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Additional file 2. Supplementary Figures.

Additional file 1. Supplementary Protocol.

Drug resistance in Plasmodium falciparum remains a challenge for the malaria eradication programmes around the world. With the emergence of artemisinin resistance, the efficacy of the partner drugs in the artemisinin combination therapies (ACT) that include quinoline-based drugs is becoming critical. So far only few resistance markers have been identified from which only two transmembrane transporters namely PfMDR1 (an ATP-binding cassette transporter) and PfCRT (a drug-metabolite transporter) have been experimentally verified. Another P. falciparum transporter, the ATP-binding cassette containing multidrug resistance-associated protein (PfMRP2) represents an additional possible factor of drug resistance in P. falciparum. In this study, we identified a parasite clone that is derived from the 3D7 P. falciparum strain and shows increased resistance to chloroquine, mefloquine and quinine through the trophozoite and schizont stages. We demonstrate that the resistance phenotype is caused by a 4.1 kb deletion in the 5' upstream region of the pfmrp2 gene that leads to an alteration in the pfmrp2 transcription and thus increased level of PfMRP2 protein. These results also suggest the importance of putative promoter elements in regulation of gene expression during the P. falciparum intra-erythrocytic developmental cycle and the potential of genetic polymorphisms within these regions to underlie drug resistance.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.).

The main objective of this project is to explore methods of high throughput functional genomics to analyze molecular mechanisms associated with the invasion machinery by which the human malaria parasites Plasmodium falciparum invade its host erythrocytes. These mechanisms are highly unique to this organism and thus can be explored as new targets for malaria intervention strategies. For these studies we chose the approach of chemical genomics in which we analyze the genome-wide transcriptional responses of the parasite cells to small molecular inhibitors that affect the parasite invasion process. ARC 11/05

DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation of the Plasmodium falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development., Background The worldwide persistence of drug-resistant Plasmodium falciparum, the most lethal variety of human malaria, is a global health concern. The P. falciparum sequencing project has brought new opportunities for identifying molecular targets for antimalarial drug and vaccine development. Results We developed a software package, ArrayOligoSelector, to design an open reading frame (ORF)-specific DNA microarray using the publicly available P. falciparum genome sequence. Each gene was represented by one or more long 70 mer oligonucleotides selected on the basis of uniqueness within the genome, exclusion of low-complexity sequence, balanced base composition and proximity to the 3' end. A first-generation microarray representing approximately 6,000 ORFs of the P. falciparum genome was constructed. Array performance was evaluated through the use of control oligonucleotide sets with increasing levels of introduced mutations, as well as traditional northern blotting. Using this array, we extensively characterized the gene-expression profile of the intraerythrocytic trophozoite and schizont stages of P. falciparum. The results revealed extensive transcriptional regulation of genes specialized for processes specific to these two stages. Conclusions DNA microarrays based on long oligonucleotides are powerful tools for the functional annotation and exploration of the P. falciparum genome. Expression profiling of trophozoites and schizonts revealed genes associated with stage-specific processes and may serve as the basis for future drug targets and vaccine development.

A report on the Molecular Approaches to Malaria meeting, Lorne, Australia, 4-8 February 2004., A report on the Molecular Approaches to Malaria meeting, Lome, Australia, 4-8 February 2004.

Background Gene copy number variants (CNVs), which consist of deletions and amplifications of single or sets of contiguous genes, contribute to the great diversity in the Plasmodium falciparum genome. In vitro studies in the laboratory have revealed their important role in parasite fitness phenotypes such as red cell invasion, transmissibility and cytoadherence. Studies of natural parasite populations indicate that CNVs are also common in the field and thus may facilitate adaptation of the parasite to its local environment. Results In a survey of 183 fresh field isolates from three populations in Eastern Africa with different malaria transmission intensities, we identified 94 CNV loci using microarrays. All CNVs had low population frequencies (minor allele frequency 0.3) and nine exhibited significant clines in population frequency across a gradient in transmission intensity. The clearest example of this was a large deletion on chromosome 9 previously reported only in laboratory-adapted isolates. This deletion was present in 33% of isolates from a population with low and highly seasonal malaria transmission, and in

Due to their remarkable parasitocidal activity, artemisinins represent the key components of first-line therapies against Plasmodium falciparum malaria. However, the decline in efficacy of artemisinin-based drugs jeopardizes global efforts to control and ultimately eradicate the disease. To better understand the resistance phenotype, artemisinin-resistant parasite lines were derived from two clones of the 3D7 strain of P. falciparum using a selection regimen that mimics how parasites interact with the drug within patients. This long term in vitro selection induced profound stage-specific resistance to artemisinin and its relative compounds. Chemosensitivity and transcriptional profiling of artemisinin-resistant parasites indicate that enhanced adaptive responses against oxidative stress and protein damage are associated with decreased artemisinin susceptibility. This corroborates our previous findings implicating these cellular functions in artemisinin resistance in natural infections. Genomic characterization of the two derived parasite lines revealed a spectrum of sequence and copy number polymorphisms that could play a role in regulating artemisinin response, but did not include mutations in pfk13, the main marker of artemisinin resistance in Southeast Asia. Taken together, here we present a functional in vitro model of artemisinin resistance that is underlined by a new set of genetic polymorphisms as potential genetic markers.