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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Artemether resistance in vitro is linked to mutations in PfATP6 that also interact with mutations in PfMDR1 in travellers returning with Plasmodium falciparum infections

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Dylan R Pillai, Rachel Lau, Krishna Khairnar, Rosalba Lepore, Allegra Via, Henry M Staines, Sanjeev Krishna
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-131) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

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.

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.
Zusatzmaterial
Additional file 1: Pyrosequencing primers (DOC 40 KB)
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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)
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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)
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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)
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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)
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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)
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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)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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