Transmissibility of clinically relevant atovaquone-resistant Plasmodium falciparum by anopheline mosquitoes

Abstract

Rising numbers of malaria cases and deaths underscore the need for new interventions. Long-acting injectable medications, such as those now in use for HIV prophylaxis, offer the prospect of a malaria "chemical vaccine", combining the efficacy of a drug (like atovaquone) with the durability of a biological vaccine. Of concern, however, is the possible selection and transmission of drug-resistant parasites. We addressed this question by generating clinically relevant, highly atovaquone-resistant, Plasmodium falciparum mutants competent to infect mosquitoes. Isogenic paired strains, that differ only by a single Y268S mutation in cytochrome b, were evaluated in parallel in southeast Asian (Anopheles stephensi) or African (Anopheles gambiae) mosquitoes, and thence in humanized mice. Fitness costs of the mutation were evident along the lifecycle, in asexual parasite growth in vitro and in a progressive loss of parasites in the mosquito. In numerous independent experiments, microscopic exam of salivary glands from hundreds of mosquitoes failed to detect even one Y268S sporozoite, a defect not rescued by coinfection with wild type parasites. Furthermore, despite uniformly successful transmission of wild type parasites from An. stephensi to FRG NOD huHep mice bearing human hepatocytes and erythrocytes, multiple attempts with Y268S-fed mosquitoes failed: there was no evidence of parasites in mouse tissues by microscopy, in vitro culture, or PCR. These studies confirm a severe-to-lethal fitness cost of clinically relevant atovaquone-resistant P. falciparum in the mosquito, and they significantly lessen the likelihood of their transmission in the field.

Keywords: Anopheles; Biological Sciences; Microbiology; Plasmodium; atovaquone; cytochrome b; malaria; resistance.

Fig. 1.

Phenotypic characterization in vitro of isogenic wild type (WT) and Pfcytb Y268S mutant P. falciparum. (A) Cumulative number of asexual parasites in continuous culture. Growth rate of wild type parasites (y = 0.35x + 9.3) was 1.4-fold greater than that of mutant (y = 0.21x + 7.9); n = 36, R² >0.99. (B) Atovaquone activity against WT or Y268S asexual erythrocytic parasites (EC₅₀ 0.68 nM or 9.0 μM, respectively). Depicted are mean ± SD of quadruplicate determinations in three independent experiments (some SD are too small to extend outside the symbols); R² ≥0.994. (C) Male gametocyte exflagellation, adjusted to 1.5% gametocytemia. Median number of centers across four independent biological replicates is indicated (ranges 4 to 28 for WT, and 0 to 10 for mutant); n, total number of fields examined; ****P <0.0001. For WT cells, all 44 microscopy fields had at least one exflagellation center (100%); Y268S evidenced exflagellation in 40 of 44 fields (91%).

Fig. 2.

Evaluation of wild type and Y268S P. falciparum in An. stephensi tissues. All feeds were adjusted to 0.5% stage V gametocytemia. (A) Oocysts on mosquito midguts were counted at 9–10 d after membrane feed. Indicated is median number of oocysts per midgut in nine biological replicate experiments (ranges 0 to 120 for WT, and 0 to 5 for Y268S); n, total number of mosquitoes dissected; pie charts, percent of mosquitoes infected; ****P <0.0001. (B, C) Photomicrographs of representative midguts infected with WT or Y268S parasites, respectively. Bars, 100 μm; arrows, representative oocysts; inset, magnification of Y268S oocyst; banded black structures are Malpighian tubules. (D) Oocyst diameters were measured in six biological replicates for wild type, and seven replicates for Y268S parasites. Median diameter is indicated (WT range 9 to 63 μm, mutant range 7 to 28 μm); n, total number of oocysts measured; ****P <0.00001. (E) Salivary glands were harvested at 17–20 d after feed in eight biological replicate experiments entailing a total of 234 WT-fed or 245 Y268S-fed mosquitoes. Symbols, for each experiment, average number of sporozoites per mosquito. No sporozoites were seen in any Y268S preparation. Bars, median (values indicated) with 95% confidence interval; ***P = 0.0002.

Fig. 3.

Analysis of huHep mouse tissues for evidence of WT or Y268S P. falciparum. (A) Giemsa-stained thin smears of peripheral blood from mice bitten 7 to 7.5 d previously by An. stephensi infected with WT parasites. Intracellular ring stage parasites are evident within the engrafted human erythrocytes, which at 6.4 μm are appreciably larger than the endogenous 4.7 μm mouse cells. Samples are from two mice in each of two biological replicate experiments; bar, 10 μm. Analogous peripheral blood from mice bitten by Y268S-infected mosquitoes evidenced no parasites in ≥16,000 RBCs examined per mouse, by at least two independent observers. (B) Indicated tissues were harvested from mice mouse bitten 7.5 d previously by mosquitoes fed on WT or Y268S 0.5% gametocyte culture; liver and spleen were obtained after exsanguination and saline perfusion. DNA was isolated and analyzed by nested PCR for nuclear DNA-encoded STEVOR sequence (188 bp product). Ladder, indicated size fragments. (C) In a second independent experiment, tissues were harvested as above and isolated DNA was analyzed by nested PCR for mitochondrial DNA-encoded Pfcytb sequence (538 bp product). Ladder, indicated size markers; pos cntl, template DNA isolated from cognate asexual parasite culture; neg cntl, no template added reaction.