Induction of Multidrug Tolerance in Plasmodium falciparum by Extended Artemisinin Pressure

Tolerance is not detected by current assays and represents a major threat to antimalarial drug policy.

It appears that all P. falciparum strains whatever their genetic background, enter quiescence after exposure to artemisinins, although the rate of recovery differs among susceptible strains, suggesting that artemisinin-induced dormancy is a conserved trait in Plasmodium falciparum parasites (5,6). This baseline propensity, which is also observed after pyrimethamine or mefloquine treatment and results in late recrudescence (7,8), probably accounts for the late and low-rate recrudescence of the F32-TEM line.
The situation differs markedly in artemisinin-resistant parasites of the F32-ART lineage that have acquired a 1-3 log higher recovery rate/quiescence capacity after exposure to dihydroartemisinin than susceptible strains (9,10). We reported that F32-ART3 remained quiescent after long exposure (>96 h) to high doses of artemisinin (>70 µmol/L) and quickly recovered after removal of artemisinin (1). This finding indicates a remarkably strong capacity of parasites to withstand toxicity of artemisinin, which is consistent with modifications of metabolic composition of artemisinin-resistant parasites documented by recent transcriptome studies that indicate increased expression of unfolded protein response pathways (11). Artemisinin-resistant parasites also display a decelerated progression through the first part of the asexual intraerythrocytic development cycle (11), which might favor quiescence upon exposure to artemisinin.
Similar, increased survival rates were observed for both lines (27% and 32% for amodiaquine, and 20% and 27% for atovaquone). These results are consistent with the insensitivity of ring stages to quinolines that inhibit mature stages, are inactive on young rings, and require a long incubation time (12), and with mode of action of atovaquone that targets mature stages (13).
The recently developed ring-stage survival assay monitors susceptibility to fast-acting molecules, such as artemisinin derivatives, and is sensitive for young developmental stages.
Therefore, this assay is irrelevant for long-acting molecules and those molecules that exert their inhibitory power at later developmental stages (such as amodiaquine and atovaquone).
Specific assays should be implemented to detect multidrug tolerance in the field.
Improvements to the recrudescence assays used are warranted because they are timeconsuming and labor-intensive. These assays should be conducted in addition to those currently used in field-based settings. The novel multidrug resistance phenotype should be evaluated for possible emergence in the field alongside possible selection of genuine resistance to the partner drug.  mutation of PF3D7_1302100 (Pfg27) was acquired between cycles 56 and 120; the S292T mutation of PF3D7_1459600 (unknown function) was acquired between cycles 56 and 120; and the N1629S mutation of PF3D7_1464500 (unknown function) was acquired between cycles 56 and 120 (1). As in vitro phenotyping experiments conducted on F32-ART lineage lasted several months, parasites were cultivated under regular drug pressure to ensure maintenance of the phenotypic characteristics.
However, assays were performed once a stable concentration/duration of pressure was achieved. The windows corresponding to F32-ART3 and F32-ART5 are indicated by the solid double-headed arrows.
Black stars indicate intermediate lines from the F32-ART lineage tested by using ring-stage survival assay (RSA) 0-3 h and RSA 13-16 h (Table 2).