In some regions, we are seeing increasing reports of resistance to both components (multidrug resistance) of current first-line artemisinin-based combination therapies (ACTs), resulting in an overall reduction in the efficacy of treatment and in some cases, treatment failure.1
Dr Didier Ménard and his team have developed an in vitro assay to enable in-development antimalarials to be tested against the most resistant strains of parasite we know of today.1 Building on this work the team was also able to identify a molecular marker to identify artemisinin-resistant parasites,2 which is now being used to map artemisinin resistance globally. He explains why drug resistance is such a problem, how the assay works and what it has told us so far.
1. Why is drug resistance such a problem in the treatment of malaria?
Drug resistance is a major threat to the control and elimination of malaria. The emergence and spread of chloroquineresistant parasites illustrates the issue. The first resistant parasite emerged along the Thai-Cambodia border in the 1960s and it spread to Africa in the 80s. We saw a huge increase in mortality. Drugs are the main tools we have to fight against malaria. If you use ineffective treatment, people will die.
2. How did you develop the in vitro resistance assay?
Previously, we used a classic in vitro test, where the parasite was exposed to different concentrations of the drug (0–60 nM) during its 48 h lifecycle. There was discordance between the findings of this test and those of the clinical studies. At Institut Pasteur in Cambodia, we developed a new assay emulating the conditions the parasite actually experiences in human beings. The half-life of artesunate in humans is short, and so we exposed the parasites to the concentration we’d see in patients after a single dose (700 nM) for just a short time of 6 hours.
We developed the assay, which we call the ring-stage survival assay, RSA0-3h and were really excited to see that OZ439 is active. Further work has shown just how strongly associated the results are to clinical data. We see a high survival rate of parasites from patients with a slow parasite clearance rate and vice versa. We are now able to characterize and distinguish resistant and sensitive parasites in the lab.
3. Which molecules from MMV’s portfolio have you been able to test and what have you found?
So far, we have tested OZ439, ferroquine and a couple of preclinical molecules. We have made an extensive evaluation of OZ439. To me it’s a wonderful molecule. It’s very efficient against artemisinin-resistant parasites and works quickly and efficiently. It gives us hope. It could be a very interesting alternative to artemisinin.
We have also evaluated new partner drugs, such as ferroquine. This is a good potential partner. It is totally effective against malaria; we will need to do more tests to determine its efficacy against resistant strains.
4. How was the molecular marker for artemisinin resistance identified?
Colleagues at Institut Pasteur in Paris sequenced a parasite strain that had been cultured under pressure and become resistant to artemisinin. We then compared the genomes of this strain with its parent (non-exposed to artemisinins) and found mutations in eight genes that could be involved in drug resistance.
In Cambodia, we then checked to see if these genes were also mutated in isolates from patients. We found that only mutations in one gene (Kelch gene on the chromosome 13 [named K13], which was clearly associated with artemisinin resistance) were expressed in the new in vitro assay (RSA0-3h). In Pailin, on the Thai-Cambodia border, we have seen an increase in the mutant parasite since 2002, which coincides with the increase in artemisinin resistance. While in provinces without delayed parasite clearance time, we see parasites with no mutation in the K13 gene.
It took a combination of genomic, epidemiological, clinical and biological expertise to confirm that the mutation in the K13 gene was strongly associated with artemisinin resistance.
5. What’s it been like working with MMV?
It’s very easy to work with MMV. The people are smart and we share the same goal: to get results. The lab is tough; you can’t just push a button and get results. They understand that. If we have a problem we work together to find a solution. Our job is to characterize drug resistance, raise the issue, and develop tools to detect it. With MMV, we also have the possibility to help develop a solution: next-generation antimalarials that are effective against the resistant parasites
1. Phyo AP et al. “Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study.” Lancet 379(9830): 1960-6 (2012).
2. Bruxvoort K et al. “How patients take malaria treatment: a systematic review of the literature on adherence to antimalarial drugs.” PLoS One. 9(1):e84555 (2014)