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Abstract
BackgroundPlasmodium falciparum has developed resistance against artemisinin in Southeast Asia. Mutations in the P. falciparum Kelch-13 (Pfk13) gene are associated with artemisinin resistance in vitro and in vivo. We investigated the prevalence of mutations in PfK13 from sporozoite-stage parasites isolated from the salivary glands of Anopheles dirus mosquitoes.MethodsMosquitoes were caught by human-landing catches at two locations within the Khanh Phu commune, South-Central Vietnam. Identification of Anopheles species was performed based on morphological features and nucleotide sequence analysis. Sporozoite-infected salivary glands were stored on filter paper and at 4–6 °C. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification. Pfk13 was amplified by nested PCR, and subjected to nucleotide sequencing.ResultsFive of 33 P. falciparum sporozoite samples carried the P553L mutation at the PfK13 locus. This mutation has been recorded previously in Vietnam, but not in Khanh Hoa province, were surveys of K13 polymorphism have not previously been carried out.ConclusionThese results demonstrate the utility of mosquito-stage malaria parasite samples for studies on the molecular epidemiology of drug resistance.
Highlights
Plasmodium falciparum has developed resistance against artemisinin in Southeast Asia
Genome-wide analysis of artemisinin resistance in P. falciparum has demonstrated that mutations in the propeller domain of the gene encoding the Kelch 13 (K13) protein (Pfk13) are associated with delayed parasite
We characterized the polymorphism at the P. falciparum Kelch-13 (Pfk13) locus using parasite DNA extracted from sporozoites isolated from the salivary glands of humanbiting Anopheles dirus mosquitoes in South-central Vietnam
Summary
Collection of mosquitoes was carried out through human-baited landing catches in and around the forest near Nga Hai village in the south of Khanh Phu commune, Khanh Vinh district, Khanh Hoa province, Vietnam. Collection of mosquitoes was carried out as previously described [11, 12] from January 2008 to October 2012. Sporozoite-infected salivary glands were stored on filter paper kept in closed vials at 4–6 °C until analysis [13]. Genomic DNA (gDNA) was extracted from preserved filter paper with sporozoite-positive salivary glands [11, 14]. The 18S rRNA gene-based nested PCR was used for the detection of P. falciparum and other Plasmodium species [12, 15]. Amplification of the Pfk gene was carried out by nested PCR as previously described [8], and the products sequenced with BigDye Terminator v3.1 Cycle Sequencing Premix Kit (ABI, Foster city, CA, USA). Sequencing products were run on an ABI/Hitachi 3130x1 Genetic Analyzer (ABI) and nucleotide sequences were analysed using Genetyx (Genetyx Corporation, Tokyo, Japan)
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