Prevalence and fitness of drug resistant minority variant mutations in Plasmodium

描述（由申请人提供）：抗药性恶性疟原虫疟疾是一个主要的公共卫生问题。大多数类型的耐药性的分子标记已经确定，可以作为监测工具。然而，在高传播地区，如撒哈拉以南非洲，个体通常携带许多遗传多样性的恶性疟原虫亚群（“变种”）。标准PCR方法不能检测代表个体宿主中<20%的寄生虫的变体（“少数变体”）中的耐药性突变。因此，标准检测低估了感染耐药寄生虫的患者的患病率，因为它们错过了耐药少数变异的患者。我们开发了异源双链追踪试验（HTAs），可以检测耐药少数变异体，并准确测量变异群体的相对丰度。在马拉维的一项试点研究中，我们证明了少数变异pfcrt 76突变（氯喹耐药性的标志物）的高患病率，这些突变被标准PCR遗漏。我们现在已经开发了HTA的少数变异在其他3个重要的基因座（dhfr 59，dhfr 164，dhps 540，抗叶酸剂耐药性）。我们将使用这些测定来（1）确定当通过HTA测量时，耐药性恶性疟原虫的流行率是否高于通过标准PCR测量;以及（2）测量经药物治疗和未经药物治疗的个体中的抗性少数变体的宿主内适应性增益和损失。在马拉维、赞比亚、马达加斯加和刚果民主共和国的样本中，将通过HTA和标准PCR横断面测量pfcrt 76、dhfr 59、dhfr 164和/或dhps 540的患病率。Pfcrt 76和dhfr 164的适应度将在马拉维的磺胺嘧啶-乙胺嘧啶（SP）治疗患者和马达加斯加的氯喹治疗患者的配对入组和复发样本中进行测量。这项研究的结果具有重要的公共卫生意义。检测耐药少数变异体-经证明适用-可以更早地发现新出现的耐药性，并为规划药物政策的变化争取更多的准备时间。更好的抗药性疟疾监测方法可能对疟疾控制计划大有裨益。我们正试图开发一种方法来测量抗药性寄生虫，即使它们是单一宿主中的少数寄生虫。这可能有助于更早地发现新出现的耐药性。

DESCRIPTION (provided by applicant): Drug-resistant P. falciparum malaria is a major public health problem. Molecular markers for most types of drug resistance have been identified and could serve as surveillance tools. However, individuals in high-transmission areas, like sub-Saharan Africa, usually carry many genetically diverse P. falciparum subpopulations ("variants"). Standard PCR methods are incapable of detecting drug-resistance mutations in variants representing <20% of the parasites in an individual host ("minority variants"). Thus, standard assays underestimate the prevalence of patients infected with resistant parasites because they miss patients with resistant minority variants. We have developed Heteroduplex Tracking Assays (HTAs) which can detect drug-resistant minority variants and accurately measure the relative abundance of variant populations. In a Malawi pilot study, we demonstrated a high prevalence of minority variant pfcrt76 mutations (a marker for chloroquine resistance) which were missed by standard PCR. We have now developed HTA's for minority variants at 3 other important loci (dhfr59, dhfr164, dhps540, for antifolate resistance). We will use these assays to (1) determine whether the prevalence of drug-resistant P. falciparum is higher when measured by HTA than by standard PCR; and (2) measure in-host fitness gains and losses of resistant minority variants in drug- treated and untreated individuals. Prevalence of pfcrt76, dhfr59, dhfr164 and/or dhps540 will be measured by HTA and standard PCR cross-sectionally in samples from Malawi, Zambia, Madagascar, and the DR Congo. Pfcrt76 and dhfr164 fitness will be measured in paired enrollment and recrudescent samples from sulfadoxine-pyrimethamine (SP)- treated patients in Malawi and chloroquine-treated patients in Madagascar. The results of this study have important public health implications. The detection of drug-resistant minority variants - with proven fitness - could enable earlier detection of emerging drug- resistance and greater lead-time to plan changes in drug policy. PUBLIC HEALTH RELEVANCE Better surveillance methods for drug-resistant malaria could be of great benefit to malaria control programs. We are trying to develop a method to measure drug-resistant parasites even when they are the minority of parasites in a single host. This could permit the earlier detection of emerging drug resistance.