Breakthrough in understanding artemisinin resistance
Although ACTs are the recommended first line treatment for uncomplicated malaria, there is still no consensus on the mode of artemisinin action, or on the mechanism underlying the observed artemisinin resistance phenotype in Southeast Asia. In a newly-published study led by Dr Didier Menard and involving colleagues in Cambodia, Thailand, France and the World Health Organisation, the clinically-observed slow parasite clearance phenotype of artemisinin resistant parasites is seen to correlate with the increased in vitro survival of culture-adapted parasites in a Ring stage Survival Assay (RSA). This is consistent with the observation by Anderson et al, 2010 that slow parasite clearance is a heritable trait.
The collaborators culture-adapted isolates from Pailin and Ratanakiri – artemisinin resistant and susceptible areas in Cambodia, respectively. “We were unable to see any significant difference in the in vitro dihydroartemisinin (DHA) responses of the two sets of isolates using the classical 3H-hypoxanthine uptake inhibition assay, explained Dr Benoit Witkowski. “However, we did see a clear geographic clustering of survival response when isolates were exposed to a six-hour pulse of 700 nM DHA during the ring stage of their development.”
Although mature stage parasites from Pailin and Ratanakiri were equally susceptible to the six-hour DHA pulse, the susceptibility of ring stage parasites differed markedly in the Ring Survival Assay. Pailin parasite lines consistently displayed much higher survival 72 hours after the DHA pulse than parasites from Ratanakiri. Compared to parasites with low survival in the RSA, parasite lines displaying a high percentage of viable parasites showed a reduced initial slope of decaying parasitaemia over a six-day follow-up period and a quicker recovery of initial parasitaemia after drug removal.
“Our observation that mature stages of artemisinin-resistant parasites from Pailin remain fully susceptible to DHA toxicity explains why these parasites score as susceptible in the standard in vitro test, which monitors DNA synthesis and maturation,” commented Dr Odile Mercereau-Puijalon. “We conclude that artemisinin resistance reported in Pailin is associated with an increased number of rings capable of entering transient developmental arrest and subsequent exit.”
The in vitro phenotype of the Pailin lines in the RSA differs from the drug resistance phenotype that has been observed for antimalarials like chloroquine or pyrimethamine. In those assays, a higher fraction of the parasites survive exposure to the drug challenge and mature as normal. Recent mathematical modelling (Saralamba et al, 2010) also supports the conclusion of this study that DHA is active on mature stages in both in Pailin and Ratanakiri but has reduced efficacy on ring stages in Pailin.
Looking to the future, Dr Ménard commented, “We have developed a simple in vitro assay that clearly distinguishes between artemisinin resistant and susceptible laboratory lines and which is much quicker than a recovery or recrudescence assay. Although microscopy used to monitor response to DHA treatment is cheap, it is also time consuming, difficult to monitor and unsuitable for large-scale epidemiological studies. We look to the wider community to help us develop or adapt other methods, such as FACS analysis using specific viability markers, to follow the DHA susceptibility of ring stages in field-based conditions.”