It is predictable from the aforementioned mechanism justifying the use of ACT for malaria treatment that resistance of P. falciparum to the ACT partner drugs may lead to the gradual selection 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 WHO and the National Malaria Control Program in disease endemic areas. As shown in Table 2 and 3, 13% (16/120) of the patients on AL (cohort 1) still carried parasites on day 3 post-treatment compared to 4% (5/120) of those on 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 and gives credence to the clinical outcome of the study already reported by Abuaku and colleagues, (Abuaku et al., 2016) who are the original owners of the samples used in this study.
The efficacy of the partner drugs investigated in this study, amodiaquine and lumifantrine, is related 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 (Bopp et al., 2018). The Pfmdr1 function by pumping compounds out of the parasite thus making it an important protein for antimalarial drug resistance. Despite this knowledge, it must be still 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. The selection of the NFD haplotype has been seen in malaria treatment using artemether with lumefantrine. The different haplotype which is the YYY haplotype is associated with reduced susceptibility to amodiaquine (Holmgren et al., 2007). From the results of this study, Pfmdr1 codon 86 alleles (N or Y), codon 184 alleles (Y or F) and codon 1246 alleles (D or Y) were detected in 95 samples. Of this, 25 (26%) samples were collected from Navrongo, 39 (41%) from Begoro (36%), and 31 (33%) from Cape Coast. This indicate that the presence of these mutation is wide spread in Ghana and is not ecological zonal bias. For the Pfmdr1 codon 86 (N or Y allele), the prevalence of the mutant allele Y86 for all the individual sites ranged from 12% (3/25) in Navrongo, 5.5% (2/39) in Begoro, and 6.5% (2/31) in Cape Coast. All put together this observation indicate a high prevalence of the wild type allele, N86. For the Pfmdr1 codon 184 alleles (Y or F), the prevalence of the mutant allele F184 observed were 72% (18/25) in Navrongo, 59% (23/39) in Begoro and 64.5% (20/31) in Cape Coast. For the Pfmdr1 codon 1246 (D or Y allele), the prevalence of the wildtype allele D1246 was 100% for all the sites. High prevalence of N86, F184, and D1246 haplotypes were observed in this study with no record of Y86, Y184 and Y1246 haplotypes. The high prevalence of the NFD haplotype recorded in this study is consistent with that reported by Duah and colleagues (Duah et al., (2013). Although the results show some consistency with the day 3 treatment outcome, however, since in vitro test of parasite susceptibilities to lumefantrine and amodiaquine were not performed in this study the association between Pfmdr1 haplotypeamplification and resistance to the antimalarial drugs could be difficult to established.
There were similar numbers of both non-synonymous and synonymous mutations observed at low frequencies in the coastal and forest ecological zones. 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. 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.
The enzymatic biotransformation of a drug to its active metabolite or bio activation to the therapeutically relevant molecule is vital in order to be effective against its target (Kebamo et al., 2015). Variation in the genetic make-up of humans is the principal factor that defines the level of drugs available in mostly the blood to clear the parasite. The cytochrome P450 enzyme family (CYP genes) is a key enzyme involved in different antimalarial drug metabolism available for the treatment of malaria (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) which have been suggested to have poor enzyme activity (Mutagonda et al., 2017). From the results of the current study, 93 individuals were successfully genotyped for CYP3A4 of which 100% were wild type. This result is contrary to what was reported by Kudzi and colleagues (Kudzi et al., 2010). The high number of individuals with wild type CYP3A4 suggests 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 and no mutation in the CYP3A4 gene of the individuals. From this, it can be inferred that the parasite genetic factors are likely to be the cause of delayed clearance in the children treated with AL. A limitation of this study is the absence of desbutyl-lumefantrine pharmacokinetic data of the study participants to film some of our assertions. Nonetheless, these results are in agreement with findings from 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 suggesting ample concentration of DEAQ in the 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. It must be noted that this study could not provide direct evidence to support the aforementioned phenomenon. 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.