DETERMINATION OF PLASMODIUM FALCIPARUM AND HOST GENETIC FACTORS THAT AFFECT THE EFFICACY OF THE ARTEMISININ-BASED COMBINATION PARTNER DRUGS USED IN GHANA

ABSTRACT

Malaria is one of the major causes of morbidity and mortality in sub-Saharan Africa, especially in children under 5 years and pregnant women. The use of Artemisinin-based combination therapy (ACT), which is a combination of a fast-acting artemisinin derivative and a relatively slow-acting partner drug, is used for malaria treatment in disease-endemic areas. The ACT partner drugs in Ghana are lumefantrine (LUM), amodiaquine (AQ), and piperaquine (PQ). Plasmodium falciparum isolates with reduced susceptibility to these partner drugs may affect treatment outcome. Mutations in the parasite multidrug-resistant 1 (Pfmdr1) gene is linked to reduced susceptibility to amodiaquine and lumefantrine and increased copy number of plasmepsin II and III (Pfpm 2 and 3) are linked to reduced susceptibility to piperaquine. In addition, the potency of the partner drugs in vivo depends on the metabolism by the cytochrome P450 (CYP) enzyme in the host. Mutations in the CYP2C8 gene are linked to reduced metabolism of amodiaquine in vitro whiles mutations in CYP3A4 may be linked to reduced metabolism of lumefantrine and piperaquine in vitro. This study investigated the host and parasite genetic factors affecting the susceptibility of the parasite to ACT partner drugs. Archived samples from 240 patients (120 given AL and the other half given AA) aged ≤9years participating in antimalarial drug resistance survey in sites representing the three ecological areas of Ghana were used. Polymerase chain reaction (PCR) followed by Sanger sequencing was used to determine the polymorphisms in CYP2C8, CYP3A4, and pfmdr1 genes. Real-time PCR was used to determine copy numbers of plasmepsin II/III genes. Of the 93 samples successfully genotyped for CYP3A4, all had wild type alleles which are suggestive that the hosts are good metabolizers of both lumefantrine and piperaquine. Ninety-four samples were successfully genotyped for CYP2C8 of which 61% had wild type alleles, 33% heterozygous, and 5% homozygous derived alleles. The high percentage of wild type allelesobserved also suggests that amodiaquine would be metabolized efficiently by the hosts. Ninety-five samples were successfully genotyped for the Pfmdr1 gene. At codon 86, 93% were wild type (N86), 6% mutant (Y86), and 1% mixed clones (N86/86Y). For codon 184, 36% were wild type (Y184), 51% mutant (F184), and 13% mixed (Y184/184F) whiles for codon 1246, 100% were wild type (D1246). The high prevalence of N86, F184, and D1246 (NFD) haplotype suggests parasites with reduced susceptibility to lumefantrine and not amodiaquine. There were both synonymous and nonsynonymous mutations observed in the Pfmdr1 at low prevalence. For Pfpm2 and Pfpm3, 35% and 20% of the isolates respectively had increased gene copy numbers and this is indicative of parasites with reduced piperaquine susceptibility. In conclusion, the parasite's genetic factors rather than the hosts' are likely to drive resistance to ACTs in Ghana.