RESUMO
In areas highly endemic for malaria, individuals are frequently found to be infected simultaneously with multiple Plasmodium falciparum clones. This raises the question of whether all parasite clones produce gametocytes equally or whether gametocytogenesis is suppressed in some clones. In order to assess this in epidemiological studies, polymorphic genes specifically expressed in gametocytes could be analyzed by both amplification of genomic DNA from blood samples and by reverse transcribed polymerase chain reaction amplifying expressed gametocyte-specific genes only. Here we report the analysis of diversity in the three gametocyte-specific genes Pfs16, Pfs48/45, and Pfs230. In addition to the previously published data, limited polymorphism was found in the coding sequences of Pfs16 and Pfs48/45. Larger polymorphism was identified in Pfs230, which might allow the development of a discriminating PCR-based genotyping scheme for transmission studies. However, the limited polymorphism in Pfs16 and Pfs48/45 renders these molecules poorly useful for such studies.
Assuntos
Antígenos de Protozoários/genética , DNA de Protozoário/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Células Germinativas , Humanos , Dados de Sequência Molecular , Mutação , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In order to facilitate molecular epidemiological studies on the transmission of the malaria parasite Plasmodium falciparum a sensitive assay for gametocyte detection based on RT-PCR was developed. The transcription of the sexual stage-specific genes Pfs16, Pfs48/45, Pfs230, and Pfs25, as well as the sexual stage- and sporozoite-specific S 18S rRNA, was detected by RT-PCR. S 18S rRNA was present in seven of nine P. falciparum-positive blood samples, despite the lack of microscopic detection of gametocytes and a parasitemia below 0.1%. Expression of the other four gametocyte-specific genes was detected less frequently in malaria-positive blood samples. These findings indicate that RT-PCR of S 18S rRNA is a highly sensitive method for gametocyte detection and, furthermore, that gametocytes are present in the peripheral blood of most malaria carriers, even if the parasitemia is below 0.1%. To determinate the expression pattern of sexual stage-specific genes in more detail, RT-PCR was performed at consecutive time points of highly synchronized monolayer cell cultures. Transcripts of all examined genes except Pfs25 were detected directly after invasion of merozoites of the strain NF54 in red blood cells.