Plasmodium falciparum merozoite invasion ligands, linked antimalarial resistance loci and ex vivo responses to antimalarials in The Gambia

Abstract

Background: Artemether/lumefantrine is the most commonly used artemisinin-based combination treatment (ACT) for malaria in sub-Saharan Africa. Drug resistance to ACT components is a major threat to malaria elimination efforts. Therefore, rigorous monitoring of drug efficacy is required for adequate management of malaria and to sustain the effectiveness of ACTs.

Objectives: This study identified and described genomic loci that correlate with differences in ex vivo responses of natural Plasmodium falciparum isolates from The Gambia to antimalarial drugs.

Methods: Natural P. falciparum isolates from The Gambia were assayed for IC50 responses to four antimalarial drugs (artemether, dihydroartemisinin, amodiaquine and lumefantrine). Genome-wide SNPs from 56 of these P. falciparum isolates were applied to mixed-model regression and network analyses to determine linked loci correlating with drug responses. Genomic regions of shared haplotypes and positive selection within and between Gambian and Cambodian P. falciparum isolates were mapped by identity-by-descent (IBD) analysis of 209 genomes.

Results: SNPs in 71 genes, mostly involved in stress and drug resistance mechanisms correlated with drug responses. Additionally, erythrocyte invasion and permeability loci, including merozoite surface proteins (Pfdblmsp, Pfsurfin), and high-molecular-weight rhoptry protein 2 (Pfrhops2) were correlated with responses to multiple drugs. Haplotypes of pfdblmsp2 and known drug resistance loci (pfaat1, pfcrt and pfdhfr) from The Gambia showed high IBD with those from Cambodia, indicating co-ancestry, with significant linkage disequilibrium between their alleles.

Conclusions: Multiple linked genic loci correlating with drug response phenotypes suggest a genomic backbone may be under selection by antimalarials. This calls for further analysis of molecular pathways to drug resistance in African P. falciparum.

Figure 1.

Association between genomic SNPs and Gambian P. falciparum IC₅₀ to antimalarial drugs from MLM. The left panel shows Manhattan plots of P values for association of each SNP with responses to lumefantrine (LUM) in the top row, artemether (ARM) in the middle row and dihydroartemisinin (DHA) in the bottom row. The right panel shows the corresponding QQ plot for each drug, indicating the deviation of the observed P values for SNPs against each drug as against random expectations. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

LD between SNPs and association with lumefantrine (LUM) and artemether (ARM) ex vivo responses. (a) Significant LD and genotype–phenotype interaction network for LUM (pink node) and ARM (yellow node). Dark orange, orange, green, blue, grey, mustard and dark-blue nodes represent chromosomes 4, 5, 6, 7, 9, 12 and 14, respectively. Each node is labelled with the SNP position. (b) Boxplots of LD between SNPs in or around drug loci (dr) and non-drug loci (ndr) across pairs of chromosomes as indicated on the x-axis. (c) LD between pfcrt and pfdhps variants for Cambodia (Cam), Gambia 2008 (Gam08) and 2014/2015 (Gam14/15). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

Manhattan plots of IBD-based signatures of positive selection across the genome of Gambian and Cambodian P. falciparum isolates. The y-axis of each plot shows the −log₁₀ of corrected P values of iR for each SNP. The left panel shows IBD for pairs of isolates within populations: (a) Gambia 2008; (b) Gambia 2014/15; and (c) Cambodia. The right panel shows IBD between isolates from different populations: (d) Gambia 2008 against Cambodia; (e) Gambia 2014/15 against Cambodia; and (f) Gambia 2008 against Gambia 2014/15. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 4.

TCS haplotype network of DBLMSP2 amongst Gambian and Cambodian isolates. Each pie chart shows the proportion of haplotypes from each population: Gambia 2008 (red); Gambia 2014/15 (green); and Cambodia (blue). The black edges are unsampled putative haplotypes not represented in the dataset. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.