Prevalence of Plasmodium vivax VK210 and VK247 subtype in Myanmar

Patients attending the Central VBDC (Vector Borne Diseases Control) clinic in Yangon and Wet-Won station hospital (Pyin Oo Lwin District) in Mandalay, Myanmar in January 2000 were selected for this study. All of the individuals exhibited clinical symptoms associated with malaria. Thin and thick blood smears were prepared from the blood collected for microscopic examination (magnification 7 × 100) from the fingertips of individuals. A local health unit employee read the Giemsa-stained thick blood smears in the fields, and treatment was administered to those individuals who tested positive for malaria, based on the guidelines of The Department of Health, the Union of Myanmar. And thin blood smears were examined by co-authors in laboratory condition. Before treatment, additional blood samples of approximately 3 ml were collected from each individual whose infection was confirmed by microscopic examination (n = 100). The blood samples were transferred to the National Institute of Health, Korea Centers for Disease Control and Prevention, Korea, for further polymerase chain reaction (PCR), DNA hybridization genotyping, and antibody analysis. Informed consent was obtained from all patients. The study protocol was approved by the Department of Health (Upper Myanmar), the Union of Myanmar.

For the purpose of preparing variant CS protein genes, genomic DNA was extracted from the whole blood of a malaria patient using the QIAamp Blood Kit (Qiagen Co., Hilden, Germany). PCRs were performed with the AccuPower PCR Premix (Bioneer Co., Taejeon, Korea), 50 ng of purified genomic DNA, 40 pmoles each forward (F1; 5'-TCCCCACGCACTGCGGGCACAAT-3') and reverse primers (R1; 5'-TTAATATGCACCGTGAGGACGCC-3'), and the total volume was adjusted to 20 μl with distilled water. The F1 primer contained several nucleotides of the signal peptide and the first 4 amino acids of the mature protein. The R1 primer contained the region starting 21 bases downstream of the stop codon so that this primer set would amplify the entire mature CS protein gene. The cycling conditions were performed as follows: denatured at 94°C for 5 min; 35 cycles of 1 min at 94°C, 1 min at 49°C, 2 min at 72°C; and a final incubation at 72°C for 5 min. A second PCR performed under the same conditions except that the PCR products (2-5 μl) were added instead of genomic DNA and that different forward (F2; 5'-AAAAAGGATGGAAAGAAAG-3') and reverse (R2; 5'-GACTTTTCATTTGGGGCA-3') primers were included in the reaction [13]. To exclude the blood samples that were co-infected with P. falciparum, PCRs were performed with a P. falciparum specific primer set that amplifies the ring-infected erythrocyte surface antigen (RESA), as described by Wooden et al [forward (5'-GATCAAGGAGGAGAGAACC-3') and reverse (5'-CAGCATTAACACCAACACC-3')] [14]. All the PCR products were analysed on a 1.2% agarose gel, confirmed under UV transillumination and purified with the NucleoSpin Extract kit (Macherey-Nagel, Duren, Germany). The purified PCR products were ligated with the pCR2.1 cloning vector (Invitrogen Co., Carlsbad, CA, USA) and then transformed into E. coli INVαF' according to the procedures of Invitrogen.

Half of the 20 μl PCR products was blotted onto a nylon membrane by a slot blotter. Two oligonucleotides, AL116 (5'-GGTGATAGAGCAGATGGA-3') for VK210 type and AL114 (5'-ATCAACCAGGAGCAAATG-3') for VK247 type, were used as probes in DNA hybridization [15]. These probes were end-labeled with ³⁵S-α-dATP using a 3'-end labeling kit (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 hr at 37°C. The radiolabeled oligonucleotides were separated from unincorporated nucleotides on a Sephadex column (Amersham Pharmacia Biotech). Nylon membranes were soaked for 10 min in denaturation buffer and washed with distilled water, and then incubated with freshly prepared neutralization buffer two times for 15 min. After incubating the nylon membranes in 5× SSC (0.75 M sodium chloride and 0.075 M sodium citrate) for 5 min, the membranes were pre-hybridized for 1 hr at 42°C in 5× SSC, 1% SDS, 1× Denhart's solution (1% BSA, 1% Ficoll 400, and 1% polyvinyl pyrrolidone), and 5% sodium pyrophosphate. Hybridizations were performed in the same buffer with radiolabeled nucleotides for 12 hrs at 42°C. Each nylon membranes was co-reacted with anti-AL116 (5'-CCACTATCTCGTCTACCT-3') and anti-AL114 (5'-TAGTTGGTCCTCGTTTAC-3') for positive controls. After hybridization, nylon membranes were washed three times for 5 min each at 42°C in 5× SSC and 1% sodium pyrophosphate. These nylon membranes were air-dried and exposed to X-ray film (Kodak X-Omat, Sigma, St. Louis, MO, USA) with an intensification screen at -80°C for 72 hrs.

After genotyping P. vivax, several candidates that could contain the variant CS protein gene were selected for DNA sequencing. After gel extraction with a Qiagen gel extraction kit, PCR products were ligated into the pCR2.1 cloning vector and transformed into Escherichia coli INVαF'. The PCR product inserted into E. coli was selected on ampicillin and 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) containing medium. To determine whether the proper DNA was in E. coli, restriction enzyme digestion was done with Eco RI after plasmid isolation with a Qiagen plasmid isolation kit according to the method supplied by manufacture. The variant CS protein gene sequence was determined using the ABI PRISM dye terminator cycle sequencing ready reaction kit FS (Perkin Elmer, Cambridge, MA, USA) according to the supplied manual. M13 reverse and M13 forward (-20) primers were used in sequencing. Nucleotide and deduced amino acid sequences were analysed using EditSeq and Clustal in the Megalign program, and the multiple alignment programs in the DNASTAR package (DNASTAR, Madison, WI, USA). The internet-based BLAST search program of the National Center for Biotechnology Information (NCBI) was used to search protein databases.

To test for antibodies against malaria, an indirect immunofluorescence antibody test (IFAT) was performed with whole blood antigens for P. vivax and P. falciparum as described by Sulzer et al[16]. Briefly, 10 ml of malaria parasite infected blood was collected by venopuncture from P. vivax and P. falciparum patients. After removing the plasma, the cells were suspended in phosphate buffered saline (PBS, pH 7.2) and centrifuged for 5 min at 2,500 rpm. The supernatant was discarded the cells were suspended in fresh PBS and the wash step was repeated three more times. Finally, an appropriate amount of PBS was added to maintain the parasitaemia at no less than 1%. The cells were dropped onto each well of Teflon coated slides. After drying them in room temperature for 12 hrs, the slides were kept at -70°C until required. To determine the antibody titres of each patient against P. vivax and P. falciparum, the antigen slides were fixed in pre-cooled acetone (-20°C) for 10 min, washed with PBS and added 20 μl diluted sera from 1:32 to 1:8,192 (vol/vol) was added to each well. The positive and negative controls were dropped on each slide and incubated in a moisture chamber for 30 min at 37°C. The reactions were stopped by washing out the reacted sera with PBS. The slides were immerged in PBS for 6 min and then dried at room temperature. Diluted FITC conjugated anti-human IgG (Sigma, 1:32 vol/vol in PBS) was added to each well and incubated and washed using the same method described above. Then several drops of buffered glycerol were added to the samples and covered with coverslips. The slides were examined under a 40× fluorescence objective.

To verify that the blood samples had antibodies against the synthetic VK210 and VK247 antigens obtained from R.A Wirtz (Centers for Disease Control and Prevention, Atlanta, GA, USA), an enzyme-linked immunosorbent assay was performed with these antigens. Briefly, 50 μl of capture antigen solution (1 μl antigen/50 μl) was placed in a 96-well plate (Corning, Lowell, MA, USA) and incubated for 12 hrs at room temperature. The cells were aspirated and filled with blocking buffer (1% BSA, 0.05% PBS-Tween 20) and incubated for 1 hr at room temperature. After washing the wells with 0.05% PBS-Tween 20 three times, the human serum samples in blocking buffer at a dilution of 1:100 (vol/vol) were added to each of the wells. The four positive and four negative control serum samples were also added to each plate. After a 2-hr incubation at room temperature, the plates were washed with 0.05% PBS-Tween 20 three times and then the peroxidase conjugated anti-human IgG (Sigma, 1:2,000, vol/vol) diluted in blocking buffer was added and incubated again for 1 hr at room temperature. The reaction was completed by washing the plates as described above. To develop the color, 100 μl of 2.2'-azino-di-(3-ethyl-benzthiozoline-6-sulfonic acid) (ABTS) peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) was added and incubated for 30 min. The absorbance of the mixture was measured at 405 nm, and the cut-off value was taken as the mean ± 2 standard deviations of the negative samples.

To confirm malaria parasite infection from symptomatic patients, thin blood films were examined in fields and thin blood films were examined in the laboratory. As a results of the thick smear examination in the field, 73 patients (n = 100) were found infected with P. vivax, 14 with P. falciparum, and 13 with both species. By thin smear, 53 patients were infected with P. vivax, eight with P. falciparum and 16 with both species. Seven patients were shown to be negative by the thin smear technique.

Most of the collected blood samples were shown to be P. vivax positive (n = 95), and only five were negative by PCR examination. All patients who had P. falciparum also had P. vivax (n = 43). However, 52 patients were infected only with P. vivax (Table 1).

In genotyping by DNA hybridization, 47 patients showed a positive reaction with VK210 (dominant form) specific probe, 23 patients were positive with both probes, and only one patient was positive for the VK247 subtype (variant form) (Table 2).

When patient serum was analysed by IFAT that used P. vivax or P. falciparum-infected red blood cells as antigen, the titre against P. vivax ranged from 1:64 to 1:4,096. Ninety four patients showed high titre over 1:256 which represented current infection. Only six cases showed under 1:128. Seven samples did not have antibodies to P. falciparum, whereas the other samples had titres ranging from 1:32 to 1:4,096. Antibody titres of patients were distributed evenly rather than P. vivax cases and sixty seven cases showed over 1:256 (Table 3).

With regards to the proportion of antibodies against the variant form, 12 patients had antibodies for the VK210 subtype only, 21 patients had them for both and 4 patients only had VK247 subtype antibodies. Of the patients tested, 42 did not have any antibodies for either subtype, even they showed positive reaction in IFAT (Table 4).