DEVELOPMENT OF METHODS FOR THE DIAGNOSIS OF LEISHMANIASIS AND ADVENTITIOUS AGENTS

全世界有近1500万人感染利什曼原虫属锥虫。流行地区的诊断很困难，因为目前的禽流感病毒检测方案不够灵敏，缺乏时间密集性。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。根据我们在基于PCR的检测方案方面的经验，我们希望将这项技术推广到检测寄生虫疫苗中的外来病原体。利什曼原虫的许多物种持续感染双链RNA病毒，称为利什曼病毒(LRV)。报道了两个来自新世界和旧世界毒株的LRV分离株的完整核苷酸序列。我们计划鉴定菌株间的保守序列，并开发可用于基于聚合酶链式反应的检测分析的引物。利用这些引物，我们将建立聚合酶链式反应方法，在世界不同地区的各种分离株中检测病毒，这些分离株可供我们使用。我们还想测试从世卫组织获得的各种疫苗制剂，以测试其中是否存在病毒序列。

Trypanosomatid parasites of the genus Leishmania infect close to 15 million people world-wide. Diagnosis is difficult in endemic areas because of the currently avialbe detection schemes are inadequate because of lack of sensitivity, 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 want to expolite this techique to detect adventious agents in the parasite vaccines. Many species of Leishmania are persistently infected with double stranded RNA virus named as Leishmainavirus (LRV). The complete nucleotide sequences of two LRV isolates from both new world and old world strains have been reported. We plan to identify sequences which are conserved among the strains and develop primers which can be used in the PCR based detection assay. Using these primers we will develop PCR assay to detect the virus in wide range of isolates form different regions of the world which are available to us. We would also like to test various vaccine preprartions, obtained from WHO, to test the presence of viral sequences in them.