Extensive alterations of blood metabolites in pediatric cerebral malaria

Abstract

Cerebral malaria (CM) presents as an encephalopathy and is due to infection with Plasmodium falciparum. Patients are comatose, often with fever, recurrent seizures and this condition is associated with a high mortality rate. The etiology of the coma and seizures are poorly understood. Circulating small molecules and lipids have bioactive functions and alterations in their concentrations have been implicated in seizure disorders and other forms of encephalopathy. We carried out a comprehensive analysis of blood metabolites during CM to explore a biochemical basis of this encephalopathy. A paired metabolomics analysis was performed on the plasma samples of Malawian children (n = 11) during CM and at convalescence thirty days later, to identify differentially abundant molecules associated with CM. We also report plasma molecules associated with CM mortality (n = 4) compared to survival (n = 19). Plasma metabolites were identified through ultra high performance liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry to maximize compound detection and accuracy and then compared to a library for identification. We detected a total of 432 small molecules in the plasma and 247 metabolites were significantly differentially abundant between CM and convalescence (p < 0.05, FDR < 0.10). These represented global changes across many classes of molecules including lipids, amino acids and hemoglobin metabolites. We observed significant changes in molecules that could impact neurologic function during CM; these include increased levels of kynurenate and decreased indolepropionate, glutamate, arginine and glutamine. Moreover, 1-methylimidazoleacetate, kyurenate, arachidonic acid and dimethylarginine were associated with mortality (p < 0.05, fold change > 1.2). These results highlight the broad changes in blood chemistry during CM. We have identified metabolites that may impact central nervous system physiology and disease outcomes and can be further explored for their mechanistic roles into the pathophysiology of CM.

Fig 1. Hierarchical clustering of differentially abundant plasma metabolites during CM and convalescence in a paired analysis of eleven Malawian children.

A heatmap of a heirarchical clustering of 247 differentially abundant (p < 0.05, FDR < 0.10, paired t test) plasma metabolites between CM and convalescence (one month later). Unsupervised hierchical clustering segregates the samples by clinical state. The asterisk notes one child with mild malaria diagnosed during the convalescent visit.

Fig 2. Plasma amino acid concentrations during CM compared to convalescence.

Selected plasma amino acid concentrations during CM and at convalescence (one month later) in eleven children. (A) Enriched amino acids during CM. (B) Depleted amino acids during CM. Scatter plots show median and interquartile ranges. A Wilcoxon matched-pairs signed rank test was performed. * represents P<0.05; ** represents P<0.01.

Fig 3. Enrichment in fatty acids, sterols, bile acids and other lipids during CM compared to convalescence.

A bar graph of the number of differentially abundant lipids (paired t test, p-value < 0.05, FDR < 0.10) by lipid class during CM compared to convalescence (n = 11 paired samples). We found marked reduction in the number of phospholipids and lysolipids during infection and enrichment in fatty acids. * denotes data obtained from a quantitative metabolomic method (n = 8 paired samples).