Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum.
BACKGROUND
Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers.


METHODS
This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP.


RESULTS
Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA.


CONCLUSIONS
FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular.
Mens
Petra F
PF
Prince Leopold Institute of Tropical Medicine, Department of Parasitology, Nationalestraat 155, B2000, Antwerp, Belgium. p.mens@kit.nl
van Overmeir
Chantal
C
Bonnet
Maryline
M
Dujardin
Jean-Claude
JC
d'Alessandro
Umberto
U
D000818:Animals; D015023:Democratic Republic of the Congo; D004094:Dihydropteroate Synthase / Q000235:genetics*; D004351:Drug Resistance; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology*; D009154:Mutation; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D004094:Dihydropteroate Synthase