Human blood samples and Plasmodium DNA extraction
A total of 46 human blood samples infected with P. knowlesi were collected from symptomatic malaria patients with informed consent in Sabah, Malaysian Borneo, from 2008 to 2011 (Table 1). The sample areas included Sandakan Division (13 samples from Telupid Health Clinic), Interior Division (6 samples from Keningau Hospital, 3 samples from Nabawan Health Clinic, 5 samples from Tambunan Hospital, and 6 samples from Tenom Hospital), and the University of Malaya Medical Centre, Peninsular Malaysia (N = 13). The presence of P. knowlesi parasites in the samples was confirmed by an experienced pathologist and verified using the PlasmoNexTM diagnostic system [19]. Plasmodium DNA was extracted using a previously described approach [20]. Ethical approval for this study was obtained from the Ethics Committee of University of Malaya Medical Centre (reference no. 709.2).
Polymerase chain reaction (PCR) amplification of csp gene
PCR amplification was carried out using a 20-μL reaction mixture containing 1X GoTaq® buffer (Promega, USA), 2.0 mM of MgCl2 solution, 0.2 mM of dNTPs, 0.2 μM of each primer, and 1.25 units of GoTaq® DNA polymerase (Promega, USA). The primers used for csp gene amplification were Pkcsp-F: 5’-TCC TCC ACA TAC TTA TAT ACA AGA-3’ and Pkcsp-R: 5’-GTA CCG TGG GGG ACG CCG-3’, which were derived from a previous study [3]. The PCR conditions were 94oC for 4 min followed by 40 amplification cycles at 94oC for 30 s, 55oC for 50 s, and 72oC for 2 min, and a final extension step at 72oC for 10 min. PCR amplicons were subjected to electrophoresis and analysed in 1% agarose gel stained with ethidium bromide.
Molecular cloning and sequencing
A QIAquick Gel Extraction Kit (Qiagen, Germany) was used to isolate PCR products from the agarose gel, and the purified PCR products were cloned into the pJET1.2/blunt vector using a CloneJET PCR Cloning Kit (Thermo Scientific, USA) according to manufacturer’s instructions. The ligased vectors were transformed into E. coli strain JM109 using a conventional heat-shock method. The desired plasmid containing the csp gene from a single colony was extracted using a QIAprep Spin Miniprep Kit (Qiagen, Germany) according to the manufacturer’s recommendations and subjected to sequencing using pJET1.2 forward sequencing primer.
Sequence alignment and phylogenetic analysis of csp gene
In addition to the 46 csp sequences obtained in this study, a total of 168 csp sequences (Additional file 1) were retrieved from the GenBank database, including 33 sequences from macaques in Sarawak, 24 sequences from macaques in Singapore, 62 sequences from humans in Peninsular Malaysia, 33 sequences from humans in Sarawak, 14 sequences from humans in Singapore, and 2 sequences as an outgroup. Alignment was performed using the CLUSTAL-W tool in Molecular Evolutionary Genetic Analysis 6 (MEGA6) software [21]. Next, the central repeat region of the csp gene was trimmed, whereas the N-terminal (first 195 bp of the coding csp gene) and the C-terminal (last 261 bp of the coding csp gene with exception of the stop codon) non-repeat regions were combined (total length = 453 bp) for the analysis. The maximum likelihood method was used to construct a phylogenetic tree with 1000 bootstrap replicates to test the robustness and reliability of the tree.
Csp sequence diversity, natural selection, and haplotype analyses
Variation in the combined csp sequences was determined using DnaSP ver. 5.10.01 [22]. The data obtained included the average number of pairwise nucleotide differences (K), number of haplotypes (h), haplotype diversity (Hd), and nucleotide diversity (π). An advanced analysis of π was also performed on a sliding window of 100 bases with a step size of 25 bp to estimate the step-wise diversity of csp. In addition, the rates of synonymous (dS) and non-synonymous (dN) mutations were obtained and compared with a Z-test in MEGA6 using Nei and Gojobori’s approach [23] with Jukes and Cantor correction. For testing the neutral theory of evolution, Tajima’s D test [24] as well as Fu and Li’s D and F tests [25] were performed using DnaSP ver. 5.10.01. The median-joining star contraction approach in the software NETWORK ver. 4.6.1.3 [26] was used to generate the relationship of csp haplotypes for isolates obtained in this study.