed assay which consists of a combination of two pairs of primers, targeted to different areas of the kinetoplast DNA of Leishmania spp., specific for Leishmania infantum, Leishmania donovani and Leishmania chagasi, showed optimum performance on our test samples, and detected 41.9% Leishmania-positive dogs from our 160 clinical cases. From the same number of cases, 46.25% were positive by IFAT (titre > or =200), and 19% by microscopic examination of lymph node aspirates.
利什曼病是一种由利什曼原虫属成员引起的人畜共患寄生虫病。传统方法的缺点使得聚合酶链反应（PCR）成为目前该疾病实验室诊断最可靠的替代方法。过去已经描述了几种相关方案，但在大多数情况下，它们的应用仅限于研究用途。后者加上不同利什曼原虫菌株的遗传变异性或PCR抑制剂的存在可能导致的诊断问题，表明应该采用另一种方法来开发利什曼病的标准诊断工具。在本研究中，我们评估了几种基于PCR的方案，以确定一种引物组合，该组合能够从临床材料中可靠地检测利什曼原虫DNA并验证其结果，其方式甚至适用于常规使用。评估包括对先前描述的引物特异性进行BLAST验证、PCR测试以及反应条件的优化。我们通过对从疑似感染利什曼病的犬只采集的临床样本上，所提出的PCR检测法、光学显微镜检查和间接荧光抗体技术（IFAT）所产生的结果进行比较评估，完成了我们的评估。所提出的检测法由两对引物组合而成，靶向利什曼原虫属动基体DNA的不同区域，对婴儿利什曼原虫、杜氏利什曼原虫和恰加斯利什曼原虫具有特异性，在我们的测试样本上显示出最佳性能，在我们的160例临床病例中检测出41.9%的利什曼原虫阳性犬只。在相同数量的病例中，IFAT检测阳性率为46.25%（滴度≥200），淋巴结穿刺液显微镜检查阳性率为19%。
