W. bancrofti infection diversity and treatment response

描述(由申请人提供)：班克罗夫氏伍氏杆菌(WB)是淋巴丝虫病(LF)的主要病原体。淋巴丝虫病是一种通过媒介传播的毁容和衰弱疾病，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，有可能通过大规模用药(MDA)降低人类和蚊子感染中WB的流行率，但在没有持续的药物压力的情况下，传播已显示出恢复到MDA之前水平的迹象。这些结果表明，用来杀死WB的药物并不完全有效。这暴露了用于监测低频消除计划的战略的一个重大差距--到目前为止，世界银行还没有可用的常规群体遗传工具。因此，WB的遗传多样性尚未被测量，没有关于其种群大小、其变异潜力或其逃避或从MDA恢复的能力的信息。通过最近成功完成WB线粒体(MT)基因组序列，我们已经开始解决通过群体遗传策略监测WB消除的有限能力。在这里，我们描述了通过下一代测序技术分析mt全基因组序列的方法，使我们能够对多个个体样本中的序列多样性进行深入分析。通过以下具体目标，我们的建议提供了创新的、新的量化战略，以监测消除低频电磁场对人口的影响。目的1：确定在预期抗丝虫病药物临床试验的地区，个人和人群中WB感染的多样性。我们将从个体患者中扩增出三个重叠的WB mt基因组片段，并生成带有包含唯一6核苷酸识别符的引物条形码的测序文库。然后我们将汇集文库，并在HiSeq 2000的一条车道上对它们进行排序。这种方法产生了必要的覆盖率，以解决合并的固有变异，识别真正的多态，并识别多个寄生虫单倍型。由此得到的数据可以用来估计WB以前未知的生活史参数(例如，遗传多样性、有效感染数量、有效繁殖率(Ro)和迁移率)。由此产生的数据库还将使丙二醛治疗前后的比较能够监测消除计划并跟踪耐药菌株的出现。目的2：通过对治疗后患者样本的血清评估，评估个体感染对抗丝虫病药物的反应。我们与James Kazura博士的合作将提供在治疗前以及治疗后14天、1个月、3个月、6个月和12个月时间点收集的WB感染患者的样本。通过对治疗期间的患者感染进行测序，我们可以收集有关治疗有效性的额外数据，而不仅仅是寄生虫负载，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世界银行流行病学和消除计划中的一个重要差距，我们的结果有可能加强对未来丙二醛努力的设计，而不仅仅是巴新。

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.