Diversity and Phenotype of Artemisinin Resistance Mutations in Central Africa

中部非洲青蒿素耐药突变的多样性和表型

• 批准号: 9301336
• 负责人: Jonathan J Juliano
• 金额: $ 19.35万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-20 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9301336
• 关键词: Adopted; Africa; African; Alleles; Antimalarials; Artemisinins; Asia; Asians; Biological Assay; Biology; Blood; Central Africa; Clinical; Clinical Markers; Combined Modality Therapy; Congo; Containment; Country; DNA; Democratic Republic of the Congo; Demographic and Health Surveys; Development; Disasters; Failure; Falciparum Malaria; Frequencies; Genes; Genetic; Genetic Drift; Genetic Engineering; Genetic Polymorphism; Genetic Variation; Hybrids; In Vitro; Investigation; Literature; Location; Malaria; Molecular; Molecular Epidemiology; Monitor; Mutation; Parasites; Phenotype; Plasmodium falciparum; Population; Predisposition; Public Health; Reporting; Research; Resistance; Sampling; Site; Spottings; Surveys; Time; Work; base; demographics; efficacy study; epidemiology study; genome sequencing; in vivo; innovation; insight; killings; molecular marker; novel; novel strategies; pressure; resistance mutation; reverse genetics; spelling; time use; tool; whole genome; zinc finger nuclease

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.

抽象的： 青蒿素组合疗法是控制疟疾的关键组成部分。青蒿素的出现 非洲的抵抗会造成全球公共卫生灾难，有可能扭转控制中的收益 努力。不幸的是，现在有几个小组报告寄生虫清除时间的增加，临床 抵抗的标志，在非洲的多个地点。最近，K13螺旋桨区域的多个突变 PF3D7_1343700与亚洲的青蒿素耐药性有关。重要的是，这些突变是 证明已经独立出现了多次出现，引起了人们对从头抗性突变的关注 非洲。这一发现促使我们在非洲寄生虫分离株中寻找K13基因中的突变。使用 一种合并的测序方法，我们确定了K13螺旋桨中的一系列新型突变，其中包括一些 在惊人的高频，但很少与亚洲共享。这提出了有关影响的关键问题 K13中的非洲突变和青蒿素耐药性的发展。为了更好地了解 这些发现的意义，在这里我们提出了一种研究非洲K13等位基因选择的方法 通过利用先前收集的样品和新的体外工具来表现和表型这些突变， 不必进行大型且昂贵的体内功效研究。使用先前收集的样品 从2007年和2013年的人口健康调查（DHS）开始，我们将混合选择和整个基因组 合并分离株的测序以评估DRC中突变频率的变化，因为ACT使用的使用已有 增加并变得普遍，过去四年中，有60％的病例接受ACT的治疗。增加 突变的频率在控制寄生虫人口统计和自然遗传变异时会增加 担心突变正在选择。但是，需要直接证据来确定是否 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（我们以前的工作中已经处于高频的影响，而那些可疑的 在其他研究中的抗性）或在2007年至2013年之间在这项研究中增加频率的突变 通过将突变转染为寄生虫线并使用最近开发的环生存测定法（RSA）来体外。 该测定与体内表型有关。该提议具有创新性，因为它将是第一个 时间我们在抗药性出现期间早期使用抗疟疾抗性的分子标记 使用几种新型方法，包括我们从血斑中捕获疟疾DNA的混合动力。也是如此 利用当前的寄生虫基因工程的进步。这个项目很重要，因为 对中非阿秒抗药性抗药性的当前状况提供批判性洞察力 对描述多态性的含义的理解越来越多，在抗药性的背景下意味着。这些发现会 对我们对寄生虫生物学的理解以及控制和消除都有重要影响 策略。