We confirmed that a proportion of children carries P. falciparum during the dry season without presenting symptoms, while clinical cases occur in the wet season.
Aim i) We profiled genetic diversity and gene expression of P. falciparum, as well as the parasite’s ability to replicate, and produce sexual forms. After excluding that host immunity is actively detecting and attacking the infected erythrocytes, we investigated how efficiently P. falciparum replication occurs in the dry vs transmission seasons. In Mali, we cultured parasites after blood collection form subclinical children carrying infections at the beginning, mid and end of the dry season, or at their first clinical malaria episode in the wet season. Parasites during the dry season were not impaired in their replicative ability, and seemed instead to be reaching the end of a 48h replicative cycle faster than parasites collected from malaria-causing infections in the transmission season. We then measured development of parasites collected straight from the arm of children in the dry season and in malaria cases in the transmission season.
We have analysed the metabolomic and proteomic analysis of plasma samples of the dry season vs malaria cases in the wet season and vs asymptomatic in the wet season and found that plasma composition is unrelated to time of year in non-infected children, and that carrying P. falciparum only minimally affects plasma constitution in asymptomatic hosts.
We have determined the ability of different antimalaria drugs to kill dry and wet season parasistes in vitro. We tested their in vitro susceptibility to existing and under-development antimalarials, and found that all parasites tested were susceptible to the tested drugs.
Aim ii) expression of var genes was analysed in blood samples collected in Mali during the dry and wet seasons. DNA and RNA was extracted and the samples were amplified by PCR using degenerate primers designed to target var gene sequences of interest. Amplified products were then sent for sequencing. We used a recently developed analytical pipeline to detect motifs identifying PfEMP1 coding genes in most of our analysed samples, and were able to annotate full-length var genes, but we did not see statistically significant enrichments.
Aim iii) with a multi-color qRT-PCR we showd that sexual commitment transcripts were unaffected throughout the year or clinical presentation, but clinical malaria cases in the wet season had higher gametocyte densities than infections maintained asymptomatic during the dry season, while the proportion of gametocytes relative to total parasite load was higher in dry season. Lysophosphatidylcholine (Lyso-PC) variations detected throughout the year were not sufficient to impose differences in gametocytogenesis in vitro.
We adapted a within-host mathematical model to infer the dynamics of gametocyte number and proportion as infections progress. We introduced increased clearance of infected erythrocytes in persisting parasitaemias compared to recently transmitted parasites. Keeping a constant sexual commitment rate throughout time, our model predicted higher number and lower proportion of gametocytes in the early acute phase of infection, but this trend was inverted in the chronic phase, when parasitaemias remain constant or are slowly decreasing.
