Methods for the Diagnosis of Leishmania Infection

摘要：全世界有近1500万人感染利什曼原虫属锥虫。在流行地区很难诊断，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够充分。最近对格尔夫瓦氏综合征和利什曼病之间可能联系的担忧，加剧了对患者和血液产品更好的诊断方式的缺乏和必要性。更可靠的检测目标是寄生虫自身的线粒体DNA(KDNA)，它在整个生命周期中保持不变，存在于每个细胞的数千个拷贝中。我们已经确定了8个不同利什曼原虫分离株的kDNA序列，并使用了基于PCR的方案来设计针对几个物种的检测系统。这种技术可以在大量人类DNA存在的情况下，从单个细胞中很容易地检测出利什曼原虫的kDNA。这些聚合酶链式反应方案的实用性进一步被证明，它们能够从黑热病患者和皮肤感染的个人中肯定地识别寄生虫分离株。根据我们对基于聚合酶链式反应的检测方案的经验，我们开发了一种聚合酶链式反应方法，能够在东半球利什马尼亚人中以物种特异性的方式扩增杜氏乳杆菌的kDNA。对于印度菌株和杜诺瓦尼乳杆菌分离株，该检测方法足够灵敏，可以检测到相当于一种寄生虫或更少的kDNA。该方法的极端敏感性反映在它能够从少量黑热病患者的外周血和黑热病后皮肤利什曼病患者的皮肤皮损中检测到寄生虫DNA。对107例利什曼病临床标本进行了分析。其中102例(95.3%)为阳性。使用患者全血样本而不是通过侵入性程序获得的骨髓或脾抽吸物，该检测提供了96%的敏感性诊断KA。本方法对48例PKDL中45例诊断成功(93.8%)。可以排除与流行地区流行的病原体如结核分枝杆菌、麻风分枝杆菌和疟疾的交叉反应。81份对照样本，包括正常部位的皮肤刮片，均为阴性。用聚合酶链式反应检测20名地方病对照中有2名阳性，但这两人很可能是无症状携带者，因为他们的KA血清学检测是阳性的。因此，这种聚合酶链式反应方法是一种非侵入性的诊断KA和PKDL的工具，同时可以在临床样本中鉴定寄生虫的种类。

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.