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Multiplicity and diversity of Plasmodium falciparum gametocytes
Malaria Journalvolume 11, Article number: P100 (2012)
Discrimination of gametocyte-producing P. falciparum clones depends on high expression of one or more polymorphic stage-specific markers and on the genetic diversity of these markers in the study area. Pfs230 and pfg377 are classical length-polymorphic markers for differentiation of gametocytes. Because of variable PCR fragment sizes, these markers are particularly well suited to distinguish gametocytes of multiple P. falciparum clones within a patient. We aimed at improving the resolution of both markers by creating amplicons spanning several polymorphic domains of these genes and by increasing the sizing accuracy by capillary electrophoresis using an automated sequencer.
Material and methods
We assessed the genetic diversity and the multiplicity of pfs230 and pfg377 in 80 DNA samples from Papua New Guinea by nested-PCR and following sizing by capillary electrophoresis. We also investigated novel size-polymorphic gametocyte markers, such as PF11.1 (PF10_0374), PF11_0214, PFI0205w and PFL0105w, as well as SNP-based genotyping approaches.
We observed high 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 pfs230 and pfg377 was compared with the asexual MOI by marker msp2 for each sample.
Gametocyte typing of field samples requires RNA sampling and high gametocyte-specific expression of the genotyping marker. A gametocyte trendline was used to evaluate the detection limit of the nested-RT-PCR of pfs230 and pfg377 in comparison to the standard marker for gametocyte detection, pfs25. We discuss the application of high-resolution gametocyte genotyping for studies on malaria transmission.