Artemisinin-resistant Plasmodium falciparum parasites exhibit altered patterns of development in infected erythrocytes

Abstract

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance.

FIG 1

Reduced susceptibility of artemisinin-resistant P. falciparum in a modified [³H]hypoxanthine assay (T₀ assay). (A) Fifty percent inhibitory concentrations (IC₅₀s) of isolates and clones from Cambodia and Thailand to artemisinin, artesunate, artelinic acid, and mefloquine were determined in the T₀ assay, in which the label was added at the same time as drug. The largest shift in dose response was to artelinic acid and artemisinin. (B) Clones from isolates exhibited a range of responses to artemisinin drugs. For example, several clones of ARC08-88 (11H, 10G, and 9D) exhibited higher tolerance to artemisinin than the parent isolates. In addition, sensitive clones (5C and 7E) were isolated. Following initial drug susceptibility testing of clones, at least three clones from each isolate were selected for more detailed evaluation and repeat drug susceptibility testing (see Fig. S3 to S5 in the supplemental material). (C) Drug resistance phenotypes in the T₀ assay were stable over 1 year of continuous culture, including several rounds of freeze and thaw. The results shown are averages from three independent replicates conducted at the start of culture and after a year in (mostly) continuous culture with multiple cryopreservation and thaw cycles. ARC08-88 clones are listed in descending order of IC₅₀ to artemisinin for each graph.

FIG 2

Delayed parasite clearance of P. falciparum clones following dihydroartemisinin (DHA) exposure. Three clones per isolate plus the control W2 clone were tested in duplicate. Highly synchronous ring-stage parasites were exposed to three 6-h pulses of 700 nM DHA (indicated in gray) in the delayed clearance assay (DCA). Delayed parasite clearance of morphologically normal rings, trophozoites, or schizonts was observed for all artemisinin-resistant clones compared to artemisinin-susceptible W2. In contrast, parasite clearance of W2 was observed by hour 18 of the experiment, whereas resistant clones took as long as 48 h to clear parasites. Dead and dormant parasites were excluded from this analysis.

FIG 3

Parasite development was monitored following exposure to DHA. Parasites (n = 100) were categorized as ring, trophozoite/schizont, or dormant/dead for each time point. In the delayed parasite clearance assay (DCA), P. falciparum clones were exposed to three 6-h pulses of 700 nM DHA. (A, C, E, and G) Only ring-stage parasites were observed in W2, whereas mature-stage parasites were observed in artemisinin-resistant clones following exposure to DHA. DCA results for additional clones are shown in Fig. S1 in the supplemental material. (B, D, F, and G) In similar studies, the clones were exposed to one 6-h pulse of 30 nM DHA (10× IC₅₀ for W2) and monitored for parasite development. W2 progressed to mature stages, yet after one asexual cycle all parasites remaining were either dead or in a ring-arrested dormant state. In contrast, the artemisinin clones progressed through two cycles at an accelerated rate, especially compared to 4G in the absence of drug (see Fig. 5).

FIG 4

Correlation of in vitro artemisinin resistance phenotypes with in vivo parasite clearance half-lives and mutations in the Kelch (K13) propeller domains of Pf3D7_1343700. (A) The results from the delayed clearance assay (DCA) were used to determine parasite reduction ratios (PRR) during the first 12 h following exposure to 700 nM DHA. There was a significant correlation between the in vitro phenotype (PRR_{1–12 h}) and in vivo parasite clearance half-life. (B) The delayed clearance phenotype in vitro also was positively correlated with mutations in Pf3D7_1343700 that were previously shown to be markers for artemisinin resistance (TF, treatment failure). (C) Although mefloquine-resistant clones were prevalent (see Fig. 1), there was an inverse correlation between mefloquine resistance and the delayed clearance phenotype in vitro (PRR_{1–12 h}). Color symbols in each panel correspond to the absence or presence of Pf3D7_1343700 (K13) mutations as annotated in panel B.

FIG 5

Altered patterns of intraerythrocytic development of artemisinin-resistant clones in the absence of drug pressure. Tightly synchronized parasites were prepared for artemisinin-resistant clones, and the timing of asexual stage development was compared with that of the control clone W2. (A) ARC08-22 (4G) exhibited a prolonged ring-phase phenotype with rings observed up to 30 h of development. (B) The trophozoite stages of 4G were temporally compressed, and the schizogony of 4G was completed simultaneously with that of W2. (C) Microscopic examination of Giemsa-stained blood smears revealed the rings of 4G were smaller and persisted ∼14 h longer than those of W2. This prolonged ring phase then was followed by rapid progression through the trophozoite stage. Although most artemisinin-resistant clones expressed the prolonged ring-phase phenotype, one clone (5C) from PL08-009 completed it full asexual life cycle in only 36 h.

FIG 6

Rapid progression through trophozoite stage in artemisinin-resistant P. falciparum. The unusually rapid progression of artemisinin-resistant clones through the trophozoite stage was examined by quantifying the percentage of rings, trophozoites, and schizonts present throughout the asexual life cycle. The trophozoite prevalence data from two independent experiments then were fit to a polynomial function and, for comparison purposes, were plotted with time at 0 h as the peak of trophozoite prevalence. (A and B) Both of the artemisinin-resistant clones obviously progressed through the trophozoite stage at an accelerated rate compared to sensitive parasites (W2 and CB132). (C) Trophozoite prevalence data then were used to estimate the AUC_{(Troph h)}; both artemisinin-resistant clones had similar AUCs and spent approximately half the time that W2 and CB132 did in the trophozoite stage per asexual life cycle. CB132 is an artemisinin-sensitive isolate from Pailin, Cambodia, that was adapted to culture in 1992 (21).

FIG 7

Stage specificity of artemisinin resistance in P. falciparum. The relative susceptibility of each stage of asexual parasite development was assessed by exposing tightly synchronous parasites to 3-h pulses of 700 nM DHA at 3-h increments throughout one cycle (duplicate experiments). Resistance then was assessed by parasite viability of each stage of development, with viability defined as the ability to grow logarithmically within 14 days of drug exposure. Initially there were no differences in viability of 4G and CB132, and a brief window of hypersusceptibility was observed for both clones. In the presence of drug, the ring-to-trophozoite transition for 4G was accelerated, and in this brief trophozoite window both clones were equally susceptible. Resistance to artemisinin was observed in both ring stages and throughout schizogony in the artemisinin-resistant 4G clone, and this was expressed as an ∼1 log increase in viability.

FIG 8

Recrudescence from DHA-induced ring-stage dormancy in P. falciparum. Three 6-h pulses of 700 nM DHA induced dormant ring stages in both susceptible (W2) and artemisinin-resistant clones (also see Fig. S3 to S5 in the supplemental material). There was a divergent pattern of recrudescence observed, with some clones recovering up to 2 days prior to W2, whereas other resistant clones remained dormant and only recovered later than 20 days (n = 2; bars represent ±1 SD).