Sequential analysis of biomarkers in cerebrospinal fluid and serum during invasive meningococcal disease.

Abstract

The aim of the present study was to determine the profile of different inflammatory molecules in serum and cerebrospinal fluid (CSF) during invasive meningococcal disease (IMD). Their relationship with IMD severity was also assessed. A cohort of 12 patients with IMD was investigated. Paired serum and CSF samples were obtained at the time of diagnostic and follow-up lumbar puncture and were examined using Luminex analysis. IMD severity correlated with serum interleukin-6 (IL-6) and interleukin-1 receptor antagonist (IL-1 ra) on admission. Furthermore, the CSF levels of IL-1 beta, IL-1 ra, IL-6, IL-8, macrophage inflammatory protein-1 beta (MIP-1 beta), and monocyte chemoattractant protein-1 (MCP-1) were significantly higher than their respective serum levels. The strongest correlations were found between serum concentrations of IL-1 beta and IL-1 ra, IL-6, IL-8, and MIP-1 beta, whereas the strongest correlations in CSF were found between endotoxin and IL-8, IL-17, MIP-1 beta, and MCP-1. As was expected, the concentrations of inflammatory molecules in both serum and CSF significantly decreased after antibiotic treatment. With regard to kinetics, a severe course of IMD correlated positively with rapid declines of CSF IL-6 and cortisol levels. Sequential multiple analyses revealed patterns of inflammatory responses that were associated with the severity of IMD, as well as with the compartmentalization and kinetics of the immune reaction.

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ARTICLE
Sequential analysis of biomarkers in cerebrospinal fluid
and serum during invasive meningococcal disease
O. Beran & D. A. Lawrence & N. Andersen &
O. Dzupova & J. Kalmusova & M. Musilek & M. Holub
Received: 1 October 2008 /Accepted: 15 January 2009 /Published online: 10 February 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract The aim of the present study was to determine
the profile of different inflammatory molecules in serum
and cerebrospinal fluid (CSF) during invasive meningococ-
cal disease (IMD). Their relationship with IMD severity
was also assessed. A cohort of 12 patients with IMD was
investigated. Paired serum and CSF samples were obtained
at the time of diagnostic and follow-up lumbar puncture
and were examined using Luminex® analysis. IMD severity
correlated with serum interleukin-6 (IL-6) and interleukin-1
receptor antagonist (IL-1ra) on admission. Furthermore, the
CSF levels of IL-1β, IL-1ra, IL-6, IL-8, macrophage
inflammatory protein-1β (MIP-1β), and monocyte chemo-
attractant protein-1 (MCP-1) were significantly higher than
their respective serum levels. The strongest correlations
were found between serum concentrations of IL-1β and IL-
1ra, IL-6, IL-8, and MIP-1β, whereas the strongest
correlations in CSF were found between endotoxin and
IL-8, IL-17, MIP-1β, and MCP-1. As was expected, the
concentrations of inflammatory molecules in both serum
and CSF significantly decreased after antibiotic treatment.
With regard to kinetics, a severe course of IMD correlated
positively with rapid declines of CSF IL-6 and cortisol
levels. Sequential multiple analyses revealed patterns of
inflammatory responses that were associated with the
severity of IMD, as well as with the compartmentalization
and kinetics of the immune reaction.
Introduction
Invasive meningococcal disease (IMD) still remains a life-
threatening infection with significant morbidity and mor-
tality. This remains true even in developed countries, in
spite of the availability of efficient antimicrobial therapy
and intensive care treatment. IMD can present in four
different clinical forms: benign meningococcemia (mortal-
ity rate <1%), meningitis (mortality rate ∼5%), meningitis
with sepsis (mortality rate ∼10%), and fulminant meningo-
coccal sepsis (FMS; mortality rate 40–50%). Important
mechanisms involved in IMD pathogenesis include the
massive production of inflammatory mediators (i.e., com-
plement factors, cytokines, etc.) and excessive activation of
the coagulation and fibrinolysis pathways [1, 2]. Exagger-
ated production of these mediators during the initial course
of IMD is associated with high levels of meningococcal
lipooligosaccharides (LOS) released into body fluids by
Neisseria meningitidis, the causative agent of IMD [3].
High LOS concentrations are believed to be responsible for
Eur J Clin Microbiol Infect Dis (2009) 28:793–799
DOI 10.1007/s10096-009-0708-6
O. Beran:M. Holub
1st Medical Faculty, Teaching Hospital Bulovka,
3rd Department of Infectious and Tropical Diseases,
Charles University in Prague,
Prague, Czech Republic
D. A. Lawrence:N. Andersen
Wadsworth Center, New York State Department of Health,
Albany, USA
O. Dzupova
3rd Medical Faculty, Teaching Hospital Bulovka,
Department of Infectious Diseases, Charles University in Prague,
Prague, Czech Republic
J. Kalmusova:M. Musilek
National Institute of Public Health,
National Reference Laboratory for Meningococcal Infections,
Prague, Czech Republic
M. Holub (*)
3rd Department of Infectious and Tropical Diseases,
Teaching Hospital Bulovka,
Budínova 2,
Prague 8 180 81, Czech Republic
e-mail: michal.holub@lf1.cuni.cz

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the over-stimulation of innate immunity through toll-like
receptors (TLRs) and coagulation systems, leading to
potentially fatal complications of IMD, which include
multiple organ failure, septic shock, and disseminated
intravascular coagulation with bleeding complications [4].
The principles of IMD management have been almost
the same over the last few decades: rapid clinical
diagnostics, immediate administration of efficient anti-
biotics, and transfer to a specialized intensive care unit
(ICU). Novel therapies that can be used in the ICU setting
include the administration of recombinant human activated
protein C (rhAPC), which is indicated especially in adult
patients with FMS without bleeding complications [5]. A
significant effort has also been devoted to the neutralization
of LOS. Unfortunately, the studies that have been con-
ducted were not powerful enough to demonstrate improved
survival [6, 7]. Thus, no new therapies for IMD have been
approved since the advent of rhAPC in 2001. Moreover, a
clinical trial in children with IMD treated with rhAPC,
which has both anti-inflammatory and anticoagulant prop-
erties, was inconclusive [8]. This lack of progress in the
development of new therapies is partly due to our
insufficient knowledge about the complexities of innate
immunity responses during the course of IMD.
Therefore, the aim of our study was to better characterize
the profiles of cytokines and other inflammatory mediators
in the serum and cerebrospinal fluid (CSF) during IMD
using a multiplex assay. We also assessed the relationship
between the investigated mediators in both compartments
and their correlation with IMD severity. Additionally,
changes in these profiles after starting IMD therapy were
evaluated.
Patients and methods
This study was conducted at the Teaching Hospital
Bulovka, a 1,209-bed, tertiary care community hospital in
Prague, Czech Republic. Enrolled patients were admitted to
the Department of Infectious Diseases between the years
2004 and 2006. The study was approved by the local ethics
committee, and it was performed in compliance with the
Helsinki Declaration (1996 revision).
Patients
A cohort of 12 patients (five females and seven males) with
IMD was analyzed. The mean age of the study subjects was
21 years (range 14–52 years). Table 1 presents the patient
demographics and clinical parameters, as well as the
characteristics of N. meningitidis strains determined by
classical and molecular methods (i.e., polymerase chain
reaction [PCR], genosubtyping, porA sequencing, and
Table 1 Demographic and clinical characteristics of the patients with invasive meningococcal disease
Patient
no.
Sex
Age
(years)
Neisseria meningitidis strain characteristics
APACHE
II (points)
SOFA
(points)
GCS
(points)
Hosp.
(days)
ICU stay(days)
Clinical
form
Outcome
Serogroup
PorA, FetAa
ST
Clonal complex
1
F
14
B
B:22:P1.22,14:F3-4
1001
ST-18 complex
25
12
14
1
1
FMS
poor
2
F
16
B
B:N.d.:N.d.
3
0
15
13
0
Men
good
3
M
18
B
B:N.d.:N.d.
6
3
14
16
5
Men
good
4
M
18
B
B:4:P1.7,14:F1-14
112
ST-41/44/Lineage
11
4
10
25
12
M+S
good
5
M
15
C
C:21:P1.17,16-4:F3-9
3346
ST-41/44/Lineage
8
1
13
25
3
M+S
good
6
F
14
B
B:N.d.:N.d.
4
2
14
26
0
Men
good
7
M
21
B
B:NT:P1.17,16-3:F5-2
919
17
6
7
25
16
M+S
good
8
M
24
B
B:4:P1.19,15:F5-1
33
ST-32/ET-5
16
5
14
21
7
M+S
good
9
F
22
C
C:NT:P1.5,2:F1-2
11
ST-11/ET-37
14
3
8
18
5
M+S
good
10
F
52
B
B:15:P1.7-2,4:F1-2
5126
11
3
8
16
0
Men.
good
11
M
19
B
B:1:P1.7-2,14:F1-14
112
ST-41/44/Lineage
10
4
11
11
0
M+S
good
12
M
19
C
C:2a:P1.5,2:F3-6
11
ST-11/ET-37
12
6
7
26
8
M+S
good
APACHE = Acute Physiological and Chronic Health Evaluation; SOFA = Sequential Organ Failure Assessment; GCS = Glasgow Coma Scale; Hosp. = hospitalization; ICU = intensive care unit;
ST = sequence type; F = female; M = male; FMS = fulminant meningococcal sepsis; Men = meningitis; M+S = meningitis + sepsis; N.d. = not determined
aouter membrane proteins
794Eur J Clin Microbiol Infect Dis (2009) 28:793–799

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multilocus sequence typing) described elsewhere [9–12].
Patients’ specimens (i.e., paired serum and CSF samples)
were obtained at the time of diagnostic (day 1) and follow-
up (day 3–5) lumbar puncture. Table 2 shows the patients’
laboratory results from routine blood and CSF tests at day
1. CSF samples were collected in polystyrene tubes closed
with screw-caps (Sarstedt AG, Germany) and venous blood
was collected using an S-Monovette® (Sarstedt AG)
collection system for blood count determination in tubes
with K3-EDTA (Sarstedt AG). All samples were centri-
fuged immediately after collection, aliquoted, and stored at
−80°C until further analyses were performed. The patients
were treated according to the national standard protocol,
which consists of antibiotics (third-generation cephalospor-
ins for meningitis and for sepsis + meningitidis or penicillin
G for FMS), corticosteroids (for meningitis), and intensive
care treatment, if required [13]. The disease severity was
evaluated using the APACHE II (Acute Physiological and
Chronic Health Evaluation), SOFA (Sequential Organ
Failure Assessment), and GCS (Glasgow Coma Scale)
scoring systems.
Laboratory methods
In addition to routine analyses (i.e., differential blood
count, CSF cytology and chemistry, and serum level of C-
reactive protein [S-CRP]), serum and CSF concentrations
of 14 biomarkers (i.e., interleukin-1β [IL-1β], IL-1 receptor
antagonist [IL-1ra], IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-
12, IL-17, tumor necrosis factor α [TNF-α], monocyte
chemoattractant protein 1 [MCP-1], macrophage inflamma-
tory protein 1β [MIP-1β], and leptin) were analyzed using
the Luminex® methodology with reagents from R&D
Systems, Inc. (USA). Endotoxin concentrations were
measured using the Kinetic LAL assay (Cambrex, USA).
Analyses of cortisol levels in the CSF and serum were
performed by radioimmunometric assay using a commer-
cial DSL-2000 kit (Diagnostic Systems Laboratories, USA)
with a detection limit of 5 nmol/l.
Statistical methods
Statistical analyses were performed using SPSS software™
(Jandel Scientific, USA). The data are presented as means
(standard deviation). The analyses consisted of two-tailed tests
with an α-level below 0.05. The differences between serum
and CSF levels were tested using paired t-tests and Wilcox-
on’s signed rank test for nonparametric data. Spearman’s and
Pearson’s correlation tests were employed to determine
whether correlations existed between clinical and laboratory
parameters.
Results
Cytokines, chemokines, leptin, and endotoxin in serum
and CSF at admission
IL-1β was detected in the serum in 41% of patients (5/12),
whereasitwasdetectedintheCSFin100%ofpatients(10/10).
IL-1ra was detected in 100% of serum specimens (12/12), as
well as in 100% of CSF samples (10/10). IL-6 was detected in
91% of serum samples (11/12), whereas CSF concentrations
were detectable in 100% of specimens (10/10). IL-10 was
detectable in 75% of patients (9/12) in the serum and in 90%
of patients in the CSF (9/10). IL-17 was detected in 41% of
serum specimens (5/12) and in 50% of CSF samples (5/10).
TNF-α levels were detectable in the serum of only 25% of
patients (3/12) and in the CSF in only 10% of the specimens
(1/10). IL-8 was detectable in 91% of serum specimens
Patient
no.
S-CRP
(mg/l)
B-WBC
(cells/mm3)
B-lympho
(cells/mm3)
CSF-leuko
(cells/3 mm3)
CSF-protein
(g/l)
CSF-glucose
(mmol/l)
1
2
3
4
5
6
7
8
9
10
11
12
102
204
346
92
246
225
226
233
271
159
165
265
2,700
17,600
25,300
4,700
17,100
21,500
10,900
25,200
20,700
16,900
16,700
21,500
1,026
1,232
759
376
171
215
218
252
414
507
835
N.d.
5
8,704
5,153
230
1,585
10,000
11,666
3,331
7,253
3,413
3,840
10,000
0.2
2.7
3.4
2.4
5.7
2.6
7.7
1.8
2.7
2.6
2.8
4.9
3.1
2.5
3.4
2
3.5
4.4
0.5
0.5
1.9
3.3
1.6
0.6
Table 2 Inflammatory markers
in (B) the blood and in the
serum (S) and cerebrospinal
fluid (CSF) obtained at the
time of diagnostic lumbar
puncture from the patients with
invasive meningococcal
disease
WBC = white blood cells;
Lympho = lymphocytes;
Leuko = leukocytes; N.d. = not
determined
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(11/12) and in 100% of CSF samples (10/10). MCP-1 and
MIP-1β were detected in 100% of serum samples (12/12) and
in 100% of CSF specimens (10/10). Similarly, leptin was
detectable in 100% of serum specimens (12/12) and 100% of
CSF samples (10/10). In addition, endotoxin was detected in
25% of serum specimens (3/12) and in 90% of CSF samples
(9/10), and cortisol levels were detectable in all serum and
CSF specimens. The serum and CSF concentrations and their
statistical differences are shown in Table 3.
Correlation of serum and CSF inflammatory mediators
with IMD severity
The APACHE II score on day 1 correlated with increased
serum concentrations of IL-6 (r=0.774, P=0.004) and IL-
1ra (r=0.648, P=0.031). The SOFA score on day 1
correlated only with IL-6 serum levels (r=0.773, P=
0.004). In addition, the serum concentration of endotoxin
correlated with GCS (r=0.662, P=0.023). Unlike the
concentrations of inflammatory molecules in the serum,
the CSF concentrations were not associated with IMD
severity.
Correlation between inflammatory mediators in the serum
at admission
IL-1β serum levels correlated significantly with IL-1ra (r=
0.694, P=0.017), IL-6 (r=0.758, P=0.006), IL-8 (r=0.773,
P=0.004), IL-10 (r=0.656, P=0.026), and MIP-1β (r=
0.877, P<0.001). IL-1ra demonstrated a significant corre-
lation with IL-6 (r=0.691, P=0.012), IL-10 (r=0.606, P=
0.043), and MCP-1 (r=0.809, P<0.001). IL-6 serum
concentrations correlated significantly with IL-8 (r=0.773,
P=0.004), IL-10 (r=0.656, P=0.023), and MCP-1 (r=
0.827, P<0.001). MCP-1 levels correlated with IL-8 and
IL-10 serum concentrations (r=0.709, P=0.013; r=0.715,
P=0.011, respectively). In addition, the serum concentra-
tions of endotoxin correlated with the cortisol levels (r=
0.695, P=0.017).
Correlation between inflammatory mediators in the CSF
at admission
IL-1β showed a significant correlation with the CSF levels
of IL-10 (r=0.636, P=0.043). IL-1ra demonstrated a
significant correlation with IL-6 (r=0.867, P<0.001), IL-
10 (r=0.721, P=0.012), and MCP-1 (r=−0.636, P=0.043).
CSF IL-6 levels correlated with IL-10 (r=0.758, P=0.009).
Intrathecal levels of IL-8 demonstrated a significant
correlation with MCP-1 (r=0.879, P<0.001). Endotoxin
concentrations in the CSF correlated with IL-8 (r=0.778,
P=0.005), IL-17 (r=−0.623, P=0.048), MCP-1 (r=0.669,
P=0.029), and MIP-1β (r=0.681, P=0.025). In addition,
CSF cortisol levels correlated with IL-1ra (r=0.636,
P=0.043), IL-6 (r=0.721, P=0.016), and IL-10 (r=0.697,
P=0.022).
Kinetics of serum and CSF inflammatory mediators
and the correlation with IMD severity
Significant decreases in serum concentrations 3–5 days
after the start of the antibiotic treatment were detected for
the following mediators: IL-1ra (P=0.001), IL-6 (P=
0.016), IL-10 (P=0.023), and MIP-1β (P=0.022). In
addition, two routine biomarkers demonstrated significant
changes during this interval: serum CRP levels decreased
(P<0.001), whereas the number of circulating lymphocytes
increased (P=0.002).
With regard to the kinetics of cytokines in CSF during
the course of IMD, significant decreases were demonstrated
in the concentrations of IL-1β (P=0.02), IL-1ra (P<0.001),
IL-6 (P<0.001), IL-8 (P=0.008), MCP-1 (P=0.001), and
MIP-1β (P=0.003). The kinetics of serum as well as the
CSF levels of IL-1ra, IL-6, MCP-1, and MIP-1β are shown
in Fig. 1. Similar to cytokines and chemokines, the
endotoxin levels decreased over time (P=0.016). As
expected, the number of leukocytes in the CSF as well as
the CSF protein concentrations declined (P=0.003 for both
markers), and the CSF glucose level increased (P=0.049).
Unlike serum parameters, the kinetics of some CSF
biomarkers demonstrated a significant correlation with the
IMD severity: Δ cortisol correlated with APACHE II score
(r=0.690, P=0.04) and Δ glucose and Δ IL-6 correlated
with SOFA score (r=−0.860, P=0.003; r=0.733, P=0.039,
respectively).
Table 3 Difference between serum and CSF concentrations of
inflammatory molecules in specimens obtained at admission to the
hospital
Parameter SerumCSFP-valuea
IL-1b (pg/ml)
IL-1ra (pg/ml)
IL-6 (pg/ml)
IL-8 (pg/ml)
IL-10 (pg/ml)
IL-17 (pg/ml)
MCP-1 (pg/ml)
MIP-1b (pg/ml)
Leptin (pg/ml)
Endotoxin (EU)
Cortisol (nmol/ml)
38 (112)
27,978 (14,234)
7,549 (12,895)
2,263 (5,325)
2,432 (4,762)
6 (6)
2,139 (2,592)
653 (1,657)
6,496 (8,846)
13 (43)
247 (358)
974 (1,118)
58,710 (33,025)
59,575 (28,462)
33,760 (19,118)
986 (1,457)
45 (109)
23,025 (14,134)
49,207 (42,336)
53 (42)
133 (140)
157 (108)
0.031
0.029
<0.001
<0.001
N.s.
N.s.
<0.001
0.006
0.002
N.s.
N.s.
N.s. = not significant; EU = endotoxin unit
aPaired t-test and Wilcoxon signed rank test, as appropriate
Data are presented as means (standard deviation)
796Eur J Clin Microbiol Infect Dis (2009) 28:793–799

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Discussion
The inflammatory response elicited by N. meningitidis is
considered to be the major factor responsible for the
outcome of IMD. Therefore, we analyzed the concentra-
tions and kinetics of different molecules that are known to
be related to inflammation in the CSF and serum during the
course of IMD.
Our results demonstrated significant compartmentaliza-
tion of the immune response in the subarachnoid space,
which has been already reported in meningococcal menin-
gitis, as well as in community-acquired bacterial meningitis
of other etiology [14, 15]. Moreover, cytokines and
chemokines that were concentrated in the CSF were also
detectable in the serum in the majority of IMD patients. It is
well known that IL-6 and IL-8 can be used in the laboratory
Fig. 1 The kinetics of serum
and cerebrospinal fluid (CSF)
cytokine levels during invasive
meningococcal disease
Eur J Clin Microbiol Infect Dis (2009) 28:793–799797