Diversity and Phenotype of Artemisinin Resistance Mutations in Central Africa

摘要： 青蒿素联合疗法是疟疾控制的关键组成部分。青蒿素的出现 非洲的抗药性将导致一场全球公共卫生灾难，可能会逆转在控制方面取得的成果 努力。不幸的是，几个组织现在报告寄生虫清除时间略有增加，临床 抗性的标志，在非洲的几个地点。最近，卡介苗病毒K13螺旋体区域的多个突变 在亚洲，PF3D7_1343700与青蒿素耐药性有关。重要的是，这些突变是 被证明独立地多次出现，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。vbl.使用 一种混合测序方法，我们在K13推进器中发现了一系列新的突变，包括一些 频率高得惊人，但很少有人与亚洲分享。这引发了关于以下影响的关键问题： 非洲K13基因突变与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 并通过利用先前收集的样本和新的体外工具来对这些突变进行表型分析， 而不必进行大规模且昂贵的体内疗效研究。使用以前收集的样本 从2007年和2013年的人口健康调查(DHS)中，我们将混合选择和全基因组 联合分离株的测序，以评估ACT使用的DRC突变频率的变化 增加并变得普遍，在过去的4年中，60%的病例接受了ACT治疗。渐增 突变的频率，在控制寄生虫人口统计和自然遗传变异的同时，将提高 担心突变正在被选择中。然而，需要直接证据来确定这些 突变会改变对青蒿素的易感性。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(那些在我们之前的工作中已经出现频率很高的突变和那些可疑的 其他研究中的耐药性)或在2007至2013年间频率增加的突变 通过将突变基因导入寄生虫系并使用最近开发的Ring Survival Assay(RSA)进行体外检测。 这项检测与体内的表型相关。这项提议是创新的，因为它将是第一个 在耐药性出现的早期，我们使用抗疟疾耐药性的分子标记，并应 到使用几种新的方法，包括我们从血点中混合捕获疟疾DNA。它还 利用目前在寄生虫反向基因工程方面的进展。这个项目意义重大，因为它将 提供对中非青蒿素耐药性现状的重要洞察和 增加了对所描述的多态在抗性背景下的含义的理解。这些发现将 对我们了解寄生虫生物学以及控制和消除寄生虫都有重要影响 战略。

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.