In this genotyping method, the genes that code the surface antigen loci of merozoite surface protein 1 and 2 ( msp1, msp2) and glutamate rich protein ( glurp) are amplified using sequence specific primers in a nested-PCR 6. One tool commonly used to distinguish between newly emergent and recrudescent parasites is conventional nested-PCR and gel electrophoresis detection. This requires the identification of clones in paired samples, before and after treatment, and determining whether they are the same clones 5. In addition, ex vivo and in vitro studies to identify drug resistant parasites in complex infections also need to identify the constituent parasite clones in an infection or isolate in order to accurately capture the emergence of minor clones in the subsequent growth of parasites 2, 3, as is also seen in vivo 4. A PCR-correction method is generally deployed to distinguish between the two. Further, to accurately estimate true drug efficacy in clinical trials of antimalarial drugs, recurrent infections seen after drug treatment need to be identified either as new infections arising from the liver or as recrudescent parasites persisting from the original infection. Efforts to control and eliminate malaria have been hampered by the emergence of drug resistant parasites 1, but our ability to track and control resistance is also hampered by the high genetic diversity of P. Plasmodium falciparum is the most virulent of the six Plasmodium species that cause malaria in humans, being responsible for high mortality and morbidity, particularly in Africa.
Assay performance in large-scale studies utilizing DNA samples derived from filter-paper bloodspots should now be evaluated. The HRM assays offer significant gains in simplicity, speed and interpretation of results, and reduced analysis cost, for studies that require discrimination of parasite clones. These rapid assays are performed in a closed-tube system, and so avoid cross-contamination while increasing throughput, which are two major advantages. The msp1 and msp2 profiles of both laboratory and clinical isolates were reproducibly differentiated by HRM. In this study, the HRM dissociation profiles of msp1 and msp2 amplicons were determined and validated against parasite isolates from malaria patients. The high-resolution melt (HRM) assay was developed with pairs of conserved primers targeting blocks of merozoite surface protein 1 and 2 ( msp1 and msp2) genes, and polymorphisms were compared using sequence-confirmed Plasmodium falciparum DNA samples from laboratory isolates. There is risk of both false positive and false negative results, leading to misclassification errors. Nested PCR is used to distinguish these two possibilities and the technique is difficult to standardise. Recurrent parasitaemia during follow up of clinical trials of antimalarial drug efficacy results from either recrudescence of parasites surviving treatment or from parasites newly emerging from the hepatic stage of infection.
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