Abstract
The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC50 (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1SLM, obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC50 of the parental strain. However, after two weeks of culturing V1SLM in drug-free medium, the IC50 returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1SLM. Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance – they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.
Highlights
Chemotherapy is a key strategy in the control of malaria
The current WHO recommendation is that different antimalarials be employed in combinations that include an Artemisinin derivative; this strategy is known as Artemisinin Combination Therapy (ACT)
In South East Asian laboratory and field isolates, pfmdr1 amplification to up to 5 copies is associated with decreased susceptibility to MFQ and LM [13,14,15,16,17,18,19]; on the other hand LM resistance in African isolates has been linked with CQ-sensitive PfMDR1 alleles (86N alone, or together with 184F, 1246D or 76K) [20,21,22], with no apparent pfmdr1 copy number changes
Summary
Chemotherapy is a key strategy in the control of malaria. To delay drug resistance, the current WHO recommendation is that different antimalarials be employed in combinations that include an Artemisinin derivative; this strategy is known as Artemisinin Combination Therapy (ACT). The exact mechanisms of drug action and development of resistance to this group of antimalarials remain poorly understood, and are sometimes contradictory. Both chloroquine (CQ) and the quinoline-methanol MFQ target the parasite food vacuole, in vitro selection of MFQ resistance has been shown to result in an increase in pfmdr copy number; at the same time an increase in CQ sensitivity was observed [10,11,12]. In South East Asian laboratory and field isolates, pfmdr amplification to up to 5 copies is associated with decreased susceptibility to MFQ and LM [13,14,15,16,17,18,19]; on the other hand LM resistance in African isolates has been linked with CQ-sensitive PfMDR1 alleles (86N alone, or together with 184F, 1246D or 76K) [20,21,22], with no apparent pfmdr copy number changes
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