Genetic Diversity of Plasmodium vivax in Clinical Isolates from Southern Thailand using PvMSP1, PvMSP3 (PvMSP3α, PvMSP3β) Genes and Eight Microsatellite Markers

  • Received : 2019.04.18
  • Accepted : 2019.09.21
  • Published : 2019.10.31


Plasmodium vivax is usually considered morbidity in endemic areas of Asia, Central and South America, and some part of Africa. In Thailand, previous studies indicated the genetic diversity of P. vivax in malaria-endemic regions such as the western part of Thailand bordering with Myanmar. The objective of the study is to investigate the genetic diversity of P. vivax circulating in Southern Thailand by using 3 antigenic markers and 8 microsatellite markers. Dried blood spots were collected from Chumphon, Phang Nga, Ranong and, Surat Thani provinces of Thailand. By PCR, 3 distinct sizes of $PvMSP3{\alpha}$, 2 sizes of $PvMSP3{\beta}$ and 2 sizes of PvMSP1 F2 were detected based on the length of PCR products, respectively. PCR/RFLP analyses of these antigen genes revealed high levels of genetic diversity. The genotyping of 8 microsatellite loci showed high genetic diversity as indicated by high alleles per locus and high expected heterozygosity ($H_E$). The genotyping markers also showed multiple-clones of infection. Mixed genotypes were detected in 4.8% of $PvMSP3{\alpha}$, 29.1% in $PvMSP3{\beta}$ and 55.3% of microsatellite markers. These results showed that there was high genetic diversity of P. vivax isolated from Southern Thailand, indicating that the genetic diversity of P. vivax in this region was comparable to those observed other areas of Thailand.


Supported by : Prince of Songkla University


  1. World Health Organization. World Malaria Report 2016. Geneva, Switzerland. World Health Organization. 2016.
  2. World Health Organization. World Malaria Report 2015. Geneva, Switzerland. World Health Organization. 2015.
  3. Wongsrichanalai C, Meshnick SR. Declining artesunate-mefloquine efficacy against falciparum malaria on the Cambodia-Thailand border. Emerg Infect Dis 2008; 14: 716-719.
  4. Rijken MJ, Boel ME, Russell B, Imwong M, Leimanis ML, Phyo AP, Muehlenbachs A, Lindegardh N, McGready R, Renia L, Snounou G, Singhasivanon P, Nosten F. Chloroquine resistant vivax malaria in a pregnant woman on the western border of Thailand. Malar J 2011; 10: 113.
  5. Howes RE, Piel FB, Patil AP, Nyangiri OA, Gething PW, Dewi M, Hogg MM, Battle KE, Padilla CD, Baird JK, Hay SI. G6PD deficiency prevalence and estimates of affected populations in malaria endemic countries: a geostatistical model-based map. PLoS Med 2012; 9: e1001339.
  6. Kitchen SF. The infection of reticulocytes by Plasmodium vivax. Am J Trop Med Hyg 1938; 18: 347-359.
  7. Mons B CJ, van der Star W, van der Kaay HJ. Erythrocytic schizogony and invasion of Plasmodium vivax in vitro. Int J Parasitol 1988; 18: 307-311.
  8. Orjuela-Sanchez P, Sa JM, Brandi MC, Rodrigues PT, Bastos MS, Amaratunga C, Duong S, Fairhurst RM, Ferreira MU. Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia. Exp Parasitol 2013; 134: 318-326.
  9. Ferreira MU, Karunaweera ND, da Silva-Nunes M, da Silva NS, Wirth DF, Hartl DL. Population structure and transmission dynamics of Plasmodium vivax in rural Amazonia. J Infect Dis 2007; 195: 1218-1226.
  10. Gunawardena S, Karunaweera ND, Ferreira MU, Phone-Kyaw M, Pollack RJ, Alifrangis M, Rajakaruna RS, Konradsen F, Amerasinghe PH, Schousboe ML, Galappaththy GN, Abeyasinghe RR, Hartl DL, Wirth DF. Geographic structure of Plasmodium vivax: microsatellite analysis of parasite populations from Sri Lanka, Myanmar, and Ethiopia. Am J Trop Med Hyg 2010; 82: 235-242.
  11. Lee SA, Yeka A, Nsobya SL, Dokomajilar C, Rosenthal PJ, Talisuna A, Dorsey G. Complexity of Plasmodium falciparum infections and antimalarial drug efficacy at 7 sites in Uganda. J Infect Dis 2006; 193: 1160-1163.
  12. Zakeri S, Barjesteh H, Djadid ND. Merozoite surface protein-3alpha is a reliable marker for population genetic analysis of Plasmodium vivax. Malar J 2006; 5: 53.
  13. Imwong M, Pukrittayakamee S, Gruner AC, Renia L, Letourneur F, Looareesuwan S, White NJ, Snounou G. Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1. Malar J 2005; 4: 20.
  14. Kim JR, Imwong M, Nandy A, Chotivanich K, Nontprasert A, Tonomsing N, Maji A, Addy M, Day NPJ, White NJ, Pukrittayakamee S. Genetic diversity of Plasmodium vivax in Kolkata, India. Malar J 2006; 5: 71.
  15. Rayner JC, Huber CS, Feldman D, Ingravallo P, Galinski MR, Barnwell JW. Plasmodium vivax merozoite surface protein PvMSP-3 beta is radically polymorphic through mutation and large insertions and deletions. Infect Genet Evol 2004; 4: 309-319.
  16. Yang Z, Miao J, Huang Y, Li X, Putaporntip C, Jongwutiwes S, Gao Q, Udomsangpetch R, Sattabongkot J, Cui L. Genetic structures of geographically distinct Plasmodium vivax populations assessed by PCR/RFLP analysis of the merozoite surface protein 3beta gene. Acta Trop 2006; 100: 205-212.
  17. Gomez JC, McNamara DT, Bockarie MJ, Baird JK, Carlton JM, Zimmerman PA. Identification of a polymorphic Plasmodium vivax microsatellite marker. Am J Trop Med Hyg 2003; 69: 377-379.
  18. Imwong M, Nair S, Pukrittayakamee S, Sudimack D, Williams JT, Mayxay M, Newton PN, Kim JR, Nandy A, Osorio L, Carlton JM, White NJ, Day NP, Anderson TJ. Contrasting genetic structure in Plasmodium vivax populations from Asia and South America. Int J Parasitol 2007; 37: 1013-1022.
  19. Thanapongpichat S, McGready R, Luxemburger C, Day NP, White NJ, Nosten F, Snounou G, Imwong M. Microsatellite genotyping of Plasmodium vivax infections and their relapses in pregnant and non-pregnant patients on the Thai-Myanmar border. Malar J 2013; 12: 275.
  20. Kittichai V, Koepfli C, Nguitragool W, Sattabongkot J, Cui L. Substantial population structure of Plasmodium vivax in Thailand facilitates identification of the sources of residual transmission. PLoS Negl Trop Dis 2017; 11: e0005930.
  21. Kosaisavee V, Hastings I, Craig A, Lek-Uthai U. The genetic polymorphism of Plasmodium vivax genes in endemic regions of Thailand. Asian Pac J Trop Med 2011; 4: 931-936.
  22. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, Thaithong S, Brown KN. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasitol 1993; 61: 315-320.
  23. Bruce MC, Galinski MR, Barnwell JW, Snounou G, Day KP. Polymorphism at the merozoite surface protein-3alpha locus of Plasmodium vivax: global and local diversity. Am J Trop Med Hyg 1999; 61: 518-525.
  24. Karunaweera ND, Ferreira MU, Hartl DL, Wirth DF. Fourteen polymorphic microsatellite DNA markers for the human malaria parasite Plasmodium vivax. Mol Ecol Notes 2007; 7: 172-175.
  25. Anderson TJ, Su XZ, Bockarie M, Lagog M, Day KP. Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples. Parasitology 1999; 119: 113-125.
  26. Goudet J. Fstat version 1.2: a computer program to calculate F-statistics. J Heredity. 86: 485-486.
  27. Hudson RR. Analytical results concerning linkage disequilibrium in models with genetic transformation and conjugation. J Evol Biol 1994; 7: 535-548.
  28. Smith JM, Smith NH, Rourke M, Spratt BG. How clonal are bacteria? Proc Natl Acad Sci USA 1993; 90: 4384.
  29. Haubold B, Hudson RR. LIAN 3.0: detecting linkage disequilibrium in multilocus data. Bioinformatics 2000; 16: 847-849.
  30. Sattabongkot J, Tsuboi T, Zollner GE, Sirichaisinthop J, Cui L. Plasmodium vivax transmission: chances for control? Trends Parasitol 2004; 20: 192-198.
  31. Boyd MF, Kitchen SF. Simultaneous Inoculation with Plasmodium vivax and Plasmodium falciparum. Am J Trop Med Hyg 1937; 17: 855-861.
  32. Boyd MF, Kitchen SF. Vernal vivax activity in persons simultaneously inoculated with Plasmodium vivax and Plasmodium falciparum. Am J Trop Med Hyg 1938; 18: 505-514.
  33. James SP. Some general results of a study of induced malaria in England. Trans R Soc Trop Med Hyg 1931; 24: 477-525.
  34. Nigatu W, Abebe M, Dejene A. Plasmodium vivax and P. falciparum epidemiology in Gambella, south-west Ethiopia. Trop Med Parasitol 1992; 43: 181-185.
  35. Somboon P, Suwonkerd W, Lines JD. Susceptibility of Thai zoophilic Anophelines and suspected malaria vectors to local strains of human malaria parasites. Southeast Asian J Trop Med Public Health 1994; 25: 766-770.
  36. Limrat D, Rojruthai B, Apiwathnasorn C, Samung Y, Prommongkol S. Anopheles barbirostris/campestris as a probable vector of malaria in Aranyaprathet, Sa Kaeo Province. Southeast Asian J Trop Med Public Health 2001; 32: 739-744.
  37. Rayner JC, Corredor V, Feldman D, Ingravallo P, Iderabdullah F, Galinski MR, Barnwell JW. Extensive polymorphism in the plasmodium vivax merozoite surface coat protein MSP-3alpha is limited to specific domains. Parasitology 2002; 125: 393-405.
  38. Cui L, Mascorro CN, Fan Q, Rzomp KA, Khuntirat B, Zhou G, Chen H, Yan G, Sattabongkot J. Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand. Am J Trop Med Hyg 2003; 68: 613-619.
  39. Zakeri S, Safi N, Afsharpad M, Butt W, Ghasemi F, Mehrizi AA, Atta H, Zamani G, Djadid ND. Genetic structure of Plasmodium vivax isolates from two malaria endemic areas in Afghanistan. Acta Trop 2010; 113: 12-19.
  40. Zakeri S, Raeisi A, Afsharpad M, Kakar Q, Ghasemi F, Atta H, Zamani G, Memon MS, Salehi M, Djadid ND. Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3alpha and pvcsp genes as molecular markers. Parasitol Int 2010; 59: 15-21.
  41. Cui L, Escalante AA, Imwong M, Snounou G. The genetic diversity of Plasmodium vivax populations. Trends Parasitol 2003; 19: 220-226.
  42. Prajapati SK, Joshi H, Valecha N. Plasmodium vivax merozoite surface protein-3 alpha: a high-resolution marker for genetic diversity studies. J Vector Borne Dis 2010; 47: 85-90.
  43. Veron V, Legrand E, Yrinesi J, Volney B, Simon S, Carme B. Genetic diversity of msp3alpha and msp1_b5 markers of Plasmodium vivax in French Guiana. Malar J 2009; 8: 40.
  44. Khatoon L, Baliraine FN, Bonizzoni M, Malik SA, Yan G. Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan. Malar J 2010; 9: 112.
  45. Rungsihirunrat K, Chaijaroenkul W, Siripoon N, Seugorn A, Na-Bangchang K. Genotyping of polymorphic marker (MSP3alpha and MSP3beta) genes of Plasmodium vivax field isolates from malaria endemic of Thailand. Trop Med Int Health 2011; 16: 794-801.
  46. Gupta P, Pande V, Eapen A, Singh V. Genotyping of $MSP3{\beta}$ gene in Indian Plasmodium vivax. J Vector Borne Dis 2013; 50: 197-201.
  47. Iwagami M FM, Hwang SY, Kim SH, Kho WG, Kano S. Population structure and transmission dynamics of Plasmodium vivax in the Republic of Korea based on microsatellite DNA analysis. PLoS Negl Trop Dis 2012; 6: e1592.
  48. Menegon M, Bardaji A, Martinez-Espinosa F, Botto-Menezes C, Ome-Kaius M, Mueller I, Betuela I, Arevalo-Herrera M, Kochar S, Kochar SK, Jaju P, Hans D, Chitnis C, Padilla N, Castellanos ME10, Ortiz L, Sanz S, Piqueras M, Desai M, Mayor A, Del Portillo H, Menendez C, Severini C. Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries. PLoS One 2016; 11: e0152447.
  49. Havryliuk T, Ferreira MU. A closer look at multiple-clone Plasmodium vivax infections: detection methods, prevalence and consequences. Mem Inst Oswaldo Cruz 2009; 104: 67-73.