Development a point-of-care lateral flow test for artemisinin-resistant malaria

青蒿素为基础的联合疗法是目前世界卫生组织推荐的治疗无并发症恶性疟疾的方法。然而，青蒿素耐药性正在威胁这些抗疟药物的有效性，并已在东南亚迅速蔓延[1]。如果青蒿素耐药性疟疾（ARM）从该地区传播到非洲，就像对老抗疟药的耐药性一样，那么在降低疟疾流行率方面取得的大部分进展都可能被逆转。虽然ARM的机制仍然只是部分了解，但位于13号染色体上的kelch基因螺旋桨结构域中的多个单核苷酸多态性已被证明与青蒿素耐药性有关[2]。C580Y和R539T是与ARM相关的最常见的SNP，在东南亚占85%，有些地区的患病率高达90% [3]。自从发现ARM的分子标记以来，基于核酸的检测如聚合酶链反应（PCR）已被用于检测ARM。但基于PCR的技术需要复杂的仪器，培训人员和外部力量，因此在资源贫乏的环境中不容易获得。这种方法对流行病学研究很有用，但不允许将结果用于病例管理，因为分子检测是在从患者采集血液后很长时间内进行的。为了克服这些挑战，其他技术，如环介导等温扩增（LAMP）方法已经开发出来。LAMP由于其低成本和简单性而具有许多优点，并且越来越多地被临床医生用作其他传染病的即时检测。它已被证明是用户友好和成本效益[4]。微流体迄今为止，还没有即时（POC）检测可用于检测ARM。在疟疾流行地区进行AMR的POC检测有助于告知临床医生对患者进行适当的病例管理。目前正在评估ARM的替代药物方案，如使用伴侣药物和更长的治疗时间，然而，这可能导致药物毒性和耐多药疟疾[5]。因此，对ARM进行POC测试将使临床医生能够提供适当的治疗，并限制现有抗疟药物的过度使用。POC测试也将提供真实的时间数据epidemiologicalstudy.ObjectivesThe主要目标是这个项目是开发一种新的低成本的测试，将结合联合收割机microfluidicsand手指针刺采样，使第一次ARM的测试在POC。该测试将使用LAMP来测试最常见的赋予ARM的SNPs，并将使用侧向流和微流体技术。该测试应具有尽可能接近PCR的灵敏度和特异性，同时适用于低资源环境，对额外设备的要求最低。

Artemisinin-based combination therapy is currently recommended by WHO for treatment ofuncomplicated falciparum malaria. However, artemisinin resistance is threatening the effectivenessof these antimalarials and has spread rapidly in South-East Asia [1]. Should artemisinin-resistantmalaria (ARM) spread from this region to Africa, as did resistance to older antimalarials, much of theprogress made in reducing the prevalence of malaria could be reversed.While the mechanism of ARM is still only partially understood, multiple single-nucleotidepolymorphisms in the propeller domain of a kelch gene, located on chromosome 13, have beenshown to be associated with artemisinin resistance [2]. Two single nucleotide polymorphisms,C580Y and R539T, are the most prevalent SNPs associated with ARM and account for 85% in SouthEast Asia, with some areas having a prevalence as high as 90% [3].Since the discovery of the molecular markers for ARM, nucleic-acid based tests such as polymerasechain reaction (PCR) have been used to test for ARM. However PCR-based techniques requirecomplex apparatus, trained personnel and external power, and therefore are not readily available inresource-poor settings. This method is useful for epidemiological studies but does not allow forresults to be used in case-management as the molecular testing is conducted long after thecollection of blood from the patient.To overcome these challenges, other techniques such as loop-mediated isothermal amplification(LAMP)-based methods have been developed. LAMP has many advantages due to its low cost andsimplicity, and is increasingly used as point-of-care tests by clinicians for other infectious diseases. Ithas been shown to be user-friendly and cost-effective [4]. MicrofluidicsTo date there is no point-of-care (POC) test available to detect ARM. A POC test for AMR in malariaendemic areas could help to inform clinicians about appropriate case management for patients.Currently alternative drug regimens such as the use of partner drugs and longer treatments arebeing evaluated for ARM, however this can lead to drug toxicity and multidrug-resistant malaria [5].POC tests for ARM would therefore enable clinicians to provide appropriate treatment and limit theoveruse of existing antimalarials. POC tests would also provide real time data for epidemiologicalstudies.ObjectivesThe main objective of this project is to develop a new low cost test that will combine microfluidicsand finger prick sampling to enable for the first time the testing of ARM at the POC. The test woulduse LAMP to test for the most common SNPs conferring ARM and would use lateral flow andmicrofluidic technology. The test should have sensitivity and specificity as close as possible to PCR,while being suitable for use in low-resource environments, with the minimal requirements foradditional equipment.