Mechanisms of immune evasion in P. falciparum malaria revealed by integrative gene expression profiling [miRNA]

We performed a genome-wide profiling of miRNAs and mRNA, DNA sequencing of human whole blood sample to identify miRNAs responsive to P.falciparum (P.f) infection and/or associated with parasite load. We identified a set of 72 miRNAs that are positively or negatively associated with parasitemia. We also performed expression quantitative trait loci of miRNAs(miR-eQTL) of P.f infected individuals. The miR-eQTL dataset allowed us to determine relevant genetic variant responsible for miRNAs expression differences between P.f infected individuals and find novel malaria-associated miRNAs Overall design: A longitudinal pediatric cohort of 150 healthy children was recruited in Banfora, Burkina Faso. Enrolment took place at the end of the dry season and none of the children were Plasmodium. -infected at the time of the first sample collection. The children were of age 2 to 10 years, had no disease symptoms or chronic disease history (including sickle cell disease) based on medical records and thorough physical assessment. In this study, a subset of 68 children were included. All of them were not P. falciparum-infected when initially sampled and were subsequently followed on a weekly basis for up to 6 months. For the discovery cohort, clinical phenotyping, sampling and genomic profiling were done at four time points: (i) before infection (at enrolment = V1), (ii) during asymptomatic parasitemia (=V2), (iii) at the beginning of symptomatic parasitemia (=V3), and (iv) three weeks after treatment (=V4). When asymptomatic parasitemia was detected, a closer follow-up of the children was done to sample the early stage of symptomatic infection and immediately followed by Artemether/lumefantrine treatment, as per Burkina Faso's national malaria treatment guidelines. A replication set of infected children was sampled the following year during the wet season. miRNAs and mRNA expression profiling were performed using whole-blood RNA-sequencing of 68 samples of the discovery. Whole blood was taken and immediately store in a Tempus Blood RNA tube at room temperature for 2h and then stored at -20 °C until RNA extraction. The total RNA was extracted using the Tempus Spin RNA Isolation Kit (Thermo scientific). miRNAs and mRNA libraries were constructed using Truseq small rna library prep kit (Illumina) and KAPA Stranded RNA-Seq kit with RiboErase (Kapa Biosystems), respectively. Small RNA libraries were sequenced using HiSeq Rapid kit (50 cycles, Illumina). mRNA libraries were 100 bp paired-end sequenced using the TruSeq SBS Kit v3 – HS kit using Illumina HiSeq 2500 platform. Genomic DNA libraries were constructed using the TruSeq Nano DNA Library Prep Kit (Illumina) and sequenced at 30X coverage on the HiSeq X platform (Illumina). The libraries of the discovery set were prepared in 3 differents batches. Those of the replication were prepared in one batch.