Early prediction of cerebral malaria by (1)H NMR based metabolomics

Abstract

Background: Cerebral malaria (CM) is a life-threatening disease, caused mainly by Plasmodium falciparum in humans. In adults only 1-2% of P. falciparum-infected hosts transit to the cerebral form of the disease while most exhibit non-cerebral malaria (NCM). The perturbed metabolic pathways of CM and NCM have been reported. Early marker(s) of CM is(are) not known and by the time a patient exhibits the pathological symptoms of CM, the disease has progressed. Murine CM, like the human disease, is difficult to assign to specific animals at early stage and hence the challenge to treat CM at pre-clinical stage of the disease. This is the first report of prediction of CM in mice using a novel strategy based on (1)H nuclear magnetic resonance (NMR)-based metabolomics.

Methods: Mice were infected with malarial parasites, and serum was collected from all the animals (CM/NCM) before CM symptoms were apparent. The assignment of mice as NCM/CM at an early time point is based on their symptoms at days 8-9 post-infection (pi). The serum samples were subjected to (1)H NMR-based metabolomics. (1)H NMR spectra of the serum samples, collected at various time points (pi) in multiple sets of experiments, were subjected to multivariate analyses.

Results: The results from orthogonal partial least square discriminant analyses (OPLS-DA) suggest that the animals with CM start to diverge out in metabolic profile and were distinct on day 4 pi, although by physical observation they were indistinguishable from the NCM. The metabolites that appeared to contribute to this distinction were serum lipids and lipoproteins, and 14-19% enhancement was observed in mice afflicted with CM. A cut-off of 14% change of total lipoproteins in serum predicts 54-71% CM in different experiments at day 4 pi.

Conclusion: This study clearly demonstrates the possibility of differentiating and identifying animals with CM at an early, pre-clinical stage. The strategy, based on metabolite profile of serum, tested with different batches of animals in both the sex and across different times of the year, is found to be robust. This is the first such study of pre-clinical prognosis of CM.

Keywords: Cerebral malaria; Lipids; Metabolomics; NMR; OPLS-DA; Prediction.

Fig. 1

The scheme of retro-orbital bleeding from the group of mice in a set of different experiments. The down arrows refer to the early time point in which mice were bled. A Scheme for retro-orbital bleeding in different experiments. B Typical 700 MHz ¹H NMR spectrum of serum of a control C57Bl/6. 1 Lipids [1a –CH₃, 1b –(CH₂)_n–, 1c –CH₂CH₂CO, 1d –CH₂C=C, 1e –CH₂=CO–, 1f =C–(CH₂)–C=], 2 leucine, 3 isoleucine, 4 valine, 5 lactate, 6 alanine, 7 acetate, 8 glutamine, 9 glutamate, 10 methionine, 11 acetate, 12 citrate, 13 lysine, 14 creatine, 15 choline, 16 phosphocholine, 17 GPC, 18 glucose

Fig. 2

Pairwise OPLS-DA scores and coefficients plot of ¹H NMR spectra of serum of CM, NCM and control at day 4 post-infection in experiment 4. A OPLS-DA scores plot of CM and NCM at day 4 post infection, B OPLS-DA coefficient plot of A, C OPLS-DA scores plot of NCM and control, D OPLS-DA coefficient plot of C, E OPLS-DA scores plot CM and control, F OPLS-DA coefficient plot of E. The red, black and blue symbols denote CM, NCM and control, respectively. The ellipse in the scores plot is a 95 % Hotelling T². The colour bar indicates the correlation of the metabolites in the segregation between two concerned classes

Fig. 3

OPLS-DA scores and coefficient plot of CM and NCM at day 4 post infection for experiment 5. A OPLSDA scores plot of CM and NCM for female mice, B OPLS-DA coefficient plot of A, C OPLSDA scores plot of CM and NCM for male mice, D OPLS-DA coefficient plot of C. The red and black symbols denote CM and NCM, respectively. The ellipse in the scores plot is a 95 % Hotelling T². The colour bar indicates the correlation of the metabolites in the segregation between two concerned classes

Fig. 4

OPLS-DA scores and co-efficient plot of CM and NCM at different day post infection for experiment 6. A Scores plot of CM and NCM at day 3 post infection, B OPLS-DA coefficient plot of A, C scores plot of CM and NCM at day 4 post infection, D OPLS-DA coefficient plot of C, E scores plot of CM and NCM at day 5 post infection, F OPLS-DA coefficient plot of E. The red and black symbols denote CM and NCM, respectively. The ellipse in the scores plot is a 95 % Hotelling T². The colour bar indicates the correlation of the metabolites in the segregation between two concerned classes