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

以青蒿素为基础的联合疗法目前被世界卫生组织推荐用于治疗非复杂性恶性疟疾。然而，青蒿素耐药性正在威胁这些抗疟疾药物的有效性，并已在东南亚迅速蔓延[1]。如果青蒿素抗药性疟疾(ARM)从该地区传播到非洲，就像对较老的抗疟疾药物的耐药性一样，在降低疟疾流行率方面取得的大部分进展可能会逆转。虽然对ARM的机制仍然只有部分了解，但位于13号染色体的海带基因螺旋桨区域的多个单核苷酸多态已被证明与青蒿素耐药性有关[2]。两个单核苷酸多态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ARM的分子标记被发现以来，聚合酶链式反应(PCR)等基于核酸的检测方法被用于ARM的检测。然而，基于聚合酶链式反应的技术需要复杂的仪器、训练有素的人员和外部电源，因此在资源匮乏的环境中并不容易获得。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，因为分子测试是在从患者采集血液后很长一段时间才进行的。为了克服这些挑战，已经开发了其他技术，如基于环介导的等温扩增(LAMP)方法。LAMP由于其低成本和简单的优点，越来越多地被临床医生用作其他感染性疾病的临床点检测。它被证明是用户友好的和成本效益的[4]。微流体到目前为止，还没有可用的护理点(POC)测试来检测ARM。在疟疾流行地区进行AMR的POC测试可以帮助临床医生了解对患者的适当病例管理。目前正在评估ARM的替代药物方案，如使用伙伴药物和更长时间的治疗，但这可能导致药物毒性和多药耐药疟疾[5]。因此，ARM的POC测试将使临床医生能够提供适当的治疗，并限制现有抗疟疾药物的过度使用。POC检测还将为流行病学研究提供实时数据。目标该项目的主要目标是开发一种新的低成本检测方法，该检测方法将结合微流体和手指点刺采样，以实现首次在POC对ARM进行检测。这项测试将使用LAMP来检测最常见的SNPs授权臂，并将使用侧向流动和微流控技术。该检测应具有尽可能接近于聚合酶链式反应的灵敏度和特异度，同时适用于低资源环境，对附加设备的要求最低。

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.