# Determination of Interleukin-6 (IL-6) in Cerebrospinal Fluid: Potential Role for the Evaluation of the Vital Prognosis in Bacterial Meningitis

Ibrahim Dan Dano, Odile Missi Oukem-Boyer, Mamane Ali El-Hadji, Ali El-Hadji Mamane

► To cite this version:

Ibrahim Dan Dano, Odile Missi Oukem-Boyer, Mamane Ali El-Hadji, Ali El-Hadji Mamane. Determination of Interleukin-6 (IL-6) in Cerebrospinal Fluid: Potential Role for the Evaluation of the Vital Prognosis in Bacterial Meningitis. Journal of Biological Sciences, 2016, 10.3923/jbs.2016.10330469

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Research Article

# Determination of Interleukin-6 (IL-6) in Cerebrospinal Fluid: Potential Role for the Evaluation of the Vital Prognosis in Bacterial Meningitis

¹Ibrahim Dan Dano, ¹Odile Ouwe Missi Oukem-Boyer, ¹Ali Elhadji Mahamane and ²Hassimi Sadou

## Abstract

**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 L⁻<fnref n="1" />). 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 mL⁻<fnref n="1" />) 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 mL⁻<fnref n="1" />). **Conclusion:** Interleukin-6 is a potential biomarker for identifying bacterial meningitis patients with a high risk of death who require intensive care.

**Key words:** Interleukin-6, cerebrospinal fluid, prognosis, bacterial meningitis

Received:

Accepted:

Published:

**Citation:** Ibrahim Dan Dano, Odile Ouwe Missi Oukem-Boyer, Ali Elhadji Mahamane and Hassimi Sadou, 2016. Determination of interleukin-6 (IL-6) in cerebrospinal fluid: Potential role for the evaluation of the vital prognosis in bacterial meningitis. J. Biol. Sci., CC: CC-CC.

**Corresponding Author:** Ibrahim Dan Dano, Centre de Recherche Medicale et Sanitaire (CERMES), 634 Boulevard de la Nation YN034, B.P. 10887, Niamey, Niger Tel: +227 20 75 20 40 Fax: +227 20 75 31 80

**Copyright:** © 2016 Ibrahim Dan Dano et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

**Competing Interest:** The authors have declared that no competing interest exists.

**Data Availability:** All relevant data are within the paper and its supporting information files.

<footnote n="1">¹Centre de Recherche Medicale et Sanitaire (CERMES), 634 Boulevard de la Nation YN034, B.P. 10887, Niamey, Niger</footnote>

<footnote n="2">²Faculte des Sciences et Techniques, Universite Abdou Moumouni, B.P. 10662, Niamey, Niger</footnote>
INTRODUCTION

Acute meningitis is responsible for rampant epidemics
each year in Africa in the "African meningitis belt"¹. 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 mm⁻³ 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 mL⁻¹ and requires 50 µL of CSF supernatant. The results are expressed in pg mL⁻¹.

**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* (*IytA* 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.

RESULTS

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 mL⁻¹ versus
332.51±470.69 pg mL⁻¹), CSF glucose (1.31±1.59 mmol L⁻¹
versus 3.01±2.09 mmol L⁻¹) and CSF protein levels
(3.09±2.79 g L⁻¹ versus 1.70±1.79 g L⁻¹). Biomarker values
associated with various clinical symptoms were determined:
(1) Mean CSF protein levels were significantly higher in
<table><caption>Table 1: Population characteristics and biomarkers of patients included in the study</caption><thead><tr><th>Variable</th><th>Aseptic meningitis</th><th>Bacterial meningitis</th></tr></thead><tbody><tr><td>Patients<sup>a</sup></td><td>35 (29.2%)</td><td>85 (70.8%)</td></tr><tr><td>Male<sup>a</sup></td><td>17 (14.2%)</td><td>42 (35%)</td></tr><tr><td>Female<sup>a</sup></td><td>18 (15%)</td><td>43 (35.8%)</td></tr><tr><td>Age (years)<sup>b</sup></td><td>5.43 (0-14)</td><td>8.43 (1-15)</td></tr><tr><td>CSF protein level<sup>c</sup></td><td>1.70±1.79 (n = 18)</td><td>3.09±2.79 (n = 65)</td></tr><tr><td>CSF glucose level (mmol L<sup>-1</sup>)<sup>c</sup></td><td>3.01±2.09 (n = 18)</td><td>1.31±1.59 (n = 65)</td></tr><tr><td>CSF IL-6 (pg mL<sup>-1</sup>) level<sup>c</sup></td><td>332.51±470.69 (n = 35)</td><td>3,538.69±2560.78 (n = 85)</td></tr></tbody></table>

<sup>a</sup>Results are expressed as the number of patients (% of total number of patients),

<sup>b</sup>Mean (minimum-maximum), <sup>c</sup>Mean ± SD (number of patients) and N: Number of patients

bacterial meningitis patients with alertness problems (3.41±2.26 g L⁻¹, p≤0.05) (Table 2) and (2) IL-6 levels were higher in aseptic meningitis patients with convulsions (1104.79±1534.16 pg mL⁻¹, 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 mL⁻¹ versus 2,983.28±2,612.13 pg mL⁻¹, p≤0.05) (Table 4).

<table><caption>Table 2: CSF IL-6, glucose and total protein values according to clinical signs of patients with bacterial meningitis</caption><thead><tr><th>Variable</th><th>CSF IL-6 (pg mL<sup>-1</sup>)</th><th>CSF glucose (mmol L<sup>-1</sup>)</th><th>CSF protein (g L<sup>-1</sup>)</th></tr></thead><tbody><tr><td><strong>Duration of the disease</strong></td><td></td><td></td><td></td></tr><tr><td>≤3 days</td><td>3,611.16±2554.29 (n = 71)</td><td>1.26±1.44 (n = 56)</td><td>2.78±2.15 (n = 56)</td></tr><tr><td>>3 days</td><td>2,835.92±2350.75 (n = 5)</td><td>0.07±0.04 (n = 2)</td><td>11.21±9.59 (n = 2)</td></tr><tr><td><strong>Fever</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3,832.37±2611.03 (n = 44)</td><td>1.44±1.79 (n = 36)</td><td>3.38±3.26 (n = 36)</td></tr><tr><td>No</td><td>2,893.78±2308.59 (n = 27)</td><td>0.88±0.94 (n = 19)</td><td>3.00±2.29 (n = 19)</td></tr><tr><td><strong>Neck stiffness</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3,600.09±2579.48 (n = 57)</td><td>1.34±1.42 (n = 43)</td><td>3.220±3.11 (n = 43)</td></tr><tr><td>No</td><td>3,235.05±2580.09 (n = 24)</td><td>1.24±1.96 (n = 20)</td><td>2.670±2.13 (n = 20)</td></tr><tr><td><strong>Convulsion</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3,644.35±2699.52 (n = 35)</td><td>1.43±1.82 (n = 23)</td><td>3.47±2.49 (n = 23)</td></tr><tr><td>No</td><td>3,375.96±2488.92 (n = 40)</td><td>1.24±1.47 (n = 40)</td><td>2.80±3.01 (n = 40)</td></tr><tr><td><strong>Alertness problems</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>3,541.42±2691.91 (n = 43)</td><td>1.24±1.40 (n = 35)</td><td>3.41±2.26* (n = 35)</td></tr><tr><td>No</td><td>3,435.93±2457.17 (n = 38)</td><td>1.40±1.84 (n = 28)</td><td>2.59±3.40 (n = 28)</td></tr><tr><td><strong>Purpura</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>1.36±0.92 (n = 6)</td><td>2.31±2.12 (n = 6)</td><td>2,894.130±3273.18 (n = 6)</td></tr><tr><td>No</td><td>1.31±1.66 (n = 57)</td><td>3.12±2.90 (n = 57)</td><td>3,557.440±2449.44 (n = 73)</td></tr></tbody></table>

*p≤0.05 and n: Number of patients

<table><caption>Table 3: CSF IL-6, glucose and total protein values according to clinical signs of patients with aseptic meningitis</caption><thead><tr><th>Variable</th><th>CSF IL-6 (pg mL<sup>-1</sup>)</th><th>CSF glucose (mmol L<sup>-1</sup>)</th><th>CSF protein (g L<sup>-1</sup>)</th></tr></thead><tbody><tr><td><strong>Duration of the disease</strong></td><td></td><td></td><td></td></tr><tr><td>≤3 days</td><td>830.30±1308.69 (n = 27)</td><td>3.03±2.20 (n = 16)</td><td>1.59±1.79 (n = 16)</td></tr><tr><td>>3 days</td><td>17.37 (n = 1)</td><td>3.74 (n = 1)</td><td>0.24 (n = 1)</td></tr><tr><td><strong>Fever</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>816.74±1257.22 (n = 17)</td><td>2.92±2.0 (n = 8)</td><td>1.17±1.49 (n = 8)</td></tr><tr><td>No</td><td>276.55±397.96 (n = 9)</td><td>3.30±1.92 (n = 6)</td><td>1.09±1.43 (n = 6)</td></tr><tr><td><strong>Neck stiffness</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>1,344.25±1585.50 (n = 11)</td><td>2.29±1.76 (n = 5)</td><td>2.12±1.76 (n = 5)</td></tr><tr><td>No</td><td>435.9100±860.36 (n = 21)</td><td>3.39±2.26 (n = 12)</td><td>1.25±1.78 (n = 12)</td></tr><tr><td><strong>Convulsion</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>1,104.79±1534.16* (n = 13)</td><td>2.49±2.74 (n = 8)</td><td>2.15±2.13 (n = 8)</td></tr><tr><td>No</td><td>504.1400±929.97 (n = 19)</td><td>3.58±1.38 (n = 9)</td><td>0.94±1.22 (n = 9)</td></tr><tr><td><strong>Alertness problems</strong></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>760.16±1279.82 (n = 14)</td><td>3.31±2.41 (n = 8)</td><td>1.00±1.10 (n = 8)</td></tr><tr><td>No</td><td>738.81±1219.90 (n = 18)</td><td>2.85±1.98 (n = 9)</td><td>1.96±2.16 (n = 9)</td></tr></tbody></table>

*p≤0.05 and n: Number of patients

<table><caption>Table 4: IL-6 values according to disease outcome of patients with bacterial meningitis</caption><thead><tr><th>Variable</th><th>CSF IL-6 (pg mL<sup>-1</sup>)</th><th>CSF glucose (mmol L<sup>-1</sup>)</th><th>CSF protein (g L<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Death</td><td>4,472.00±2494.52* (n = 21)</td><td>1.78±2.18 (n = 13)</td><td>4.47±4.82 (n = 13)</td></tr><tr><td>Survival</td><td>2,983.28±2612.13 (n = 48)</td><td>0.87±1.05 (n = 33)</td><td>3.19±2.0 (n = 33)</td></tr></tbody></table>

*p≤0.05 and n: Number of patients
DISCUSSION

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, Hsieh et al⁴
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 dL⁻¹ 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.

CONCLUSION

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.

ACKNOWLEDGMENTS

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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