Diversity and Phenotype of Artemisinin Resistance Mutations in Central Africa

摘要： 青蒿素综合疗法是疟疾控制的一个关键组成部分。青蒿素的出现 非洲的耐药性将导致全球公共卫生灾难，可能会逆转在控制艾滋病毒方面取得的进展。 努力不幸的是，现在有几个研究小组报告说，寄生虫清除时间略有增加， 在非洲的几个地方，最近，在K13螺旋桨区的多个突变， PF3D7_1343700与亚洲的青蒿素耐药性有关。重要的是，这些突变是 显示已独立出现多次，引起了对新生耐药突变的关注， 非洲这一发现促使我们在非洲寄生虫分离株中寻找K13基因的突变。使用 通过合并测序方法，我们在K13螺旋桨中发现了一系列新的突变，包括一些 但很少与亚洲分享。这就提出了关于 非洲K13突变和青蒿素耐药性的发展。为了更好地理解 这些发现的意义，在这里，我们提出了一种方法来研究非洲K13等位基因的选择， ACT并通过利用先前收集的样本和新的体外工具对这些突变进行表型， 而不必进行大型和昂贵的体内功效研究。使用以前收集的样本 从2007年和2013年人口健康调查（DHS），我们将混合选择和全基因组 对合并分离株进行测序，以评价使用ACT后DRC中突变频率的变化 增加并变得普遍，在过去4年中，>60%的病例接受ACT治疗。增加 在控制寄生虫人口统计学和自然遗传变异的同时， 担心突变是在选择之下。然而，需要直接证据来确定这些 突变改变了对青蒿素的敏感性。为了提供这一点，我们将使用反向遗传学来评估 候选突变的影响（那些在我们以前的工作中已经处于高频率的突变和那些可疑的突变） 其他研究中的耐药性）或本研究中2007年至2013年间频率增加的突变， 通过将突变体导入寄生虫系并使用最近开发的环存活测定（RSA）进行体外测定。 该测定与体内表型相关。这是一项创新，因为它将是第一个 我们应该在抗药性出现的早期使用抗疟药抗药性的分子标记， 几种新方法的使用，包括我们从血斑中混合捕获疟疾DNA。它还 利用了寄生虫反向基因工程的最新进展。这个项目意义重大，因为它将 提供关于中部非洲青蒿素耐药性现状的重要见解， 增加了对所描述的多态性在耐药性背景下的含义的理解。这些发现将 对我们理解寄生虫生物学以及控制和消除都有重要影响 战略布局

Abstract: Artemisinin combination therapies are a critical component to malaria control. The emergence of artemisinin resistance in Africa would spell a global public health disaster, potentially reversing gains made in control efforts. Unfortunately, several groups are now reporting small increases in parasite clearance times, the clinical marker of resistance, at several sites in Africa. Recently, multiple mutations in the K13 propeller region of PF3D7_1343700 have been associated with artemisinin resistance in Asia. Importantly, these mutations were shown to have arisen multiple times independently, raising concern for de novo resistance mutations arising in Africa. This discovery pushed us to look for mutations in the K13 gene among African parasite isolates. Using a pooled sequencing approach, we identified an array of novel mutations in the K13 propeller, including some at startlingly high frequency, but few shared with Asia. This raised critical questions concerning the impact of the African mutations in K13 and the development of artemisinin resistance. In order to better understand the significance of these findings, here we propose an approach to study the selection of African K13 alleles by ACTs and to phenotype these mutations by leveraging previously collected samples and new in vitro tools, without having to conduct large and expensive in vivo efficacy studies. Using previously collected samples from the 2007 and 2013 Demographic Health Surveys (DHS), we will hybrid selection and whole genome sequencing of pooled isolates to evaluate for changes in frequency of mutations in the DRC as ACT use has increased and became common, with >60% of cases being treated with ACT over the last 4 years. Increasing frequency of mutations, while controlling for parasite demographics and natural genetic variation, would raise concerns that mutations are under selection. However, direct evidence would be required to determine if these mutations alter susceptibility to artemisinin. In order to provide this, we will use reverse genetics to assess the impact of candidate mutations (those already at high frequency in our previous work and those suspicious for resistance in other studies) or mutations that increase in frequency between 2007 and 2013 in this study in vitro by transfecting mutations into parasite lines and using the recently developed Ring Survival Assay (RSA). This assay has been correlated to the in vivo phenotype. This proposal is innovative because it will be the first time we use a molecular marker of antimalarial resistance early during the emergence of resistance, and due to the use of several novel approaches, including our hybrid capture of malaria DNA from blood spots. It also leverages current advances in reverse genetic engineering of the parasite. This project is significant as it will provide critical insight into the current situation concerning artemisinin resistance in Central Africa and an increased understanding on what described polymorphism mean in context of resistance. These findings will have important impacts for both our understanding of parasite biology, as well as for control and elimination strategies.