在马来西亚一个疟疾共流行地区，采用环介导等温扩增技术（LAMP）检测诺氏疟原虫、恶性疟原虫和间日疟原虫。
Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia.
作者信息
Piera Kim A, Aziz Ammar, William Timothy, Bell David, González Iveth J, Barber Bridget E, Anstey Nicholas M, Grigg Matthew J
机构信息
Global and Tropical Health Division, Menzies School of Health Research and Charles Darwin University, Darwin, NT, Australia.
Infectious Diseases Society Sabah-Menzies School of Health Research Clinical Research Unit, Kota Kinabalu, Sabah, Malaysia.
出版信息
Malar J. 2017 Jan 13;16(1):29. doi: 10.1186/s12936-016-1676-9.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
背景
诺氏疟原虫是马来西亚疟疾最常见的病因。然而，显微镜诊断不准确，快速诊断检测（RDT）的敏感性不足。聚合酶链反应（PCR）灵敏且特异，但在地区层面不可行。环介导等温扩增技术（LAMP）仅需基本条件，显示出应用潜力。一种市售的LAMP检测方法，荣研公司环介导等温扩增疟疾全检测试剂盒，对恶性疟原虫和间日疟原虫敏感，但此前尚未针对诺氏疟原虫进行评估。本研究旨在确定该LAMP检测方法对检测诺氏疟原虫感染的敏感性。
方法
研究参与者包括73例经PCR确诊疟原虫种类的非重症疟疾患者：50例诺氏疟原虫患者、20例恶性疟原虫患者和3例间日疟原虫患者。还纳入了19例疟疾阴性的非流行区对照。评估了荣研公司环介导等温扩增疟疾全检测试剂盒（全LAMP）检测每种疟原虫种类的敏感性。还确定了荣研公司环介导等温扩增恶性疟原虫检测试剂盒（Pf LAMP）对恶性疟原虫的敏感性和特异性。评估了每种LAMP检测方法的检测限，并将结果与PCR进行比较。所有诺氏疟原虫患者也通过CareStart™（Pf/VOM）和OptiMAL-IT™（全/Pf）RDT进行检测。
结果
全LAMP检测方法对诺氏疟原虫（95%可信区间92.9 - 100）、恶性疟原虫（95%可信区间83.2 - 100）和间日疟原虫（95%可信区间29.2 - 100）的敏感性均为100%。Pf LAMP对恶性疟原虫检测的敏感性和特异性均为100%，所有诺氏疟原虫样本反应均为阴性。LAMP的敏感性优于两种RDT，诺氏疟原虫样本中分别只有10%和28%对CareStart™和OptiMAL-IT™检测呈阳性。使用全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，全LAMP和Pf LAMP的检测限均为20个寄生虫/μL。
结论
荣研公司环介导等温扩增疟疾全检测试剂盒对低疟原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的检测均敏感。然而，一种更简单形式的诺氏疟原虫特异性现场检测方法将有助于正确的种类鉴定，并为所有疟疾患者启动最佳治疗。
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