Molecular typing of Mycobacterium leprae

说明(申请人提供)：1991年世界卫生大会的一个目标是到2000年消除麻风病这一公共卫生问题。其目标是将麻风病的患病率降低到每10,000人中不到1人。尽管实施了多种药物疗法(MDT)计划，但在最流行的国家尚未实现这一目标，全球80%以上的麻风病例发生在这些国家。发现的新病例数量也没有减少，这表明麻风分枝杆菌的传播尚未得到控制。 在对传播的了解方面存在根本差距，而且缺乏追踪麻风的敏感方法。麻风杆菌宿主的性质、感染途径和传播方式仍存在疑问。为了解决这些问题，根据麻风杆菌基因组序列的可用性和多位点可变数目串联重复序列分析(MLVA)的原理，提出了一种用于其他几种感染性生物的麻风杆菌DNA分型方法。麻风杆菌不能在实验室中培养，用于研究的细菌主要来自易受感染的螳螂或裸鼠。因此，这项先导性研究有两个特别的目的：1.利用一组来自犰螂的麻风杆菌分离物建立一种标准的分子分型方法；2.将优化的方法应用于一组从患者的活检和鼻拭子中提取的麻风杆菌DNA。这些方法包括对麻风杆菌每个分离物的含有短串联重复序列的多个基因组座位进行PCR扩增，并将每个基因座的片段长度与其他分离物的片段长度进行比较。将使用自动化的电泳、数据收集和分析。这项研究的长期目标是提供适合于未来流行病学研究的工具和技术，例如人类、非人类和环境来源在麻风通过鼻腔或皮肤途径传播麻风病中的作用，以及病原体的释放和患者复发与再感染的区分。

DESCRIPTION (PROVIDED BY APPLICANT): An objective of the 1991 World Health Assembly was the elimination of leprosy as a public health problem by the year 2000. The goal was to achieve a reduction in prevalence of leprosy to less than 1 per 10,000 population. This goal has not been achieved in the most endemic countries, where more than 80% of the global leprosy cases occur, despite the implementation of the Multi Drug Therapy (MDT) program. The number of new cases detected has also not reduced indicating that the transmission of the causative agent Mycobacterium leprae has not been controlled. There are fundamental gaps in the understanding of transmission, and a lack of sensitive methods to track leprosy. Questions remain regarding the nature of the reservoir of M. leprae, the route of infection, and mode of its spread. To address these concerns, a DNA typing method for M. leprae is proposed based on the availability of the genome sequence and the principle of multiple locus variable-number of tandem repeat analysis (MLVA), which has been developed for several other infectious organisms. M. leprae cannot be cultivated in a laboratory and bacteria for research applications is mainly derived from susceptible armadillo or nude mice. This pilot study is therefore designed with two specific aims: 1. Development of a standard molecular typing method using a panel of armadillo derived M. leprae isolates and 2. Application of the optimized methods in a panel of M. leprae DNA derived from biopsies and nasal swabs from patients. The methods involve the PCR amplification of multiple genomic loci containing short tandem repeats for each isolate of M. leprae and the comparison of the fragment length of each locus with that obtained from other isolates. Automated electrophoresis, data collection and analysis will be used. The long-term goal of the study is to provide the tools and techniques suitable for future epidemiological studies such as the role of human, non-human and environmental sources of M. leprae in the transmission of leprosy via nasal or dermal routes of ntry and release of the pathogen and the differentiation of relapse from reinfection in patients.