Artikel
Detection of P. falciparum and P. vivax gametocytes in field surveys
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Veröffentlicht: | 8. Januar 2013 |
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Gliederung
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Background: Measurements of the prevalence of Plasmodium sp. gametocytes may serve as a tool to monitor the success of antimalarial interventions, in particular in the context of renewed efforts to eliminate malaria. We developed tools for detection and quantification of P. vivax and P. falciparum gametocytes in field surveys, and evaluated several markers for differentiating individual gametocyte-producing clones of P. falciparum.
Methods: In cross-sectional samples from 315 Papua New Guinean children aged 5–10 years, three RNA sampling methods were tested in parallel: whole blood into RNAprotect (Qiagen), whole blood on Whatman 3MM paper stored in TRIzol reagent, and whole blood on Whatman FTA filter cards. Sequential qRT-PCR analyses were performed to gain prevalence data for asexual parasites and for sexual stages. We also assessed the genetic diversity of pfs230 and pfg377 in a set of 80 DNA samples by nested-PCR and subsequent sizing by capillary electrophoresis. We also investigated novel size-polymorphic gametocyte markers, such as PF11.1 (PF10_0374), PF11_0214, PFI1210w, PFL0545w. A gametocyte trendline was used to evaluate the detection limit of the nested-RT-PCR of theses markers.
Results: Sampling and storing finger prick blood in RNAprotect was found to be most sensitive and suitable for high sample throughput. Of 76 P. falciparum positive study participants, 31 (40.8%) carried gametocytes. Of 121 P. vivax positive samples, 44 (36.4%) were gametocyte positive. For our gametocyte-genotyping markers we observed high genetic diversity with pfs230 (He=96.3) and pfg377 (He=89.4). 17 and 13 different alleles were found for pfs230 and pfg377, respectively. The multiplicity of infection (MOI) of these stage-specific markers was lower than MOI by marker msp2, but showed the highest discriminatory power for gametocytes in literature.
Conclusion: Although prevalence of gametocytes was lower in P. vivax than in P. falciparum infections, the reservoir of asymptomatic schoolchildren carrying gametocytes was 11.2% (33/295) and 7.5% (22/295) for P. vivax and P. falciparum, respectively. The contribution of these asymptomatic children to the overall transmission needs to be considered in antimalarial interventions. Gametocyte typing of field samples requires RNA sampling and high gametocyte-specific expression of the genotyping marker. We discuss the application of high-resolution gametocyte genotyping for studies on malaria transmission.