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

DOI: 10.1186/1475-2875-9-134

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Abstract:

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.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.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.The molecular basis of chloroquine resistance (CQR) in Plasmodium falciparum is still relatively unclear, and the association of point mutations in different genes with CQR has been largely studied in the last decade. In 2000, the pfcrt gene was identified, which appears to play a crucial role in CQR. A lysine to threonine change at position 76 (K76T), which was subsequently found in every in vitro CQR parasite from around the world, was identified as an important mutation associated with CQR [1]. Although this mutation is not the sole requirement for determining of CQR, the absence of the K76T mutation is highly predictive of CQ sensitivity in vitro and CQ efficacy in vivo [2].Similarly, the molecular basis of atovaquone resistance in P. falciparum has been suggested to be due to mutations in the cytochrome bc1 gene of the parasite mitochondrial genome, which prevents binding of atovaquone to the cytochrome [3]. In particular, mutations at codon 268 are associated with atovaquone/proguanil treatment failure in vivo and can be used as possible resistance markers. Both the amino acid changes, Y268N and Y268S, result in extremely high increase in resistance levels [4]. There are also recent reports of Y268C mutant

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