Abstract
BackgroundLoop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection.MethodsThe cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR.ResultsThe LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method.ConclusionThe colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.
Highlights
Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target
LAMP analytical sensitivity and specificity Using a serial dilution of plasmid for each target, Pfr364-LAMP showed an limit of detection (LOD) of 27.3 copies of plasmid/μL and PfEMP1-LAMP showed an LOD of 28.9 copies of plasmid/μL
The results showed an LOD of 3.7 parasites/μL for Pfr364-LAMP and of 3.3 parasites/ μL for PfEMP1-LAMP
Summary
Loop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. Nolasco et al Malar J (2021) 20:225 marked active transmission in rural and remote areas with poor access to medical care, despite the effort of the Peruvian malaria control programme (i.e., Plan Malaria Cero, PMC) to provide field-diagnosis based on microscopy detection and treatment to a large part of the affected population. According to surveillance reports based on passive case detection and microscopy diagnosis, Plasmodium vivax causes four times more malaria infections than Plasmodium falciparum in the Peruvian Amazon region. Such reports are based on passive case detection and microscopy assessment, which miss a high proportion of asymptomatic and submicroscopic infections that are only detected by molecular diagnosis [3, 4]. In Peru, P. falciparum infections are treated using artemisinin-based combination therapy (ACT) with mefloquine and primaquine as partner drug, whereas P. vivax treatment is based on chloroquine to clear blood stages and primaquine to eliminate dormant liver stages (i.e., hypnozoites) that cause relapse [5]
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