Detection of novel haplotypes under treatment pressure in paediatric severe malaria.

In Africa, the clearance time for severe malaria varies significantly, likely due to the complexity of 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 in Beninese children with severe malaria. Sixty-five 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 (, THE REAL McCOIL and RoH). We then characterized the genetic complexity in WGS-identified polyclonal infections using amplicon sequencing (AmpSeq) on DNA extracted from plasma and the red blood cell pellet. 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. 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.