Development of a rapid malaria LAMP-MS assay for diagnosis of malaria infections

Abstract

Malaria remains a critical global health concern, especially in tropical and subtropical regions, where it causes substantial morbidity and mortality. Current diagnostic methods, such as microscopy and PCR-based assays, are reliable but often impractical in resource-limited settings due to their dependency on complex equipment and skilled personnel. This study developed a novel malaria diagnostic platform by combining the Chelex-100/boiling DNA extraction method with a Loop-mediated Isothermal Amplification-MicroScanner (LAMP-MS) assay. The Chelex-100/boiling method is simpler and more cost-effective than conventional DNA extraction processes, making it suitable for use in resource-limited settings. The LAMP-MS assay enables multiplex detection through a microchip design with four chambers. Each chamber of the microchip is preloaded with specific primers targeting Pan, Plasmodium falciparum (Pf), Plasmodium vivax (Pv), and an internal control, minimizing non-specific amplification in multiplex LAMP reactions. In a clinical evaluation of 260 samples, the assay demonstrated a sensitivity of 97.5% for the Pan target and 100% for the Pf-specific target in the 80 Plasmodium falciparum (Pf) clinical samples. Similarly, for the 80 Plasmodium vivax (Pv) clinical samples, the assay achieved a sensitivity of 95% for the Pan target and 94% for the Pv-specific target. Notably, in the 100 non-infected clinical samples, the assay exhibited 100% specificity, with no false positives observed. These findings suggest that LAMP-MS is a rapid and reliable alternative to PCR-based methods, especially in resource-limited environments.

Keywords: LAMP-MS; Loop-mediated isothermal amplification; Malaria; Point of care testing.

Fig. 1

Schematic illustration of the Malaria Pan/Pf/Pv/IC LAMP-MS assay.

Fig. 2

Optimization of the Chelex-100/boiling method for nucleic acid extraction from blood. Comparison of blood sample concentrations (2.5%, 5%, and 10%) for nucleic acid extraction was performed using both the Malaria Pan qPCR assay (A) and the Malaria Pan LAMP assay (B). Similarly, Chelex-100 resin concentrations (5%, 10%, and 20%) were tested for nucleic acid extraction using both the Malaria Pan qPCR assay (C) and the Malaria Pan LAMP assay (D).

Fig. 3

Detection of Malaria Using the Newly Developed LAMP-MS Assay. (A) Results of the Pan/Pf/Pv/IC LAMP assay for a Plasmodium falciparum sample. The graph shows the fluorescence amplification curves for Pan (①), Pf (②), Pv (③), and IC (④) targets. (B) Results of the Pan/Pf/Pv/IC LAMP-MS assay for a Plasmodium falciparum sample. Microscopic images show the presence of amplified DNA products for Pan (①), Pf (②), and IC (④) targets, while no amplification is observed for the Pv (③) target.