The study was conducted in Ganghwa county of Incheon Metropolitan city and Cheorwon county of Gangwon province, ROK. Ganghwa county (37°31′-45 N’, 125°33′-126°2′E) contains 179 villages (administrative village, -ri) in an area of 411 km2 that is covered with forested hilly mountains (181 km2, 44%), agricultural land (164 km2, 40%), building site (13 km2, 3%), and others (52 km2, 13%). The total population of Ganghwa county was 67,668 persons (male, 33,725; female, 38,943) living in 29,055 households in 2010 . Cheorwon county (38°05′-24 N’, 126°56′-127°24′E) contains 109 villages (administrative village, -ri) in an area of 889 km2 that is covered with forested hilly mountains (601 km2, 67%), agricultural land (209 km2, 24%), building site (8 km2, 1%), and others (71 km2, 8%). The total population of Ganghwa county was 49,463 persons (male, 25,820; female, 23,643) living in 20,316 households in 2010 .
Blood sample collection
The study locations are shown on the map in Figure 1. All of the study areas were near the DMZ, which is located along the borderline of DPRK. Blood samples were collected from participants residing in 32 villages among two administrative areas in Ganghwa county of Gyeonggi province and in 10 villages among four administrative areas in Cheorwon county of Gangwon province. It was conducted in November and December of 2010 and in November and December of 2011 in two areas of ROK: Ganghwa county of Incheon city, which contains two islands, Gyodongmyeon and Samsanmyeon (Figure 1A), and Cheorwon county of Gangwon province, which contains 3 –eup and 1 –myeon, Cheorwoneup, Dongsongeup, Gimhwaeup, and Seomyeon which is connected to DPRK by land (Figure 1B). To evaluate CSP recombinant protein as antigen for serodiagnosis, a total of 1,825 (4.77%) and 1,959 (5.12%) blood samples were collected in 2010 and 2011 from who wanted be a volunteer, respectively, from 38,288 total inhabitants in the study areas.
Blood smears were prepared for microscopic examination. Sera were separated and stored at -20°C for antibody analysis. Informed consent was obtained from all individuals. All samples were collected using human protocols that were reviewed and approved by the Human Ethics Committee of Inha University
Thin blood films were prepared to determine the infectivity of blood samples. The blood films were fixed with methanol and stained with Giemsa stain diluted with buffered water at pH 7.2 to emphasize the parasite inclusions in the red blood cells (RBCs). The fixed monolayer of RBCs in this procedure makes the morphological identification of the parasite to the species level much easier and provides greater specificity than that obtained with thick-film examination. Thin blood films are often preferred for routine estimation of parasitaemia because the organisms are easier to see and count with this method . To estimate the densities of blood-stage parasites by microscopy, it was counted the number of asexual parasites observed relative to 200 white blood cells (WBCs) and then multiplied the parasite:WBC ratio by 8,000, that is, the assumed number of WBCs per microlitre of blood .
Amplification of the CSP gene
To express the CSP gene, genomic DNA was extracted from the whole blood of a patient with malaria using a QIAamp Blood Kit (Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) was performed using AccuPower PCR PreMix (Bioneer, Daejeon, Korea), 50 ng of purified genomic DNA, and 40 pmoles each of forward (F1; 5′-CAC GTA GGA CAA AGT GCT AGC CG-3′) and reverse primer (R1; 5-′ATG GAC TCC ATG CAG TGT AA-3′). The total volume was adjusted to 50 μl with distilled water. The thermocycler conditions were as follows: denaturation at 94°C for 5 min; 35 cycles of 30 s at 94°C, 30 s at 55°C, and 45 s at 72°C; and finally, incubation at 72°C for 5 min. All PCR products were analyzed on a 1% agarose gel, confirmed under a UV transilluminator, and purified with a NucleoSpin Extract Kit (Macherey-Nagel, Duren, Germany).
DNA sequencing and analysis
To genotype the CSP gene of P. vivax, the PCR product of the CSP gene was ligated into a pGEM-T Easy Vector (Promega, Madison, WI, USA) and transformed into E. coli DH5α. The PCR product containing E. coli DH5α was selected on ampicillin-containing medium . To confirm the transformants, gel electrophoresis was performed with EcoRI digestion products after the plasmid was prepared with a Qiagen plasmid isolation kit according to the protocol supplied by the manufacturer. The CSP gene sequence was determined using an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit FS (Perkin Elmer, Cambridge, MA, USA) according to the manual supplied by the manufacturer. M13 reverse and M13 forward (-20) primers were used in the sequencing. Nucleotide and deduced amino acid sequences were analyzed using EditSeq and Clustal in the MegAlign program, a multiple alignment program in the DNASTAR package (DNASTAR, Madison, WI, USA). The internet-based BLAST search program of the National Center for Biotechnology Information was used to search protein databases.
Construction of the CSP expression vector
To express the CSP gene in E. coli DH5α, a CSP gene fragment was amplified from a blood sample that was confirmed to be infected with the dormant type of P. vivax as described above with the exception of the addition of the Fex2 (5′-ggatccAAAAAGGATGGAAAGAAAG-3′) and Rex2 (5′-aagcttGACTTTTCATTTGGGGCA-3′) primers, which contain BamHI and HindIII sites on their 5′ ends, respectively. The amplified PCR products were digested with BamHI and HindIII, purified with a Qiagen gel extraction kit after being run on an agarose gel, and then integrated into the BamHI and HindIII cleavage sites of the pET-28a(+) expression vector (Novagen, 5,369 bp). The resulting plasmid was subsequently used for the expression of a CSP-(His)6 fusion protein in E. coli. The transformants were confirmed by both gel electrophoresis of plasmid DNA after restriction enzyme digestion with BamHI and HindIII and DNA sequencing.
Expression and purification of recombinant CSP
Expression of the recombinant protein was induced in E. coli DH5α with isopropyl-1-thio-β-D-galactopyranoside (IPTG) . The CSP-(His)6 fusion protein was purified using immobilized metal ion affinity chromatography . The purification was performed under native conditions according to the supplier’s protocol (Novagen). Proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after each purification step.
Western blot analysis
The recombinant CSP-(His)6 fusion protein was separated on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. After the transfer, the membrane was cut into strips and blocked for nonspecific binding with 3% skim milk for 12 h at 4°C. The membrane was then washed three times for 10 min each with 0.15% Tween 20-PBS. The strips were allowed to react with sera from patients with malaria or from uninfected individuals (diluted 1:100, vol/vol) for 4 h; they were then washed using the procedure described above. The membrane was subsequently incubated with diluted peroxidase-conjugated goat anti-human IgG secondary antibody (1:1,000, v/v) (Sigma) for 3 h at room temperature. For colour development, a solution containing 0.2% diaminobenzidine and 0.02% H2O2/PBS was applied to each well [22, 23].
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assays (ELISAs) were used to determine whether the blood samples contained antibodies against the CSP VK210 of P. vivax. Briefly, the capture antigen solution (50 ml, 0.5 μg/ml) was placed on a 96-well plate (Corning, Lowell, MA, USA) and incubated for 12 h at room temperature. The wells were aspirated and filled with blocking buffer (1% BSA, 0.05% PBS-Tween 20) and incubated for 1 h at room temperature. After the wells were washed with 0.05% PBS-Tween 20 three times, human serum samples in blocking buffer at a dilution of 1:100 (vol/vol) were added to the wells. Four positive and four negative control serum samples were also added to each plate. After 2 h of incubation at room temperature, the plates were washed with 0.05% PBS-Tween 20 three times, and peroxidase-conjugated anti-human IgG (Sigma, 1:2,000, vol/vol) diluted in blocking buffer was then added. The plates were re-incubated for 1 h at room temperature. The reaction was stopped by washing the plates as described above. To develop the colour, 100 μl of 2.2′-azino-di-(3-ethyl-benzthiozoline-6-sulfonic acid) peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) was added, and the plates were incubated for 30 min. Absorbance was measured at 405 nm, and the cut-off value for positivity was defined as the mean + 3 standard deviations of the negative control samples. Negative sera were collected from volunteers among the staff of the Korea National Institute of Health (KNIH).
Calculation of the annual parasite incidence
The annual parasite incidence (API) was calculated as the number of malaria-positive patients per 1,000 inhabitants for each of the study sites using microscopy: API = (number of positive slides / total number of slides) × 1,000.
The differences in the positive CSP rates between 2010 and 2011 were determined by a Mann–Whitney test. Data analyses were performed using GraphPad software (GraphPad Software Inc., La Jolla, CA, USA). Pearson’s correlation analysis was performed to examine the relationship between seropositivity and the API of P. vivax in a given year. The data were analyzed using SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA). A P value of <0.05 was considered statistically significant. The correlation sizes were interpreted as none (0.0–0.09), small (0.1–0.3), medium (0.3–0.5), or strong (0.5–1.0) .