BackgroundPlasmodium vivax is divided into two subtypes, a dominant form, VK210 and a variant form, VK247. This division is dependent on the amino acid composition of the circumsporozoite (CS) protein. In this study, the prevalence of the VK247 variant form of P. vivax was investigated in Myanmar.MethodsThe existence of malaria parasites in blood samples was determined by microscopic examination, polymerase chain reaction (PCR) and DNA hybridization assays. To test for antibodies against P. vivax and Plasmodium falciparum in blood samples, an indirect immunofluorescence antibody test (IFAT) was performed using asexual blood antigens. An enzyme-linked immunosorbent assay with synthetic VK210 and VK247 antigens was carried out to discriminate between the P. vivax subtypes.ResultsBy thick smear examination, 73 (n = 100) patients were single infected with P. vivax, one with P. falciparum and 13 with both species. By thin smear, 53 patients were single infected with P. vivax, eight with only P. falciparum and 16 with both. Most of the collected blood samples were shown to be P. vivax positive (n = 95) by PCR. All cases that were positive for P. falciparum by PCR (n = 43) were also positive for P. vivax. However, 52 cases were single infected with P. vivax. IFAT showed antibody titres from 1:32 to 1:4,096. Additionally, using specific antibodies for VK210 and VK247, ELISA showed that 12 patients had antibodies for only the VK210 subtype, 4 patients had only VK247 subtype antibodies and 21 patients had antibodies for both subtypes. Using a DNA hybridization test, 47 patients were infected with the VK210 type, one patient was infected with VK247 and 23 patients were infected with both subtypes.ConclusionsThe proportion of the VK247 subtype in Myanmar was 43.1% (n = 25) among 58 positive cases by serodiagnosis and 25.6% (n = 24) among 94 positive cases by genetic diagnosis. In both diagnostic methods, the infection status of malaria patients is highly diverse with respect to malaria species, and multiple clonal infections are prevalent in Myanmar. Therefore, the complexity of the infection should be considered carefully when diagnosing malaria in Myanmar.

Plasmodium vivax is classified into two serotypes, VK210 [the dominant form-GDRA(D/A)GQPA repeats] and VK247 [the variant form-ANGA(G/D)(N/D)QPG repeats], based on sequence variation of the repeat region of the circumsporozoite (CS) protein gene. Genomic DNA for the variant CS protein gene was obtained from field isolate strains in Myanmar. The repetitive region has highly 19 immunogenic repeats flanked by non-repeat stretches of amino acids. The sequence including this region (717 bp) was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein has a molecular weight of about 50 kDa as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Anti-VK247 antibodies were found in malaria patients who have been exposed to variant form of P. vivax in western blot analysis. Therefore, this recombinant protein might be a useful tool in serodiagnosis of malaria patients who have been infected with variant form of P. vivax.

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