Original article. A novel reverse transcription polymerase chain reaction reveals a high prevalence of Plasmodium vivax gametocyte carriage in an endemic area of Thailand

Open access


Background: Gametocytes are precursors of malaria sexual stages that are infective to mosquito vectors and play crucial roles in maintaining cycle of infection. Microscopy cannot determine the status of gametocyte carriage in those who had submicroscopic gametocytemia that may serve as infectious reservoirs in endemic areas. Meanwhile, gametocytes possess stage-specific mRNA that can be detected by molecular methods.

Objective: To develop a sensitive method for detection of Plasmodium vivax gametocytes using reverse transcription polymerase chain reaction (RT-PCR) and determine its diagnostic performance in clinical samples.

Materials and Methods: A nested RT-PCR was devised using primers targeting Pvs25, a mature gametocytespecific mRNA transcript of P. vivax (nested Pvs25 RT-PCR). Performance of the assay was evaluated using mRNA extracted from blood samples of 180 febrile patients attending a malaria clinic in Tak Province. Total RNA was extracted from blood samples that were preserved in RNAlater and from dried blood on filter papers. Malaria species was determined by microscopy from Giemsa stained blood smears and reaffirmed by nested PCR targeting mitochondrial cytochrome b (nested mtCytb PCR).

Results: Of 180 blood samples, malaria was diagnosed in 120 patients (69 P. vivax and 51 P. falciparum) by microscopy and 125 patients by nested mtCytb PCR (69 P. vivax, 51 P. falciparum and 5 coinfections with both these species). Microscopy detected gametocytes in 30 of all 74 (40.5%) P. vivax positives by nested mtCytb PCR. Meanwhile, 67 of 74 (90.5%) P. vivax-positive isolates that were preserved in RNAlater gave positive results by nested Pvs25 RT-PCR. Therefore, nested Pvs25 RT-PCR identified mature P. vivax gametocytes more than twice as frequently as microscopy. The minimum detection threshold for nested Pvs25 RT-PCR was 10 copies of template DNA whereas no cross-reactivity with other human malaria species was observed. Dried blood collected on filter papers offered comparable results for Pvs25 mRNA detection with blood stored in RNA preservative with only 2.7% difference in positive rates.

Conclusion: The nested RT-PCR targeting Pvs25 developed in this study is sensitive and specific for diagnosing mature P. vivax gametocytes and can be efficiently applied to both blood samples kept in RNA preservative and dried blood on filter paper.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1. Hay SI Guerra CA Tatem AJ Noor AM Snow RW. The global distribution and population at risk of malaria: past present and future. Lancet Infect Dis. 2004; 4:327-36.

  • 2. Baird JK. Neglect of Plasmodium vivax malaria. Trends Parasitol. 2007; 23:533-9.

  • 3. Mendis K Sina BJ Marchesini P Carter R. The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg. 2001; 64:97-106.

  • 4. Aide P Bassat Q Alonso PL. Towards an effective malaria vaccine. Arch Dis Child. 2007; 92:476-9.

  • 5. Cui L Yan G Sattabongkot J Cao Y Chen B Chen X et al. Malaria in the Greater Mekong subregion: Heterogeneity and complexity. Acta Trop. 2012; 121: 227-39.

  • 6. Drakeley C Sutherland C Bousema JT Sauerwein RW Targett GA. The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion. Trends Parasitol. 2006; 22:424-30.

  • 7. Karl S Davis TM St. Pierre TG. A comparison of the sensitivities of detection of Plasmodium falciparum gametocytes by magnetic fractionation thick blood film microscopy and RT-PCR. Malar J. 2009; 8:98.

  • 8. Dowling MA Shute GT. A comparative study of thick and thin blood films in the diagnosis of scanty malaria parasitemia. Bull World Health Organ. 1966; 34:249-67.

  • 9. Bousema T Drakeley C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clin Microbiol Rev. 2011; 24:377-410.

  • 10. Putaporntip C Hongsrimuang T Seethamchai S Kobasa T Limkittikul K Cui L et al. Differential prevalence of Plasmodium infections and cryptic Plasmodium knowlesi malaria in humans in Thailand. J Infect Dis. 2009; 199:1143-50.

  • 11. Jongwutiwes S Buppan P Kosuvin R Seethamchai S Pattanawong U Sirichaisinthop J et al. Plasmodium knowlesi malaria in humans and macaques Thailand. Emerg Infect Dis. 2011; 17:1799-806.

  • 12. Putaporntip C Buppan P Jongwutiwes S. Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays. Clin Microbiol Infect. 2011; 17:1484-91.

  • 13. Baker DA. Malaria gametocytogenesis. Mol Biochem Parasitol. 2010; 172:57-65.

  • 14. Pradel G. Proteins of the malaria parasite sexual stages: expression function and potential for transmission blocking strategies. Parasitology. 2007; 134:1911-29.

  • 15. Lasonder E Ishihama Y Andersen JS Vermunt AM Pain A Sauerwein RW et al. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature. 2002; 419:537-42.

  • 16. Alano P Billker O. Gametocytes and gametes. In Molecular approaches to malaria. 1st edition. Edited by Sherman IW. Washington DC: ASM Press; 2005: 191-219.

  • 17. Ou☐draogo AL Bousema T Schneider P de Vlas SJ Ilboudo-Sanogo E Cuzin-Ouattara N et al. Substantial contribution of submicroscopical Plasmodium falciparum gametocyte carriage to the infectious reservoir in an area of seasonal transmission. PLoS One. 2009; 4:e8410.

  • 18. Schneider P Bousema JT Gouagna LC Otieno S van de Vegte-Bolmer M Omar SA et al. Submicroscopic Plasmodium falciparum gametocyte densities frequently result in mosquito infection. Am J Trop Med Hyg. 2007; 76:470-4.

  • 19. Babiker HA Abdel-Wahab A Ahmed S Suleiman S Ranford-Cartwright L Carter R et al. Detection of low level Plasmodium falciparum gametocytes using reverse transcriptase polymerase chain reaction. Mol Biochem Parasitol. 1999; 9:143-8.

  • 20. Bousema T Okell L Shekalaghe S Griffin JT Omar S Sawa P et al. Revisiting the circulation time of Plasmodium falciparum gametocytes: molecular detection methods to estimate the duration of gametocyte carriage and the effect of gametocytocidal drugs. Malar J. 2010; 9:136.

  • 21. Malikul S. The current situation of the anti-malaria programme in Thailand. Southeast Asian J Trop Med Pub Health. 1988; 19:355-9.

  • 22. Bharti AR Chuquiyauri R Brouwer KC Stancil J Lin J Llanos-Cuentas A et al. Experimental infection of the neotropical malaria vector Anopheles darlingi by human patient-derived Plasmodium vivax in the Peruvian Amazon. Am J Trop Med Hyg. 2006; 75: 610-6.

  • 23. Beurskens M Mens P Schallig H Syafruddin D Asih PB Hermsen R et al. Quantitative determination of Plasmodium vivax gametocytes by real-time quantitative nucleic acid sequence-based amplification in clinical samples. Am J Trop Med Hyg. 2009; 81: 366-9.

  • 24. Sattabongkot J Maneechai N Rosenberg R. Plasmodium vivax: gametocyte infectivity of naturally infected Thai adults. Parasitology. 1991; 102:27-31.

  • 25. Smalley ME Brown J Bassett NM. The rate of production of Plasmodium falciparum gametocytes during natural infections. Trans Roy Soc Trop Med Hyg. 1981; 75:318-9.

  • 26. Nacher M Carrara VI McGready R Ashley E Nguen JV Thwai KL et al. Seasonal fluctuations in the carriage of Plasmodium vivax gametocytes in Thailand. Ann Trop Med Parasitol. 2004; 98:115-20.

  • 27. Nakazawa S Culleton R Maeno Y. In vivo and in vitro gametocyte production of Plasmodium falciparum isolates from Northern Thailand. Int J Parasitol. 2011; 41:317-23.

  • 28. Maeno Y Nakazawa S Nagashima S Sasaki J Higo KM Taniguchi K. Utility of the dried blood on filter paper as a source of cytokine mRNA for the analysis of immunoreactions in Plasmodium yoelii infection. Acta Trop. 2003; 87:295-300.

  • 29. Maeno Y Nakazawa S Dao le D Yamamoto N Giang ND Van Hanh T et al. A dried blood sample on filter paper is suitable for detecting Plasmodium falciparum gametocytes by reverse transcription polymerase chain reaction. Acta Trop. 2008; 107:121-7.

Journal information
Impact Factor
5-year IMPACT FACTOR: 0.293

CiteScore 2018: 0.30

SCImago Journal Rank (SJR) 2018: 0.172
Source Normalized Impact per Paper (SNIP) 2018: 0.237

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 106 57 2
PDF Downloads 56 27 0