Study design
Blood blot filter paper used in this study were archived samples collected in 2016 from children participating in studies previously described by Abuaku et al. [15]. The study was part of routine surveillance on the therapeutic efficacy of ACT in Ghana; the efficacy of AA and AL were studied in six sentinel sites representing the forest and savannah zones of the country. Three sites representing the two ecological zones studied AA whilst the other three sites studied AL. In each site, the study was a one-arm, prospective evaluation of the clinical, parasitological and haematological responses to directly observed treatment with either AA or AL among children 6 months to 9 years old with uncomplicated falciparum malaria. The WHO 2009 protocol on surveillance of anti-malaria drug efficacy was used for the study with primary outcomes as prevalence of day 3 parasitaemia and clinical and parasitological cure rates on day 28.
An informed consent was obtained from each parent or guardian. A medical doctor prescribed either AA or AL to the study participants who were then followed up for 28 days.
The archived samples were selected from three sentinel sites, Navrongo, Begoro and Cape Coast, which represent three distinct eco-epidemiological zones in Ghana (Fig. 1). Begoro is located in the tropical forest ecological zone, Navrongo is in the northern savanna ecological zone and Cape Coast is situated in the coastal savanna ecological zone. The samples (100 μl blood) were collected on Whatman 3 filter paper (Sigma, UK), stored in plastic bags containing silica gel, and kept at room temperature until use.
The samples from a group of participants referred hereafter as ‘cohort 1’ comprised 120 recruited patients who were given AL, and a second group, cohort 2, comprised 120 patients who received AA. Of the 240 study participants’ archived samples analysed, 60 were selected from the savannah zone, 90 from the coastal zone and 90 from the forest zone.
DNA extraction
Malaria parasite DNA was extracted from the archived blood blots filter papers using a QIAamp DNA minikit (Qiagen, Germany) following the manufacturer’s protocol. Convectional PCR [16] was performed to amplify the region of interest using published protocols [17-19]. PCR products were sequenced using Sanger sequencing at Macrogen, The Netherlands.
Detection of pfmdr1 polymorphisms
The regions of pfmdr1 gene was amplified and sequenced to determine the presence of any mutation. The amplification was carried out using the protocol described by [17]. The polymorphisms were analysed at codons 86 (asparagine to tyrosine), 184 (tyrosine to phenylalanine), 1034 (serine to cysteine), 1042 (asparagine to aspartic acid) and 1246 (aspartic acid to tyrosine). A PCR followed by Sanger sequencing was used in determining these polymorphisms. Thirty microlitres aliquot of PCR products were kept on ice and shipped for sequencing at Macrogen, The Netherlands.
Detection of CYP2C8 and CYP3A4 polymorphisms
The CYP2C8 polymorphisms were analysed at codon 269 (isoleucine to phenylalanine). The CYP3A4 polymorphisms were analysed at position -392A>G of the proximal promoter region. A PCR followed by Sanger sequencing was used to determine the polymorphism in CYP2C8 as reported by Cavaco et al. [18] and CYP3A4 as described by Hodel et al. [19]. A PCR product of 120 bp and 717 bp represents a successful amplification of CYP2C8 and CYP3A4, respectively. Aliquot of the PCR products was shipped appropriately for sequencing at Macrogen, The Netherlands.
Data analysis
Data were organized using R software, SPSS software (version 20) and GraphPad Prism version 6. Sequence data were analysed with the BLAST program (http://blast.ncbi.nlm.nih.gov/) to determine the authenticity of the sequences. Multiple sequences were aligned with MAFFT (EMBL.EBI, Hinxton, Cambridge, UK) using the 3D7 wild-type as reference. Consensus sequence editing and single nucleotide polymorphism (SNP) detection was carried out using the CLC Main Workbench 7.9.1 (Qiagen, Aarhus, Denmark). The CYP2C8 sequences were aligned to CYP2C8 (ENSG 00000138115) as reference sequence while CYP3A4 sequences were aligned to CYP3A4 (ENSG 00000160868) as reference sequence from NCBI database. Genotype deviations from the Hardy-Weinberg equilibrium were also determined. The Hardy-Weinberg equilibrium determines whether or not the allele or genotype frequencies for a particular gene will remain constant from generation to generation in the absence of evolutionary influences such as genetic drift, inbreeding and founder effect [20]. All tests in this study were considered statistically significant when p-value <0.005.