A Portfolio of Geographically Distinct Laboratory-adapted Plasmodium Falciparum Clones With Consistent Infection Rates in Anopheles Mosquitoes

BackgroundThe ability to culture P. falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture adapted transmissible P. falciparum isolates, originating from distinct geographical locations.ResultsOut of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to A. stephensi or A. coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). ConclusionsThese new P. falciparum culture adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

Shifts in P. falciparum genetic structure and gametocyte markers in the transition to elimination

Large-scale programs targeting Plasmodium falciparum (Pf) elimination can exert strong selection pressures on the parasite population. To better understand the impact that elimination initiatives can have on Pf genetic structure and gametocyte carriage, we applied amplicon-based sequencing of two polymorphic Pf genes and quantitative reverse-transcription PCR targeting gametocyte-specific genes to Pf isolates collected in Magude District (Southern Mozambique) before and after an elimination initiative. The 71% reduction of Pf prevalence achieved in 2 years was followed by reductions in Pf genetic diversity and increases in between-infection similarity. These genetic shifts were accompanied by increases in the relative transcript number of the female mature gametocyte marker pfs25, the pfap2g transcription factor that drives gametocytogenesis and the sexual ring marker pfgexp02, suggesting the parasite ability of adapting its sexual investment during elimination initiatives. Reactive interventions that target Pf sexual stages may be required to achieve complete interruption of transmission.

Plasmodium falciparum sexual parasites regulate infected erythrocyte permeability

ABSTRACTTo ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These New Permeation Pathways (NPP) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPP has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPP are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPP are regulated by cyclic AMP (cAMP) signaling cascade during sexual parasite stages, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPP facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPP and increase drug uptake in mature gametocyte-infected erythrocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.