Evidence for linkage of pfmdr1, pfcrt, and pfk13 polymorphisms to lumefantrine and mefloquine susceptibilities in a Plasmodium falciparum cross

Abstract

Background: Lumefantrine and mefloquine are used worldwide in artemisinin-based combination therapy (ACT) of malaria. Better understanding of drug susceptibility and resistance is needed and can be obtained from studies of genetic crosses.

Methods: Drug response phenotypes of a cross between Plasmodium falciparum lines 803 (Cambodia) and GB4 (Ghana) were obtained as half-maximal effective concentrations (EC₅₀s) and days to recovery (DTR) after 24 h exposure to 500 nM lumefantrine. EC₅₀s of mefloquine, halofantrine, chloroquine, and dihydroartemisinin were also determined. Quantitative trait loci (QTL) analysis and statistical tests with candidate genes were used to identify polymorphisms associated with response phenotypes.

Results: Lumefantrine EC₅₀s averaged 5.8-fold higher for the 803 than GB4 parent, and DTR results were 3-5 and 16-18 days, respectively. In 803 × GB4 progeny, outcomes of these two lumefantrine assays showed strong inverse correlation; these phenotypes also correlated strongly with mefloquine and halofantrine EC₅₀s. By QTL analysis, lumefantrine and mefloquine phenotypes mapped to a chromosome 5 region containing codon polymorphisms N86Y and Y184F in the P. falciparum multidrug resistance 1 protein (PfMDR1). Statistical tests of candidate genes identified correlations between inheritance of PfK13 Kelch protein polymorphism C580Y (and possibly K189T) and lumefantrine and mefloquine susceptibilities. Correlations were detected between lumefantrine and chloroquine EC₅₀s and polymorphisms N326S and I356T in the CVIET-type P. falciparum chloroquine resistance transporter (PfCRT) common to 803 and GB4.

Conclusions: Correlations in this study suggest common mechanisms of action in lumefantrine, mefloquine, and halofantrine responses. PfK13 as well as PfMDR1 and PfCRT polymorphisms may affect access and/or action of these arylaminoalcohol drugs at locations of hemoglobin digestion and heme metabolism. In endemic regions, pressure from use of lumefantrine or mefloquine in ACTs may drive selection of PfK13 polymorphisms along with versions of PfMDR1 and PfCRT associated with lower susceptibility to these drugs.

Keywords: Artemisinin-based combination chemotherapy; Drug resistance; Halofantrine; Malaria; Quantitative trait loci analysis.

Graphical abstract

Fig. 1

Lumefantrine response phenotypes of parents and progeny from the 803 × GB4 cross. Parasites indicated in green carry the 803 pfmdr1 locus encoding N86 and Y184, whereas parasites indicated in yellow carry the GB4 pfmdr1 locus encoding 86Y and 184F. A Results from 72 h half-maximal effective concentration (EC₅₀) of growth inhibition assays for 22 progeny and parental lines using SYBR green, a sensitive fluorescence indicator of double-stranded DNA levels (Smilkstein et al., 2004). B Parasitemia of parental lines 803 and GB4 after a 24 h 500 nM LUM exposure with three biological replicates. C Days to 2% parasitemia recovery (DTR) after a 24 h 500 nM LUM exposure for 22 progeny and parental lines. D Scatter plot between the EC₅₀ and DTR values of the individual parents and progeny. 803 is indicated in green and GB4 is indicated in yellow. Data from individual phenotype determinations are provided in Table S1 and Table S2. EC₅₀ means and confidence intervals were calculated from three biological replicates for all parasites except 36H9 and 40E7 (n = 2). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) ** Parental 803 and GB4 haplotypes are indicated by ‘1’ (green) and ‘2’ (yellow), respectively. * UM DTR values are taken from Tables S1 and S2 of this report. DHA EC50s are from supplementary Table S1 of Sá et al. (2018), except for subsequently determined mean ± standard errors in the mean (SEM) values of 1.39±0.04 (37D9; n=37), 3.32±0.41 (36H9; n=3), 2.22±0.30 (43E5; n=3), 2.60±0.47 (44D4; n=3) published here. See the cited Tables for confidence intervals and SEMs. EC50 values are in nanomolar; DTR values in days. ND = not determined.

Fig. 2

Quantitative trait loci (QTL) analysis of lumefantrine phenotypes in the 803 × GB4 cross. A QTL plot from LUM 72 h half-maximal effective concentration (EC₅₀) determinations. Maximal logarithm of odds (LOD) score is at location 966,290 on Chromosome 5 (p < 0.005). B QTL plot from the parasitemia Days to Recovery (DTR) LUM data, with a maximal LOD score at location 966,290 on Chromosome 5 (p < 0.005). Dashed lines in the figure panels mark the 0.05 significance threshold.