Benassi Julia Cristina, Benvenga Graziella U, Ferreira Helena Lage, Pereira Vanessa F, Keid Lara B, Soares Rodrigo, Oliveira Tricia Maria Ferreira de Sousa
Departamento de Medicina Veterinária da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Pirassununga, SP, Brazil.
Programa de Pós-Graduação em Epidemiologia Experimental Aplicada às Zoonoses, Departamento de Medicina Veterinária Preventiva da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, São Paulo, SP, Brazil.
Exp Parasitol. 2017 Jun;177:93-97. doi: 10.1016/j.exppara.2017.04.004. Epub 2017 Apr 21.
Although some studies have investigated the potential role of cats as a reservoir for Leishmania, their role in the epidemiology of visceral leishmaniasis (VL) is still poorly understood. Molecular diagnostic techniques are an important tool in VL diagnosis, and PCR shows high sensitivity and specificity for Leishmania spp. detection. Quantitative real-time PCR (qPCR) is a method that permits quantitative analysis of a large number of samples, resulting in more sensitive, accurate, and reproducible measurements of specific DNA present in the sample. This study compared real-time PCR (qPCR) and conventional PCR (cPCR) for detection of Leishmania spp. in blood and conjunctival swab (CS) samples of healthy cats from a non-endemic area in the state of São Paulo, Brazil. Of all CS samples, 1.85% (2/108) were positive for Leishmania spp. by both cPCR as qPCR (kappa index = 1), indicating excellent agreement between the two methods. The DNA from the two CS-cPCR- and CS-qPCR-positive samples was further tested with a PCR test amplifying the Leishmania spp. discriminative rRNA internal transcribed spacer 1 (ITS 1), of which one sample generated a 300-350-bp DNA fragment whose size varies according to the Leishmania species. Following sequencing, the fragment showed 100% similarity to a GenBank L. infantum sequence obtained from a cat in Italy. In conclusion, the association of qPCR and CS proved to be effective for detection of Leishmania in cats. Conjunctival swab samples were shown to be a practical and better alternative to blood samples and may be useful in the diagnosis and studies of feline leishmaniasis.
尽管一些研究调查了猫作为利什曼原虫宿主的潜在作用，但它们在内脏利什曼病（VL）流行病学中的作用仍知之甚少。分子诊断技术是VL诊断的重要工具，PCR对利什曼原虫属的检测具有高灵敏度和特异性。定量实时PCR（qPCR）是一种能够对大量样本进行定量分析的方法，可对样本中存在的特定DNA进行更灵敏、准确和可重复的测量。本研究比较了实时PCR（qPCR）和传统PCR（cPCR）在巴西圣保罗州一个非流行地区健康猫的血液和结膜拭子（CS）样本中检测利什曼原虫属的效果。在所有CS样本中，1.85%（2/108）通过cPCR和qPCR检测利什曼原虫属均呈阳性（kappa指数 = 1），表明两种方法之间具有极好的一致性。对两个CS-cPCR和CS-qPCR阳性样本的DNA进一步进行PCR检测，扩增利什曼原虫属的鉴别性rRNA内部转录间隔区1（ITS 1），其中一个样本产生了一个300 - 350 bp的DNA片段，其大小因利什曼原虫种类而异。测序后，该片段与从意大利一只猫获得的GenBank婴儿利什曼原虫序列显示出100%的相似性。总之，qPCR与CS相结合被证明对检测猫体内的利什曼原虫有效。结膜拭子样本被证明是血液样本的一种实用且更好的替代方法，可能有助于猫利什曼病的诊断和研究。
