Rapid identification of chloroquine and atovaquone drug resistance in Plasmodium falciparum using high-resolution melt polymerase chain reaction.
BACKGROUND
Drug resistance determination for Plasmodium falciparum infections are important to determining the type of treatment to be given. Besides in vivo experiments, molecular methods, such as sequencing and PCR, are now increasingly being used. Here a cheaper alternative to sequencing or the use of multiplex 5'nuclease PCR assay for detection and differentiation of drug resistance haplotypes for chloroquine and atovaquone using polymerase chain reaction-high resolution melt (PCR-HRM) is reported.


METHODS
Separate PCR-HRM assays were designed for the detection and differentiation of chloroquine and atovaquone drug resistance haplotypes in P. falciparum. PCR was conducted on a thermal cycler and melt curves generated using a LightScanner. These were tested against reference strains of P. falciparum from MR4 as well as 53 local isolates.


RESULTS
The PCR-HRM assays are able to detect and differentiate between the various haplotypes consistently. These assays can also be used to detect new variants.


CONCLUSIONS
PCR-HRM is an inexpensive option for the determination of drug resistance profile in P. falciparum and will see increasing use as an alternative to sequencing and 5'nuclease PCR assays in reference laboratories or once PCR systems that are able to conduct HRM become commonplace.
Gan
Linda Seo Hwee
LS
Detection and Diagnostics Laboratory, Defence Medical & Environmental Research Institute, DSO National Laboratories, 17510, Singapore.
Loh
Jin Phang
JP
D000962:Antimalarials / Q000494:pharmacology*; D053626:Atovaquone / Q000494:pharmacology*; D002738:Chloroquine / Q000494:pharmacology*; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance; D006239:Haplotypes; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis / Q000188:drug therapy* / Q000469:parasitology; D026901:Membrane Transport Proteins / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D015800:Protozoan Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D000962:Antimalarials; D016054:DNA, Protozoan; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine; D053626:Atovaquone