Application of loop-mediated isothermal amplification combined with lateral flow assay visualization of Plasmodium falciparum kelch 13 C580Y mutation for artemisinin resistance detection in clinical samples

Highlights

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  PfC580Y LAMP-SNP-LFA method was developed to detect Pfkelch13 mutations.

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  This rapid and accurate tool effectively detects  Pfkelch13 mutations in clinical samples.

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  It reduces dye-induced non-specific binding and amplification inhibition in the LAMP assay.

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  It can aid in monitoring the spread of drug-resistant infections.

Abstract

Resistance to the antimalarial drug artemisinin (ART) has emerged in Greater Mekong Subregion. The molecular marker predominantly used to identify ART resistance is the C580Y mutation in Pfkelch13 of Plasmodium falciparum. Rapid and accurate detection of ART resistance in the field is necessary to guide malaria containment and elimination interventions. Our study evaluates the PfC580Y by using the loop-mediated isothermal amplification and single nucleotide polymorphism analysis visualization using a lateral flow assay (LAMP-SNP-LFA) method for detecting ART resistance in clinical samples collected from Thailand between 2014 and 2019. The optimized incubation condition for the reaction was determined as 45 min at 56 °C, followed by visual detection of positive amplicons using LFA. The assay demonstrated high analytical sensitivity and specificity, with a limit of detection of 16.8 copies of C580Y plasmid/µL of and 100% accuracy for C580Y mutation detection. The PfC580Y LAMP-SNP-LFA method is faster and simpler than conventional polymerase chain reaction/DNA sequencing and has the potential to support antimalarial management policies, malaria control, and global elimination efforts.