DEVELOPMENT OF METHODS FOR THE DIAGNOSIS OF LEISHMANIASIS AND ADVENTITIOUS AGENTS

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，因为目前的禽流感检测方案是不够的，因为缺乏灵敏度，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。根据我们的经验与PCR为基础的检测方案，我们想expolite这一技术来检测寄生虫疫苗中的外来因子。利什曼原虫病毒（Leishmainavirus，LRV）是一种双链RNA病毒，可持续感染利什曼原虫。已报道了两个新世界和旧世界LRV分离株的全核苷酸序列。我们计划鉴定菌株之间保守的序列，并开发可用于基于PCR的检测测定的引物。利用这些引物，我们将开发PCR检测方法，以检测来自世界不同地区的广泛分离株中的病毒。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.