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Background Although rapid diagnostic tests (RDTs) play a key role in malaria-control strategies, their efficacy has been threatened by deletion and genetic variability of the genes pfhrp2/3. This study aims to characterize the deletion, genetic patterns and diversity of these genes and their implication for malaria RDT effectiveness, as well as their genetic evolution in the Amhara region of Ethiopia. Methods The study included 354 isolates from symptomatic patients from the Amhara region of Ethiopia who tested positive by microscopy. Exon 1–2 and exon 2 of genes pfhrp2 and -3 were amplified, and exon 2 was sequenced to analyse the genetic diversity, phylogenetic relationship and epitope availability. Results The deletion frequency in exon 1–2 and exon 2 was 22 and 4.6% for pfhrp2, and 68 and 18% for pfhrp3, respectively. Double deletion frequency for pfhrp2 and pfhrp3 was 1.4%. High genetic diversity, lack of clustering by phylogenetic analysis and evidence of positive selection suggested a diversifying selection for both genes. The amino-acid sequences, classified into different haplotypes, varied widely in terms of frequency of repeats, with novel amino-acid changes. Aminoacidic repetition type 2 and type 7 were the most frequent in all the sequences. The most frequent epitopes among protein sequences were those recognized by MAbs 3A4 and C1-13. Conclusion Deletions and high amino acidic variation in pfhrp2 and pfhrp3 suggest their possible impact on RDT use in the Amhara region, and the high genetic diversity of these genes could be associated with a diversifying selection in Ethiopia. Surveillance of these genes is, therefore, essential to ensure the effectiveness of public health interventions in this region. Graphical Abstract

Background In 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. Methods Plasmodium 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. Results Prevalence 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. Conclusions Chloroquine 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.