TY - JOUR
T1 - Utility of Plasmodium falciparum DNA from rapid diagnostic test kits for molecular analysis and whole genome amplification
AU - Srisutham, Suttipat
AU - Suwannasin, Kanokon
AU - Mathema, Vivek Bhakta
AU - Sriprawat, Kanlaya
AU - Smithuis, Frank M.
AU - Nosten, Francois
AU - White, Nicholas J.
AU - Dondorp, Arjen M.
AU - Imwong, Mallika
N1 - Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/5/27
Y1 - 2020/5/27
N2 - 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 ®, MALBAC TM, 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 ®, MALBAC TM, 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.
AB - 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 ®, MALBAC TM, 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 ®, MALBAC TM, 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.
KW - Plasmodium falciparum
KW - RDT
KW - Whole genome amplification
UR - http://www.scopus.com/inward/record.url?scp=85085539970&partnerID=8YFLogxK
U2 - https://doi.org/10.1186/s12936-020-03259-9
DO - https://doi.org/10.1186/s12936-020-03259-9
M3 - Article
C2 - 32460780
SN - 1475-2875
VL - 19
JO - Malaria journal
JF - Malaria journal
IS - 1
M1 - 193
ER -