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

描述（由申请人提供）：耐药恶性疟原虫的出现和传播构成了巨大的全球健康威胁。虽然我们对种群中耐药疟疾的选择了解很多，但对宿主内的进化知之甚少。大多数感染恶性疟疾的个体携带多种遗传上不同的变体（“基因型”、“菌株”），这些变体不断进化并争夺资源。这种宿主内部的竞争可能与种群内部的竞争同样重要。许多变异的水平很低，旧的基因分型技术无法检测到。为了测量宿主内的竞争，我们提出了一种能够识别和定量单个宿主基因型的新方法-大规模平行Pyrosequencing （MPP）。有了这项技术，我们将在个体宿主内测量恶性疟原虫感染的真正多样性和抗疟药的选择性压力。具体来说，我们将：i)对来自两个不同传播强度区域的个体受试者的merozoite surface protein-2 （msp2）基因型进行测序和定量；坦桑尼亚（高传播）和泰国（低强度），ii)测量接受复方蒿甲醚治疗的坦桑尼亚患者个体寄生虫变异的频率变变率（选择系数），并确定竞争变异对频率的影响，以及iii)测量接受青蒿琥酯-甲氟喹治疗的泰国患者个体寄生虫变异的向上选择，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)允许精确测量混合感染中的宿主内选择和变异动态，(2)为测试关于低和高耐药性遗传基础的假设提供新的工具，(3)定义允许耐药寄生虫在种群中发展和传播的因素。

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. PUBLIC HEALTH RELEVANCE: Malaria remains the most important vector borne parasitic infection in the world today, causing nearly a million deaths annually. This project focuses on understanding the factors involved in the evolution of drug resistance within individual hosts and improving the methods for early detection of drug resistant parasites. A better understanding of these evolutionary processes will augment resistance management strategies, assist malaria control efforts, and help alleviate malaria's public health burden in the developing world.