Dataset Information

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity.

ABSTRACT: 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. Nested PCR is used to distinguish these two possibilities and the technique is difficult to standardise. There is risk of both false positive and false negative results, leading to misclassification errors. 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. In this study, the HRM dissociation profiles of msp1 and msp2 amplicons were determined and validated against parasite isolates from malaria patients. The msp1 and msp2 profiles of both laboratory and clinical isolates were reproducibly differentiated by HRM. These rapid assays are performed in a closed-tube system, and so avoid cross-contamination while increasing throughput, which are two major advantages. 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. Assay performance in large-scale studies utilizing DNA samples derived from filter-paper bloodspots should now be evaluated.

SUBMITTER: Beshir KB

PROVIDER: S-EPMC6031682 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity.

Beshir Khalid B KB Diallo Nouhoum N Sutherland Colin J CJ

Scientific reports 20180704 1

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. Nested PCR is used to distinguish these two possibilities and the technique is difficult to standardise. There is risk of both false positive and false negative results, leading to misclassification errors. The high-resolution melt (HRM) assay was developed with pairs of co ...[more]

PMID: 29973679

Similar Datasets

Project description:BackgroundChloroquine (CQ) resistance is conferred by mutations in the Plasmodium falciparum CQ resistance transporter (pfcrt). Following CQ withdrawal for anti-malarial treatment, studies across malaria-endemic countries have shown a range of responses. In some areas, CQ sensitive parasites re-emerge, and in others, mutant haplotypes persist. Active surveillance of resistance mutations in clinical parasites is essential to inform treatment regimens; this effort requires fast, reliable, and cost-effective methods that work on a variety of sample types with reagents accessible in malaria-endemic countries.MethodsQuantitative PCR followed by High-Resolution Melt (HRM) analysis was performed in a field setting to assess pfcrt mutations in two groups of clinical samples from Southwestern Uganda. Group 1 samples (119 in total) were collected in 2010 as predominantly Giemsa-stained slides; Group 2 samples (125 in total) were collected in 2015 as blood spots on filter paper. The Rotor-Gene Q instrument was utilized to assess the impact of different PCR-HRM reagent mixes and the detection of mixed haplotypes present in the clinical samples. Finally, the prevalence of the wild type (CVMNK) and resistant pfcrt haplotypes (CVIET and SVMNT) was evaluated in this understudied Southwestern region of Uganda.ResultsThe sample source (i.e. Giemsa-stained slides or blood spots) and type of LCGreen-based reagent mixes did not impact the success of PCR-HRM. The detection limit of 10-?5 ng and the ability to identify mixed haplotypes as low as 10?% was similar to other HRM platforms. The CVIET haplotype predominated in the clinical samples (66?%, 162/244); however, there was a large regional variation between the sample groups (94?% CVIET in Group 1 and 44?% CVIET in Group 2).ConclusionsThe HRM-based method exhibits the flexibility required to conduct reliable assessment of resistance alleles from various sample types generated during the clinical management of malaria. Large regional variations in CQ resistance haplotypes across Southwestern Uganda emphasizes the need for continued local parasite genotype assessment to inform anti-malarial treatment policies.

Dataset Information

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity.

Publications

Identifying Recrudescent Plasmodium falciparum in Treated Malaria Patients by Real-time PCR and High Resolution Melt Analysis of Genetic Diversity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure