Prevalence and fitness of drug resistant minority variant mutations in Plasmodium

描述(申请人提供)：抗药性恶性疟原虫是一个主要的公共卫生问题。大多数耐药类型的分子标记已经被确定，并可以作为监测工具。然而，在高传播率地区，如撒哈拉以南非洲地区，个人通常携带许多遗传多样性的恶性疟原虫亚群(“变种”)。标准的聚合酶链式反应方法不能检测代表个体宿主中20%寄生虫的变异体(“少数变异体”)的抗药性突变。因此，标准检测低估了感染抗药性寄生虫患者的流行率，因为它们漏掉了携带抗药性少数变异体的患者。我们已经建立了异双链跟踪分析(HTAS)，它可以检测耐药少数变异，并准确地测量变异群体的相对丰度。在马拉维的一项试点研究中，我们证明了少数人变异的pfcrt76突变(氯喹耐药性的标志)的高流行率，标准PCR没有发现这些突变。我们现在已经建立了针对其他3个重要基因座(dhfr59、dhfr164、dhps 540，用于抗叶酸抗性)的少数变异的HTA。我们将使用这些检测方法来(1)确定使用HTA测量抗药性恶性疟原虫的流行率是否高于标准PCR；以及(2)测量接受药物治疗和未治疗的个体中耐药少数变异体在宿主内的适合度获得和损失。Pfcrt76、dhfr59、dhfr164和/或dhps540的患病率将在马拉维、赞比亚、马达加斯加和刚果民主共和国的样本中用HTA和标准聚合酶链式反应进行横断面测量。将在马拉维接受磺胺多辛-乙胺(SP)治疗的患者和马达加斯加接受氯喹治疗的患者的配对登记和复发样本中测量Pfcrt76和dhfr164的适合度。这项研究的结果具有重要的公共卫生影响。检测到具有抗药性的少数变异--具有已证实的适合性--可以更早地检测到新出现的耐药性，并为规划药物政策的变化提供更多的准备时间。公共卫生相关性更好的抗药性疟疾监测方法可能对疟疾控制项目大有裨益。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。这可能使及早发现新出现的耐药性成为可能。

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.