Study site
This study was carried out in August 2014 in two communities - Badagry (Lagos State) and Alajue (Ede, Osun State). Badagry (6o25’N 2053’E), is a coastal town with an area of 170m2 and a human population of 241,093 (NPC, 2006) that borders the Republic of Benin. Alajue village (7040’N 4030’E), is an ancient Yoruba town with a total area of 130m2 and a human population of 159,866 (NPC, 2006). Both towns are located in the south western part of Nigeria, with similar environmental conditions, occupation and lifestyle of the people.
Sampling
A total of 83 symptomatic malaria patients were recruited for this study. Following informed consent of the participants, parent or guardian, they were tested with malaria rapid diagnostic test (RDT) kit and 2 ml blood samples were collected into RNAlater. Dried blood spots (DBS) of each sample was made on 3MM Whatman filter paper.
Nucleic acid extraction
DNA from DBS was extracted using the Qiagen Mini Kit (Qiagen) according to manufacturer’s instructions and stored at minus 20 oC until needed. Total RNA was isolated from whole blood stored in RNAlater using PureLinkTM RNA Mini Kit (Invitrogen) following the manufacturer’s instructions.
Plasmodium falciparum molecular detection
DNA from DBS of each sample was used for molecular speciation of P. falciparum by nested PCR through amplification of the 18S rRNA following established protocols (13). Nest 1 amplified a large part of the 18S rRNA common to the Plasmodium genus, while the Nest 2 amplified a region in the genus specific for that species of Plasmodia. Differentiation of the species was based on amplicon band size with P. falciparum having a size of 205bp. PCR fragments were detected and sized on the QIAXCEL automated electrophoresis system.
Quantitative PCR
To determine the gene expression levels of Pfk13 and PfATPase, total RNA was treated to remove genomic DNA by digesting with 2 µl of DNaseI (Fermentas) and 5 µl of reaction buffer, incubated at 37 oC for 30 minutes and inactivated with 5 µl, 25 mM EDTA, 65 oC for 10 minutes. RNA purity and concentration were determined using a NanoDrop 1000TM (Thermo Scientific). cDNA was synthesised using the RNA Reverse Transcriptase kit (Invitrogen). Synthesized cDNA was quantified by qPCR on a CFX 96 (Bio-Rad) with the following cyclic conditions: 95 oC, 10 minutes, 49 cycles of 95 oC, 15 seconds and 60oC for 90 seconds. Relative fold increase of specific mRNA transcripts in samples was compared to P. falciparum (3D7) wildtype control, normalised using the 18S rRNA housekeeping gene. Expression levels were calculated using 2- ΔΔ Ct method. Data was analysed using at least 3 independent experiments.
pfk13 and PfATPase genotyping
Alleles of Pfk13 propeller domain polymorphisms (Y493H, R539T, I543T, C580Y), and PfATPase6 (S679S, M699V, S769M) associated with delayed clearance were determined by Taqman allelic discrimination and sequencing and list of primers used provided is supplementary materials (Tables S1&2). For each sample a working Master Mix was prepared to include 1x of Taqman Universal PCR Master Mix (Life Technologies), 300nM of the forward and reverse primers, 200 nM of each allele specific probes (Table S2) and at least 5 ng of DNA from DBS in 25 µl reaction volume. Amplification was done on the Bio-Rad CFX96 real-time thermocycler set to detect fluorescent emissions for 6-carboxyfluorescein (6-FAM) (mutant) and hexachloro-6-carboxyfluorescein (HEX) (wild type). Each SNP was amplified in a thermocycle of 50 oC for 2 min, 10 min of initial template denaturation and enzyme activation at 95 oC followed by 50 cycles of 92 oC for 15 seconds and 60 oC for 1 minute. Allelic discrimination analysis was performed with the Bio-Rad CFX manager with parameters set to subtract background and correct for fluorescent drift prior to clustering of wild or mutant amplicons. All PCRs included DNA from P. falciparum 3D7 as wildtype control.
Sequencing
Pfk13, PfATPase6, Pfcrt and Pfmdr1 amplicons were purified from a 0.8% agarose gel and subjected to cycle sequencing using BigDye V3.1 (details in supplementary methods section). Sequencing was done on ABI3130XL DNA analyser.
Data analysis
Descriptive statistics was carried out in Microsoft Excel 2010. Sequence alignment was done on CLC Main Workbench Version 6.7.1 and translation of nucleotide sequence to amino acid sequences and editing were done using MEGA 7.0.4 software. A P-value of ≤ 0.05 was considered statistically significant. Transcript level determination and allelic discrimination analyses were done with the Bio-Rad CFX96 manager software.