Copy number variation of plasmepsins 2 and 3 genes in Plasmodium falciparum isolates and implication for dihydroartemisinin-piperaquine resistance in Ghana

Background: In 2008, dihydroartemisinin-piperaquine and artemether-lumefantrine were introduced to supplement artesunate-amodiaquine for the treatment of uncomplicated malaria in Ghana. Drug pressure over the years enhances the development of parasite resistance to drugs. The World Health Organization recommends the detection of copy number variations of plasmepsins 2 (PfPm2) and plasmepsins 3 (PfPm3) genes linked to dihydroartemisinin-piperaquine resistance in treatment efficacy studies.Objective: This study investigated the copy number variations of PfPm2 and PfPm3 genes in the malaria parasite population in Ghana.Methods: Overall, 313 blood samples from children ≤ 9 years presenting with uncomplicated malaria at three sentinel sites used for monitoring antimalarial drug efficacy and resistance in Ghana were used for genetic investigations. The samples were collected in the malaria transmission seasons of 2015 and 2016. Malaria parasite DNA extraction from the blood samples followed by real-time quantitative PCR was used to determine the copy number of the PfPm2 and PfPm3 genes. The gene copy number was calculated by the relative expression formula 2-ΔΔCt for quantification, where ΔΔ is the relative delta-delta, and Ct is the cycle threshold. ΔΔCt was calculated as (Ctβ-tubulin − Ctpfpm2/3) - (Ctβ-tubulin cal − Ctpfpm2/3 cal), where cal is the calibration control of genomic 3D7 DNA with one copy of both the β-tubulin endogenous control and pfpm2 and pfpm3. A change in Ct (ΔCt = Ct PfPm2/3 - Ct Pfβ-tubulin) where is the difference in Ct values for the target gene of interest PfPm2 and PfPm3 and the reference gene Pfβ-tubulin. Statistical significance was defined as p < 0.05.Results: Of the parasites analyzed, 79.2% (n = 228/288) and 80.5% (n = 227/282) had one gene copy for PfPm2 and PfPm3, respectively. For PfPm2, 14.9% (n = 43/288), 3.8% (n = 11/288), and 2.1% (n = 6/288) of the isolates had copy numbers 2, 3 and 4 respectively. For PfPm3, gene copies of 2, 3 and 4 were observed in 16.3% (n = 46/282), 2.1% (n = 6/282), and 1.1% (n = 3/282) of isolates. Analysis of the copy number variation across the three study sites in Cape-Coast, Begoro, and the Navrongo areas showed no significant difference for PfPm2 (p = 0.93) and PfPm3 (p = 0.94) genes.Conclusion: After over a decade of the use of dihydroartemisinin-piperaquine, the mutations associated with resistance to the drug have been observed in Ghanaian P. falciparum isolates. This serves as baseline data for further monitoring of this molecular marker extensively as part of ongoing surveillance of antimalarial drug efficacy studies in Ghana

Datasheet1_Putative molecular markers of Plasmodium falciparum resistance to antimalarial drugs in malaria parasites from Ghana.pdf

IntroductionAntimalarial drugs including artemisinin-based combination therapy (ACT) regimens and sulphadoxine-pyrimethamine (SP) are used in Ghana for malaria therapeutics and prophylaxis respectively. The genetic basis of Plasmodium falciparum development of drug resistance involves single nucleotide polymorphisms in genes encoding proteins for multiple cellular and metabolic processes. The prevalence of single nucleotide polymorphisms in nine P. falciparum genes linked to ACT and SP resistance in the malaria parasite population was determined.MethodsArchived filter paper blood blot samples from patients aged 9 years and below with uncomplicated malaria reporting at 10 sentinel sites located in three ecological zones for the Malaria Therapeutic Efficacy Studies were used. The samples used were collected from 2007-2018 malaria transmission seasons and mutations in the genes were detected using PCR and Sanger sequencing.ResultsIn all 1,142 samples were used for the study. For falcipain-2 gene (pffp2), Sanger sequencing was successful for 872 samples and were further analysed. The prevalence of the mutants was 45% (392/872) with pffp2 markers V51I and S59F occurring in 15.0% (128/872) and 3.0% (26/872) of the samples respectively. Prevalence of other P. falciparum gene mutations: coronin (pfcoronin) was 44.8% (37/90); cysteine desulfurase (pfnfs) was 73.9% (68/92); apicoplast ribosomal protein S10 (pfarps10) was 36.8% (35/95); ferredoxin (pffd) was 8.8% (8/91); multidrug resistance protein-1 (pfmrp1) was 95.2.0% (80/84); multidrug resistance protein-2 (pfmrp2) was 91.4% (32/35); dihydrofolate reductase (pfdhfr) was 99.0% (84/85); dihydropteroate synthase (pfdhps) was 72% (68/95).DiscussionThe observation of numerous mutations in these genes of interest in the Ghanaian isolates, some of which have been implicated in delayed parasite clearance is of great interest. The presence of these genotypes may account for the decline in the efficacies of ACT regimens being used to treat uncomplicated malaria in the country. The need for continuous monitoring of these genetic markers to give first-hand information on parasite susceptibility to antimalarial drugs to inform policy makers and stakeholders in malaria elimination in the country is further discussed.</p