Drug-Resistant Genotypes and Multi-Clonality in Plasmodium falciparum Analysed by Direct Genome Sequencing from Peripheral Blood of Malaria Patients


Robinson, T; Campino, SG; Auburn, S; Assefa, SA; Polley, SD; Manske, M; MacInnis, B; Rockett, KA; Maslen, GL; Sanders, M; Quail, MA; Chiodini, PL; Kwiatkowski, DP; Clark, TG; Sutherland, CJ; (2011) Drug-Resistant Genotypes and Multi-Clonality in Plasmodium falciparum Analysed by Direct Genome Sequencing from Peripheral Blood of Malaria Patients. PLoS One, 6 (8). e23204. ISSN 1932-6203 DOI: 10.1371/journal.pone.0023204

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Abstract

: Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity.<br/>

Item Type: Article
Keywords: merozoite surface-antigen, polymerase-chain-reaction, genetic diversity, structural variation, children, transmission, complexity, pcr, polymorphisms, pyrimethamine, merozoite surface-antigenpolymerase-chain-reactiongenetic diversitystructural variationchildrentransmissioncomplexitypcrpolymorphismspyrimethamine
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Immunology and Infection
Faculty of Epidemiology and Population Health > Dept of Infectious Disease Epidemiology
Faculty of Infectious and Tropical Diseases > Dept of Pathogen Molecular Biology
Faculty of Infectious and Tropical Diseases > Dept of Clinical Research
Research Centre: Antimicrobial Resistance Centre (AMR)
Malaria Centre
PubMed ID: 21853089
Web of Science ID: 293953400032
URI: http://researchonline.lshtm.ac.uk/id/eprint/92

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