DEVELOPMENT OF METHODS FOR THE DIAGNOSIS OF LEISHMANIA P

利什曼原虫属的锥虫寄生虫感染全世界近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是不够的，因为缺乏敏感性和时间密集。最近对海湾综合征和利什曼病之间可能存在联系的关注，突出了缺乏和需要更好的诊断模式，为病人和血液制品。一个更可靠的检测目标是寄生虫自身的线粒体DNA（kDNA），它在其生命周期中保持不变，每个细胞有数千个拷贝。我们已经表征了来自8种不同利什曼原虫分离株的kDNA序列，并使用了基于PCR的方案来设计几个物种的检测系统。该技术可以在存在大量人类DNA的情况下容易地从单细胞中检测利什曼原虫kDNA。这些PCR方案的实用性已被进一步证明，他们的能力，积极确定寄生虫分离黑热病患者和个人皮肤感染。基于我们在PCR检测方案方面的经验，我们开发了一种能够扩增L.在东半球利什曼原虫中以物种特异性的方式发现了donovani。用印度株和分离株进行了比较。该检测方法的极端灵敏度反映在其从黑热病（KA）患者的小体积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的能力。共分析了107例利什曼病临床样本。PCR阳性102例（95.3%）。该测试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来诊断KA，灵敏度为96%。48例PKDL中45例（93.8%）诊断成功。与流行区流行病原体的交叉反应，M. tuberculosis，M.麻风病和疟原虫可以排除81个对照样品，包括来自PKDL患者的正常皮肤部分的皮肤刮片，均为阴性。2/20例地方病对照组PCR检测结果为阳性，但由于KA血清学阳性，这2例患者很可能是无症状携带者。因此，这种PCR检测代表了一种以非侵入性方式诊断患者KA和PKDL的工具，同时对临床样本中的寄生虫进行物种鉴定。

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.