Sem Ezinmegnon

Background. In Africa, the clearance time for Plasmodium falciparum severe malaria varies significantly, likely due to the complexity of P. falciparum infections and the sequestration phenomenon exhibited by this parasite. This study aims to evaluate different methods to study the intra-host dynamics of polyclonal infections during parasite clearance under antimalarial treatment. Additionally, it seeks to determine the association between parasite clearance rate following artesunate or quinine treatment and the genetic complexity of P. falciparum in Beninese children with severe malaria. Methods. Sixty-five P. falciparum severe malaria individuals diagnosed by microscopy and treated with artesunate or quinine were sampled every 8 h for 24 h. Using whole-genome sequencing (WGS) data, we estimated the multiplicity of infection (MOI) with three algorithms ( Fws , THE REAL McCOIL and RoH). We then characterized the P. falciparum genetic complexity in WGS-identified polyclonal infections using amplicon sequencing (AmpSeq) on DNA extracted from plasma and the red blood cell pellet. Results. AmpSeq demonstrated greater sensitivity in detecting multiple genomes within isolates compared to WGS methods. The MOI from AmpSeq was significantly higher in red blood cell pellets compared to plasma (2.4 vs. 1.8 distinct microhaplotypes per isolate). However, at parasitaemia over 1,000 parasites per microlitre, the same MOI was detected in both plasma and pellet samples in 85.4% of the isolates. We observed a high variability in parasite clearance rate among participants, but it was not associated with parasite MOI at diagnosis. Interestingly, in 60.9% of participants, previously undetected microhaplotypes appeared in circulation 16 h after treatment initiation. Conclusion. These findings demonstrate that combining different haplotyping techniques effectively determines parasite genetic complexity. Additionally, plasma can be effectively used for parasite genotyping at sufficient parasitaemia levels. The parasite clearance rate of severe malaria is independent of parasite MOI. However, genotyping a single blood sample upon hospital admission does not capture the full spectrum of parasite genotypes present in the infection.

Cytoadherence of Plasmodium falciparum- infected erythrocytes (IEs) in deep microvasculature endothelia plays a major role in the pathogenesis of cerebral malaria (CM). This biological process is thought to be mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP-1) and human receptors such as CD36 and ICAM-1. The relationship between the expression of PfEMP-1 variants and cytoadherence phenotype in the pathology of malaria is not well established. Cytoadherence phenotypes of IEs to CD36, ICAM-1, CSPG and the transcription patterns of A, B, var2csa, var3, var gene groups and domain cassettes DC8 and DC13 were assessed in parasites from children with CM and uncomplicated malaria (UM) to determine if cytoadherence is related to a specific transcription profile of pfemp-1 variants. Parasites from CM patients bind significantly more to CD36 than those from UM patients, but no difference was observed in their binding ability to ICAM-1 and CSPG. CM isolates highly transcribed groups A, B, var2csa, var3, DC8 and DC13 compared to UM parasites. The high transcription levels of var genes belonging to group B positively correlated with increased binding level to CD36. CM isolates bind significantly more to CD36 than to ICAM-1, which was correlated with high transcription level of group B var genes, supporting their implication in malaria pathogenesis.