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Quantitative Determination of Plasmodium vivax Gametocytes by Real-Time Quantitative Nucleic Acid Sequence-Based Amplification in Clinical Samples

Microscopic detection of Plasmodium vivax gametocytes, the sexual life stage of this malaria parasite, is insensitive because P. vivax parasitaemia is low. To detect and quantify gametocytes a more sensitive, quantitative real-time Pvs25-QT-NASBA based on Pvs25 mRNA was developed and tested in two clinical sample sets from three different continents. Pvs25-QT-NASBA is highly reproducible with low inter-assay variation and reaches sensitivity approximately 800 times higher than conventional microscopic gametocyte detection. Specificity was tested in 104 samples from P. vivax-, P. falciparum-, P. malariae-, and P. ovale-infected patients. All non-vivax samples were negative in the Pvs25-QT-NASBA; out of 74 PvS18-QT-NASBA positive samples 69% were positive in the Pvs25-QT-NASBA. In a second set of 136 P. vivax microscopically confirmed samples, gametocyte prevalence was 8%, whereas in contrast 66% were positive by Pvs25-QT-NASBA. The data suggest that the human P. vivax gametocyte reservoir is much larger when assessed by Pvs25-QT-NASBA than by microscopy.

Received January 13, 2009. Accepted for publication May 5, 2009.

Acknowledgments: We thank the community in West Sumba and West Papua, Indonesia for their cooperation and Adrian Luty for critical reading of the manuscript.

Financial support: This work was in part financially supported by a grant from The Netherlands Foundation for the Advancement of Tropical Research (WOTRO contract 01.83.2004.006 and W1293-465).

^* Address correspondence to Rob Hermsen, Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail: R.Hermsen{at}ncmls.ru.nl

Authors’ addresses: Martijn Beurskens, Rob Hermsen, and Robert Sauerwein, Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands, E-mails: martijnbeurskens{at}gmail.com, r.hermsen{at}ncmls.ru.nl, and r.sauerwein{at}mmb.umcn.nl. Pètra Mens and Henk Schallig, KIT Biomedical Research, Royal Tropical Institute, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands/Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Meiberg-dreef 9, 1105 AZ Amsterdam, The Netherlands, E-mails: p.mens{at}kit.nl and h.schallig{at}kit.nl. Din Syafruddin and Puji Budi Setia Asih, Eijkman Institute for Molecular Biology, Jl. Diponegoro No. 69, Jakarta 10430, Indonesia, E-mails: din{at}eijkman.go.id and puji_bsa{at}yahoo.com.