Phenotypic and Genotypic Analysis of \u3ci\u3eIn Vitro\u3c/i\u3e Selected Artemisinin Resistant \u3ci\u3ePlasmodium falciparum\u3c/i\u3e

Artemisinin and its derivatives provide faster clearance of parasitemia than any other antimalarial drugs and these drugs are part of frontline combination therapies in areas where drug-resistant Plasmodium falciparum exists. Clinical resistance to artemisinins is emerging on the Thailand-Cambodia border, making it imperative to investigate mechanisms of artemisinin resistance. Previous work in our laboratory showed ring-stage parasites enter a dormant state after exposure to artemisinin. We hypothesize that this period of dormancy is directly related to recrudescence and prolonged parasite clearance times in patients, and possibly resistance. The target of artemisinin is currently unknown, and potential resistance mechanisms are not well described. Our laboratory previously selected artemisinin resistance in P. falciparum clones D6 (Africa), W2 (southeast Asia), and a patient isolate from Thailand, TM91c235. Studies were attempted in order to characterize artemisinin resistant phenotypes and molecular mechanisms of resistance in these lines. W2 lines resistant to 40 ng/ml artemisinin (W2.QHS40) and 80 ng/ml artelinic acid (W2.AL80) were exposed to AL and transcriptionally profiled. Analysis of results found genes that were significantly differentially expressed (such as pfmdr1, pfmdr2, PF11_0466, PFE1050w) in resistant vs. parental lines. It was hypothesized that the differential expression of genes may be due to novel single nucleotide polymorphisms (SNPs). Further studies found that the P. falciparum multidrug resistance transporter-1 gene (pfmdr1) is involved in resistance in W2 and TM91, but not D6. Resistant parasites also exhibited resistance to other artemisinin drugs than those used to originally select resistance in these strains. We expanded on earlier selection of D6, W2, and TM91 artemisinin selection to produce high level resistance to concentrations of artemisinin and artelinic acid that are considered clinically relevant. Parental and resistant parasites were characterized for differences in recovery after drug, growth rates, and in vitro susceptibility to antimalarial drugs. During the generation of D6 resistant lines, it was determined that parental D6 could tolerate up to 1500 ng/ml QHS, but D6.QHS340x3 tolerated 2400 ng/ml of artemisinin. Resistant D6 parasites recrudesced before parental strains in these assays. Recovery assays also found D6 and W2 resistant lines treated with 200 ng/ml dihydroartemisinin recrudesced before parent strains after drug treatment. In vitro susceptibility testing with various antimalarial drugs found that resistant D6, W2, and TM91c235 parasites all exhibited reduced susceptibility to artemisinin drugs compared to parental parasites, with marked resistance to QHS and AL. A novel hypoxanthine incorporation assay showed that resistant progeny and parental lines of D6 and W2 both entered dormancy following treatment with various artemisinin drugs, but resistant parasites tolerated higher drug concentrations. These results have clinical relevance with artemisinin resistance that may be occurring in patients. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly less merozoites than parent strains, whereas W2 resistant progeny had significantly more. However, this only coincided with a slower growth rate in the D6 resistant parasite, marked by a decrease in progression from ring to trophozoite. Through these methods of characterization, we defined a phenotype for artemisinin resistance and have made strides toward the relationship of dormancy, resistance, and recrudescence. We investigated potential molecular markers of artemisinin resistance using a variety of drug selected lines. Next generation approaches that included proteomics, whole genome sequencing, and microarrays allowed us to identify putative resistance mechanisms in the highest artemisinin-selected D6 and W2 lines. SNPs in D6.QHS2400x5 vs. D6 were identified and an amplification event on chromosome 10 in QHS-resistant D6 and W2 were identified. Microarray analyses found ring stage parasites of D6 and D6.QHS2400x5 were transcriptionally arrested at ring stage, but the resistant parasite exited the transcriptional arrest before the parent parasite. We also identified genes that were differentially expressed in both parent and resistant parasites during dormancy, along with genes that were constitutively expressed in resistant vs. parent strains before drug was added. Genes identified in the amplification of chromosome 10 in D6.QHS2400x5 were up-regulated in the resistant parasite before drug was added. Future studies will focus on validating transcriptional data by real-time PCR and analyzing early selected parasites to determine when markers of resistance accumulated. Our molecular analyses have identified high-probability markers of artemisinin resistance in strains from different locations in the world which may be useful in surveillance of artemisinin resistance