Plasmodium falciparum Gametocyte Persistence and Within-host Dynamics in an Area of Unstable Malaria in Eastern Sudan- Gedaref

A reverse transcriptase-polymerase chain reaction (RT-PCR) protocol has been developed for the specific detection of Plasmodium falciparum gametocytes. This technique was established on laboratory clone cultures of asexual forms and gametocytes of the parasite. Gametocyte-producing clones 3D7 and HB3, non-gametocyte producing clones 3D7-F12 and T9-94 in addition to 3D7 cultured to produce predominantly asexual forms were used. This method, which combines RT-PCR and nested PCR of mRNA of the Pfs25 (zygote-ookinete surface protein) gene, expressed exclusively in mature gametocytes, was found to detect 1-2 gametocytes μl–1 blood with total specificity with respect to asexual stages. The dynamics of gametocyte production in P. falciparum clones was studied in inhabitants of Asar village, Gedaref State, which is an area of highly seasonal malaria transmission in eastern Sudan. Five cross-sectional surveys were conducted over a 12- month period, from the transmission season of October 1998 through the dry season until the next transmission season. All samples collected during these surveys were examined by microscopy and RT-PCR for the presence of gametocytes. RT-PCR was used to detect the expression of 2 genes that encode gametocyte–specific proteins, Pfs25 and Pfg377 (gamete surface protein), in parasites sampled from individuals throughout the one year period. Pfs25 which has a higher sensitivity for the detection of mature gametocytes than other gametocyte specific proteins was initially used to detect the presence of gametocytes in an infection. Pfg377, being highly polymorphic, was subsequently used to examine the diversity of alleles among the detected gametocytes. All the isolates with microscopically detectable gametocytes produced RT-PCR products for Pfs25. Patent gametocytaemia was very low during the dry season; however, a high proportion of people who contracted the infection during the transmission season maintained sub-patent gametocytaemia detectable by RT-PCR through the following dry season in the apparent absence of mosquito transmission. Consistent gametocytaemia was retained as a chronic infection for more than 9 months. Genotyping of parasites in multiclonal infections showed considerable fluctuation of gametocyte production by individual clones within a single infection. Nevertheless, not all multiple clones produced gametocytes simultaneously. The gametocytes present at the end of the dry season provide the most probable source of the genetically complex cyclical malaria outbreaks following the rainy season in this region. The studied parasite population was genotyped by PCR-amplification of the polymorphic regions of the merozoite surface proteins 1 and 2 (MSP1 and MSP2) and the glutamine rich protein (GLURP) genes. The majority of the non-gametocyte carriers harboured single clone infections (57.6%) and only 5.9% harboured 3 clones. On the other hand, in most of the gametocyte carriers 2 clones were detected which represented 47.6% while 8.7% harboured 3 clones. The mean minimum number of clones (MNC) was significantly higher (P< 0.05) in gametocyte carriers (1.65) compared to non-gametocyte carriers (1.48). These active multiclonal gametocytes are likely to infect mosquitoes that emerge at the start of the rainy season allowing recombination between clones to produce novel genotypes that may circumvent control strategies