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. 2010 May 21:9:134.
doi: 10.1186/1475-2875-9-134.

Rapid identification of chloroquine and atovaquone drug resistance in Plasmodium falciparum using high-resolution melt polymerase chain reaction

Linda Seo Hwee Gan  1 , Jin Phang Loh
Affiliations

Rapid identification of chloroquine and atovaquone drug resistance in Plasmodium falciparum using high-resolution melt polymerase chain reaction

Linda Seo Hwee Gan et al. Malar J. .

Abstract

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.

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Figures

Figure 1
Figure 1
High resolution melt profile of the 3 pfcrt haplotypes.
Figure 2
Figure 2
High resolution melt profile for the pfcrt sensitivity assay using MRA-102G.
Figure 3
Figure 3
High resolution melt profile of different proportions of CVMNK:CVIET mixes to assess the detection threshold of the minority haplotype.
Figure 4
Figure 4
High resolution melt profile of the 'unknown' haplotype with respect to the 3 common pfcrt haplotypes.
Figure 5
Figure 5
"Unknown" haplotype has a point mutation (A->G) in codon 71 just upstream of the SVMNT signature.
Figure 6
Figure 6
High resolution melt profile of the 4 haplotypes for detection of atovaquone resistance using the cytB protocol.
Figure 7
Figure 7
High resolution melt profile for the cytB sensitivity assay using MRA-102G.
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