Malaria diagnosis using a combined system of a simple and fast extraction method with a lyophilised Dual-LAMP assay in a non-endemic setting.

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.

Usefulness of Malachite-Green LAMP for Diagnosis of Plasmodium and Five Human Malaria Species in a Nonendemic Setting.

Molecular methods are necessary to detect low-density malaria infections. The purpose of this study was to assess the diagnostic performance of six malachite-green loop-mediated amplification method (MG-LAMP) assays (MG-LAMP-Pf, MG-LAMP-Pv, MG-LAMP-Po, MG-LAMP-Pm, MG-LAMP-Pk, and MG-LAMP-Pspp) for the species-specific detection of each human Plasmodium, including P. knowlesi, and the Plasmodium genus compared with the nested-multiplex malaria polymerase chain reaction (NM-PCR), using 161 malaria-positive and 274 malaria-negative samples. MG-LAMP-Pspp assay detected the five human Plasmodium species and each species-specific MG-LAMP assay detected only its corresponding species. Sensitivity, specificity, and predictive values of MG-LAMP assays, compared with NM-PCR, were > 90%, except in the case of the MG-LAMP-Pm assay, which dropped to 47%. Limit of detection for MG-LAMP-Pspp assay ranged from 0.1 parasites/µL for P. falciparum to 16.9 parasites/µL for P. malariae samples, and it was similar for the rest of MG-LAMP assays except for the MG-LAMP-Pm assay. Turnaround time was estimated to be 2 hours and 35 minutes for one MG-LAMP assay and 4 hours and 15 minutes if all species-specific MG-LAMP is set up, whereas for the NM-PCR, turnaround time was ∼6 hours and 15 minutes. Costs per determination ranged from 1 to 6 euros for MG-LAMP assays and 5 euros for NM-PCR. Therefore, MG-LAMP assays appear to have good concordance compared with the reference method, except for the MG-LAMP-Pm assay. They can detect low parasitemia and identify malaria species, with lower costs and shorter time to obtain results, and they are suitable tools to be used in endemic and non-endemic countries for malaria detection.