title: Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.
abstract: Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance.
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia.
authors.forename: Zuleima
authors.initials: Z
authors.lastname: Pava
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia.
authors.forename: Irene
authors.initials: I
authors.lastname: Handayuni
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia.
authors.forename: Grennady
authors.initials: G
authors.lastname: Wirjanata
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia.
authors.forename: Sheren
authors.initials: S
authors.lastname: To
authors.affiliation: Eijkman Institute for Molecular Biology, Jakarta, Indonesia.
authors.forename: Leily
authors.initials: L
authors.lastname: Trianty
authors.affiliation: Eijkman Institute for Molecular Biology, Jakarta, Indonesia.
authors.forename: Rintis
authors.initials: R
authors.lastname: Noviyanti
authors.affiliation: Papuan Health and Community Development Foundation (PHCDF), Timika, Papua, Indonesia Department of Paediatrics, Faculty of Medicine, Gadjah Mada University, Yogyakarta, Indonesia.
authors.forename: Jeanne Rini
authors.initials: JR
authors.lastname: Poespoprodjo
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia.
authors.forename: Sarah
authors.initials: S
authors.lastname: Auburn
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Ric N
authors.initials: RN
authors.lastname: Price
authors.affiliation: Global and Tropical Health Division, Menzies School of Health Research, Charles Darwin University, Darwin, Australia jutta.marfurt@menzies.edu.au.
authors.forename: Jutta
authors.initials: J
authors.lastname: Marfurt
mesh_terms: D000655:Amodiaquine / Q000494:pharmacology; D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology; D000077332:Artesunate; D002738:Chloroquine / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D004912:Erythrocytes / Q000187:drug effects / Q000469:parasitology; D005634:Fructose-Bisphosphate Aldolase / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D020128:Inhibitory Concentration 50; D008018:Life Cycle Stages / Q000187:drug effects* / Q000235:genetics; D016780:Malaria, Vivax / Q000188:drug therapy / Q000469:parasitology; D015767:Mefloquine / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D010966:Plasmodium vivax / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology; D014158:Transcription, Genetic; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D010879:Piperazines
chemical_list: D000655:Amodiaquine; D000962:Antimalarials; D037621:Artemisinins; D000077332:Artesunate; D002738:Chloroquine; D005634:Fructose-Bisphosphate Aldolase; D015767:Mefloquine; D026901:Membrane Transport Proteins; D015800:Protozoan Proteins; D011804:Quinolines; D014404:Tubulin; C541685:Crt-o protein, Plasmodium vivax; C034759:piperaquine; D010879:Piperazines