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-3α, and PvMSP-3β—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-3α and PvMSP-3β 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-3α and PvMSP-3β were identified based on the
distinct banding patterns observed following RFLP digestion. The allelic
diversity within the PvMSP-3α and PvMSP-3β 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-3α and PvMSP-3β 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-3α
and PvMSP-3β, three distinct types (A, B, and C) were observed. RFLP analysis
revealed 28 and 14 allelic variants of PvMSP-3α and 36 and 20 allelic variants of
PvMSP-3β identified during the first and second sampling periods, respectively.
PvMSP-3β exhibited higher genetic diversity and a higher frequency of
multiple-genotype infections than PvMSP-3α.
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-3β displayed a higher level of genetic
diversity and was associated with multiple-genotype infections, indicating the
complexity of P. vivax populations in this region.
Author
(s) Details
Abdifatah Abdullahi
Jalei
Chulabhorn International College of Medicine, Thammasat University (Rangsit
Campus), Pathumthani 12121, Thailand.
Wanna Chaijaroenkul
Drug Discovery and Development Center, Thammasat University (Rangsit
Campus), Pathum Thani 12121, Thailand.
Kesara Na-Bangchang
Chulabhorn International College of Medicine, Thammasat University (Rangsit
Campus), Pathumthani 12121, Thailand, Drug Discovery and Development Center,
Thammasat University (Rangsit Campus), Pathum Thani 12121, Thailand and Center
of Excellence in Pharmacology and Molecular Biology of Malaria and
Cholangiocarcinoma, Chulabhorn International College of Medicine (Rangsit
Campus), Thammasat University, Pathum Thani 12121, Thailand.
Please see the book here:- https://doi.org/10.9734/bpi/acmms/v8/3512
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