School of Veterinary Medicine, Hebrew University, Rehovot, Israel.
PLoS Negl Trop Dis. 2010 Jan 12;4(1):e581. doi: 10.1371/journal.pntd.0000581.
Three major forms of human disease, cutaneous leishmaniasis, visceral leishmaniasis and mucocutaneous leishmaniasis, are caused by several leishmanial species whose geographic distribution frequently overlaps. These Leishmania species have diverse reservoir hosts, sand fly vectors and transmission patterns. In the Old World, the main parasite species responsible for leishmaniasis are Leishmania infantum, L. donovani, L. tropica, L. aethiopica and L. major. Accurate, rapid and sensitive diagnostic and identification procedures are crucial for the detection of infection and characterization of the causative leishmanial species, in order to provide accurate treatment, precise prognosis and appropriate public health control measures.
METHODS/PRINCIPAL FINDINGS: High resolution melt analysis of a real time PCR product from the Internal Transcribed Spacer-1 rRNA region was used to identify and quantify Old World Leishmania in 300 samples from human patients, reservoir hosts and sand flies. Different characteristic high resolution melt analysis patterns were exhibited by L. major, L. tropica, L. aethiopica, and L. infantum. Genotyping by high resolution melt analysis was verified by DNA sequencing or restriction fragment length polymorphism. This new assay was able to detect as little as 2-4 ITS1 gene copies in a 5 microl DNA sample, i.e., less than a single parasite per reaction.
CONCLUSIONS/SIGNIFICANCE: This new technique is useful for rapid diagnosis of leishmaniasis and simultaneous identification and quantification of the infecting Leishmania species. It can be used for diagnostic purposes directly from clinical samples, as well as epidemiological studies, reservoir host investigations and vector surveys.
三种主要形式的人类疾病，皮肤利什曼病、内脏利什曼病和黏膜皮肤利什曼病，是由几种利什曼原虫引起的，这些利什曼原虫的地理分布经常重叠。这些利什曼原虫有不同的储存宿主、白蛉媒介和传播模式。在旧世界，导致利什曼病的主要寄生虫物种是利什曼原虫婴儿、L. donovani、L. tropica、L. aethiopica 和 L. major。准确、快速和敏感的诊断和鉴定程序对于检测感染和鉴定致病利什曼原虫种类至关重要，以便提供准确的治疗、精确的预后和适当的公共卫生控制措施。
方法/主要发现：使用实时 PCR 产物的高分辨率熔解分析内部转录间隔区-1 rRNA 区域，用于鉴定和量化来自人类患者、储存宿主和白蛉的 300 个样本中的旧世界利什曼原虫。L. major、L. tropica、L. aethiopica 和 L. infantum 表现出不同的特征高分辨率熔解分析模式。通过高分辨率熔解分析进行基因分型通过 DNA 测序或限制性片段长度多态性得到验证。该新测定法能够在 5 微升 DNA 样本中检测到低至 2-4 ITS1 基因拷贝，即每个反应少于一个寄生虫。
结论/意义：这项新技术可用于快速诊断利什曼病，并同时鉴定和定量感染的利什曼原虫。它可用于直接从临床样本进行诊断，以及流行病学研究、储存宿主调查和媒介调查。
