Maria Isabel Veiga

In Vivo Selection of Plasmodium falciparum Parasites Carrying the Chloroquine-Susceptible pfcrt K76 Allele after Treatment with Artemether-Lumefantrine in Africa

BACKGROUND Artemether-lumefantrine (AL) is a major and highly effective artemisinin-based combination therapy that is becoming increasingly important as a new first-line therapy against Plasmodium falciparum malaria. However, recrudescences occurring after AL treatment have been reported. Identification of drug-specific parasite determinants that contribute to treatment failures will provide important tools for the detection and surveillance of AL resistance. METHODS The findings from a 42-day follow-up efficacy trial in Tanzania that compared AL with sulfadoxine-pyrimethamine (SP) were analyzed to identify candidate markers for lumefantrine tolerance/resistance in the chloroquine resistance transporter gene (pfcrt) and multidrug resistance gene 1 (pfmdr1). The findings were corroborated in vitro with genetically modified isogenic P. falciparum parasite lines. RESULTS Treatment with AL selected for the chloroquine-susceptible pfcrt K76 allele (P < .0001) and, to a lesser extent, the pfmdr1 N86 (P = .048) allele among recurrent infections. These genotypes were not selected during SP treatment. No pfmdr1 gene amplifications were observed. Isogenic pfcrt-modified parasite lines demonstrated a 2-fold increase in susceptibility to lumefantrine, which was directly attributable to the K76T mutation. CONCLUSIONS Our findings suggest that the pfcrt K76T mutation is a drug-specific contributor to enhanced P. falciparum susceptibility to lumefantrine in vivo and in vitro, and they highlight the benefit of using AL in areas affected by chloroquine-resistant P. falciparum malaria.

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance.

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.

Diversity of the sarco/endoplasmic reticulum Ca2+-ATPase orthologue of Plasmodium falciparum (PfATP6)

The sarco/endoplasmic reticulum Ca(2+)-ATPase orthologue of Plasmodium falciparum (PfATP6) has been suggested to be involved in the mechanism of action and resistance to artemisinins, the main constituent of artemisinin-based combination therapy (ACT). In previous studies only six single-nucleotide polymorphisms (SNPs) have been described in clinical samples and field isolates. Our aim was to sequence a large number of clinical samples with different geographical origins to further explore the natural diversity of PfATP6. We sequenced three genetic regions of PfATP6 in 388 samples from 17 countries, mainly Zanzibar and Tanzania, and identified 33 SNPs, of which 29 were non-synonymous and 4 synonymous. To our knowledge 29 of these SNPs have not been described previously. Three mutations were found in high frequency in Zanzibar and Tanzania; E431K, N569K and A630S were present in respectively 31% (95% CI, 26-37%), 36% (95% CI, 30-42%), and 2% (95% CI, 1-5%) of Zanzibar samples and in 39% (95% CI, 29-51%), 29% (95% CI, 16-45%) and 7% (95% CI, 1-22%) of the Tanzania Mainland samples. No variation was found in position 263, suggested to be involved in artemisinin binding to PfATP6, or in position 769, proposed to be related to decreased sensitivity to artemether in vitro. A considerable difference in diversity was observed between the three genetic regions. In conclusion our findings show that PfATP6 is a more diverse gene than previously demonstrated. This natural variation may constitute a starting ground for artemisinin-driven progressive selection of resistant parasites.

PfMDR1: mechanisms of transport modulation by functional polymorphisms.

ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.

Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America

BackgroundIn Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras.MethodsBlood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods.ResultsThirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples.ConclusionThe results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence of drug resistant/tolerant P. falciparum and P. vivax is therefore essential also in Honduras.

Prevalence of resistance associated polymorphisms in Plasmodium falciparum field isolates from southern Pakistan

BackgroundScarce data are available on Plasmodium falciparum anti-malarial drug resistance in Pakistan. The aim of this study was, therefore, to determine the prevalence of P. falciparum resistance associated polymorphisms in field isolates from southern Pakistan.MethodsBlood samples from 244 patients with blood-slide confirmed P. falciparum mono-infections were collected between 2005-2007. Single nucleotide polymorphisms in the P. falciparum chloroquine resistance transporter (pfcrt K76T), multi drug resistance (pfmdr1 N86Y), dihydrofolate reductase (pfdhfr A16V, N51I, C59R, S108N, I164L) and dihydropteroate synthetase (pfdhps A436S, G437A and E540K) genes and pfmdr1 gene copy numbers were determined using PCR based methods.ResultsThe prevalence of pfcrt 76T and pfmdr1 86Y was 93% and 57%, respectively. The prevalence of pfdhfr double mutations 59R + 108N/51R + 108N was 92%. The pfdhfr triple mutation (51I, 59R, 108N) occurred in 3% of samples. The pfdhfr (51I, 59R, 108N) and pfdhps (437G, 540E) quintuple mutation was found in one isolate. Pfdhps 437G was observed in 51% and 540E in 1% of the isolates. One isolate had two pfmdr1 copies and carried the pfmdr1 86Y and pfcrt 76T alleles.ConclusionsThe results indicate high prevalence of in vivo resistance to chloroquine, whereas high grade resistance to sulphadoxine-pyrimethamine does not appear to be widespread among P. falciparum in southern Pakistan.

Antimalarial exposure delays Plasmodium falciparum intra-erythrocytic cycle and drives drug transporter genes expression.

Background Multi-drug resistant Plasmodium falciparum is a major obstacle to malaria control and is emerging as a complex phenomenon. Mechanisms of drug evasion based on the intracellular extrusion of the drug and/or modification of target proteins have been described. However, cellular mechanisms related with metabolic activity have also been seen in eukaryotic systems, e.g. cancer cells. Recent observations suggest that such mechanism may occur in P. falciparum. Methodology/Principal Findings We therefore investigated the effect of mefloquine exposure on the cell cycle of three P. falciparum clones (3D7, FCB, W2) with different drug susceptibilities, while investigating in parallel the expression of four genes coding for confirmed and putative drug transporters (pfcrt, pfmdr1, pfmrp1 and pfmrp2). Mefloquine induced a previously not described dose and clone dependent delay in the intra-erythrocytic cycle of the parasite. Drug impact on cell cycle progression and gene expression was then merged using a non-linear regression model to determine specific drug driven expression. This revealed a mild, but significant, mefloquine driven gene induction up to 1.5 fold. Conclusions/Significance Both cell cycle delay and induced gene expression represent potentially important mechanisms for parasites to escape the effect of the antimalarial drug.

Polymorphism of antimalaria drug metabolizing, nuclear receptor, and drug transport genes among malaria patients in Zanzibar, East Africa.

Artemisinin-based combination therapy is a main strategy for malaria control in Africa. Zanzibar introduced this new treatment policy in 2003. The authors have studied the prevalence of a number of functional single nucleotide polymorphisms (SNPs) in genes associated with the elimination of the artemisinin-based combination therapy compounds in use in Zanzibar to investigate the frequencies of subgroups potentially at higher drug exposure and therefore possible higher risk of toxicity. One hundred three unrelated children with uncomplicated malaria from the Unguja and Pemba islands of Zanzibar were enrolled. With use of polymerase chain reaction (PCR)-restriction fragment length polymorphism and real-time PCR-based allele discrimination methods, the CYP2B6 (G15631T), CYP3A4 (A-392G), CYP3A5 (A6986G, G14690A, 27131-132 insT, C3699T) SNPs and MDR1 SNPs C3435T, G2677T/A, and T-129C were analyzed. PCR product sequencing was applied to regulatory regions of MDR1, the CYP3A4 proximal promoter, and to exons 2 and 5 of PXR, a gene coding for a nuclear factor activated by artemisinin antimalarials and associated with the transcription induction of most of the studied genes. Homozygous subjects for alleles coding for low activity proteins were found at the following frequencies: 1) MDR1: 2.9%; 2) CYP2B6: 9.7%; 3) CYP3A5: 14.1%; and 4) CYP3A4: 49.5%. No functionally relevant allele was found in the analyzed regions of PXR. A new MDR1 SNP was found (T-158C), located in a putative antigen recognition element. Ten (10.1%) subjects were predicted to be low metabolizers simultaneously for CYP3A4 and CYP3A5. This fraction of the population is suggested to be under higher exposure to certain antimalarials, including lumefantrine and quinine.

pfmdr1 Amplification Is Related to Increased Plasmodium falciparum In Vitro Sensitivity to the Bisquinoline Piperaquine

ABSTRACT The 4-aminoquinoline bisquinoline piperaquine is an important partner drug in one of the presently recommended artemisinin combination therapies. Recent clinical trials have confirmed its high efficacy in combination with dihydroartemisinin. Resistance to piperaquine alone has, however, been documented. Amplification in copy number of the Plasmodium falciparum multidrug resistance locus on chromosome 5, containing the pfmdr1 gene, has been shown to confer resistance to structurally unrelated antimalarials. Through the determination of the 50% inhibitory concentrations (IC50s) and IC90s for piperaquine and chloroquine in a set of 46 adapted P. falciparum cultures originating from the Thai-Burmese border, we have characterized the regions around the pfmdr1 gene and identified a significant association between the presence of pfmdr1 duplications and enhanced sensitivity to piperaquine (P = 0.005 for IC50 and P = 0.002 for IC90) and chloroquine, reaching statistical significance at IC90s (P = 0.026). These results substantiate the potential importance of pfmdr1 copy number amplifications in the efficacy of the combination therapy piperaquine-dihydroartemisinin. It supports the rational use of 4-aminoquinolines and artemisinin-based compounds, as they independently select for mutually incompatible combinations of mutations.

Complex Polymorphisms in the Plasmodium falciparum Multidrug Resistance Protein 2 Gene and Its Contribution to Antimalarial Response

ABSTRACT Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasites in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.