The online version of this article (doi:10.1186/1475-2875-11-131) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
DRP, RL and KK derived the dataset. AV and RL undertook the modelling studies. HMS undertook statistical analyses and helped daft the text. SK conceived the study and drafted the text. All authors read and approved the final manuscript.
Monitoring resistance phenotypes for Plasmodium falciparum, using in vitro growth assays, and relating findings to parasite genotype has proved particularly challenging for the study of resistance to artemisinins.
Plasmodium falciparum isolates cultured from 28 returning travellers diagnosed with malaria were assessed for sensitivity to artemisinin, artemether, dihydroartemisinin and artesunate and findings related to mutations in pfatp6 and pfmdr1.
Resistance to artemether in vitro was significantly associated with a pfatp6 haplotype encoding two amino acid substitutions (pfatp6 A623E and S769N; (mean IC50 (95% CI) values of 8.2 (5.7 – 10.7) for A623/S769 versus 623E/769 N 13.5 (9.8 – 17.3) nM with a mean increase of 65%; p = 0.012). Increased copy number of pfmdr1 was not itself associated with increased IC50 values for artemether, but when interactions between the pfatp6 haplotype and increased copy number of pfmdr1 were examined together, a highly significant association was noted with IC50 values for artemether (mean IC50 (95% CI) values of 8.7 (5.9 – 11.6) versus 16.3 (10.7 – 21.8) nM with a mean increase of 87%; p = 0.0068). Previously described SNPs in pfmdr1 are also associated with differences in sensitivity to some artemisinins.
These findings were further explored in molecular modelling experiments that suggest mutations in pfatp6 are unlikely to affect differential binding of artemisinins at their proposed site, whereas there may be differences in such binding associated with mutations in pfmdr1. Implications for a hypothesis that artemisinin resistance may be exacerbated by interactions between PfATP6 and PfMDR1 and for epidemiological studies to monitor emerging resistance are discussed.
Additional file 1: Pyrosequencing primers (DOC 40 KB)12936_2012_2142_MOESM1_ESM.doc
Additional file 2: Patient information, demographic details, symptoms, parasitemia, results of individual drug sensitivity assays and mutations determined for 28 P. falciparum isolates collected from returning travellers. Excel file (XLS 58 KB)12936_2012_2142_MOESM2_ESM.xls
Additional file 3: Mean differences and 95% CI of mean differences between comparisons of IC50 values that are significantly associated with particular parasite genotypes (see Results and Additional files 4 and 5) (DOCX 87 KB)12936_2012_2142_MOESM3_ESM.docx
Additional file 4: Association of pfmdr1 haplotypes, Y184F (A), N86Y (B) and copy number (CN1/2; C) and IC50 values for artemisinin (ART; circles), artesunate (AS; squares), DHA (triangles) and artemether (AM; diamonds). Mean individual IC50 values are shown for non-mutant (open symbols) and mutant (closed symbols) containing parasite isolates. The horizontal lines illustrate the mean IC50 values for each group. *, p < 0.05 (DOCX 143 KB)12936_2012_2142_MOESM4_ESM.docx
Additional file 5: Association of (A) pfatp6 haplotype at 623/769 versus pfmdr1 copy number (CN), (B) pfatp6 haplotype at 623/769 versus pfmdr1haplotype at 86 and (C) pfatp6 haplotype at 623/769 versus pfmdr1 haplotype at 86 and IC50 values for artemether (ART), artesunate (AS), DHA and artemether (AM). Mean individual IC50 values are shown for non-mutant (open circles) and mutant parasite isolates containing either single mutation (closed squares and triangles) or both (closed diamonds). The horizontal lines illustrate the mean IC50 values for each group. **, p < 0.01; *, p < 0.05 (DOC 1 MB)12936_2012_2142_MOESM5_ESM.doc
Additional file 6: Sequence alignments of PfATP6 with the human, rabbit, and schistosomal homologues. The two positions at which SNPs were observed (623 and 769) are highlighted with red-filled circles and the artemether binding regions are in grey boxes (DOC 294 KB)12936_2012_2142_MOESM6_ESM.doc
Additional file 7: Multiple sequence alignments of the ATP N-domain of PfATP6 with its homologues.The top part of the alignment presents the conservation level along the PfATP6 sequence observed in PfATP6 variants. The bottom part of the alignment presents the conservation level along the PfATP6 sequence observed with its homologues. The higher the column, the more conserved a residue is in the corresponding position. Columns are coloured according to the residue’s chemical properties: cysteines in yellow, aliphatic hydrophobics (V, L, I, M) in green, aromatic amino acids (Y, F, W) in dark green, small amino acids (G, A, S, T) in light grey, negatively charged (D, E) in blue, polar (N, Q) in magenta, histidines in orange, positively charged (K, L) in red, prolines in grey. The black bar in the middle shows the column score between the query sequence and the homologous amino acid distributions with |, +, ., -, = indicating scores from 'very good' to 'very bad'. The ATP binding residue positions are indicated with red arrows and positions 623 and 769 with black arrows. The insertions unique to P. falciparum can be easily recognized where the alignment lacks the bottom part and the black bar (the homologue information (DOC 366 KB)12936_2012_2142_MOESM7_ESM.doc
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Jambou R, Legrand E, Niang M, Khim N, Lim P, Volney B, Ekala MT, Bouchier C, Esterre P, Fandeur T, Mercereau-Puijalon O: Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet. 2005, 366: 1960-1963. 10.1016/S0140-6736(05)67787-2. CrossRefPubMed
Jambou R, Martinelli A, Pinto J, Gribaldo S, Legrand E, Niang M, Kim N, Pharath L, Volnay B, Ekala MT, Bouchier C, Fandeur T, Berzosa P, Benito A, Ferreira ID, Ferreira C, Vieira PP, Alecrim MG, Mercereau-Puijalon O, Cravo P: Geographic structuring of the Plasmodium falciparum sarco(endo)plasmic reticulum Ca2+ ATPase (PfSERCA) gene diversity. PLoS One. 2010, 5: e9424-10.1371/journal.pone.0009424. PubMedCentralCrossRefPubMed
Tanabe K, Zakeri S, Palacpac NM, Afsharpad M, Randrianarivelojosia M, Kaneko A, Marma AS, Horii T, Mita T: Spontaneous mutations in the Plasmodium falciparum sarco/endoplasmic reticulum Ca2+-ATPase (PfATP6) gene among wide geographical parasite populations unexposed to artemisinin-based combination therapies. Antimicrob Agents Chemother. 2010, 55: 94-100. PubMedCentralCrossRefPubMed
Karunajeewa HA: Artemisinins: artemisinin, dihydroartemisinin, artemether, arteether and artesunate. Treatment and prevention of malaria: antimalarial drug chemistry, action and use. Edited by: Staines HM, Krishna S. 2012, Springer, Basel, 157-190.
Nelson AL, Purfield A, McDaniel P, Uthaimongkol N, Buathong N, Sriwichai S, Miller RS, Wongsrichanalai C, Meshnick SR: pfmdr1 genotyping and in vivo mefloquine resistance on the Thai-Myanmar border. Am J Trop Med Hyg. 2005, 72: 586-592. PubMed
Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, Patel R, Laing K, Looareesuwan S, White NJ, Nosten F, Krishna S: Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet. 2004, 364: 438-447. 10.1016/S0140-6736(04)16767-6. PubMedCentralCrossRefPubMed
Lim P, Alker AP, Khim N, Shah NK, Incardona S, Doung S, Yi P, Bouth DM, Bouchier C, Puijalon OM, Meshnick SR, Wongsrichanalai C, Fandeur T, Le Bras J, Ringwald P, Ariey F: Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia. Malar J. 2009, 8: 11-10.1186/1475-2875-8-11. PubMedCentralCrossRefPubMed
Price RN, Uhlemann AC, van Vugt M, Brockman A, Hutagalung R, Nair S, Nash D, Singhasivanon P, Anderson TJ, Krishna S, White NJ, Nosten F: Molecular and pharmacological determinants of the therapeutic response to artemether-lumefantrine in multidrug-resistant Plasmodium falciparum malaria. Clin Infect Dis. 2006, 42: 1570-1577. 10.1086/503423. PubMedCentralCrossRefPubMed
Sidhu AB, Uhlemann AC, Valderramos SG, Valderramos JC, Krishna S, Fidock DA: Decreasing pfmdr1 copy number in Plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis. 2006, 194: 528-535. 10.1086/507115. PubMedCentralCrossRefPubMed
Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC: The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol. 2000, 108: 13-23. 10.1016/S0166-6851(00)00201-2. CrossRefPubMed
Noedl H, Bronnert J, Yingyuen K, Attlmayr B, Kollaritsch H, Fukuda M: Simple histidine-rich protein 2 double-site sandwich enzyme-linked immunosorbent assay for use in malaria drug sensitivity testing. Antimicrob Agents Chemother. 2005, 49: 3575-3577. 10.1128/AAC.49.8.3575-3577.2005. PubMedCentralCrossRefPubMed
Zhou Z, Poe AC, Limor J, Grady KK, Goldman I, McCollum AM, Escalante AA, Barnwell JW, Udhayakumar V: Pyrosequencing, a high-throughput method for detecting single nucleotide polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes of Plasmodium falciparum. J Clin Microbiol. 2006, 44: 3900-3910. 10.1128/JCM.01209-06. PubMedCentralCrossRefPubMed
WHO World Malaria Report 2010. http://www.who.int/malaria/world_malaria_report_2010/en/index.html,
Ecker A, Lehane A, Fidock DA: Treatment and prevention of malaria: antimalarial drug chemistry, action and use. Treatment and prevention of malaria: antimalarial drug chemistry, action and use. Edited by: Staines HM, Krishna S. 2012, Springer, Basel, 249-280.
Gadalla NB, Adam I, Elzaki SE, Bashir S, Mukhtar I, Oguike M, Gadalla A, Mansour F, Warhurst D, El-Sayed BB, Sutherland CJ: Increased pfmdr1 copy number and sequence polymorphisms in Plasmodium falciparum isolates from Sudanese malaria patients treated with artemether-lumefantrine. Antimicrob Agents Chemother. 2011, 55: 5408-5411. 10.1128/AAC.05102-11. PubMedCentralCrossRefPubMed
Uhlemann AC, McGready R, Ashley EA, Brockman A, Singhasivanon P, Krishna S, White NJ, Nosten F, Price RN: Intrahost selection of Plasmodium falciparum pfmdr1 alleles after antimalarial treatment on the northwestern border of Thailand. J Infect Dis. 2007, 195: 134-141. 10.1086/509809. PubMedCentralCrossRefPubMed
Borges S, Cravo P, Creasey A, Fawcett R, Modrzynska K, Rodrigues L, Martinelli A, Hunt P: Genomewide scan reveals amplification of mdr1 as a common denominator of resistance to mefloquine, lumefantrine, and artemisinin in Plasmodium chabaudi malaria parasites. Antimicrob Agents Chemother. 2011, 55: 4858-4865. 10.1128/AAC.01748-10. PubMedCentralCrossRefPubMed
Chaijaroenkul W, Wisedpanichkij R, Na-Bangchang K: Monitoring of in vitro susceptibilities and molecular markers of resistance of Plasmodium falciparum isolates from Thai-Myanmar border to chloroquine, quinine, mefloquine and artesunate. Acta Trop. 2010, 113: 190-194. 10.1016/j.actatropica.2009.10.016. CrossRefPubMed
Veiga MI, Ferreira PE, Jornhagen L, Malmberg M, Kone A, Schmidt BA, Petzold M, Bjorkman A, Nosten F, Gil JP: Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance. PLoS One. 2011, 6: e20212-10.1371/journal.pone.0020212. PubMedCentralCrossRefPubMed
Sanchez CP, Rotmann A, Stein WD, Lanzer M: Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum. Mol Microbiol. 2008, 70: 786-798. PubMed
Saliba KJ, Lehane AM, Kirk K: A polymorphic drug pump in the malaria parasite. Mol Microbiol. 2008, 70: 775-779. PubMed
- Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections
Dylan R Pillai
Henry M Staines
- BioMed Central
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