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

描述(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巨大威胁。虽然我们对耐药疟疾在人群中的选择了解很多，但对宿主内的进化知之甚少。大多数感染恶性疟疾的人携带多种不同的遗传变异(“基因类型”、“菌株”)，这些变异不断进化和竞争资源。这种东道主内部的竞争可能与人群内部的竞争一样重要。许多变异存在于低水平，无法被旧的基因分型技术检测到。为了测量宿主内的竞争，我们建议使用一种新的方法来唯一地识别和定量单个宿主的基因类型-大规模并行焦磷酸测序(MPP)。利用这项技术，我们将在单个宿主内测量恶性疟原虫感染的真实多样性和抗疟疾药物的选择压力。具体地说，我们将：i)对来自两个不同传播强度地区的个体受试者的裂殖子表面蛋白2(Msp2)基因型进行测序和定量，这两个地区是：坦桑尼亚(高传播)和泰国(低传播)；ii)测量接受Coartem治疗的坦桑尼亚患者的单个寄生虫变体的频率变化率(选择系数)，并确定竞争变体对频率的影响；iii)测量对使用青蒿琥酯-甲氧喹治疗的泰国患者的单个寄生虫变体的上调选择，以确定选择系数，并更好地确定用于研究青蒿素耐药性的表型。这种定量研究个体内变异多样性的新方法将：1)准确测量混合感染中宿主内选择和变异的动态；2)提供新的工具来检验关于低水平和高水平耐药的遗传基础的假说；3)确定允许在人群中发展和传播耐药寄生虫的因素。

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.