Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification

dc.contributor.authorSuttipat Srisuthamen_US
dc.contributor.authorKanokon Suwannasinen_US
dc.contributor.authorVivek Bhakta Mathemaen_US
dc.contributor.authorKanlaya Sriprawaten_US
dc.contributor.authorFrank M. Smithuisen_US
dc.contributor.authorFrancois Nostenen_US
dc.contributor.authorNicholas J. Whiteen_US
dc.contributor.authorArjen M. Dondorpen_US
dc.contributor.authorMallika Imwongen_US
dc.contributor.otherShoklo Malaria Research Uniten_US
dc.contributor.otherChulalongkorn Universityen_US
dc.contributor.otherMahidol Universityen_US
dc.contributor.otherNuffield Department of Medicineen_US
dc.contributor.otherMyanmar Oxford Clinical Research Uniten_US
dc.contributor.otherMedical Action Myanmaren_US
dc.description.abstract© 2020 The Author(s). Background: Rapid diagnostic tests (RDTs) have become the most common diagnostic tool for detection of Plasmodium falciparum malaria, in particular in remote areas. RDT blood spots provide a source of parasite DNA for molecular analysis. In this study, the utility of RDTs for molecular analysis and the performance of different methods for whole genome amplification were investigated. Methods: Positive P. falciparum RDTs were collected from Kayin, Myanmar from August 2014 to January 2016. The RDT samples were stored for 6 months, 9 months, 20 months, 21 months, and 32 months before DNA extraction and subsequent molecular analysis of P. falciparum kelch 13 (pfkelch13) mutations, P. falciparum multidrug resistance 1 (pfmdr1), and P. falciparum plasmepsin 2 (pfplasmepsin2) gene amplification. In addition, performance of four whole genome amplification (WGA) kits were compared, including REPLI-g®, MALBACTM, PicoPLEX®, and GenomePlex®, for which DNA quantity and quality were compared between original DNA and post-WGA products. Results: The proportion of successful amplification of the different molecular markers was similar between blood spots analysed from RDTs stored for 6, 9, 20, 21, or 32 months. Successful amplification was dependent on the molecular markers fragment length (p value < 0.05): 18% for a 1245 bp fragment of pfkelch13, 71% for 364 bp of pfkelch13, 81% for 87 bp of pfmdr1, 81% for 108 bp of pfplasmepsin2. Comparison of the four WGA assay kits showed that REPLI-g®, MALBACTM, and PicoPLEX® increased the quantity of DNA 60 to 750-fold, whereas the ratio of parasite DNA amplification over human DNA was most favourable for MALBAC®. Sequencing results of pfkelch13, P. falciparum chloroquine resistance transporter (pfcrt), P. falciparum dihydrofolate reductase (pfdhfr) and six microsatellite markers assessed from the post-WGA product was the same as from the original DNA. Conclusions: Blood spots from RDTs are a good source for molecular analysis of P. falciparum, even after storage up to 32 months. WGA of RDT-derived parasite DNA reliably increase DNA quantity with sufficient quality for molecular analysis of resistance markers.en_US
dc.identifier.citationMalaria Journal. Vol.19, No.1 (2020)en_US
dc.rightsMahidol Universityen_US
dc.subjectImmunology and Microbiologyen_US
dc.titleUtility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplificationen_US