Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia.
Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia.
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DOI：
10.1186/s12936-016-1676-9
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发表时间：
2017-01-13
期刊：
影响因子：
3
通讯作者：
Grigg MJ
中科院分区：
文献类型：
--
作者：
Piera KA;Aziz A;William T;Bell D;González IJ;Barber BE;Anstey NM;Grigg MJ
诺氏疟原虫是马来西亚最常见的疟疾病因。然而，显微镜诊断是不准确的，快速诊断测试（RDTs）不够敏感。聚合酶链反应是敏感和特异的，但在地区一级不可行。环介导等温扩增（LAMP）显示出潜力，只有基本的要求。市售的LAMP检测试剂盒Eiken Loopamp™ MALARIA Pan Detection试剂盒对恶性疟原虫和间日疟原虫敏感，但之前未对诺氏疟原虫进行过评价。本研究旨在确定该LAMP检测法检测诺氏疟原虫感染的灵敏度。研究参与者包括73名PCR物种确认的无并发症疟疾患者：50名诺氏疟原虫，20名恶性疟原虫和3名间日疟原虫。还包括19个疟疾阴性、非流行区对照。评价了Eiken Loopamp™ MALARIA Pan Detection试剂盒（Pan LAMP）检测每种疟原虫的灵敏度。还测定了Eiken Loopamp™ MALARIA Pf检测试剂盒（Pf LAMP）对恶性疟原虫的灵敏度和特异性。评价了每种LAMP测定的检测限，并将结果与PCR进行比较。所有诺氏毕赤酵母菌患者还通过CareStart™（Pf/VOM）和OptiMAL-IT™（Pan/Pf）RDT进行了检测。Pan LAMP测定法对诺氏疟原虫（95% CI 92.9-100）、恶性疟原虫（95% CI 83.2-100）和间日疟原虫（95% CI 29.2-100）的灵敏度为100%。Pf LAMP检测恶性疟原虫的灵敏度和特异性均为100%，所有诺氏疟原虫样品均为阴性反应。LAMP灵敏度上级两种RDT，分别只有10%和28%的诺氏毕赤酵母样品对CareStart™和OptiMAL-IT™检测呈阳性。使用Pan LAMP对诺氏疟原虫和间日疟原虫的检测限均为2个寄生虫/μL，与PCR相当。对于恶性疟原虫，Pan LAMP和Pf LAMP的检测限均为20个寄生虫/μL。Eiken Loopamp™ MALARIA Pan Detection试剂盒对于检测低寄生虫血症临床感染中的诺氏疟原虫以及恶性疟原虫和间日疟原虫具有灵敏度。然而，以更简单的形式进行的诺氏疟原虫特异性现场测定将有助于正确的物种鉴定和对所有疟疾患者的最佳治疗。
Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection. Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs. The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9–100), P. falciparum (95% CI 83.2–100), and P. vivax (95% CI 29.2–100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL. The Eiken Loopamp™ MALARIA Pan Detection kit is sensitive for detection of P. knowlesi in low parasitaemia clinical infections, as well as P. falciparum and P. vivax. However, a P. knowlesi-specific field assay in a simpler format would assist correct species identification and initiation of optimal treatment for all malaria patients.