Temporal Analysis of Genetic Diversity in Plasmodium vivax Field Isolates from the Thai-Myanmar Border (2006-2016)

Background: High-level genetic variants of Plasmodium vivax have previously been reported in Thailand. Circumsporozoite surface protein (CSP), merozoite surface protein (MSP), and microsatellite markers have been used to determine the genetic polymorphisms in P. vivax. This study aimed to investigate the molecular epidemiology of P. vivax populations at the Thai-Myanmar border.

Methodology: A total of 440 Plasmodium vivax clinical isolates were collected from two districts along the Thai-Myanmar border: Mae Sot and Sai Yok, during two sampling periods: 2006–2007 and 2014–2016. The isolates were collected from patients diagnosed with P. vivax malaria by microscopy. Blood samples were preserved on filter papers for subsequent DNA extraction. Genotyping of three P. vivax genes—PvCSP, PvMSP-3\(\alpha\), and PvMSP-3\(\beta\)—was performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. PCR was carried out using primers designed to amplify the CSP gene, which is known to be polymorphic with two major genotypes: VK210 and VK247. The PCR products were analyzed using gel electrophoresis to identify band size variations. The presence of specific alleles was determined by comparing the observed band sizes with the known standards for VK210 and VK247. The PvMSP-3\(\alpha\) and PvMSP-3\(\beta\) genes were genotyped using PCR primers specific to these regions. PCR amplification was followed by RFLP analysis, in which the amplicons were digested with restriction enzymes that produce distinct fragment patterns depending on genetic polymorphisms within these genes. This allowed for the classification of alleles into distinct types. Three genotypic variants (A, B, and C) of both PvMSP-3\(\alpha\) and PvMSP-3\(\beta\) were identified based on the distinct banding patterns observed following RFLP digestion. The allelic diversity within the PvMSP-3\(\alpha\) and PvMSP-3\(\beta\) genes was quantified by determining the number of different alleles present in the population for each gene. The frequency of each allele and its distribution between the two sampling periods (2006–2007 and 2014–2016) were also assessed. A detailed comparison of genetic diversity between PvMSP-3\(\alpha\) and PvMSP-3\(\beta\) was made to evaluate the relative levels of polymorphisms and multiple-genotype infections. Allelic frequencies were calculated, and diversity indices such as the Shannon index were used to quantify genetic variation within the study population. The data were compared between the two time periods to assess any temporal changes in genetic diversity. All statistical analyses were conducted using appropriate software, with significance at p<0.05.

Results: Fourteen different PvCSP alleles were identified based on PCR band size variations: 8 for VK210 and 6 for VK247. The VK210 genotype was the dominant variant during both sample collection periods. For both PvMSP-3\(\alpha\) and PvMSP-3\(\beta\), three distinct types (A, B, and C) were observed. RFLP analysis revealed 28 and 14 allelic variants of PvMSP-3\(\alpha\) and 36 and 20 allelic variants of PvMSP-3\(\beta\) identified during the first and second sampling periods, respectively. PvMSP-3\(\beta\) exhibited higher genetic diversity and a higher frequency of multiple-genotype infections than PvMSP-3\(\alpha\).

Conclusion: The study found high genetic variability of P. vivax at the Thai-Myanmar border, with significant diversity in both PvCSP and PvMSP-3 genes. Among these, PvMSP-3\(\beta\) displayed a higher level of genetic diversity and was associated with multiple-genotype infections, indicating the complexity of P. vivax populations in this region.