Evolution of Antimalarial Drug Resistance Markers in the Reservoir of Plasmodium falciparum Infections in the Upper East Region of Ghana

Abstract

Background: The majority of Plasmodium falciparum infections, constituting the reservoir in all ages, are asymptomatic in high-transmission settings in Africa. The role of this reservoir in the evolution and spread of drug resistance was explored.

Methods: Population genetic analyses of the key drug resistance-mediating polymorphisms were analyzed in a cross-sectional survey of asymptomatic P. falciparum infections across all ages in Bongo District, Ghana.

Results: Seven years after the policy change to artemisinin-based combination therapies in 2005, the pfcrt K76 and pfmdr1 N86 wild-type alleles have nearly reached fixation and have expanded via soft selective sweeps on multiple genetic backgrounds. By constructing the pfcrt-pfmdr1-pfdhfr-pfdhps multilocus haplotypes, we found that the alleles at these loci were in linkage equilibrium and that multidrug-resistant parasites have not expanded in this reservoir. For pfk13, 32 nonsynonymous mutations were identified; however, none were associated with artemisinin-based combination therapy resistance.

Conclusions: The prevalence and selection of alleles/haplotypes by antimalarials were similar to that observed among clinical cases in Ghana, indicating that they do not represent 2 subpopulations with respect to these markers. Thus, the P. falciparum reservoir in all ages can contribute to the maintenance and spread of antimalarial resistance.

Keywords: Plasmodium falciparum; Ghana; antimalarials; artemisinin-based combination therapies; asymptomatic reservoir; drug resistance; malaria; population genetics.

Figure 1.

A, Prevalence of wild-type and mutant sequences at the indicated alleles for pfcrt (n = 170), pfmdr1 (n = 198), pfdhfr (n = 177), and pfdhps (n = 202) in the asymptomatic Plasmodium falciparum reservoir (see Supplementary Table 7 for additional details on the unique haplotypes identified). (Note: pfmdr1 codon 1246 was not polymorphic. All isolates genotyped carried the D1246 wild-type allele.) B, Pairwise linkage disequilibrium (LD) between all loci (codons) for pfcrt, pfdhfr, and pfdhps. Gray shading along the horizontal is used to denote within codon comparisons, for which the calculation of LD is not possible. For each gene, the number of unique haplotypes (h) and heterozygosity (H_e) are provided, as well as the number of isolates included. Pfmdr1 is not included, since there was no significant pairwise LD between codons 86 and 184. (For pfcrt and pfmdr1 previous studies with clinical isolates have observed LD between codons pfcrt 76 and pfmdr1 86 due to directional selection by chloroquine [25, 26]. No significant LD was detected between the codon pair (${\overline{r}}_{\text{d}}$ = 0.0121; P = .62)).

Figure 2.

Inferred phylogeny of the pfk13 sequences from Bongo District, Ghana. An unrooted neighbor-joining phylogenetic tree was constructed using sequences from West Africa (Bongo District, Ghana [n = 418] and Mali [n = 206]), East Africa (Uganda [n = 66] and Kenya [n = 70]) (GenBank accession nos. KT901412-KT901454 and KT955978-KT956003), and Southeast Asia (n = 60). Asterisks at major nodes indicate bootstrap values ≥65%.

Figure 3.

Patterns of expected heterozygosity (H_e) in the microsatellite loci flanking (selected) pfcrt (A) and pfmdr1 (B) alleles/haplotypes. On the x-axis, microsatellite locus distance (kb) upstream and downstream of the drug resistance genes are indicated with negative and positive values, respectively. Dashed line crossing the y-axis represents the mean H_e of the 12 neutral microsatellite loci (unselected); error bars indicated standard deviation. (For additional details on the number of alleles and the H_e for each locus, see Supplementary Table 13.)

Figure 4.

The genetic relatedness networks constructed for pfcrt (A) and pfmdr1 (B). The multilocus microsatellite haplotypes (−4.9, −4.6, 1.6, and 4.2-kb loci flanking pfcrt and −1.6, −1.4, −1.2, −0.9, −0.2, 0.3, and 0.6 kb loci flanking pfmdr1) were constructed for each gene to generate the networks. Each node represents an isolate, with the color of each node signifying the pfcrt 76 allele (K/T) or pfmdr1 haplotype (NY/NF/YF). The edges in the networks denote the pairwise relatedness between isolates at the selected pairwise allele sharing (P_AS) ≥0.70 threshold (ie, identical at ≥3 loci for pfcrt and ≥5 loci for pfmdr1). This threshold was selected to visualize the genetic similarity between isolates that likely share a recent transmission history. For further details on the pfcrt networks with the pfcrt haplotypes defined and comparisons to the Ghanaian isolates collected 3-years after the introduction of artemisinin-based combination therapies, see Supplementary Figure 5. The P_AS networks were constructed using the ggraph and tidygraph R software packages.