Plasmodium falciparum sulfadoxine resistance is geographically and genetically clustered within the DR Congo

Abstract

Understanding the spatial clustering of Plasmodium falciparum populations can assist efforts to contain drug-resistant parasites and maintain the efficacy of future drugs. We sequenced single nucleotide polymorphisms (SNPs) in the dihydropteroate synthase gene (dhps) associated with sulfadoxine resistance and 5 microsatellite loci flanking dhps in order to investigate the genetic backgrounds, genetic relatedness, and geographic clustering of falciparum parasites in the Democratic Republic of the Congo (DRC). Resistant haplotypes were clustered into subpopulations: one in the northeast DRC, and the other in the balance of the DRC. Network and clonal lineage analyses of the flanking microsatellites indicate that geographically-distinct mutant dhps haplotypes derive from separate lineages. The DRC is therefore a watershed for haplotypes associated with sulfadoxine resistance. Given the importance of central Africa as a corridor for the spread of antimalarial resistance, the identification of the mechanisms of this transit can inform future policies to contain drug-resistant parasite strains.

Figure 1. Distributions of wildtype (A), single-mutant (B), and double- and triple-mutant (C) dhps haplotypes across the DRC.

Size of circles is proportional to the number of samples in that location. Haplotypes are indicated by the amino acids at codons 436, 437, 540, and 581 of DHPS; mutant amino acids are underlined and bolded.

Figure 2. Clustering of provincial falciparum parasite populations.

(A) Predicted clustering of microsatellite profiles between DRC provinces into two distinct parasite subpopulations from western DRC (Bandundu, Equateur, Katanga, Bas-Congo/Kinshasa, Kasaï Occidental, Kasaï Oriental) and eastern DRC (North/South Kivu, Orientale, Maniema). Computed by UniFrac with jackknifing and 1000 replicates. (B) Predicted provincial subpopulations in the DRC. The parasites in the hatched provinces were partitioned by the analysis in (A) to be distinct from those of the un-hatched provinces.

Figure 3. Reductions in mean heterozygosity by dhps haplotype.

Haplotypes are indicated by the amino acids at codons 436, 437, 540, and 581 of DHPS; mutant amino acids are underlined and bolded. Diamonds are point estimates of the mean, and bars represent standard error. Calculated with GenAlEx v6.5 .

Figure 4. Correlations of genetic and geographic distance for all parasites (A), wildtype parasites (SAKA, AAKA, CAKA) only (B), and mutant parasites (SGKA, AGKA, SGEA, SGEG) only (C) in the DRC.

Black dots represent pairwise comparisons, gray line is the best-fit regression line, and dotted gray line is the 95% confidence interval of best-fit line. Mantel values were 0.098 (p = 0.010) for all parasites, 0.046 (p = 0.213) for wildtype parasites only, and 0.277 (p < 0.001) for mutant parasites only. Pearson product-moment correlation coefficient (PMCC) values were 0.09828 (p < 0.0001) for all parasites, 0.04580 (p = 0.0158) for wildtype parasites only, and 0.2771 (p < 0.0001) for mutant parasites only. Genetic and geographic distances and Mantel tests computed using 999 permutations with GenAlex v6.5, and PMCC computed with GraphPad Prizm.

Figure 5. Median-joining network of dhps haplotypes in the DRC.

“SQ” numbers designate microsatellite profile, circles are proportional to the number of parasites bearing that microsatellite profile, and dhps haplotypes are indicated by the color key and are indicated by the amino acids at codons 437, 540, and 581 of DHPS; mutant amino acids are underlined and bolded. Small red nodes are hypothetical median vectors created by the program to connect sampled haplotypes into a parsimonious network. Distances between nodes are arbitrary. Constructed in NETWORK v4.6.10 .

Figure 6. Hypothesized lineages of dhps haplotypes in the DRC.

Numbers designate microsatellite profile, nodes are proportional to the number of parasites bearing that profile, and dhps haplotypes are indicated by the color key and are indicated by the amino acids at codons 437, 540, and 581 of DHPS. Nodes with more than one color indicate that the microsatellite profile was borne by more than one type of dhps haplotype. Connected haplotypes are clonal complexes that are hypothesized by the algorithm to have descended from the same founder haplotype. Haplotypes that are connected vary in their microsatellite profile at one locus; unconnected haplotypes are those that do not share 4 loci with any other haplotype. Calculated with eBURST v3 .