JOURNAL OF TROPICAL PEDIATRICS, VOL. 59, NO.

4, 2013

Brief Report
Oxidative Stress in Children with Bacterial Meningitis
by Ragni Srivastava,1 Rajeev Lohokare,1 and Rajniti Prasad2
1
Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India
2
Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India

Correspondence: Rajniti Prasad, Department of Pediatrics, Institute of Medical Sciences, Banaras Hindu University,
Varanasi 221005. Tel/Fax: 0091-542-2367677. E-mail <rajnitip@gmail.com>.

Summary

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Bacterial meningitis is a common cause of morbidity and mortality in children. The oxidative stress in
bacterial meningitis is barely determined. Forty children with bacterial meningitis were studied for their
oxidants and antioxidants status in serum and cerebrospinal fluid. Fever (95%) was commonest pres-
entation followed by seizure and vomiting. Neck rigidity and Kernig’s sign were present in 37.5% and
27.5% cases, respectively. Plasma and cerebrospinal fluid malondialdehyde, protein carbonyl and nitrite
levels were significantly raised in cases (p < 0.001). Plasma and cerebrospinal fluid ascorbic acid,
glutathione and superoxide dismutase levels were significantly decreased in children with septic menin-
gitis (p < 0.001). Significantly elevated malondialdehyde, nitrite and protein carbonyl levels reflect
increased oxidative stress, whereas decreased concentrations of glutathione, ascorbic acid and super-
oxide dismutase indicates utilization of the antioxidants in septic meningitis. Thus, changes in oxidants
and antioxidants observed suggest production of reactive oxygen species and their possible role in
pathogenesis of septic meningitis.

Key words: meningitis, bacterial, oxidants, antioxidants.

Introduction of free radicals in the pathogenesis of bacterial

Bacterial meningitis is the most severe infection of the
central nervous system of children in developing coun-
tries including India. It leads to serious neurological
sequelae, including cognitive impairment [1, 2].
According to World Health Organization, 171 000
children die annually from bacterial meningitis and
10–20% suffer from sequelae such as mental retard-
ation, deafness or epilepsy [1].
In bacterial meningitis, neutrophils and macro-
phages use reactive oxygen species (ROS) as part of
their host defense mechanisms. In this process, these
cells consume molecular oxygen, which is converted
into toxic superoxide anion (O2), hydrogen peroxide
(H2O2) and hydroxyl radical (OH) [3–5]. Under
normal circumstances, ROS are eliminated by cellular
enzymatic system, i.e. superoxide dismutase (SOD)
and catalase, and non-enzymatic system, glutathione,
ascorbic acid and uric acid antioxidants defenses [6].
Thus, generated ROS result in peroxidation of mem-
brane lipids and denaturation of proteins and DNA [6,
7]. The natural or synthetic antioxidants may prevent
disease progression, tissue damage and disturbed
blood–brain barrier [8]. ROS may contribute to the
pathophysiology of bacterial meningitis [9]. The role
meningitis has been shown in experimental animals,
but studies in humans, especially in children, are lack-
ing. The present study was done to observe the con-
centration of oxidants and antioxidants in serum and
cerebrospinal fluid (CSF) in children and their role in
pathogenesis of bacterial meningitis.
Patients and Methods
This prospective hospital-based cross-sectional study
was conducted from December 2010 to June 2012 in
children admitted in pediatric ward of a tertiary-care
hospital. Informed consent was obtained by the par-
ents, and study protocol was approved by Institute
Ethics Committee.
Inclusion criteria
Cases: children, aged 2 months to 14 years, having
CSF white blood count >50/ml with predominant
neutrophils; CSF: blood glucose ratio <0.5, elevated
CSF protein >50 mg/dl and positive gram’s stain
and/or CSF culture.
Controls: children admitted in pediatric ward and
have undergone lumbar puncture with normal CSF.

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doi:10.1093/tropej/fmt013 Advance Access published on 22 February 2013
 BRIEF REPORT

Exclusion criteria TABLE 1

Children with viral meningitis and encephalitis, fetal Basic characteristics of cases and controls
malformations, chromosomal abnormalities, im-
munodeficiency, endocrine diseases, neuromuscular Characteristics Cases Controls
disorders and who had received prior antibiotics (n ¼ 40) (n ¼ 40)
were excluded from the study. Mean age (years) 5.8  4.2 4.5  3.57
Sex
Blood and cerebrospinal fluid sample collection and Male 26 (65.0) 27 (67.5)
estimation Female 14 (35.0) 13 (32.5)
About 10 ml of blood was drawn by venipuncture Fever 38 (95.0) 18 (45.0)
with a heparinized syringe and transferred to sterile Seizure 22 (55.0) 2 (5)
Vomiting 15 (37.5) 4 (10)
deionized polyethylene vials. Plasma was separated
Neck rigidity 15 (37.5) 0 (0)
immediately by centrifugation at 2000 rpm for 5 min Bulging anterior fontanelle 13 (32.5) 0 (0)
and was stored in separate deionized vials at 20 C. Unconsciousness 11 (27.5) 7 (17.5)
Two milliliters of whole blood was also stored separ- Kernig’s sign 11 (27.5) 0 (0)

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ately for glutathione estimation. Complete blood
count and renal functions were also done in all pa-
tients. Under aseptic precaution, CSF was collected
in deionized polyethylene vials stored at 20 C for
analysis of malondialdehyde, protein carbonyl, ni-
trite, SOD, ascorbic acid, glutathine peroxidase till
estimation. CSF was also subjected to routine cyto-
chemical examinations including Gram’s staining
and aerobic culture.
Plasma malondialdehyde (MDA) was estimated by
thiobarbituric acid test [10], glutathione by 5,5
dithiobis method, whereas protein carbonyl, nitrite,
ascorbic acid and SOD were analyzed by the methods
of Reznick and Packer [11], Moshage et al. [12], Roe
[13] and Marklund and Marklund [14], respectively.
Treatment and follow-up
All children were treated with intravenous ceftriax-
one (100 mg/kg/d) for 14 days but prolonged to
3 weeks in Escherichia coli bacterial meningitis.
Intravenous lorazepam and phenytoin sodium were
used to control seizure in cases.
Statistical analysis
Data were analyzed using SPSS version 16 software.
Data were presented as mean  standard deviation
(SD). Differences between groups were analyzed for
statistical significance by paired t-test.
Results
Out of 40 cases of bacterial meningitis, 26 (65%)
were male. Fever (95%) was the commonest presen-
tation followed by seizure (55%) and vomiting
(37.5%). Neck rigidity, bulging anterior fontanelle
and Kernig’s sign were present in 37.5, 32.5 and
27.5% cases, respectively (Table 1). Laboratory find-
ings of cases and controls are mentioned in Table 2.
Significant neutrophillic leukocytosis in blood and
CSF pleocytosis was present in cases. CSF protein
was significantly increased but CSF glucose and
CSF glucose and plasma glucose ratio were signifi-
cantly decreased in cases (p < 0.001). CSF Gram’s
Headache 8 (20.0) 5 (12.5)
stain had shown Gram’s positive diplococcic in
three cases. Blood culture was sterile in all cases,
whereas CSF culture was positive in five cases. Out
of five CSF culture positive cases, Streptococcus
pneumoniae was grown in four cases and one had
E. coli, which were sensitive to ceftriaxone.
The serum MDA, protein carbonyl, SOD, gluta-
thione, ascorbic acid, nitrite in children with bacterial
meningitis are shown in Table 3. The mean serum
levels of MDA, protein carbonyl and nitrite were sig-
nificantly higher in patients with bacterial meningitis
as compared with control (p < 0.001 each), while the
mean serum concentrations of glutathione and ascor-
bic acid were significantly lower in cases (p < 0.001).
The mean serum SOD level was also significantly
lower in children with meningitis.
Oxidant concentrations (Malondialdehyde,
Protein carbonyl, Nitrite) were markedly increased
in CSF in cases compared to controls (Table 4).
Antioxidant (glutathione, ascorbic acid, SOD) levels
were significantly reduced in cases (p-value < 0.001).
The observed changes in serum and CSF concentra-
tion of oxidants and antioxidants signify increased
production of oxidants in bacterial meningitis with
utilization of antioxidants as reflected by their
decreased concentration.
Discussion
ROS may contribute to the pathophysiology of
bacterial meningitis [9]. Neutrophils and macro-
phages consume molecular oxygen, which is con-
verted into the toxic oxygen metabolites O2 and
H2O2 [7]. Recent studies have suggested that gen-
erated ROS play an important role in several
pathologic processes including vascular damage,
brain edema and CSF pleocytosis [5, 9, 15–17].
Significant increases in CSF ROS levels in patients
with bacterial meningitis have been reported by
other researchers [7, 18]. Koedel and Pfister [15]

306 Journal of Tropical Pediatrics Vol. 59, No. 4

BRIEF REPORT

TABLE 2
Laboratory findings of cases and controls

Cases (mean  SD) (range) Control (mean  SD) (range) p-value

Blood TLC (/ml) 19686.4  8102.6 16 548  3805.7 0.015

(3900–49 800) (6000–20 900)
Blood neutrophil (%) 72.46  14.67 54.2  18.6 0.006
(15–95) (10–95)
CSF TLC (/ml) 1602  4314.5 2.8  2.1 <0.001
(36–23 040) (0–6)
CSF polymorphs (%) 58.2  29.92 0 <0.001
(4–98)
CSF protein (mg/dl) 243.9  116.2 41.2  18.7 <0.001
(60–698) (6–80)
CSF glucose (mg/dl) 36.25  25.4 68.2  17.4 <0.001
(0–102) (37–102)

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CSF glucose: plasma 0.38  0.24 0.78  0.12 <0.001
glucose ratio (0.0–0.97) (0.51–0.97)

TABLE 3
Antioxidants and oxidants concentration in serum of cases and controls

Parameters Cases (n ¼ 40) Controls (n ¼ 40) t-value p-value

Glutathione (mg/ml) 0.43  0.12 0.7  0.18 7.89 <0.001

Ascorbic acid (mg/dl) 15.02  2.01 21.52  5.46 7.066 <0.001
SOD (mg/ml) 0.25  0.09 0.4  0.14 5.7 <0.001
MDA (mmol/l) 3.17  0.54 1.02 0.32 21.66 <0.001
Protein carbonyl (nmol/l) 3.42  1.75 1.79  0.09 5.883 <0.001
Nitrite (mmol/l) 81.3  14.9 20.6  2.7 25.35 <0.001

TABLE 4
Antioxidants and oxidants concentration in cerebrospinal fluid of cases and controls

Parameters Cases Controls t-value p-value

(n ¼ 40) (n ¼ 40)

Glutathione (mg/ml) 0.19  0.06 0.4  0.11 11.1 <0.001

Ascorbic acid (mg/dl) 10.3  1.3 16.64  1.4 20.86 <0.001
SOD (mg/ml) 14.73  3.01 5.62  1.43 17.29 <0.001
MDA (mmol/l) 2.76  0.38 0.67  0.12 33.17 <0.001
Protein carbonyl (nmol/l) 2.132  1.06 1.12  0.23 5.88 <0.001
Nitrite (mmol/l) 14.73  3.01 5.62  1.43 17.29 <0.001

had concluded that malondialdehyde could be an
important factor in the pathophysiology of a
number of nervous diseases and ageing [19].
Elevated malondialdehyde has also been reported
in CSF of patients with pneumococcal meningitis
[5, 20]. The significant increases in serum and CSF
protein carbonyl and nitrite levels in patients with
bacterial meningitis had been reported by other
workers [7, 18]. The increased levels of CSF nitrites
are thought to result from the inflammatory pro-
cess and neurologic damage [21]. ROS and reactive
nitrogen species are potential targets for therapy of
meningitis.
Glutathione has a role in scavenging ROS and
reactive nitrogen species. Kawakami et al. [22] had
also observed low glutathione, and glutathione per-
oxidase activities in CSF as in our study. Serum and
CSF SOD and ascorbic acid concentration was found
to be significantly decreased in present study.
Endothelial cell injury by ROS and reactive nitrogen
species is ameliorated by SOD, ascorbic acid and toc-
opherol in vitro, highlighting the potential thera-
peutic benefit of antioxidants as endothelial cell
protectors [23].
Thus, therapy with agents that scavenge free
radicals and augment endogenous antioxidant

Journal of Tropical Pediatrics Vol. 59, No. 4 307


capacity may be a useful treatment option to
block destructive alteration of lipids, proteins
and nucleic acids by oxygen-derived free radicals to
prevent neurological insult and disease
complications.
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