Molecular determinants of P. falciparum resistance to ACTs in Nigeria.

描述（由申请人提供）：耐药恶性疟原虫的迅速发展和传播是一个严重的全球健康问题，并导致疟疾引起的死亡率和发病率上升。这种情况导致包括尼日利亚在内的许多非洲国家采用以青蒿素为基础的联合疗法来管理疟疾感染。然而，许多非洲国家的抗疟药物政策变化是在没有大规模监测恶性疟原虫对新药耐药性的发展和传播的工具的情况下进行的。此外，人们还担心，一旦将青蒿素衍生物或伙伴药物引入非洲用于疟疾管理，寄生虫可能很快对这些药物产生耐药性。因此，迫切需要开展研究，确定寄生虫对青蒿素衍生物（ARTs）和联合治疗中使用的配合药物反应的分子决定因素，因为它们可能揭示对有效监测耐药寄生虫和新的化疗靶点至关重要的标志物。我们假设恶性疟原虫的转运体和pfATPase6基因的不同突变（snp）和/或差异表达的组合可能是对青蒿素和伴侣药物的体外和体内反应差异的原因。此外，多种药物反应决定因素可能以不同的组合作用，导致寄生虫表型之间的差异。这项研究的主要目的是鉴定和验证新鲜恶性疟原虫患者分离株对青蒿素衍生物、lumemantrine （LUM）和amodiaquine （AQ）耐药的分子决定因素。该项目确定的疟原虫对抗疟药反应的分子决定因素将用于开发一种快速、简单、高通量的基于PCR-ELISA的工具/技术，用于检测恶性疟原虫pfATPase6和转运体基因以及其他疟原虫基因的snp，用于大规模监测疟原虫对青蒿素衍生物和其他抗疟药的耐药性。为实现这些目标，将在3年期间开展以下具体活动：1)在尼日利亚西南部评估恶性疟原虫对蒿甲醚-氨芳啶和青蒿琥酯-阿莫地喹组合的疗效和体外敏感性；2)确定从患者身上获得的新鲜恶性疟原虫分离株的寄生虫种群多样性，并评估基因（选择转运蛋白或pfATPase6）中snp的存在；3)评估和比较患者分离物和新克隆寄生虫在药物（AQ、LUM和ARTs）压力前后选定的恶性疟原虫转运体和pfATPase6基因的snp模式；4)将转运体基因和pfATPase6的snp模式与体外敏感性谱、治疗方式进行比对，以确定ARTs、LUM或AQ的分子决定因素，并形成独特的抗性表型；5)利用恶性疟原虫对art、LUM或AQ反应的分子决定因素（snp），开发一种简单的高通量方法，用于大规模监测尼日利亚疟疾联合治疗中对青蒿素和伴用药的耐药寄生虫。该研究项目提供了一个独特的机会，可以利用FIRCA第1号赠款在尼日利亚开发的研究基础设施和能力。NIH RO3TW006298，以便更好地了解对ACTs和其他抗疟疾药物的耐药性的发展和传播。该项目获得的数据和开发的工具将提供给尼日利亚国家疟疾控制规划，以促进该国的循证抗疟药物政策。这个研究项目与尼日利亚和非洲的耐药性问题直接相关。它提供了一个独特的机会，利用FIRCA资助下在尼日利亚开发的研究基础设施和能力。NIH RO3TW006298以及将在当前项目下开发的技术，以更好地了解寄生虫对ACTs和其他抗疟疾药物的耐药性的发展和传播。该项目获得的数据和开发的工具将提供给尼日利亚国家疟疾控制规划，以促进循证抗疟药物政策。

DESCRIPTION (provided by applicant): The rapid development and spread of drug resistant Plasmodium falciparum is a serious global health problem and has contributed to increased mortality and morbidity caused by malaria. This situation has led to the adoption of artemisinin-based combination therapies (ACTs) for the management of malaria infections in many African countries including Nigeria. However, changes in antimalarial drug policies in many African countries were done without the availability of tools for large-scale monitoring of development and spread of P. falciparum resistance to the new drugs. In addition, there are concerns that parasites may soon become resistant to artemisin derivatives or partners drugs once they are introduced for management of malaria in Africa. Thus, researches leading to the definition of molecular determinants of parasites responses to artemisinin derivatives (ARTs) and partner drugs used in combination therapy are crucially needed, as they may unveil markers that are critical for efficient monitoring of drug resistant parasites as well as new chemotherapeutic targets. We hypothesized that combination of different mutations (SNPs) and/or differential expression in transporters and pfATPase6 genes of P. falciparum may be responsible for the differential in vitro or in vivo responses to artemisinin and partner drugs. Furthermore, multiple drug response determinants may be acting in different combinations resulting in differences between parasites phenotypes. This research is primarily directed at identifying and validating molecular determinants of resistance to artemisinin derivatives, lumefantrine (LUM) and amodiaquine (AQ) in fresh patients isolates of Plasmodium falciparum. Molecular determinants of parasites responses to antimalarials identified under this project will be used to develop a quick and simple high-throughput PCR-ELISA based tool/technique that detects SNPs in P. falciparum pfATPase6 and transporters genes as well as other parasites genes, for large- scale monitoring of parasites resistance to artemisinin derivatives and other antimalarials. To achieve these goals the following specific activities will be carried out over a 3 year period: 1) Evaluate the efficacy and in vitro susceptibility of P. falciparum to artemether-lumefantrine and artesunate- amodiaquine combinations in Southwest Nigeria; 2) determine parasites population diversity and assess the presence of SNPs in genes (selected transporters or pfATPase6) in fresh isolates of P. falciparum obtained from patients; 3) evaluate and compare SNPs patterns in selected P. falciparum transporters and pfATPase6 genes in patients isolates and freshly cloned parasites before and following drug (AQ, LUM and ARTs) pressure; 4)Collate SNPs patterns of transporters genes and pfATPase6 with in vitro susceptibility profile, treatment modalities in order to Identify molecular determinants of ARTs, LUM or AQ, and the development of unique resistant phenotypes; 5) use molecular determinants (SNPs) of P. falciparum response to ARTs, LUM or AQ to develop a simple high- throughput method for large-scale monitoring/surveillance of drug resistant parasites to artemisinin and partner drugs used in combination therapy for malaria in Nigeria. This research project provides a unique opportunity to use Research Infrastructures and Capacities developed in Nigeria under the FIRCA Grant no. NIH RO3TW006298, for a better understanding of the development and spread of resistance to ACTs and other antimalarial drugs. Data obtained and tools developed by this project will be made available to the Nigerian National Malaria Control Program in order to promote evidence-based antimalarial drugs policy in the country. . This research project has a direct relevance to the problem of drug resistance in Nigeria and Africa. It provides a unique opportunity to use Research Infrastructures, Capacities Developed in Nigeria under the FIRCA Grant no. NIH RO3TW006298 as well as the technologies to be developped under the current project to better understand the development and spread of parasites resistance to ACTs and other antimalarial drugs. Data obtained and tools developed by this project will be made available to the Nigerian National Malaria Control Program in order to promote evidence-based antimalarial drugs policy.