Used RDTs provide a valuable source of parasite DNA that can be used for molecular analysis [
2‐
4,
7,
8]. The current study investigated the utility of DNA preps from RDT samples for
pfkelch13 SNPs,
pfmdr1 CNV and
pfplasmepsin2 CNV analysis. Even though theDNA preps were of limited quantity and with degradation, molecular typing was successful in 17.59–81.48% of samples. Storage up to 32 months did not negatively affect the accuracy of typing, showing that stored RDTs can be used as a source for
Plasmodium DNA typing as also suggested in earlier studies [
2,
4,
7,
8]. Comparing short (364 bp) and long (1,245 bp) fragment
Pfkelch13 amplicons suggested that PCR product size affects the success rate of molecular analysis using RDT blood spots.
Inadequate amounts of parasite DNA is a limiting factor for conducting molecular assays from RDT blood spots of around 5 µl, in particular when several markers are evaluated [
25,
26]. RDT samples were collected from symptomatic cases from the area where the observed geometric mean
P. falciparum parasitaemia was reported as 6
Plasmodium genome equivalents/µL (95% CI 2 to 16), so around 30 (95% CI 10 to 80) genomes per blood spot [
27]. The purity and amount of DNA will affect outcomes of downstream molecular analysis. Although the WGA using any of the four kits increased parasite DNA, only WGA with REPLI-g
® and MALBAC
TM yielded sufficient
P. falciparum DNA purity and quantity needed for further molecular analysis. MALBAC
TM yielded higher concentrations of parasite DNA compared to REPLI-g
®, which makes this kit preferable for amplification using RDT blood spots with low parasites content. A high concordance in microsatellite marker and SNP typing was previously reported using the MDA
® kit to amplify parasite DNA from whole blood samples of
P. falciparum-infected patients [
13]. The current study shows reliable results for molecular marker typing, DNA sequencing and microsatellite marker analysis using REPLI-g
® and MALBAC
TM WGA of RDT blood spots. However, CNV estimates for
pfmdr1 and
Pfplasmepsin2 were less reliable, suggesting that WGA amplified DNA should not be used for the purpose of CNV assessment. A previous study showed that PicoPLEX
® and MALBAC
TM provided reliable CNVs estimates based on an ion proton platform, whereas CNV estimates were not accurate using MDA or GenomePlex
® [
16]. As expected from the working principles of WGA and confirmed by the current study, WGA interferes with the assessment of CNV in the parasite genome. This study showed that the success rate of molecular marker analysis was affected by the initial parasite DNA concentration, and WGA with MALBAC
TM achieved a higher success rate for molecular marker typing than REPLI-g
® in concordance. Although an estimated cost of MALBAC
TM is higher than REPLI-g
®, the use of MALBAC
TM was less time consuming compared to REPLI-g
® and provided higher yield of parasite DNA. Therefore, MALBAC
TM shall be recommended for wide use to generate high-quality parasite DNA in sufficient quantities using stored RDT blood spots containing low parasite DNA quantities. Undoubtedly, good laboratory practices and good laboratory facilities are important to implement these techniques.