Background: To analyze whether the determination of interleukin-6 levels in cerebrospinal fluid is useful as a biomarker for the severity of bacterial meningitis. Methodology: Cerebrospinal fluid was obtained from 120 patients aged 0-15 years with meningitis. They were classified as having bacterial meningitis (n = 85) or aseptic meningitis (n = 35) according to the cerebrospinal fluid white blood cell count, microbiological culture and molecular methods. Interleukin-6 levels were determined by enzyme-linked immunosorbent assay. Results: No significant change in the mean interleukin-6 level on the basis of clinical signs was observed in patients with bacterial meningitis. However, the cerebrospinal fluid total protein level was elevated in patients with alertness problems (3.41±2.26 g L1). There was a significant correlation (Pearson correlation p≤0.05) between the cerebrospinal fluid glucose and total protein levels in patients with bacterial meningitis. The mean cerebrospinal fluid interleukin-6 level (4,472.0±2,494.52 pg mL1) in patients with bacterial meningitis whose disease outcome was fatal was significantly higher (p≤0.05) than that of those who survived the disease (2,983.28±2,612.13 pg mL1). Conclusion: Interleukin-6 is a potential biomarker for identifying bacterial meningitis patients with a high risk of death who require intensive care.
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Acute meningitis is responsible for rampant epidemics each year in Africa in the "African meningitis belt1. This meningitis, primarily due to a meningococcus, remains a public health problem. The establishment of new biomarkers that allow the rapid and precise diagnosis of the disease while also indicating its severity could improve care and reduce mortality. Certain cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF) and interferon (IFN) may be responsible for the severe inflammatory reaction associated with acute meningitis. Thus, there may be a correlation between cytokine concentrations in the cerebrospinal fluid (CSF) and morbidity and mortality2,3. The IL-6 is a potential marker for the diagnosis of bacterial meningitis4-7. However, inflammatory and anti-inflammatory cytokines are rarely measured for the diagnosis or prognosis of meningitis8. A new CSF biomarker that permits the differential diagnosis of meningitis and prognostic evaluation of the disease could improve the management of meningitis.
MATERIALS AND METHODS
This is a prospective, observational, transversal and non-blinded study. Patient inclusion was conducted at the Emergency Pediatric department of the National Hospital of Niamey (HNN) from February-April, 2015. Clinical patient information and microbiological culture results for patients and their CSF glucose and CSF protein levels, determined by the medical biology and biochemistry laboratories of the HNN were collected from a data collection sheet. Aliquots of the CSF supernatant obtained after centrifugation at 4000 rpm for 5 min was conserved at -20°C for IL-6 measurements and bacterial identification by molecular biology at the Medical and Health Research Center (CERMES).
Patients: This study included 120 patients with an average age of 6.8±4.67 years of which 35 had aseptic meningitis (leukocyte count >10 cells mm3 and no bacteria identified by culture or molecular biology) and 85 had bacterial meningitis identified by both microbiological culture and molecular biology. The CSF sampling was performed at admission before the start of antibiotic therapy with ceftriaxone.
Measurement of CSF biomarkers: Glucose (Cypress Diagnostics, Langdorp, Belgium) and protein (Sprinreact, Sant Esteve de Bas, Spain) contents of the CSF supernatant were determined using an enzymatic, colorimetric method9-14.
An enzyme-linked immunosorbent assay (ELISA) method using the Human IL-6 ELISA (EH2IL65) kit (Thermo Scientific, USA) was used to measure IL-6 in the CSF supernatant. Each sample was analyzed in duplicate and the absorbance of the plates measured at 450 nm. The test has a sensitivity <1 pg mL1 and requires 50 μL of CSF supernatant. The results are expressed in pg mL1.
Bacteriological and molecular identification: Bacteriological identification involved culture on polyvitex chocolate agar or blood agar using an API NH gallery for Neisseria meningitidis and Haemophilus influenzae and the Alere BinaxNOW® Streptococcus pneumoniae Antigen Card Kit (Alere Inc, USA) for Streptococcus pneumoniae.
A conventional Multiplex Polymerase Chain Reaction (PCR) was used to identify the three principle bacteria responsible for bacterial meningitis: Neisseria meningitidis (crgA gene), Streptococcus pneumoniae (lytA gene) and Haemophilus influenzae (bexA gene). The genogroup of Neisseria meningitidis was determined by multiplex PCR that first identifies the A, X and W genogroups and then the Y and C genogroups for those that are negative in the first PCR15-17.
Statistics: The IBM SPSS Statistics vs 20 software was used for statistical analysis. The Mann-Whitney U test was performed to compare the groups. The Pearson correlation was determined only for patients with results for the three biomarkers. The p≤0.05 was considered to be significant.
The distribution of the patients according to age and gender and the mean values of the biomarkers depending on the type of meningitis, are summarized in Table 1. The principal bacterium identified in the cases of bacterial meningitis was Neisseria meningitides (97.6% of cases): 77.6% were serogroup C, 11.8% serogroup W and 8.2% undetermined. Only two cases of S. pneumoniae (2.4%) were identified. There was a significant difference (p<0.01) between the patients with bacterial meningitis and those with aseptic meningitis for the CSF IL-6 concentration (3,538.69±2,560.78 pg mL1 versus 332.51±470.69 pg mL1), CSF glucose (1.31±1.59 mmol L1 versus 3.01±2.09 mmol L1) and CSF protein levels (3.09±2.79 g L1 versus 1.70±1.79 g L1).
|Table 1:||Population characteristics and biomarkers of patients included in the study|
|aResults are expressed as the number of patients (% of total number of patients),|
bMean (minimum-maximum), cMean±SD (number of patients) and N: Number of patients
Biomarker values associated with various clinical symptoms were determined: (1) Mean CSF protein levels were significantly higher in bacterial meningitis patients with alertness problems (3.41±2.26 g L1, p≤0.05) (Table 2) and (2) IL-6 levels were higher in aseptic meningitis patients with convulsions (1104.79±1534.16 pg mL1, p≤0.05) (Table 3). There was a significant correlation (Pearson correlation, p<0.05) between CSF glucose and protein levels, but not IL-6, in patients with bacterial meningitis. The mean concentration of IL-6 at admission was higher in patients for whom the disease outcome was death than in patients who survived the disease (4,472.0±2,494.52 pg mL1 versus 2,983.28±2,612.13 pg mL1, p≤0.05) (Table 4).
|Table 2:||CSF IL-6, glucose and total protein values according to clinical signs of patients with bacterial meningitis|
|*p≤0.05 and n: Number of patients|
|Table 3:||CSF IL-6, glucose and total protein values according to clinical signs of patients with aseptic meningitis|
|*p≤0.05 and n: Number of patients|
|Table 4:||IL-6 values according to disease outcome of patients with bacterial meningitis|
|*p≤0.05 and n: Number of patients|
The diagnosis of meningitis is primarily based on clinical examination and the analysis of CSF cytology and CSF glucose and protein levels18,19. Other biomarkers of CSF including cytokines have been explored to increase the number of available biological examinations that are informative about the intensity of inflammation in the subarachnoid space20. The central hypothesis of this study is a possible link between the IL-6 concentration in the CSF and both the clinical signs and outcome of the disease. The results obtained show that the clinical signs of bacterial meningitis are independent of the IL-6 concentration. In contrast, CSF protein is higher in patients with than without alertness problems. The increase in CSF protein levels following inflammation in the subarachnoid space is one of the physiopathological mechanisms that leads to nerve damage21. This nerve damage may be the direct consequence of bacterial toxicity or due to the indirect effect of cytokine secretion22,23. Alertness problems associated with bacterial meningitis are a consequence of the increased protein concentration in the CSF and convulsions in aseptic meningitis a consequence of increased IL-6 in the CSF. Nevertheless, the CSF IL-6 concentration is significantly higher in bacterial than aseptic meningitis21,24,25. A link between the increase in the CSF IL-6 concentration in aseptic meningitis and nerve damage has been reported21, this high IL-6 concentration does not correlate with CSF glucose and protein levels. This study did not find a relation between the CSF IL-6 concentration and the duration of the illness and fever. In contrast, Hseih et al.4 found a significant increase in the CSF IL-6 concentration if the fever lasted longer than three days for both bacterial and aseptic meningitis. In this study, a link between high mean CSF IL-6 concentration and a fatal outcome of the disease was found. According to Vazquez et al.26, an IL-6 concentration in the CSF above 1000 pg dL1 is associated with high morbidity and mortality independent of the clinical signs of the disease. In contrast, Misra et al.27 reported that the CSF IL-6 concentration does not correlate with the stage of the disease, the severity, or the outcome in patients with tuberculin meningitis. The IL-6, TNFα and IL-1β are the principal cytokines secreted first and act in synergy to stimulate a cascade of inflammatory mediators28. This inflammatory reaction is partially responsible for the physiopathological consequences of bacterial meningitis such as the stimulation of protein secretion (exudation), fever, leukocyte infiltration29 and neurological damage: Cerebral edema, cerebral hypoperfusion, nerve damage23. The association between the severity of inflammation in the subarachnoid space and the mortality of meningitis has already been reported in experimental animal models of bacterial meningitis30,31. There is also a correlation between the CSF IL-6 concentration and neurological damage in cases of aseptic meningitis21,32. Consequently, the use of dexamethasone has been suggested to reduce the intensity of the inflammatory reaction in the subarachnoid space and the negative effects33.
This study coincided with a major meningitis epidemic34. The capacity of the National Hospital of Niamey is limited and some patients were redirected to other centers such that we do not know the outcome, also, in these conditions of overload, CSF was not collected or not tested for some patients. Medical follow-up of patients is not considered in this study. However, follow-up of these patients would be valuable to evaluate the clinical value of assaying CSF IL-6.
The IL-6 is a potential biomarker for the identification of bacterial meningitis patients with an elevated risk of dying and who need intensive care. The IL-6 testing may identify patients who do not require hospitalization and could be treated at home, thereby freeing-up hospital beds for serious cases, particularly during epidemics.
We warmly thank the personnel of the Bacteriology-Virology Unit of CERMES, the personnel of the Pediatric Emergency Department, the Medical Biology Laboratory, the Biochemistry Laboratory and the Epidemiology Department of the National Hospital of Niamey, as well Monsieur Ibrahim Oumarou Sadou of the National Institute of Statistics, for their contributions.
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