It is obvious that resistance of P. falciparum to the ACT partner drugs may lead to the gradual evolution of strains of the parasites with reduced susceptibility to the artemisinins. The failure of the partner drugs should therefore be of great concern to the National Malaria Control Program in disease endemic areas. Since both the host and the parasite genome play a role in metabolism of the ACTs, a key question often asked is: what drives parasites resistance to the ACT partner drugs? Before we attempt to address this question let us systematically examine the clinical data generated in this study, which is a subset of a bigger data published elsewhere. The clinical data indicates that 13% (16/120) of the participants treated with AL (cohort 1) still carried parasites on day 3 post-treatment compared to 4% (5/120) of those given AA (cohort 2). However all parasites were cleared by day 7 post treatment. This observation, indicate a better rate of parasite clearance with AA than AL. This observation is not surprising, as it gives credence to previous report by Abuaku and colleagues (Abuaku et al., 2016) inferring from the entire population data from which our samples set was drawn.
The efficacy of the partner drugs investigated in this study, amodiaquine and lumifantrine, is linked to Pfmdr1 gene which is part of the ATP-Binding Cassette (ABC) transporters (Ferreira et al., 2011). This gene encodes a transporter which is found in the digestive vacuole of the parasite (Bopp et al., 2018). The Pfmdr1 is thought to function by pumping compounds out of the parasite thus making it an important protein for antimalarial drug resistance. It must be emphasized that the true mechanistic role of the Pfmdr1 in initiating antimalarial drug resistance is poorly understood (Chen et al., 2010) although certain mutations in the gene have been associated with resistance to different antimalarial drugs (Sá et al., 2009; Sisowath et al., 2007). Polymorphisms in the Pfmdr1 have been linked to differential susceptibility to amodiaquine (Sá et al., 2009) and lumefantrine (Sisowath et al., 2007). The polymorphic Pfmdr1 alleles mostly found in Africa are N86Y, F184Y, and D1246Y. The P. falciparum NFD haplotype is associated with decreased susceptibility to lumefantrine while the other haplotype, YYY, is associated with reduced susceptibility to amodiaquine (Holmgren et al., 2007). There were high prevalence of N86, F184, and D1246 haplotypes in this study with no record of Y86, Y184 and Y1246 haplotypes. This observation is consistent with that reported by Duah and colleagues (Duah et al., (2013) and strongly support the difference observed in the rate of parasite clearance between treatment with AL or AA. The results also showed the widespread presence of these mutations in Ghana which are not ecological zonal bias.
The cytochrome P450 enzyme family (CYP genes) is a key enzyme involved in the metabolism of different antimalarial drug (Zanger & Schwab, 2013). Lumefantrine is metabolized to desbutyl-benflumetol mainly by CYP3A4 (Lefevre & Thomseadn, 1999). Mutation in the gene proximal promoter region which results from a change from adenine (A) to guanine (G) at the position 392 results in CYP3A4*1B (Lamba, Lin, Schuetz, & Thummel, 2012) have been observed to have poor enzyme activity (Mutagonda et al., 2017). From the results obtained in the current study, 93 individuals were successfully genotyped for CYP3A4 of which 100% had the wild type gene. This observation suggest that lumefantrine is well metabolized in the participants. Again, delayed clearance observed in patients treated with AL were seen to have one or more mutations in the Pfmdr1 gene of the P. falciparum clinical isolates rather than mutation in the CYP3A4 gene of the individuals (Table 2). Based on these observations we are quick to infer that the parasite genetic factors could be the driving force behind drug efficiency in the children treated with AL and this could possibly be the determinant of clinical resistance to the ACT in future. However in saying this, we still tread on the side of caution due to our inability to conduct any pharmacokinetic studies of desbutyl-lumefantrine in the children in order to back our assertion. Interestingly, similar findings have been reported by the group of Kiaco (Kiaco et al., 2017).
The CYP2C8 is the main enzyme that metabolizes amodiaquine to desethyl amodiaquine (DEAQ) (Li et al., 2002). The wild type CYP2C8*1 and the mutant CYP2C8*2 are the most predominant in Ghana (Kudzi et al., 2009). A change from adenine (A) to thymine (T) at nucleotide position 895 on exon 5 results in the CYP2C8*2 mutant. CYP2C8*2 has been shown to be associated with decreased enzyme activity in vitro and reduced intrinsic clearance of amodiaquine (Parikh et al., 2007). From the results of the study, 94 individuals were successfully genotyped for CYP2C8 of which 60% (56/94) had wild type alleles, 35% (33/94) heterozygous and 5% (5/94) homozygous recessive alleles. This result is contrary to what has been reported by Kudzi et al., (2009). The high number of individuals with wild type CYP2C8 suggests that amodiaquine was well metabolized in the participants. It must however be emphasized that delayed clearance was observed in individuals who reported with high parasitemia (parasitemia > 100,000) on day 0 and with one or more mutation(s) in the Pfmdr1 gene. These individuals had either wild type or heterozygous CYP2C8 genotype (Table 3) suggesting ample concentration of DEAQ in their plasma. Thus it was expected that their parasites should have been easily cleared. There was no delayed clearance observed in CYP2C8*2 individuals. This may imply that the CYP2C8 genotype of an individual may not alter the metabolism of the drug significantly, hence the plasma concentration of DEAQ may be adequate to clear the parasite. The absence of delayed clearance in CYP2C8*2 individuals can also be explained by the fact that dihydroartemisinin (DHA) which is a metabolite of artesunate in the patients clears most of the parasites and leaves only a few supposedly ‘weakened parasite’ residues making the presence of a suboptimal concentration of DEAQ enough to clear the parasite residue in these individuals. For the few cases of delayed parasite clearance using AA, the lack of association between the wild type enzyme and the cases indicate that the hosts’ gene-type of the enzyme couldn’t be responsible for the delayed parasite clearance. Therefore this observation suggest that the parasite genetic factor among others could be responsible for the delayed clearance rather than the host genetic factors.
There were similar numbers of both non-synonymous and synonymous mutations observed at low frequencies in the coastal and forest ecological zones (Table 1). The synonymous mutations may not have any significant effect on the susceptibility of the parasite to the antimalarial drugs since it does not lead to change in amino acids. However, the novel non-synonymous mutations observed in this study may suggest the possible emergence of new mutations that may lead to reduced parasites susceptibility to ACTs in Ghana sooner than later.