Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model

Abstract

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t_1/2s (>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, t_1/2 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 (76H10^C580Rev) 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.

Keywords: artemisinin-based combination chemotherapy; genetic cross; malaria; parasite clearance time; recrudescence.

Fig. 1.

Design of the P. falciparum 803×GB4 cross and in vitro drug responses. (A) Mixed gametocytes of the parents were fed to Anopheles mosquitoes, and resulting sporozoites were inoculated into three Sp− NHPs: A. nancymaae (night or owl monkey), P. troglodytes (chimpanzee), and S. boliviensis (squirrel monkey). Blood-stage recombinant progeny were recovered only from the chimpanzee; after cultivation in human erythrocytes, some progeny were able to infect Aotus. (B) DHA IC₅₀ values from 803 and GB4 parents (gray), 20 progeny (black) including two pairs of isogenic clones (curly braces), a GB4 self-mating product 34F5, and the engineered 76H10^C580Rev revertant (checkerboard pattern). K13 types are indicated. (C) RSA results from 803×GB4 parasites and the engineered 76H10^C580Rev revertant. Details of the data are listed in SI Appendix, Table S1.

Fig. 2.

AS responses of 803×GB4 parasites in Aotus. Information in these panels corresponds to entries in Table 1 except for the data from FVO infections. (A–C) Primary and recrudescent parasitemias in Aotus monkeys receiving AS treatment: 87E7, 76H10, and 85G7 infections (K13 C580Y-type); GB4 and 61D3 infections (K13 C580-type); and 76H10^C580Rev revertant infections (K13 C580-type). Numbers after parasite names identify individual Aotus. AS1, AS treatments of primary infections; AS2–AS14, treatments of recrudescences after AS1; CS, cleared to subpatent levels without treatment. “T2” identifies treatment of a 61D3 recrudescence with a different antimalarial regimen; “MEF” indicates administration for cure as a single 25-mg/kg oral dose. (D) Ring-stage parasite clearance curves from AS-treated Aotus infections presented as percentage of initial count. C580Y-type primary clearance profiles are shown in red, and C580-type primary and recrudescence clearances are in gray and blue, respectively. Few trophozoite and schizont stages were observed in the circulation, and clearance results were similar when these were included with the ring-stage counts for analysis (total parasitemia; Dataset S3). (E) Plot of ring-stage parasite clearance t_1/2 values obtained by the WWARN (“W”), joinpoint (“J”), and AHL95 (“A”) methods for 803×GB4 parasites carrying the C580Y (red) or C580 polymorphism (gray, primary clearances; blue, recrudescence clearances). Dotted line represents the 5-h t_1/2 threshold proposed by WHO (6).