Malaria & Parasitic Emerging Diseases Laboratory. National Microbiology Center, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
Centro de Investigación Biomédica En Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain.
Pathog Glob Health. 2024 Feb;118(1):80-90. doi: 10.1080/20477724.2023.2232595. Epub 2023 Jul 6.
Malaria is a parasitic disease distributed in tropical areas but with a high number of imported cases in non-endemic countries. The most specific and sensitive malaria diagnostic methods are PCR and LAMP. However, both require specific equipment, extraction procedures and a cold chain. This study aims to solve some limitations of LAMP method with the optimization and validation of six LAMP assays, genus and species-specific, using an easy and fast extraction method, the incorporation of a reaction control assay, two ways (Dual) of result reading and reagent lyophilization. The Dual-LAMP assays were validated against the Nested-Multiplex Malaria PCR. A conventional column and saline extraction methods, and the use of lyophilized reaction tubes were also assessed. A new reaction control Dual-LAMP-RC assay was designed. Dual-LAMP-spp assay showed no cross-reactivity with other parasites, repeatability and reproducibility of 100%, a significant correlation between parasite concentration and time to amplification and a LoD of 1.22 parasites/µl and 5.82 parasites/µl using column and saline extraction methods, respectively. Sensitivity and specificity of the six Dual-LAMP assays reach values of 100% or close to this, being lower for the Dual-LAMP-. The Dual-LAMP-RC assay worked as expected. Lyophilized Dual-LAMP results were concordant with the reference method. Dual-LAMP malaria assays with the addition of a new reaction control LAMP assay and the use of a fast and easy saline extraction method, provided low limit of detection, no cross-reactivity, and good sensitivity and specificity. Furthermore, the reagent lyophilization and the dual result reading allow their use in most settings.
疟疾是一种寄生虫病，分布在热带地区，但在非流行国家有大量输入病例。最特异和敏感的疟疾诊断方法是 PCR 和 LAMP。然而，这两种方法都需要特定的设备、提取程序和冷链。本研究旨在通过优化和验证六种属和种特异性的 LAMP 检测方法，解决 LAMP 方法的一些局限性，这些方法使用了一种简单、快速的提取方法，整合了反应控制检测方法，采用了两种结果读取方式（双重）和试剂冻干。双重-LAMP 检测方法与巢式多重疟疾 PCR 进行了验证。还评估了常规柱和盐水提取方法，以及冻干反应管的使用。设计了一种新的反应控制双重-LAMP-RC 检测方法。双重-LAMP-spp 检测方法与其他寄生虫无交叉反应，重复性和再现性为 100%，寄生虫浓度与扩增时间之间存在显著相关性，使用柱和盐水提取方法时的 LoD 分别为 1.22 个寄生虫/µl 和 5.82 个寄生虫/µl。六种双重-LAMP 检测方法的敏感性和特异性均达到 100%或接近 100%，双重-LAMP-除外。反应控制双重-LAMP-RC 检测方法按预期工作。冻干双重-LAMP 结果与参考方法一致。添加新的反应控制 LAMP 检测方法并使用快速、简单的盐水提取方法的双重-LAMP 疟疾检测方法具有较低的检测限、无交叉反应、良好的敏感性和特异性。此外，试剂冻干和双重结果读取允许它们在大多数环境中使用。
