Abstract
BackgroundThe recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer.ResultsA LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia.ConclusionsAn innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.
Highlights
The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health
Plasmid construction The templates used for the analytical loop-mediated isothermal amplification (LAMP) reactions were the pEX-A2J1 plasmids harbouring partial sequences within the k13 of P. falciparum, which were constructed by Eurofines Genomics Co., Ltd. (Tokyo, Japan) based on the reference sequences (GenBank accession numbers: P. falciparum, KT956001.1 and KR537460.1)
Clinical evaluation of LAMP assays Parasitemia was confirmed in 40 blood samples by nested-polymerase chain reaction (PCR)
Summary
The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. The spread of artemisinin (ART)-resistant Plasmodium falciparum malaria, which is defined as delayed parasite clearance from the blood following appropriate treatment with ART monotherapy or ART-based combination therapy (ACT) [1], has gained global attention, in Southeast Asia and southern China [2,3,4]. Amino acids substitutions N458Y, Y493H, R539T, I543T, R561H, and C580Y could all be associated with ART-resistance and reduce the cure rate following ACT treatment, C580Y is thought to be the dominant mutation and a potential drug resistance marker, especially in Southeast Asia [6,7,8,9]. There is an urgent need to strengthen the surveillance and elimination of ART-resistant P. falciparum C580Y mutation in Southeast Asia
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