Browsing by Author "Rohlwink, Ursula"
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- ItemOpen AccessAn Examination of Lumbar and Ventricular Cerebrospinal Fluid Findings in Children with Tuberculous Meningitis and Hydrocephalus(2019) Mwenda, Lona Albertha; Figaji, Anthony; Rohlwink, Ursula; Diedericks, RalphBackground: Childhood tuberculous meningitis (TBM) has poor outcomes. These are often associated with delayed diagnosis because early diagnosis and treatment is challenging. Existing diagnostic criteria use CSF characteristics to suspect TBM. However, lumbar and ventricular CSF may differ. These differences have not been well characterised Sometimes only ventricular CSF is available and decisions about surgical treatment may be influenced by CSF characteristics. This study examined CSF parameters from lumbar and ventricular compartments in patients with TBM and hydrocephalus who required neurosurgical procedures, their CSF temporal profiles, differentials between compartments, and factors that may influence these results. Methodology: A descriptive cross-sectional study was conducted including data from two prospective TBM studies. Children treated for TBM and hydrocephalus at Red Cross War Memorial Children’s Hospital with lumbar and/ or ventricular samples were selected. Pooled lumbar verses ventricular samples and paired time-linked samples in individual patients were analysed. Differences in CSF cell counts and biochemistry parameters across compartments were analyzed using Wilcoxon signed rank test, and temporal profiles graphically presented. Associations between laboratory, clinical and radiological data were analyzed using Mann-Whitney’s U test. To test for associated factors, results of the nature of hydrocephalus (level of CSF obstruction) and spinal imaging were analyzed where available. Association between CSF parameters and morbidity was analyzed. Results: Eighty-one patients were studied, 29 had time-linked paired CSF. The mean patient age was 36 months (2- 156 months), 93% were HIV-uninfected, and the mortality rate was 13.6%. Seventy-two percent had communicating hydrocephalus, 16% non-communicating, and 12% uncertain (unable to demonstrate level of block). Medians of admission lumbar CSF showed low glucose (2.2 mmol/L), low chloride (112 mmol/L), raised protein (2g/L) and elevated white cell count (165 x 106 /L). Corresponding values for admission ventricular CSF were minimally affected glucose (3mmol/L), mildly low to normal chloride (114.5mmol/L), normal to mildly raised protein (0.5g/L) and less elevated white cell count (22 x 106 /L). In paired samples, all parameters were significantly different between lumbar and ventricular CSF. Ventricular CSF showed milder aberrations than lumbar CSF: lower protein and total white cell count, higher glucose and chloride. All paired samples showed higher lumbar CSF protein; lower lumbar CSF chloride in almost 80%; lower lumbar CSF glucose in 96%. Analysis of possible factors was limited by the small patient numbers who had full brain and spine imaging, and also paired CSF samples (n=17). However, maximum lumbar CSF protein was associated with severity of spinal disease on imaging. The lymphocyte ratio between lumbar and ventricular CSF was higher in patients with non-communicating and uncertain hydrocephalus. CSF parameters normalized slowly. White cell count and lymphocyte CSF differential were associated with favorable outcome in survivors. Conclusion: Lumbar CSF depicted a typical TBM pattern. Ventricular CSF differed: CSF parameters were less abnormal in both pooled analysis and across individual paired samples. Spinal disease severity and nature of hydrocephalus may affect this differential. The CSF compartment sampled is therefore clinically relevant when interpreting CSF characteristics for diagnostic and treatment decisions. Studies of TBM diagnosis, pathophysiology, biomarkers and drug concentrations should consider these differences.
- ItemOpen AccessDescribing the immune cell profile of ventricular cerebrospinal fluid (CSF) in paediatric central nervous system (CNS) infections(2025) Morris, Kate; Rohlwink, Ursula; Figaji, AnthonyIntroduction: Paediatric central nervous system (CNS) infections are associated with high mortality rates and neurological disability in survivors due to brain injury caused by cerebral inflammation. Because the brain is difficult to study, the unique characteristics of the neuroinflammatory response are poorly understood. A better understanding of the immune response to these infections could lead to improved host-directed therapies. An important method to study this is the analysis of infected ventricular cerebrospinal fluid (CSF), but there is often a paucity of cells in CSF samples, especially in conditions like tuberculous meningitis (TBM), and these undergo rapid immune cell death after sampling. Consequently, the cell populations in CSF are not well described. Cryopreservation of CSF and flow cytometric analysis have improved the ability to study immune cells in CSF; therefore, these techniques were employed in this study. Aims: This project aimed to 1) describe the cellular immunophenotype and inflammatory mediators in CSF samples from patients with common CNS infections through flow cytometric and Luminex® analysis respectively, and 2) explore changes in immune cells and analytes over time. Methods: CSF samples were prospectively collected during clinically indicated procedures, the cell pellets and supernatant were cryopreserved. Flow cytometric analysis was performed after two weeks of storage at -80°C. Different populations of major peripheral immune cells (CD45+: lymphocytes, monocytes and granulocytes) and CNS-derived immune cells (microglia (CD45-TMEM119+) and astrocytes (CD45-ACSA+) were examined along with their respective sub-groups. The sample supernatants were batch analysed for inflammatory biomarkers including interleukin (IL)-1, IL-6, IL-8, IL-10, IL-1 receptor antagonist (IL-1Ra), tumour necrosis factor (TNF)-, interferon (IFN)-, IFN-, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory proteins (MIP)-1 and interferon-inducible protein 10 (IP-10) using Luminex® technology. Cell proportions and concentrations (using Flow-Count Fluorospheres) and cytokine concentrations were described in admission and serial samples. Results: This study recruited 30 children with CNS infections (including tuberculous and other bacterial meningitis, shunt infections, and ventriculitis) in whom 61 samples were collected (30 admission, 31 serial samples). Microglia (CD45-TMEM119+) were the most abundant cell population on admission and over time. Lymphocytes (CD45+CD3+ and CD45+CD3-) were the most abundant peripheral immune cell, population above granulocytes (CD45+) and monocytes (CD45+CD14+). Cytokines with the highest concentration included IL-1Ra, MCP-1 and IP-10. MCP-1 remained elevated over time whereas overall cytokine concentrations were highest on admission and decreased over time. Cytokine and cell data were influenced by the aetiology of the CNS infection (70% of the cohort comprised patients with TBM). Conclusions: Brain-resident immune cells are important contributors to the neuroinflammatory response to CNS infection, particularly microglia, which are the most abundant immune cell present in the ventricular CSF of these patients. The techniques used in the study could be used at scale to characterise the unique characteristics of the neuroinflammatory response in different CNS infections and inflammatory conditions, which could lead to the development of novel immunomodulatory therapies. The role of microglia in inflammation as well as neurodevelopment is important to consider when studying children.
- ItemOpen AccessDrug recovery in paediatric tuberculous meningitis(2025) Tshavhungwe, Mvuwo phophi; Figaji, Anthony; Rohlwink, UrsulaBACKGROUND: Tuberculosis (TB) remains the single leading infectious disease killer across the world. Infection of the central nervous system (CNS) is the most lethal form of the disease. There has been little adjustment to the standard drug regimens over several decades, but there remain many unknowns about their effectiveness, especially because penetration across the blood-brain barrier (BBB) has been difficult to study. Rifampicin (RIF) is a leading drug in the management of TBM but concentrations in ventricular cerebrospinal fluid (VCSF) have not been studied in TBM patients, and emerging evidence suggests that lumbar spinal CSF (LCSF) concentrations of substances may not fully reflect brain concentrations. Finally, there are no data on brain extracellular fluid (ECF) concentrations of TB drugs in humans. AIMS AND OBJECTIVES The aim was to produce the first pilot data of RIF concentrations in VCSF in children with TBM and to explore the use of microdialysis (MD) to detect RIF in brain ECF. The primary objective was to measure RIF total concentrations in multiple compartments, namely plasma, LCSF, VCSF, and brain ECF. The secondary objectives were 1) to examine RIF concentrations in paired, time-linked L- and VCSF of TBM patients (samples taken at the same time), and 2) to determine total protein concentrations in time-linked L-and VCSF. METHODS: This study prospectively recruited children with definite or probable TBM in a descriptive cross-sectional study design at a university-affiliated paediatric hospital in Cape Town. Patients were treated with standard TB drug regimens. Sampling was performed after standard drug administration. Blood samples were taken from routine procedures or long lines or arterial lines. LCSF was sampled from scheduled clinically indicated procedures. VCSF was accessed from external ventricular drains (EVD) or CSF shunt insertion. In a subgroup of patients, VCSF and LCSF were taken at the same time (usually from clinically indicated column tests). A subgroup of patients underwent bedside MD monitoring of brain chemistry for clinical purposes. Remnant fluid was stored and examined offline for RIF concentrations in brain ECF. RESULTS: A total of 61 children who fulfilled the definition of definite or probable TBM and who had samples analysed for RIF were recruited to the study; the median age was 2.3 years and 7% were human immunodeficiency (HIV) positive. All patients had hydrocephalus (HCP) and most presented in stage 2 and 3. Plasma concentrations peaked at 2 hours (hrs) post-dose while CSF values peaked after 4 hrs. Below level of quantification percentages were 8%, 15%, 16% and 22% respectively for plasma, LCSF, VCSF, and ECF respectively. The median peak concentration in plasma was 5.2 μg/mL. By comparison, LCSF concentrations at 4 and 6 hrs were 0.23 and 0.14 μg/mL and VCSF concentrations were 0.15 μg/mL and 0.14 μg/mL respectively. CSF concentrations were at most 5% of peak plasma concentrations. RIF was detectable in MD samples (n=74) but showed the lowest concentrations. ECF concentrations ranged from 0.01-0.04 μg/mL. In the first 10 hrs of sampling, brain ECF RIF concentrations were 13-26% of corresponding VCSF concentrations. In a subgroup of patients with time-linked LCSF and VCSF samples (n=28), median RIF concentrations were significantly lower in the VCSF:133 ng/mL (range, 7.40-937 ng/mL) versus LCSF: 299 ng/mL (range, 5-1080 ng/mL) as were protein concentrations: 1.3 g/L (0.17-7.44 g/L) and 6.0 g/L (range, 0.68-51.8 g/L). LCSF showed higher RIF concentrations in 64% of paired samples, and higher protein concentrations in all paired samples. There were no apparent differences in the time-to-peak drug concentrations in the two CSF compartments. CONCLUSION: Our data adds to the limited knowledge of CNS distribution of RIF, an important drug in TBM management. We report RIF concentrations in VCSF for the first time in patients with TBM. Intriguingly, the paired sample analysis shows higher concentrations in LCSF, which may reflect a sump effect or differential permeability in the spinal blood vessels. Given that the current knowledge of RIF in TBM patients is largely based on spinal CSF studies, it is important to note that this may overestimate concentrations in VCSF. We also demonstrated for the first time the capacity to serially sample RIF directly from brain ECF, where concentrations were lower than in VCSF. This may be technical but also may reflect a difference between total and free drug concentrations, given the differential barrier properties of the BBB (former) and the blood-CSF barrier (BCSFB) (latter). This study contributes timely and important data in an era of focus on maximising RIF exposure by increasing drug dosing.
- ItemOpen AccessInvestigating cerebrovascular pressure reactivity in a large cohort of children with severe traumatic brain injury(2021) Smith, Claudia Ann; Figaji, Anthony; Rohlwink, UrsulaIntroduction: Traumatic brain injury (TBI) contributes to worldwide death and disability more than any other traumatic event, but it is of particular concern in children in developing resource-scarce countries. Cerebral autoregulation (CA) is a homeostatic mechanism that aims to maintain constant cerebral blood flow within a range of systemic blood pressures, and the loss of this mechanism has been associated with mortality and worse outcomes in adult TBI. Paediatric studies of CA disturbance are few and consist of small cohorts. Given the differences between adult and paediatric TBI pathophysiology, CA needs examination in a larger cohort of paediatric TBI. This study aimed to describe the profile of PRx, a mathematical indicator of cerebrovascular pressure reactivity, in a large cohort of children with severe TBI. The specific aims were to 1) describe the characteristics of PRx; 2) examine associations between PRx, clinical and physiological variables, and 3) examine associations between PRx and mortality at 6 months, and PRx and dichotomized outcome (as well as survivors only) at ≥ 6 months post-injury. Methods: Patient demographics, clinical and monitoring data were recorded, and the temporal profile of median PRx was plotted by outcome groups. The associations between PRx, Glasgow Coma Score (GCS), intracranial pressure (ICP), and cerebral perfusion pressure (CPP) were examined with both summary measures and correlation analysis using high frequency data. Associations between PRx and mortality/outcome were examined with multiple regression analysis, and the prognostic ability of PRx, ICP and CPP was investigated with receiver operating curve analysis. Results: We examined 196 children with severe TBI. Mortality rate was 10.7%, and 70.4% of the cohort had unfavourable outcome. PRx was consistently higher in patients with poor outcome when examined by various summary statistics and over time. Hourly analysis showed that PRx had a moderate positive correlation with ICP (r = 0.35; p < 0.001) and a weak negative correlation with MAP (r = -0.10; p < 0.001) and CPP (r = -0.27; p < 0.001). PRx had a strong and independent association with mortality. Conclusion: This study calculated, described, and analysed PRx in the largest known cohort of children with severe TBI. PRx had a strong association with outcome (particularly mortality) that was independent of ICP, CPP and GCS. However, a combination of several PRx and ICP-related variables will likely be important for overall prognostication in paediatric severe TBI. Whether CA should be incorporated into clinical care, and if so, how, requires separate investigation.
- ItemOpen AccessThe influence of fixation and cryopreservation of cerebrospinal fluid on antigen expression and cell percentages by flow cytometric analysis(2022) Singh, Gabriela; Rohlwink, Ursula; Figaji, AnthonyIntroduction: Infections of the central nervous system (CNS) remain a major burden of disease. Cerebrospinal fluid (CSF) is an essential sample for the investigation of CNS pathologies and flow cytometry enables detailed immunophenotyping of cells present in CSF.However, the pauci-cellular nature of CSF, and the rapid cell death following sampling, incumbers the use of flow cytometric analysis of these samples. Immediate processing and analysis of CSF for flow cytometry is not feasible in busy clinical environments where sample collection is unpredictable, and flow cytometers are not readily available. Therefore, developing a method to easily store CSF samples is highly desirable for clinically relevant research on CNS pathologies. Thus, the objective of this study was to examine 2 methods of long-term storage of CSF samples which ensured reliable measurement of cell percentages and relative proportion of cell subsets using flow cytometry. Aims: To examine percentages and relative proportion of subsets of selected peripheral leukocytes and brain derived cells in 1) cryopreserved CSF in comparison to freshly processed CSF, and 2) Transfix-treated CSF in comparison to freshly processed CSF. Method: CSF samples were prospectively collected and processed as follows 1) Freshwithin 24 hours (the current gold standard); 2) Cryopreserved- analysed after 1 month storage at..............Percentages of numerous white blood cell populations and brain-derived immune cells were analysed using flow cytometry and compared across these methods. The median fluorescent intensity of select markers was also compared across these methods. Results: The majority of cell percentages were not statistically significantly different between Fresh and Cryopreserved CSF, and cell proportions were comparable. Conversely, loss of marker expression of various lymphocyte sub-populations was observed in Transfixtreated CSF compared to Fresh, and certain cell populations could not be clearly distinguished in Transfix-treated CSF. Conclusion: Cryopreservation is a feasible option for long-term storage of CSF and allows quantification of cell percentages and immunophenotyping of peripheral and brain-derived cell populations by flow cytometry. This offers valuable opportunities for clinical research across a broad spectrum of CNS conditions (infectious and non-infectious). Further, this work highlights the potential to cryopreserve other surgical specimens for which the application of flow cytometry is currently limited by resource constraints and low cell counts.