FIG 6.

Differential expression profiling of parasite transcripts in freshly collected infections in Malian children with uncomplicated malaria with HbAA or HbAS. (A) Proportion of parasite stage-specific transcripts expressed in each patient’s infection (rows) in HbAA (left) and HbAS (right) children. Expression of stage-restricted transcripts for ring (red), trophozoite (blue), and schizont (green) stages were measured as TPM, summed within each stage, and expressed as a proportion of stage for each infection. We observed a nearly complete absence of schizont-specific transcript expression, and most infections were predominantly either rings or trophozoites with little admixture. See Jupyter Notebook “Genes_by_stage.ipynb” on our GitHub for the full workflow. (B) Principal-component analysis of transcript expression from in vivo isolates following variance-stabilizing transformation based on the 500 transcripts with the highest variance across all samples. Clusters separate according to parasite stage identified from stage-specific transcript expression. (C and D) Volcano plots of differential transcript expression conditioned by parasite stage. The x axis shows the log₂ fold change of HbAS versus HbAA children, with positive values indicating increased expression in HbAS. The y axis shows −log₁₀ P values of each transcript, and horizontal dashed lines indicate P value thresholds that capture the transcripts with adjusted P values of <0.05. (E) Scatterplots of the log₂ fold change of individual transcripts for severe-malaria parasites compared to uncomplicated-malaria parasites in Gambian children (y axes) and HbAS compared to HbAA parasites (x axes). Points in the upper right and lower left quadrants signify transcripts that are differentially expressed in the same direction between studies; i.e., those in the upper right quadrant are upregulated in both severe malaria and HbAS, and those in the lower left quadrant are downregulated in both severe malaria and HbAS. All transcripts included were statistically significant in severe malaria versus uncomplicated malaria (26). Red points have an adjusted P value of <0.05 in trophozoite parasites from HbAS versus HbAA Malian children.