Origin and evolution of sulfadoxine resistant Plasmodium falciparum

Abstract

The Thailand-Cambodia border is the epicenter for drug-resistant falciparum malaria. Previous studies have shown that chloroquine (CQ) and pyrimethamine resistance originated in this region and eventually spread to other Asian countries and Africa. However, there is a dearth in understanding the origin and evolution of dhps alleles associated with sulfadoxine resistance. The present study was designed to reveal the origin(s) of sulfadoxine resistance in Cambodia and its evolutionary relationship to African and South American dhps alleles. We sequenced 234 Cambodian Plasmodium falciparum isolates for the dhps codons S436A/F, A437G, K540E, A581G and A613S/T implicated in sulfadoxine resistance. We also genotyped 10 microsatellite loci around dhps to determine the genetic backgrounds of various alleles and compared them with the backgrounds of alleles prevalent in Africa and South America. In addition to previously known highly-resistant triple mutant dhps alleles SGEGA and AGEAA (codons 436, 437, 540, 581, 613 are sequentially indicated), a large proportion of the isolates (19.3%) contained a 540N mutation in association with 437G/581G yielding a previously unreported triple mutant allele, SGNGA. Microsatellite data strongly suggest the strength of selection was greater on triple mutant dhps alleles followed by the double and single mutants. We provide evidence for at least three independent origins for the double mutants, one each for the SGKGA, AGKAA and SGEAA alleles. Our data suggest that the triple mutant allele SGEGA and the novel allele SGNGA have common origin on the SGKGA background, whereas the AGEAA triple mutant was derived from AGKAA on multiple, albeit limited, genetic backgrounds. The SGEAA did not share haplotypes with any of the triple mutants. Comparative analysis of the microsatellite haplotypes flanking dhps alleles from Cambodia, Kenya, Cameroon and Venezuela revealed an independent origin of sulfadoxine resistant alleles in each of these regions.

Figure 1. The map of Cambodia showing study sites.

The pie-diagrams show the frequency distribution of the single, double, triple and quadruple mutant dhps alleles in Cambodia. PL, Pailin; KS, Kampong Seila; CK, Chumkiri; MM, Memut; RK, Rattanakiri.

Figure 2. Selective sweeps around dhps alleles in Cambodia.

(A) Comparison of the wild type and three mutant (single, double and triple) groups. (B) Comparison of the single (SGKAA) and double mutant (AGKAA, SGEAA, SGKGA) dhps alleles. (C) Comparison of the three triple mutant (AGEAA, SGEGA, SGNGA) dhps alleles. The dashed line crossing the y-axis indicates the mean heterozygosity (H_e) at 8 neutral microsatellite loci on chromosomes 2 and 3. The error bars indicate ±1SD (standard deviation).

Figure 3. Comparison of F_ST across selected (dhps) and unselected (neutral) loci.

(A) Histogram plotting F_ST (±SD) values at 8 neutral (gray bars) and 10 dhps (black bars) microsatellite loci. Asterisk indicates significant values obtained after 1000 random permutations. Populations were defined according to their dhps genotype (i.e. wild type, single, double, or triple mutant) for calculations of the overall (global) F_ST. The overall F_ST for dhps after jackknifing over loci was 0.20 (95% CI: 0.15–0.26) whereas the overall F_ST for 8 neutral loci was 0.01 (95% CI: 0.006–0.019). The difference between F_ST values of dhps and neutral groups was highly significant as assessed by Mann-Whitney U test (Z = 3.55, P = 0.0003). (B) Plot of F_ST versus H_e to disentangle selection on dhps and neutral loci. Black circles represent 10 dhps loci and white diamonds represent 8 neutral loci. The gray shading indicates the region of neutral expectations at a confidence level of 95%. The markers above the gray region are considered to be under directional selection while those inside the gray region are considered to be neutral. The P values (simulated F_ST<sample F_ST) for each locus are given as supporting information (Table S4).

Figure 4. Genetic relationships among dhps alleles in Cambodia.

(A) A median-joining network diagram depicting three independent origins for double mutant and two independent origins for triple mutant dhps alleles in Cambodia. The 141 P. falciparum isolates were classified into 85 haplotypes (H1 to H85; see individual haplotypes profiles in Table S5) based on the 10-loci microsatellite profile around dhps gene. The size of the circle is proportional to the number of isolates showing particular 10-loci microsatellite haplotype, the proportion of dhps alleles on that haplotype background are depicted by the pie charts. The red dots are hypothetical median vectors generated by the software to connect existing haplotypes within the network with maximum parsimony. Box 1, 2 and 3 indicate three major origins for dhps mutant alleles. (B) Scheme of plausible evolution of dhps alleles inferred from the microsatellite haplotypes data. Three major and independent lineages are proposed for the double mutants, one each for SGKGA, SGEAA and AGKAA. The SGKGA is the progenitor allele for SGEGA and SGNGA triple mutants, whereas AGKAA is the progenitor for AGEAA triple mutant. Most likely, the AGKAA double mutant could also be the progenitor of AGNAA triple mutant found on the Andaman and Nicobar Islands, given its wide occurrence on the islands.

Figure 5. Genetic relationships among the dhps alleles of Southeast Asia (Cambodia), Africa (Kenya and Cameroon) and South America (Venezuela).

An eBURST diagram showing major lineages for Cambodian dhps alleles. Two major lineages exist in Cameroon (SGKAA and AGKAA), one major lineage in Kenya (SGEAA), and one in Venezuela (SGKGA and SGEGA). Each lineage is distinct from the others, suggesting multiple, independent evolutionary histories.