Variation in Resistance and Fitness to Artemisinins in African Malaria

 描述(由申请人提供) 抗药性疟疾寄生虫的出现一直是控制恶性疟原虫的主要障碍，恶性疟原虫对几乎所有使用中的抗疟疾药物都产生了抗药性。一个主要的担忧是，对当前一线青蒿素衍生品明显出现抗药性，这可能引发全球健康危机。为了确保持续的有效性， 这些药物、及早发现耐药性、良好的监测和遏制是必要的。目前临床现场检测寄生虫表型和监测疗效的工具 青蒿素是寄生虫的清除曲线。清除时间较长(半衰期较长)的寄生虫被认为具有更强的“抵抗力”。在感染复杂性相对较高的地区，寄生虫清除曲线提供的信息较少。这是因为寄生虫清除曲线是整个感染水平的衡量标准，代表该样本中不同寄生虫清除表型的加权平均值。在非洲，大多数恶性疟疾感染是多克隆的，这意味着抗药性寄生虫可能与敏感寄生虫共享宿主，特别是在耐药性传播的早期。因此，在这些地区，抗药性克隆对抗药性水平估计的影响可能会被感染中敏感寄生虫的绝对数量所掩盖。为了克服这一局限性，我们以前提出了一种新的方法，利用第二代测序来揭示在多克隆感染的药物治疗过程中相对丰度上升的单个寄生虫克隆的耐药表型特征。使用这些新的深度测序方案，我们证实了这一假设，在坦桑尼亚分离株中发现了与来自亚洲的青蒿素抗性寄生虫类似的清除半衰期的克隆。因此，非洲出现的青蒿素耐药性可能不会被标准的临床定义检测到。在坦桑尼亚发现的抗性克隆中，没有一个包含最近与亚洲高水平青蒿素抗性有关的海带(K13)基因的多态。因此，这些抗性克隆甚至不会被分子监测发现。这些发现表明，对青蒿素具有低水平抗性的克隆存在于 并将不被发现，直到它们在多克隆感染中占优势。在这项提议中，我们寻求更好地描述这些非洲抗性克隆的特征。我们的深度测序方法为在早期阶段研究抗疟疾耐药性以及非洲青蒿素耐药性的长期演变提供了一个新的框架。我们将用它来证实这些寄生虫克隆清除缓慢是由于耐药性而不是其他临床因素。随着对这些低频率慢清除克隆的进一步研究以及新描述的可能影响耐药性的新的非洲K13突变的功能意义，我们有机会在非洲重大临床失败之前检测到耐药性，并了解可能增强或减缓这种传播的生态因素。

 DESCRIPTION (provided by applicant) The emergence of drug resistant malaria parasites has been a major hurdle in the control of Plasmodium falciparum, which has evolved resistance to nearly every antimalarial drug in use. A major concern is the apparent emergence of resistance to the current front-line artemisinin derivatives, which could spark a global health crisis. In order to ensure the continued efficacy of these drugs, early detection of resistance, good surveillance, and containment are required. The current clinical tool for phenotyping parasites in the field and monitoring therapeutic efficacy of artemisinins is the parasite clearance curve. Parasites that have longer slower clearance times (longer half-lives) are considered more "resistant". Parasite clearance curves are less informative in regions with relatively high complexity of infection. This is because the parasite clearance curve is a whole-infection level measure that represents a weighted average of the clearance phenotypes of different parasites within that sample. In Africa, the majority of falciparum malaria infections are polyclonal meaning that resistant parasites are likely to share their host with sensitive parasites, particularly early during the spread of resistance. In these areas, the effects of resistant clones on infection-level estimates of resistance may therefore be obscured by the sheer number of sensitive parasites in an infection. In order to overcome this limitation, we previously proposed a novel approach that capitalizes on second generation sequencing in order to reveal phenotypic signatures of resistance among individual parasite clones that rise in relative abundance over the course of drug treatment in polyclonal infections. Using these new deep sequencing protocols, we confirmed this hypothesis, finding clones with clearance half-lives similar to artemisinin resistant parasites from Asia among Tanzanian isolates. Thus, emerging artemisinin resistance in Africa might go undetected using the standard clinical definition. None of the resistant clones identified in Tanzania contained polymorphisms in the kelch (K13) gene recently associated with high-level artemisinin resistance in Asia. Accordingly, these resistant clones would even go undetected by molecular surveillance. These findings suggest that clones with low-level resistance to artemisinins exist in Africa and will go undetected until they become dominant in a polyclonal infection. In this proposal, we seek to better characterize these African resistant clones. Our deep sequencing approach provides a novel framework for studying antimalarial resistance at this early stage and potentially the long-term evolution of artemisinin resistance in Africa. We will use it to confirm that slow clearance by these parasite clones is due to resistance rather than other clinical factors. With further investigation of these low frequency slow clearing clones and the functional significance of newly described novel African K13 mutations that may impact resistance, we have the chance to detect resistance prior to significant clinical failures in Africa and understan the ecological factors that may enhance or retard that spread.