Differentiating between types of meningitis in a paediatric population using ¹H-NMR metabolomics
Meningitis is a disease characterized by the inflammation of the meninges and spinal cord that is most commonly caused by viruses and bacteria and can be classified into two categories. 1) Acute meningitis (rapid onset), the most common form of meningitis, is caused by infectious agents such as viruses (i.e. enteroviruses, herpes simplex viruses, and mumps virus) and bacteria (i.e. Streptococcus pneumoniae, Neisseria meningitidis). 2) Chronic meningitis is characterized by gradual (delayed) symptoms over weeks to months. The most common form of chronic meningitis in the South African paediatric population of the Western Cape province is tuberculous meningitis (TBM), caused by the endemic bacillus Mycobacterium tuberculosis (M.tb). Metabolomics is the science of analysing the small molecules (metabolites) in a biological system. Proton nuclear magnetic resonance (¹H-NMR) spectroscopy is a method that does not require prior separation of metabolites, and is able to simultaneously detect and determine the concentration of all types of metabolites, above the detection limit of 1 µM, that are present in a complex biological sample. It is well-known that the 1H-NMR analytical platform is highly repeatable, due to calibration steps performed prior to each sample analysed. Since the technical variation can be considered negligible, the analytical variation – analyst repeatability – was assessed (Chapter 3). An artificial cerebrospinal fluid (CSF) sample was created by mimicking the natural salt and buffering capacity and spiking with nine of the most common CSF metabolites. Repeat analyses was performed on three levels – low (50%), medium (100%) and high (150%) concentrations of normal reference ranges. From the acceptable repeatability results it was concluded that the analyst (myself) is competent and repeatable, capable of producing reliable ¹H-NMR data. ¹H-NMR metabolomics can be a valuable approach to expand upon existing knowledge of meningitis, however, only a few ¹H-NMR metabolomics studies are available in the literature to aid in the metabolic characterization of meningitis. A gap exists in the literature where the possibility of a CSF metabolic profile can be used to identify differences between types of meningitis. Hence, ¹H-NMR metabolomics was used in this study to analyse the CSF of a South African paediatric cohort to better characterise cases with acute and chronic meningitis. Firstly, CSF of a TBM sample set was compared to that of CSF from a control sample set (Chapter 4). After strict exclusion criteria, quality control checks and data filtering, statistical analyses identified markers that differentiated TBM from controls. Herein lies the strength of our study – we started with ~100 TBM and ~97 control cases, more than any other study, and we were able to refine them into a well-defined TBM group (n=23) and a homogenous control group (n=33). Initial analysis revealed that the dominating discriminators were decreased glucose and elevated lactate in TBM cases. Removal of the NMR spectral regions representing glucose and lactate allowed us to examine the remaining metabolic profile more closely. The main differentiating metabolites identified were: acetate, alanine, choline, citrate, creatinine, isoleucine, lysine, myo-inositol, pyruvate, valine, 2-hydroxybutyrate, carnitine, creatine, creatine phosphate, glutamate, glutamine, guanadinoacetate, and proline. Of these 18 metabolites, the first 10 overlap with other studies in the literature, while the last eight metabolites are unique to this study. These eight unique metabolites led to the identification of five metabolic pathways that are significantly altered in a brain infected by M.tb, namely: uncontrolled glucose metabolism, increased carnitine, upregulated proline and creatine metabolism, and disrupted glutamate-glutamine cycle TBM cases. Associated with oxidative stress and chronic neuroinflammation, our findings cumulatively contribute toward destabilization of the blood brain barrier (BBB); hence, increasing BBB permeability, which is associated with increased intra-cranial pressure â€“ a clinical hallmark of advanced meningitis, particularly in TBM. Lastly, a comparison was also made between the CSF metabolic profile for viral (acute) meningitis (VM) to that of a control CSF sample set and that of a TBM sample set (Chapter 5). Following the same 1H-NMR metabolomics workflow, it was found that the VM and control groups did not distinguish form each other and led to the postulation that, in our paediatric cohort, VM has the same CSF metabolic profile as controls. This postulation was supported by the findings when comparing TBM and VM metabolic profiles, which were very similar to the comparison of TBM to controls. The metabolites differentiating TBM from VM were: valine, alanine, glutamine, lysine, choline, carnitine, creatine, isoleucine, proline, myo-inositol and guanadinoacetate. Thus, VM has the same metabolic profile as controls, as very similar metabolites were identified to be of statistical significance when cross comparison was done. The metabolic insights gained from this investigation improve upon our understanding of TBM, and contribute toward the metabolic characterization of TBM to aid in future diagnostic and possibly therapeutic research. Furthermore, this study opens up a vast number of future directives for TBM research, some of which are listed at the end of my thesis.