RESUMO
Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Animais , Aotidae , Cruzamentos Genéticos , Resistência a Medicamentos , Regulação da Expressão Gênica , Mutação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
Plasmodium vivax is a major cause of malaria morbidity worldwide yet has remained genetically intractable. To stably modify this organism, we used zinc-finger nucleases (ZFNs), which take advantage of homology-directed DNA repair mechanisms at the site of nuclease action. Using ZFNs specific to the gene encoding P. vivax dihydrofolate reductase (pvdhfr), we transfected blood specimens from Saimiri boliviensis monkeys infected with the pyrimethamine (Pyr)-susceptible Chesson strain with a ZFN plasmid carrying a Pyr-resistant mutant pvdhfr sequence. We obtained Pyr-resistant parasites in vivo that carried mutant pvdhfr and additional silent mutations designed to confirm editing. These results herald the era of stable P. vivax genetic modifications.
Assuntos
Desoxirribonucleases/genética , Genoma de Protozoário , Plasmodium vivax/genética , Dedos de Zinco , Animais , Antimaláricos/farmacologia , Resistência a Medicamentos , Genes de Protozoários/genética , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Proteínas de Protozoários/genética , Pirimetamina/farmacologia , Saimiri/parasitologia , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.