Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea

Abstract

BackgroundIn Plasmodium, the high level of genetic diversity and the interactions established by co-infecting parasite populations within the same host may be a source of selection on pathogen virulence and drug resistance. As different patterns have already been described in humans and mosquitoes, parasite diversity and population structure should be studied in both hosts to properly assess their effects on infection and transmission dynamics. This study aimed to characterize the circulating populations of Plasmodium spp and Plasmodium falciparum from a combined set of human blood and mosquito samples gathered in mainland Equatorial Guinea. Further, the origin and evolution of anti-malarial resistance in this area, where malaria remains a major public health problem were traced.MethodsPlasmodium species infecting humans and mosquitoes were identified by nested-PCR of chelex-extracted DNA from dried blood spot samples and mosquitoes. Analysis of Pfmsp2 gene, anti-malarial-resistance associated genes, Pfdhps, Pfdhfr, Pfcrt and Pfmdr1, neutral microsatellites (STR) loci and Pfdhfr and Pfdhps flanking STR was undertaken to evaluate P. falciparum diversity.ResultsPrevalence of infection remains high in mainland Equatorial Guinea. No differences in parasite formula or significant genetic differentiation were seen in the parasite populations in both human and mosquito samples. Point mutations in all genes associated with anti-malarial resistance were highly prevalent. A high prevalence was observed for the Pfdhfr triple mutant in particular, associated with pyrimethamine resistance.Analysis of Pfdhps and Pfdhfr flanking STR revealed a decrease in the genetic diversity. This finding along with multiple independent introductions of Pfdhps mutant haplotypes suggest a soft selective sweep and an increased differentiation at Pfdhfr flanking microsatellites hints a model of positive directional selection for this gene.ConclusionsChloroquine is no longer recommended for malaria treatment in Equatorial Guinea but sulphadoxine-pyrimethamine (SP) remains in use in combination with artesunate and is the only drug recommended in preventive chemotherapy in pregnancy. The high prevalence of point mutations in Pfdhfr and Pfdhps points to the danger of an eventual reduction in the efficacy of SP combined therapy in P. falciparum populations in Equatorial Guinea and to the essential continuous monitoring of these two genes.

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

“…The H5 haplotype was previously reported in São Tomé and Príncipe (67) as a possible African lineage of the Pfdhfr triple mutant; in Equatorial Guinea, it is associated with the single 108N mutation (3). The present data support the hypothesis that the triple mutant H11 haplotype is derived from H5.…”

“…Some moderate positive impact was attained, especially for the group of children aged 2 to 14 years, reducing the incidence of P. falciparum infection from 72% to 61% between 2007 and 2011, but the program was discontinued (4). Considering only this age group, the present study reports a much higher prevalence of Plasmodium infection (87% [55/63 samples]) that is comparable to the overall prevalence of 87% in 2005, before the introduction of ACT in this region (3).…”

“…In 2005, the majority of STR haplotypes for the Pfdhps gene showed the AGK/SGK 1 lineage (117/105 bp at the loci at 0.8/4.3 kb); the double mutant SGE lineage was not detected (3). In the present analysis, at least four independent introductions of mutant alleles in P. falciparum populations in both villages were identified.…”

“…Nevertheless, little attention has been given to the monitoring of evolution and changes in genetic diversity of P. falciparum populations regarding drug resistance markers. The last publication on the subject, to our knowledge, was in 2005, before the introduction of ACT (3).…”

“…Regarding SP, the prevalence of a Pfdhfr triple mutant (N51I/C59R/S108N [mutant amino acids are underlined]) (IRN mutant) was nearly 80%, and a quintuple mutant (Pfdhfr triple mutant plus Pfdhps A437G/K540E double mutant [GE mutant]) was absent (3). The analysis of Pfdhfr-and Pfdhps-flanking STRs revealed a decrease in genetic diversity, and this finding, along with multiple independent introductions of Pfdhps mutant haplotypes, suggested a soft selective sweep.…”