Cerebrospinal fluid samples were retrospectively collected from a historical cohort of paediatric patients between 2009 and 2018. The paediatric sample group used in this investigation reside in an area endemic for TB–the Western Cape province of South Africa. Each participant was referred from local regional clinics to the paediatric service at the Tygerberg Academic Hospital in Cape Town, based on clinical signs and symptoms of meningitis. Upon admission to the hospital, each patient was assessed by the paediatric neurology team, and, when they were stable enough, a CSF sample was collected via lumbar puncture for routine differential diagnostic purposes. Cerebrospinal fluid was collected from a total of 195 participants: 88 controls, 40 cases of viral meningitis (VM), and 67 cases of tuberculous meningitis (TBM). The control group consists of patients who were suspected of having meningitis based on clinical symptoms but proved to be negative for meningitis upon final diagnosis. Within the control group, there were two subgroups–controls without any neurological symptoms (“Nonneuro”, n = 40) and controls exhibiting neurological symptoms (“Neuro”, n = 44), such as various forms of seizures, brain injury, and encephalopathy. Both the VM and TBM cases were subdivided into “probable” and “definite” classifications. For VM, a “definite” VM diagnosis (n = 16) was assigned based upon a positive viral PCR test, whereas a “probable” VM diagnosis (n = 24) was given if the PCR test was negative, but the patient presented with clinical symptoms of VM and CSF pleocytosis (>5 leucocytes/mm³). For TBM, a “definite” (n = 30) and “probable” (n = 37) TBM diagnosis was assigned based upon a uniform case definition given by Marais et al. (2010).

Written and informed consent and/or assent were obtained for CSF samples for research purposes. This study was approved by the Health Research Ethics Committee (HREC) of Stellenbosch University (ethics approval no. N16/11/142), the Western Cape Department of Health and Wellness, and the HREC of the North-West University, Potchefstroom campus (ethics approval no. NWU-00063-18-A1-05)Reagents and quantification standards.

We used UPLC-grade solvents (water and acetonitrile) from Anatech (Burdick and Jackson) and formic acid (Merck South Africa) to prepare the mobile phases. Acetyl chloride and 1-butanol were obtained from Sigma-Aldrich (South Africa) and were used to prepare butanolic-HCl for the derivatisation of acylcarnitines. Acylcarnitine standards were purchased from Cerilliant (Sigma-Aldrich) and were used to create external calibration curves. The composition of each acylcarnitine mix was as follows: Acylcarnitine mix 1 [ L-carnitine (C0) and acetyl-L-carnitine (C2)] and acylcarnitine mix 2 [butyryl-L-carnitine (C4), isobutryryl-L-carnitine (iC4), isovaleryl-L-carnitine (iC5), valeryl-L-carnitine (C5), hexanoyl-L-carnitine (C6), octanoyl-L-carnitine (C8), decanoyl-L-carnitine (C10), dodecanoyl-L-carnitine (C12), tetradecoloyl-L-carnitine (C14) and octadecanoyl-L-carnitine (C18)]. Deuterated acylcarnitines were obtained from the Centre for Human Metabolomics (North-West University) and served as internal standards [methyl-d3-L-carnitine (d3-C0), d3-acetylcarnitine (d3-C2), d3-propionylcarnitine (d3-C3), d3-butyrylcarnitine (d3-C4), d9-isovalerylcarnitine (d9-iC5), d3-octanoylcarinitine (d3-C8), d3-decanoylcarnitine (d3-C10), d3-dodecanoylcarnitine (d3-C12), d3-tetradecanoylcarnitine (d3-C14), d3-octadecanoylcarnitine (d3-C18)].

The analytical method was optimised for the CSF sample matrix prior to sample preparation. Known concentrations of acylcarnitine standards were spiked into blank CSF to generate external calibrators and correct for matrix effects. Pooled quality control (PQC) samples were prepared by combining aliquots from individual CSF samples into a single tube, which was aliquoted into vials and analysed with each batch. Sample preparation was carried out as described previously (Plaatjie et al., 2025). Briefly, CSF samples, external calibrators and PQC were thawed to room temperature and homogenised by vortex mixing. Extraction was achieved by adding 200 µL of internal standard (containing deuterated acylcarnitines) to 10 µL of CSF samples; the same procedure was applied to calibrators and PQC. The mixtures were vortexed and centrifuged at 12,000 x g at 10 °C for 10 min. A 200 µL volume of the sample supernatant was transferred to clean Eppendorf tubes and evaporated to dryness under a gentle stream of nitrogen for 1 h. The samples were derivatised by adding 100 µL of N-butanol: acetyl chloride (25:6.25, v/v) to the dried residue and incubated for 45 min at 65 °C. The butylated samples were then evaporated to dryness under a stream of nitrogen gas. The dried residue was then reconstituted with 100 µL of freshly prepared mobile phase [acetonitrile: water (50/50) (v/v), with 0.1% formic acid]. The samples were transferred to LC vials for analysis.

The LC-MS/MS was carried out as previously described by Plaatjie et al. (2025). Acylcarnitines were analysed using high-performance liquid chromatography coupled to a triple quadrupole tandem mass spectrometer for detection and quantification. The chromatographic separation of acylcarnitines was achieved by using a C18 Zorbax reverse phase column (2.1 mm × 100 mm × 1.8 µm) fitted with a Phenomenex guard column (2.0 µm depth filter × 0.004in ID). The column temperature was kept at 30 °C during the entire run. The mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The elution gradient started with 5% B, followed by a linear gradient to 100% B. The flow rate was set at 0.3 mL/min for the first 13 min and increased to 0.35 mL/min for the rest of the run. The total run time for each sample was 30 min. The samples were delivered to the QQQ-MS via electrospray ionisation (ESI) in positive mode with the following source conditions: nitrogen drying gas temperature of 300 °C and a flow rate of 7.5 L/min for the remainder of the run. The compounds were analysed in multiple reaction monitoring (MRM) mode using enhanced sensitivity with the multiplier voltage set at 300 Delta EMV and a dwell time of 45 milliseconds for all metabolites. Data acquisition was achieved using Agilent’s Masshunter Workstation Software (v B02.01) and Masshunter Optimizer software (v B02.01).

Data was extracted using Agilent’s Masshunter Workstation software (v B06.00; Qualitative Analysis and Quantitative Analysis). Samples and metabolites were individually inspected, aligned with their respective internal standards, and integrated manually. The extracted metabolite responses were then exported to an Excel file for subsequent quantification. Blank subtraction was done before quantification to account for endogenous background signal contributions from the pooled CSF. The blank-corrected calibrators were then normalised by their corresponding internal standard response. An external calibration curve was generated for each acylcarnitine metabolite using the normalised response ratios plotted against the known standard concentrations. Absolute concentrations in the samples were determined by interpolating their normalised response ratios onto the respective calibration curves. The data were summarised using the mean, median, standard deviation (SD), and interquartile range (IQR).

A comparison analysis of acylcarnitine profiles across TBM, VM, and NMC groups of this cohort has been reported (Plaatjie et al., 2025). For this study, only the control group was used. The following clinical characteristics were mostly present upon presentation: fever (60%), vomiting (47%), diarrhoea (21%), weight loss (14%), headache (30%), seizures (37%), cough (28%), raised intracranial pressure (2%), meningeal irritation (14%), and decreased level of consciousness (19%). The main exclusion criterion was HIV-positive or HIV-unknown status. Despite presenting with clinical symptoms of disease, all control participants were ruled out for meningitis, and their CSF parameters, including glucose, protein, and leucocyte levels, remained within normal ranges (Table 1).

To ensure analytical reliability, pooled quality control samples were analysed together with the samples. The CV for each acylcarnitine was calculated from the mean values of the pooled QC samples across batches. Free carnitine (C0), propionylcarnitine (C3), and butyrylcarnitine (C4) exhibited the lowest CVs (<10%) while tetradecanoylcarnitine showed a slightly higher CV (31%) (Figure 1). All acylcarnitines were analysed together within each sample; thus, the higher CV observed for certain acylcarnitines may not be solely attributed to analytical variability but could also indicate the inherent instability of these metabolites. Furthermore, CSF’s naturally low acylcarnitine concentration may contribute to the elevated CV values (Bogumil et al., 2008). Figure 2 shows that the assignment of the acylcarnitines was confirmed in the LC chromatograms using deuterated internal standards of acylcarnitines. The retention times are represented by the metabolite peaks. The retention times increase with acylcarnitine chain length, demonstrating good chromatographic separation.

In this study, we report the mean, median, standard deviation, and interquartile range (IQR) concentrations of CSF acylcarnitine based on 57 samples from children (Table 2) and categorised into four age groups: ≤1 year old, >1 and ≤3 years old, >3 and ≤6 years old, and >6 and ≤12 years old (Table 3; Figure 3). Table 3 and Figure 3 show that acylcarnitine concentrations do not differ much across the paediatric age groups. Additionally, we compare these reference ranges to the CSF acylcarnitine values from adults reported in the literature (Table 2). Mandal et al. (2012) provided the mean concentration values from seven non-meningitis adults (aged 18–40 years). Carlsson et al. (2020) documented mean CSF acylcarnitine concentrations from 12 healthy adults. Plewa et al. (2019) reported the mean concentration of CSF acetylcarnitine from 10 individuals without nervous system disorders, but the age group was unspecified.