Methods for the Diagnosis of Leishmania Infection

摘要：利什曼属锥虫虫寄生虫感染了全世界近1500万人。在流行地区诊断是困难的，因为目前可用的检测方案由于缺乏敏感性和时间密集性而不充分。最近对海湾战争综合症和利什曼病之间可能存在的联系的关注，突出了对病人和血液制品缺乏和需要更好的诊断方式。更可靠的检测目标是寄生虫自身的线粒体DNA (kDNA)，它在其整个生命周期中保持不变，每个细胞中存在数千个拷贝。我们已经鉴定了8种不同利什曼原虫分离株的kDNA序列，并使用基于pcr的方案设计了几种利什曼原虫的检测系统。这种技术可以在大量人类DNA存在的情况下，很容易地从单个细胞中检测利什曼原虫的kDNA。这些PCR方案的效用已进一步证明，它们能够积极地识别黑热病患者和皮肤感染个体的寄生虫分离物。根据我们在基于PCR的检测方案方面的经验，我们开发了一种PCR检测方法，能够在旧大陆利什曼原虫中以特定物种的方式扩增多诺瓦利杆菌的kDNA。对于多诺瓦氏L.的印度菌株和分离株，该试验足够敏感，可以检测到相当于单个寄生虫或更少的kDNA。该检测方法的极高灵敏度反映在它能够从黑热病（KA）患者的少量外周血和黑热病后皮肤利什曼病（PKDL）患者的皮肤病变中检测寄生虫DNA。对107例临床利什曼病病例进行分析。其中PCR阳性102例（95.3%）。该测试提供了96%的灵敏度诊断KA使用患者全血样本，而不是通过侵入性手术获得骨髓或脾脏抽吸。该方法的诊断成功率为45/48（93.8%）。可以排除与流行地区流行的病原体，即结核分枝杆菌、麻风分枝杆菌和疟原虫的交叉反应。81份对照样本，包括来自PKDL患者正常皮肤部分的皮肤刮擦，均为阴性。2/20的地方病对照经PCR检测呈阳性；然而，这两人很有可能是无症状携带者，因为他们血清学上对KA呈阳性。因此，该PCR检测代表了一种非侵入性诊断患者KA和PKDL的工具，同时可以在临床样本中识别寄生虫的种类。

Summary: Trypanosomatid parasites of the genus Leishmania infect close to 15 million people world-wide. Diagnosis is difficult in endemic areas because of the currently available detection schemes are inadequate because of lack of sensitivity, and time intensiveness. Recent concern regarding possible link between the Gulfwar syndrome and Leishmaniasis has accentuated the lack of and need for better diagnostic modalities for both patients and blood products. A more reliable target for detection is the parasite's own mitochondrial DNA (kDNA) which remains constant through its life cycle and is present in thousands of copies per cell. We have characterized kDNA sequences from eight different Leishmania isolates and have used a PCR-based scheme to design detection systems for several species. This techinque can readily detect Leishmania kDNA from single cell in the presence of vast amounts of human DNA. The utility of these PCR schemes has been further shown by their ability to positively identify parasite isolates from kala-azar patients and individuals with cutaneous infections. Based on our experience with the PCR-based detection schemes, we have developed a PCR assay that is capable of amplifying kDNA of L. donovani in a species-specific manner among Old World Leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of Kala-azar (KA) patients and from skin lesions of Post kala-azar dermal leishmaniasis (PKDL) patients. A total of 107 clinical samples of Leishmaniasis were analyzed. Of these 102 (95.3%) were positive in PCR. The test provided for diagnosis of KA with 96% sensitivity using patient whole blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45/48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the endemic area viz., M. tuberculosis, M. leprae and Plasmodium could be ruled out. Eighty one control samples including dermal scrapings from normal portions of skin from PKDL patients were all negative. 2/20 endemic controls were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus this PCR assay represents a tool for the diagnosis of KA and PKDL in patients in a non-invasive manner, with simultaneous species identification of parasite in clinical samples.