W. bancrofti infection diversity and treatment response

描述（由申请人提供）：班氏吴策线虫（Wb）是淋巴丝虫病（LF）的主要病原体，LF是一种毁容和使人衰弱的媒介传播疾病，估计影响83个国家的1.2亿人。我们在巴布亚新几内亚的研究表明，通过大规模药物管理（MDA）可以降低人和蚊子感染的Wb流行率，但如果没有持续的药物压力，传播已显示出恢复到MDA前水平的迹象。这些结果表明，用于杀死Wb的药物并不完全有效。这暴露了用于监测LF消除计划的策略中的显著差距-迄今为止，常规的群体遗传工具尚未用于WB。因此，尚未测量Wb的遗传多样性，没有关于其种群大小、变异潜力以及逃避或恢复MDA的能力的信息。通过最近成功完成的Wb线粒体（mt）基因组序列，我们已经开始解决有限的能力，监测Wb消除通过人口遗传策略。在这里，我们描述了通过下一代测序技术分析完整的mt基因组序列的方法，使我们能够提供多个个体样本中序列多样性的深入分析。通过以下具体目标，我们的建议提供了创新的，新的定量战略，以监测人口的影响，LF消除。目的1：确定在预期进行抗丝虫药物临床试验的地区，个体和人群中Wb感染的多样性。我们将扩增来自个体患者的整个Wb mt基因组的三个重叠片段，并生成用含有独特6个核苷酸标识符的引物条形码化的测序文库。然后，我们将汇集文库并在HiSeq 2000的一个泳道上对其进行测序。这种方法产生的覆盖率必须考虑到合并的固有变异，以确定真正的多态性，并确定多个寄生虫单倍型。所得数据可用于估计Wb的先前未知的生活史参数（例如遗传多样性、有效感染数、有效繁殖率（Ro）和迁移率）。由此产生的数据库还将使MDA治疗前后的比较能够监测消除程序并跟踪耐药菌株的出现。目的2：通过治疗后患者样本的血清学评估，评估个体感染对抗丝虫药物的反应。我们与James Kazura博士的合作将提供在治疗前和治疗后14天、1个月、3个月、6个月和12个月时间点收集的WB感染患者样本。通过在治疗期间对患者感染进行测序，我们可以收集关于治疗有效性的额外数据，而不仅仅是寄生虫负荷，从而深入了解遗传多样性，寄生虫血症和药物疗效之间的相互作用。由于这些人群水平的研究解决了白念珠菌流行病学和消除计划中的一个重要差距，我们的研究结果有可能加强未来MDA工作的设计，超越巴布亚新几内亚。

DESCRIPTION (provided by applicant): Wuchereria bancrofti (Wb) is the primary causative agent of lymphatic filariasis (LF), a disfiguring and debilitating vector-borne disease estimated t affect 120 million people in 83 countries. Our studies in Papua New Guinea have shown that it is possible to reduce the prevalence of Wb in human and mosquito infections by mass drug administration (MDA), however without continued drug pressure, transmission has shown signs of recovering to pre-MDA levels. These results suggest that drugs used to kill Wb are not completely effective. This exposes a significant gap in strategies used to monitor LF elimination programs - to date conventional population genetic tools have not been available for Wb. It follows then that genetic diversity of Wb has not been measured, there is no information regarding its population size, its potential for variation, nor its capacity to evade or recover frm MDA. Through recent successful completion of the Wb mitochondrial (mt) genome sequence, we have begun to address the limited ability to monitor Wb elimination through population genetic strategies. Here, we describe approaches for analyzing complete mt genome sequence through next-generation sequencing technology allowing us to provide in-depth analysis of sequence diversity within multiple individual samples. Through the following Specific Aims our proposal provides innovative, new quantitative strategies to monitor the population impact of LF elimination. Aim 1: Define the diversity of Wb infections within individuals and populations in areas anticipating clinical trials of anti-filarial drugs. We will amplify three overlapping segmens of the entire Wb mt genome from individual patients and generate sequencing libraries barcoded with primers containing unique 6-nucleotide identifiers. We will then pool libraries and sequence them on one lane of HiSeq 2000. This approach generates coverage necessary to account for inherent variations to pooling, to identify true polymorphism, and identify multiple parasite haplotypes. Resulting data can be used to estimate previously unknown life history parameters for Wb, (e.g. genetic diversity, effective number of infections, effective reproductive rate (Ro), and migration rates). The resulting database will also enable pre- and post-MDA treatment comparisons to monitor elimination programs and track emergence of drug resistance strains. Aim 2: Evaluate the response of individual infections to anti-filarial drugs through seria assessment of patient samples following treatment. Our collaboration with Dr. James Kazura will provide samples from Wb-infected patients collected at pre-treatment and 14 day, 1-, 3-, 6- and 12-month post-treatment time points. By sequencing patient infections during treatment we can collect additional data on treatment effectiveness beyond just parasite load, providing insight into the interactions between genetic diversity, parasitemia, and drug efficacy. As these population-level studies address an important gap in Wb epidemiology and elimination programs, our results have potential to strengthen design of future MDA efforts beyond PNG.