Within Host Selection of P. falciparum Variants by Artemisinin Combination Therap

描述（由申请人提供）：抗药性恶性疟原虫的出现和传播对全球健康构成了巨大的威胁。虽然我们对抗药性疟疾在人群中的选择了解得很多，但对宿主内的进化却知之甚少。大多数感染恶性疟疾的个体携带多种遗传上不同的变异体（“基因型”、“菌株”），这些变异体不断进化并争夺资源。这种宿主内的竞争可能与种群内的竞争一样重要。许多变体以低水平存在，并且无法通过旧的基因分型技术检测到。为了测量宿主内的竞争，我们提出采用一种新的方法，唯一能够识别和定量基因型在一个单一的主机-大规模并行焦磷酸测序（MPP）。有了这项技术，我们将在个体宿主中测量恶性疟原虫感染的真实多样性和抗疟药物的选择压力。具体而言，我们将：i）对来自不同传播强度两个区域的个体受试者中的裂殖子表面蛋白-2（msp 2）基因型进行测序和定量：坦桑尼亚（高传输）和泰国（低强度），ii）测量频率变化率（选择系数），并确定竞争性变体对频率的影响，和iii）测量来自用青蒿琥酯-甲氟喹治疗的泰国患者的单个寄生虫变体的向上选择，以确定选择系数并更好地确定用于研究对青蒿素的药物抗性的表型。这种定量研究个体内变异多样性的新方法将i）允许准确测量混合感染中的宿主内选择和变异动态，ii）提供用于测试关于低水平和高水平抗性的遗传基础的假设的新工具，以及iii）定义允许在群体中发展和传播抗药性寄生虫的因素。

DESCRIPTION (provided by applicant): The emergence and spread of drug-resistant Plasmodium falciparum poses an immense global health threat. While we understand much about the selection of drug resistant malaria in populations, little is known about in- host evolution. Most individuals infected with falciparum malaria carry multiple genetically distinct variants ("genotypes", "strains") which continually evolve and compete for resources. This within-host competition could be as important as competition within populations. Many of the variants are present at low levels and undetectable by older genotyping technologies. In order to measure within-host competition, we propose to employ a new method uniquely capable of identifying and quantitating genotypes in a single host - Massively Parallel Pyrosequencing (MPP). With this technology, we will measure, within individual hosts, the true diversity of falciparum infections and the selective pressure of antimalarials. Specifically, we will: i) sequence and quantitate merozoite surface protein-2 (msp2) genotypes within individual subjects from two areas of different transmission intensity: Tanzania (high transmission) and Thailand (low intensity), ii) measure the rate of change in frequency (selection coefficients) for individual parasite variants from Tanzanian patients treated with Coartem and determine the effect of competing variants on frequency, and iii) measure up-selection for individual parasite variants from Thai patients treated with Artesunate-Mefloquine to define selection coefficients and better define the phenotype used to study drug resistance to artemisinins. This novel approach of quantitatively studying variant diversity within individuals will i) allow accurate measurement of within-host selection and dynamics of variants in mixed infections, ii) provide new tools for testing hypotheses about the genetic basis of low- and high-level resistance, and iii) define the factors that allow the development and spread drug resistant parasites in populations.