Department of Tropical Medicine and Malaria, Research Institute, National Center for Global Health and Medicine, Tokyo, 162-8655, Japan.
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki, 305-8575, Japan.
Malar J. 2018 Oct 22;17(1):373. doi: 10.1186/s12936-018-2527-7.
Malaria is one of the most important parasitic infectious diseases for which almost half of the world's population is at risk. Although several diagnostic methods are now available to detect the infection, more sensitive and applicable tests are still required in the field. The loop-mediated isothermal amplification (LAMP) method is a DNA amplification tool in which the DNA amplification can be achieved by incubation at a stable temperature. A malaria detection kit based on this methodology has already been commercialized and is being used in some countries. The kit includes two reaction tubes: one targeting the common Plasmodium genus (Pan tube) and the other specifically targeting Plasmodium falciparum (Pf tube). In parallel, a simple DNA extraction method, the procedure for ultra rapid extraction (PURE), which can produce a DNA solution suitable for the LAMP reaction without the use of a centrifuge, has also become available. In this study, the sensitivity of the combination of the PURE and LAMP methods (PURE-LAMP) was evaluated with archived dried clinical blood samples of imported malaria cases, including P. falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae.
Using a nested PCR as the reference, 117 samples including 46 P. falciparum, 7 P. vivax, 9 P. ovale, 4 P. malariae, and 51 negative cases were tested. The PURE-LAMP Pan correctly identified 64 of the 66 positives and the 51 negatives. Among the Pan-positive samples 45 P. falciparum were also detected with the PURE-LAMP Pf. The PURE-LAMP Pan and PURE-LAMP Pf had respective sensitivities of 96.96% (95% CI 89.47-99.63) and 97.82% (95% CI 88.47-99.94) and common specificity of 1.
The PURE-LAMP system is accurate when used with dried blood spots and extendable to the field.
疟疾是最重要的寄生虫病之一，全球近一半人口面临感染风险。目前已经有几种诊断方法可以检测到这种感染，但在现场仍然需要更敏感和适用的检测方法。环介导等温扩增（LAMP）方法是一种 DNA 扩增工具，通过在稳定温度下孵育可以实现 DNA 扩增。一种基于这种方法的疟疾检测试剂盒已经商业化，并在一些国家使用。该试剂盒包括两个反应管：一个针对常见的疟原虫属（Pan 管），另一个专门针对恶性疟原虫（Pf 管）。与此同时，一种简单的 DNA 提取方法——超快速提取程序（PURE）也已经问世，它可以在不使用离心机的情况下产生适合 LAMP 反应的 DNA 溶液。在这项研究中，我们评估了 PURE 和 LAMP 方法（PURE-LAMP）的敏感性，使用的是存档的进口疟疾病例干血斑临床样本，包括恶性疟原虫、间日疟原虫、卵形疟原虫和三日疟原虫。
使用巢式 PCR 作为参考，对 117 个样本进行了检测，其中包括 46 个恶性疟原虫、7 个间日疟原虫、9 个卵形疟原虫、4 个三日疟原虫和 51 个阴性样本。PURE-LAMP Pan 正确识别了 66 个阳性样本中的 64 个和 51 个阴性样本。在 Pan 阳性样本中，还通过 PURE-LAMP Pf 检测到 45 个恶性疟原虫。PURE-LAMP Pan 和 PURE-LAMP Pf 的灵敏度分别为 96.96%（95%CI 89.47-99.63）和 97.82%（95%CI 88.47-99.94），共同特异性为 1。
PURE-LAMP 系统在使用干血斑时准确可靠，并且可以扩展到现场使用。
