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

Martijn Beurskens Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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Pètra Mens Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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Henk Schallig Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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Din Syafruddin Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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Puji Budi Setia Asih Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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Rob Hermsen Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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Robert Sauerwein Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT)/Royal Tropical Institute, Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The Netherlands; Eijkman Institute for Molecular Biology, Jakarta, Indonesia

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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.

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