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.