(Sub)microscopic Plasmodium falciparum gametocytaemia in Kenyan children after treatment with sulphadoxine-pyrimethamine monotherapy or in combination with artesunate
Introduction
Clinical symptoms of malaria are caused by cyclical proliferation of asexual Plasmodium parasites in the patient's red blood cells. Spread of the disease depends on the presence of mature sexual stage parasites (gametocytes), which do not replicate or cause clinical symptoms, but are essential for transmission from humans to the mosquito vector.
As measures to prevent malaria are not completely efficient, early treatment of symptomatic infections is a major component of malaria control strategies. Drugs are designed to cure clinical symptoms and therefore mostly target asexual stages; the impact of drug treatment on gametocytes and transmission has long been neglected. Some drugs increase (e.g. chloroquine and sulphadoxine-pyrimethamine) or decrease (e.g. artemisinin derivatives) gametocyte prevalence (Targett et al., 2001; von Seidlein et al., 2001; Sowunmi and Fateye, 2003; Sutherland et al., 2005). Due to increasing resistance to most affordable antimalarial drugs, many countries are forced to revise drug treatment strategies. Generally, artemisinin combination therapies (ACT) are recommended to replace current first line drugs such as chloroquine and sulphadoxine-pyrimethamine (SP). Combination of artemisinin derivatives with other antimalarial drugs may protect the other drug from development of resistance. The benefit of better cure rates induced by ACT combined with its effect on transmission due to its alleged gametocytocidal effects (Dutta et al., 1989) may justify increased costs for treatment.
The key factors that trigger sexual stage development are not yet understood. Gametocytes are derived only from a small subset of asexual parasites and only a fraction of patients develop patent gametocytaemia (Shute and Maryon, 1951). However, the apparently low prevalence of gametocytes contrasts with the successful spread of malaria and the difficulties to control malaria transmission (Taylor and Read, 1997). Recently, a number of studies have addressed the effect of antimalarial drug treatment on gametocytes (Sokhna et al., 2001; Osorio et al., 2002; Sutherland et al., 2002; Tjitra et al., 2002; Sowunmi and Fateye, 2003; Suputtamongkol et al., 2003; Hallett et al., 2004). Most of these studies used microscopy for detection and quantification of gametocytes, but it has been shown that patients without microscopically detectable gametocytes can infect mosquitoes (Githeko et al., 1992; Boudin et al., 1993) and submicroscopic gametocytaemia can be common (Babiker et al., 1999; Menegon et al., 2000; Abdel-Wahab et al., 2002; Nassir et al., 2005). Detailed quantitative studies on submicroscopic gametocytes are now possible with the recently developed gametocyte-specific Pfs25 quantitative Nucleic Acid Sequence-Based Amplification (QT-NASBA) (Schneider et al., 2004). The assay is performed in real-time, and quantifies mature Plasmodium falciparum gametocytes in blood samples with a lower detection limit of 20–100 gametocytes per millilitre of blood. The objective of this study was to investigate the effect of SP and of SP plus artesunate (SP+AS) treatment on P. falciparum gametocyte dynamics using Pfs25 real-time QT-NASBA.
Section snippets
Real-time Pfs25 QT-NASBA and nucleic acid extraction
Nucleic acids were extracted from blood samples using the Guanidiumisothiocyanate (GuSCN)/silica procedure as described by Boom et al. (1990). Total parasite load was quantified by real-time 18S rRNA QT-NASBA as previously described (Schneider et al., 2005). Pfs25 mRNA QT-NASBA was performed as described by Schneider et al. (2004) with some adjustments to enable real-time detection. Briefly, real-time QT-NASBA for Pfs25 mRNA (Genbank accession number AF193769.1) was performed on a NucliSens
Detection limit and interassay variation of real-time Pfs25 QT-NASBA
Eighteen independent gametocyte series from in vitro parasite culture were collected and processed over a period of 2 months. Gametocyte densities from 102 to 106 gametocytes/ml could be detected consistently with 10 gametocytes per millilitre being close to the detection limit of 20–100 gametocytes per millilitre of blood. Interassay variability (SD/mean×100%), including variations in culture material and nucleic acid extractions, is below 10% for all measured gametocyte densities and is
Discussion
The real-time Pfs25 QT-NASBA is more sensitive for quantification of P. falciparum gametocytes compared with standard microscopy (detection limits of 20–100 and 16,000 gametocytes per millilitre of blood, respectively). Inconsistent results between gametocyte detection by microscopy or Pfs25 QT-NASBA, including both false positive and false negative results, occurred in 3.7% of all samples. Similar low percentages of inconsistent results were found during a cross-sectional study in Burkina Faso
Acknowledgements
This work was supported by the Technology Foundation STW (NFA6009) and WOTRO (2003/00702). We thank the people from Mbita for their cooperation; George Omweri, Nick Makio, Bernard Kapesa of ICIPE for their work in the clinic; Stephen Kaniaru of KEMRI for his microscopic analyses and sharing his experience in clinical studies; Marga van de Vegte-Bolmer of Radboud University Nijmegen for gametocyte production in the in vitro parasite culture and Gerard Schoone of KIT Biomedical Research for his
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