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

描述(申请人提供)：抗药性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，并导致疟疾造成的死亡率和发病率增加。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(ACTs)来管理疟疾感染。然而，许多非洲国家的抗疟疾药物政策的变化是在没有大规模监测恶性疟原虫对新药的耐药性的发展和传播的工具的情况下进行的。此外，人们担心，一旦将青蒿素衍生物或伙伴药物引入非洲用于疟疾管理，寄生虫可能很快就会对它们产生抗药性。因此，迫切需要研究确定寄生虫对青蒿素衍生物(ARTS)和联合治疗中使用的伙伴药物的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至关重要的标记以及新的化疗靶点。我们推测，恶性疟原虫转运蛋白和pfATPase6基因的不同突变(SNPs)和/或差异表达的组合可能是导致青蒿素和配伍药物在体外或体内反应不同的原因。此外，多种药物反应决定因素可能在不同的组合中发挥作用，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恶性疟原虫新鲜分离株对青蒿素衍生物、鲁米芬(Lum)和阿莫地喹(AQ)的耐药性的分子决定因素。根据该项目确定的寄生虫对抗疟疾药物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，该工具/技术检测恶性疟原虫pfATPase6和转运蛋白基因以及其他寄生虫基因中的SNPs，用于大规模监测寄生虫对青蒿素衍生物和其他抗疟疾药物的耐药性。为实现这些目标，将在3年内开展以下具体活动：1)评估尼日利亚西南部恶性疟原虫对蒿甲醚-鲁米芬和青蒿琥酯-阿莫地喹组合的疗效和体外敏感性；2)确定寄生虫种群多样性，并评估从患者那里获得的恶性疟原虫新鲜分离株基因(选定的转运子或pfATPase6)中是否存在SNPs；3)评估和比较患者分离株和新克隆的寄生虫在药物(AQ、Lum和ARTS)压力前后的SNPs图谱和pfATPase6基因；4)将转运蛋白基因和pfATPase6的SNPs模式与体外药敏情况、治疗方式进行比较，以确定ARTS、LUM或AQ的分子决定因素，以及独特耐药表型的发展；5)利用恶性疟原虫对ARTS、LUM或AQ的反应分子决定因素(SNPs)，开发一种简单、高通量的方法，用于大规模监测/监测尼日利亚疟疾联合治疗中使用的青蒿素和伙伴药物的耐药性寄生虫。这一研究项目提供了一个独特的机会，可以利用尼日利亚在FIRCA赠款编号下建立的研究基础设施和能力。NIH RO3TW006298，以更好地了解对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方案，以促进该国的循证抗疟疾药物政策。。该研究项目与尼日利亚和非洲的抗药性问题有直接关系。它提供了一个独特的机会来利用研究基础设施，即尼日利亚在FIRCA赠款编号下建立的能力。NIH RO3TW006298以及在当前项目下将开发的技术，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方案，以促进循证抗疟疾药物政策。

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.