Plasma metabolomics reveals membrane lipids, aspartate/asparagine and nucleotide metabolism pathway differences associated with chloroquine resistance in <i>Plasmodium vivax</i> malaria

<div><p>Background</p><p>Chloroquine (CQ) is the main anti-schizontocidal drug used in the treatment of uncomplicated malaria caused by <i>Plasmodium vivax</i>. Chloroquine resistant <i>P</i>. <i>vivax</i> (PvCR) malaria in the Western Pacific region, Asia and in the Americas indicates a need for biomarkers of resistance to improve therapy and enhance understanding of the mechanisms associated with PvCR. In this study, we compared plasma metabolic profiles of <i>P</i>. <i>vivax</i> malaria patients with PvCR and chloroquine sensitive parasites before treatment to identify potential molecular markers of chloroquine resistance.</p><p>Methods</p><p>An untargeted high-resolution metabolomics analysis was performed on plasma samples collected in a malaria clinic in Manaus, Brazil. Male and female patients with <i>Plasmodium vivax</i> were included (n = 46); samples were collected before CQ treatment and followed for 28 days to determine PvCR, defined as the recurrence of parasitemia with detectable plasma concentrations of CQ ≥100 ng/dL. Differentially expressed metabolic features between CQ-Resistant (CQ-R) and CQ-Sensitive (CQ-S) patients were identified using partial least squares discriminant analysis and linear regression after adjusting for covariates and multiple testing correction. Pathway enrichment analysis was performed using Mummichog.</p><p>Results</p><p>Linear regression and PLS-DA methods yielded 69 discriminatory features between CQ-R and CQ-S groups, with 10-fold cross-validation classification accuracy of 89.6% using a SVM classifier. Pathway enrichment analysis showed significant enrichment (<i>p</i><0.05) of glycerophospholipid metabolism, glycosphingolipid metabolism, aspartate and asparagine metabolism, purine and pyrimidine metabolism, and xenobiotics metabolism. Glycerophosphocholines levels were significantly lower in the CQ-R group as compared to CQ-S patients and also to independent control samples.</p><p>Conclusions</p><p>The results show differences in lipid, amino acids, and nucleotide metabolism pathways in the plasma of CQ-R versus CQ-S patients prior to antimalarial treatment. Metabolomics phenotyping of <i>P</i>. <i>vivax</i> samples from patients with well-defined clinical CQ-resistance is promising for the development of new tools to understand the biological process and to identify potential biomarkers of PvCR.</p></div

10-fold cross-validation analysis using clinical variables and top discriminatory metabolic features.

<p>10-fold cross-validation classification accuracies varied from 65% to 89.6% using platelet count, glycerophosphocholines, top 10, top 30, and all 69 discriminatory features. The average permuted accuracies (N = 1000 permutations) varied from 55–58%.</p

Demographic and laboratory characteristics of 46 patients with <i>P</i>. <i>vivax</i> infections assessed for CQ resistance in Manaus, Brazil, 2011–2012.

<p>Demographic and laboratory characteristics of 46 patients with <i>P</i>. <i>vivax</i> infections assessed for CQ resistance in Manaus, Brazil, 2011–2012.</p

Identification of metabolic features associated with CQ resistance.

<p>A) Type 1 Manhattan plot, -log₁₀ <i>p</i> vs mass-to-charge. 81 <i>m/</i>z features with a broad range of <i>m/z</i> were found significant at FDR 0.20 threshold. Green dots represent the features that were up-regulated in the CQ-Resistant group and the red dots represent the features that were higher in the CQ-Sensitive group; B) Type 2 Manhattan plot, -log₁₀ <i>p</i> vs retention time, Majority of features had retention time greater than 4 minutes, which is consistent with elution profile of lipids on a C18 column; C) Two-way hierarchical clustering analysis using discriminatory features; D) Mummichog enriched pathways.</p

Comparison of glycerophosphocholine abundance levels in NIST, pooled normal plasma (US), healthy controls (Brazil), CQ-Resistant (<i>P</i>. <i>vivax</i>), and CQ-Sensitive (<i>P</i>. <i>vivax</i>) groups along with 95% confidence intervals.

<p>The glycerophosphocholines were found to be lower in CQ-Resistant group as compared to CQ-Sensitive and other control samples (<i>p</i><0.05).</p