Identifying the Components of Acidosis in Patients With Severe Plasmodium falciparum Malaria Using Metabolomics

Stije J Leopold, Aniruddha Ghose, Erik L Allman, Hugh W F Kingston, Amir Hossain, Asok Kumar Dutta, Katherine Plewes, Kesinee Chotivanich, Nicholas P J Day, Joel Tarning, Markus Winterberg, Nicholas J White, Manuel Llinás, Arjen M Dondorp, Stije J Leopold, Aniruddha Ghose, Erik L Allman, Hugh W F Kingston, Amir Hossain, Asok Kumar Dutta, Katherine Plewes, Kesinee Chotivanich, Nicholas P J Day, Joel Tarning, Markus Winterberg, Nicholas J White, Manuel Llinás, Arjen M Dondorp

Abstract

Background: Acidosis in severe Plasmodium falciparum malaria is associated with high mortality, yet the pathogenesis remains incompletely understood. The aim of this study was to determine the nature and source of metabolic acids contributing to acidosis in patients with severe falciparum malaria.

Methods: A prospective observational study was conducted to characterize circulating acids in adults with P. falciparum malaria (n = 107) and healthy controls (n = 45) from Bangladesh using high-resolution liquid chromatography-mass spectrometry metabolomics. Additional in vitro P. falciparum culture studies were performed to determine if parasites release the acids detected in plasma from patients with severe malaria acidosis.

Results: We identified previously unmeasured plasma acids strongly associated with acidosis in severe malaria. Metabolomic analysis of P. falciparum parasites in vitro showed no evidence that these acids are released by the parasite during its life cycle. Instead, 10 of the plasma acids could be mapped to a gut microbial origin. Patients with malaria had low L-citrulline levels, a plasma marker indicating reduced gut barrier integrity. Longitudinal data showed the clearance of these newly identified acids was delayed in fatal cases.

Conclusions: These data suggest that a compromise in intestinal barrier function may contribute significantly to the pathogenesis of life-threatening acidosis in severe falciparum malaria.

Clinical trials registration: NCT02451904.

Keywords: Plasmodium falciparum; malaria; gut-barrier integrity; metabolic acidosis; metabolomics; severe.

© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.

Figures

Figure 1.
Figure 1.
Elevated levels of previously unidentified organic acids in patients with severe Plasmodium falciparum malaria. A, Standard base deficit corrected for albumin and phosphate (SBDc) in relation to outcome in patients with severe P. falciparum malaria (n = 60). B, Mean proportion of standard base deficit attributable to L-lactate among patients with mild, moderate, and severe acidosis. C, Anion gap corrected for albumin and phosphate. D, Strong ion gap. Box plots show median (interquartile range) and minimum and maximum; significance tests were done using Kruskal–Wallis tests. *P < .05, **P < .01, ***P < .001.
Figure 2.
Figure 2.
Metabolomic analysis of plasma of patients with severe malaria. A, Heat map of all organic acids that were significantly associated with the standard base deficit corrected for albumin and phosphate (false discovery rate < 0.15). Visualization was done by calculating the log2 (fold change) normalized against the levels found in patients with uncomplicated malaria, and stratified according to the degree of acidosis. Metabolomic analysis of plasma of patients with severe malaria. B, Venn diagram of the biological origin of acids identified in acidotic patients with severe Plasmodium falciparum malaria. Acids were mapped to their biological source using the online Human Metabolome Database, version 4.0. C, Correlation matrix based on Spearman correlation coefficients of clinical variables of acid-base balance, including blood pH, bicarbonate, base deficit, and 10 microbial acids implicated in acidosis in patients with severe malaria. D, Box plots of microbial acids implicated in acidosis. Diaminopemelic acid is a component of gram-negative cell walls; p-hydroxyphenylacetic acid is a product of polyphenol metabolism by gut microbiota; pipecolic acid is a product of lysine metabolism in intestinal bacteria. * = <0.05; **** = <0.0001. Abbreviations: DAP, diaminopemelic acid; HE, healthy controls; HPAA, hydroxyphenylacetic acid; ns, not significant; PfHRP2, plasma Plasmodium falciparum histidine-rich protein 2; SBDc, standard base deficit corrected for albumin and phosphate; SM, severe malaria; UM, uncomplicated malaria.
Figure 3.
Figure 3.
Metabolomic analysis of culture media of Plasmodium falciparum 3D7 strain parasites in vitro. A, Metabolites secreted by Pf3D7 in culture media during the parasite life cycle. The concentration profiles were based on the log2 normalized fold change 3% parasitemia cultures, compared to uninfected red blood cell controls. B, Secreted microbial markers of acidosis. C, Secreted glycolytic metabolites.*Metabolites previously detected in plasma of acidotic patients with malaria.†Metabolites previously identified in plasma of acidotic patients with malaria of a suspected microbial origin.
Figure 4.
Figure 4.
Delayed clearance of suspected microbial markers associated with acidosis. The study hours are displayed on the x-axis. Means and standard errors of the mean are reported of the ion intensity of candidate acids. Serial sampling was done in patients with severe malaria (n = 60) at 0, 24, and 48 hours and temporal dynamics of candidate compounds were compared between survivors (n = 40, black lines) and patients who died (n = 20, red lines). Shown here are 4 of 10 microbial acids identified in the plasma of acidotic patients with severe malaria. A, Diaminopimelic acid (DAP). B, p-Hydroxyphenylacetic acid (HPAA). C, Pipecolic acid (PIP). D, Dimethylglycine (DMG).
Figure 5.
Figure 5.
Markers of gut-wall and endothelial function in patients with malaria and healthy controls. Plasma amino acids were quantified using targeted high-performance liquid chromatography–tandem mass spectrometry in plasma of healthy controls (HE), and patients with uncomplicated malaria (UM) and severe malaria (SM). A, L-citrulline is a marker of gut wall integrity; reduced blood levels indicate intestinal wall damage. B, L-arginine is a marker of endothelial function. **** = <0.0001.

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