Plasmodium falciparum Genetic Diversity in Continental Equatorial Guinea before and after Introduction of Artemisinin-Based Combination Therapy

Abstract

Efforts to control malaria may affect malaria parasite genetic variability and drug resistance, the latter of which is associated with genetic events that promote mechanisms to escape drug action. The worldwide spread of drug resistance has been a major obstacle to controlling Plasmodium falciparum malaria, and thus the study of the origin and spread of associated mutations may provide some insights into the prevention of its emergence. This study reports an analysis of P. falciparum genetic diversity, focusing on antimalarial resistance-associated molecular markers in two socioeconomically different villages in mainland Equatorial Guinea. The present study took place 8 years after a previous one, allowing the analysis of results before and after the introduction of an artemisinin-based combination therapy (ACT), i.e., artesunate plus amodiaquine. Genetic diversity was assessed by analysis of the Pfmsp2 gene and neutral microsatellite loci. Pfdhps and Pfdhfr alleles associated with sulfadoxine-pyrimethamine (SP) resistance and flanking microsatellite loci were investigated, and the prevalences of drug resistance-associated point mutations of the Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps genes were estimated. Further, to monitor the use of ACT, we provide the baseline prevalences of K13 propeller mutations and Pfmdr1 copy numbers. After 8 years, noticeable differences occurred in the distribution of genotypes conferring resistance to chloroquine and SP, and the spread of mutated genotypes differed according to the setting. Regarding artemisinin resistance, although mutations reported as being linked to artemisinin resistance were not present at the time, several single nucleotide polymorphisms (SNPs) were observed in the K13 gene, suggesting that closer monitoring should be maintained to prevent the possible spread of artemisinin resistance in Africa.

Keywords: ACT introduction; Equatorial Guinea; Kelch propeller protein K13 polymorphisms; Pfcrt; Pfdhfr; Pfdhps; Pfmdr1; Plasmodium falciparum; drug resistance molecular markers; genetic diversity; malaria; neutral and flanking microsatellites.

FIG 1

Map of Equatorial Guinea with study areas. The country's capital is Malabo, located on Bioko Island. Sampling took place in two villages of mainland Equatorial Guinea: Ngonamanga and Miyobo (red stars). Ngonamanga (Litoral Province; 02°09′34.5″N, 009°47′54.4″E) is a coastland village, isolated from the main trade routes and with an older population, whereas Miyobo (Centro Sur Province; 01°44′56.40″N; 10°10′40.05″E) is a village in the interior, but closer to the developing city Niefang, near a main road, and with a younger population. (Adapted from http://d-maps.com/.)

FIG 2

Chronology of antimalarial drug policies adopted on Bioko Island and mainland Equatorial Guinea. On the mainland, SP was presumably adopted at the same time as that on Bioko Island (gray dashed arrow). CQ, chloroquine; SP, sulfadoxine-pyrimethamine; AS+SP, artesunate plus sulfadoxine-pyrimethamine; AS+AQ, artesunate plus amodiaquine. The figure is based on data from the work of Rehman et al. (4), Charle et al. (5), and the WHO (1).

FIG 3

Total prevalences of mutations in the Pfmdr1, Pfcrt, Pfdhfr, and Pfdhps genes in Ngonamanga (A) and Miyobo (B). The Z test for comparison of two proportions was used to compare data between villages. *, P < 0.05; **, P < 0.01; ***, P < 0.001.