Plasmodium falciparum genetic factors rather than host factors are likely to drive resistance to ACT in Ghana

Malaria caused by an infection of Plasmodium falciparum is one of the major causes of morbidity and mortality in sub-Saharan Africa, especially in children under 5 years old and pregnant women [1] The World Health Organization (WHO) recommends the use of a combination of a fast-acting artemisinin derivative and a relatively slow-acting partner drug, for the treatment of uncomplicated malaria in disease-endemic areas [2]. The recommended first-line artemisinin combination therapy (ACT) in Ghana for treating uncomplicated malaria is artesunate with amodiaquine (AA), artemether with lumefantrine (AL) or a combination of dihydroartemisinin with piperaquine [3]. The reason for combining the drugs (ACT) is to slow down the development of resistance to anti-malarial drugs by P. falciparum [4]. The fast-acting drug quickly reduces parasite load whilst the slow-acting anti-malarial gradually destroys residue parasites. The potency of ACT is dependent on the efficacy of both the artemisinin component and the partner drug [4]. Reduced susceptibility of parasites to partner drugs in ACT can potentially result in resistance to artemisinin in future as parasites that escape the fast action of artemisinin or its derivatives will not be cleared by the partner drug and this could allow for growth and expansion of a drug-resistant parasite population [4].

Variations observed in effectiveness of ACT in malaria-endemic regions are dependent on parasite genetic factors [5], as well as human genetic factors [6]. For parasite genetic factors, polymorphisms which arise due to single nucleotide changes in the pfmdr1 gene in its coding region have been linked to differential parasite susceptibility to ACT partner drugs, such as amodiaquine [7] and lumefantrine [8]. This makes pfmdr1 an important likely candidate for initiating ACT partner drug resistance [9].

The polymorphic pfmdr1 alleles that are mostly found in Africa are N86Y, F184Y and D1246Y. The P. falciparum N86-F184-D1246 haplotype (NFD haplotype) has been linked to decreased susceptibility to anti-malarial drugs, such as mefloquine and lumefantrine. The selection of the NFD haplotype has been seen in malaria treatment using AL. The different haplotype, which is the Y86-Y184-Y1246 haplotype (YYY haplotype), is associated with reduced amodiaquine susceptibility [10].

Differences in genetic make-up of humans is the principal factor that defines the level of drug availability in the blood to clear the parasites [6]. The cytochrome P450 enzyme family (CYP genes) is involved in the metabolism of different anti-malarial drugs [6]. Amodiaquine is mainly metabolized by CYP2C8 [11] whiles lumefantrine is metabolized mainly by CYP3A4 [12]. Different mutations in the promoter region, introns or exons can result in different alleles of the CYP450 genes in different individuals. The metabolism of a drug or a combination of drugs could be decreased, increased or unaffected depending on the allele(s) an individual possesses [13]. Elucidating the exact role these disparities in the genes coding for the enzymes involved in ACT metabolism is vital for understanding the inter-individual pharmacokinetic differences observed in persons using ACT [14]. This study investigated P. falciparum and host genetic factors that are likely to affect the efficacy of ACT partner drugs used in Ghana.

It is obvious that resistance of P. falciparum to ACT partner drugs may lead to the gradual evolution of strains of parasites with reduced susceptibility to artemisinin. The failure of partner drugs should therefore be of great concern to national malaria control programmes in disease-endemic areas. Since both the host and the parasite genome play a role in metabolism of ACT, a key question often asked is: what drives parasite resistance to ACT partner drugs? Before attempting to address this question, the clinical data generated in this study, which is a sub-set of data published elsewhere, must be examined. The clinical data indicate 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 indicates a better rate of parasite clearance with AA than AL. It is not surprising as this results gives credence to a previous report by Abuaku et al. [15]. The report indicate an overall PCR-corrected cure rate of 100% for AA and 97.6% (95% CI 93.1, 99.5) for AL: 97.2% (95% CI 92.0, 99.4) in the forest zone and 100% in the savannah zone [15]. The significantly high N86-F184-D146 haplotype in AL-treated individuals compared to Y84-Y184-Y1246 haplotype in AA-treated individuals observed here may explain the slight difference in efficacy between AL and AA observed in that study.

The efficacy of the partner drugs, amodiaquine and lumifantrine, investigated in this study, is linked to pfmdr1 gene, which is part of the ATP-binding cassette (ABC) transporters [21]. This gene encodes a transporter which is found in the digestive vacuole of the parasite [22]. The pfmdr1 is thought to function by pumping compounds out of the parasite, making it an important protein for anti-malarial drug resistance. The true mechanistic role of the pfmdr1 in initiating anti-malarial drug resistance is poorly understood [9], although certain mutations in the gene have been associated with resistance to different anti-malarial drugs [7, 8]. The exact mechanism in which mutation at the pfmdr1 F184 confers resistance to lumefantrine while mutations at the pfmdr1 Y86 and Y1246 confer resistance to amodiaquine is not well understood but has been observed to be mostly selected during lumefantrine and amodiaquine drug pressure, respectively [7, 8].

There was 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 et al. [23]. The results also show the widespread presence of these mutations in Ghana which are not ecological zonal bias. This was because there was no significant difference in these mutations across the different ecological zones (Additional file 1: Fig. S1 and Additional file 2: Fig. S2).

The cytochrome P450 enzyme family (CYP genes) is a key enzyme involved in the metabolism of different anti-malarial drugs [6]. Lumefantrine is metabolized to desbutyl-benflumetol mainly by CYP3A4 [12]. A change from adenine (A) to guanine (G) at position 392 of CYP3A4 gene proximal promoter region results in CYP3A4*1B allele [24].This mutant has been reported to have poor enzyme activity [25]. 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 suggests that lumefantrine is well metabolized in the participants. Again, delayed clearance observed in patients treated with AL was 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 it can be strongly inferred 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, there is the need to tread cautiously since this inference is more or less speculative as it is not backed by any pharmacokinetic studies of desbutyl-lumefantrine in the children. It must however be emphasized that similar findings have been reported [26].

The CYP2C8 is the main enzyme that metabolizes amodiaquine to desethyl amodiaquine (DEAQ) [27]. The wild-type CYP2C8*1 and the mutant CYP2C8*2 are the most predominant in Ghana [28]. 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 [11]. 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 [28]. The high number of individuals with wild-type CYP2C8 suggests that amodiaquine was well metabolized in the participants. However delayed clearance was observed in individuals who reported with high parasitaemia (> 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. It is speculated 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, clears most of the parasites and leaves only a few supposedly ‘weakened parasite’ residues making the presence of a sub-optimal concentration of DEAQ enough to clear the parasite residue in these individuals.

When Chi square test was used to determine the association between CYP2C8/ CYP3A4 and pfmdr1 genotypes and day 3 positivity, there was no significant difference. 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 host gene-type of the enzyme could not be responsible for the delayed parasite clearance. Therefore, this observation suggests 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 anti-malarial 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 parasite susceptibility to ACT in Ghana sooner than later.

Observations made in this study give enough grounds to conclude that parasite genetic factors rather than the host’s is more likely to drive resistance to ACT, especially AL in Ghana. All individuals successfully genotyped for CYP3A4 were wild-type, suggesting that lumefantrine is well metabolized in the participants. The high percentage of CYP2C8 wild-type individuals also suggests that amodiaquine is metabolized efficiently. High prevalence of N86, F184 and D1246 suggests AL is less efficacious than AA. The outcome of this study conveys a warning that malaria parasites are becoming resistant to anti-malarial drugs. Prompt monitoring of ACT is required. There is the need to find other anti-malarial drugs that could be used as ACT partner drugs.