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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.