RESUMEN
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-Pspp 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-Pm. 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.