Journal of the Neurological Sciences 267 (2008) 36 – 40

www.elsevier.com/locate/jns

Involvement of the central nervous system in patients

with dengue virus infection
Renan B. Domingues a,b,⁎, Gustavo W. Kuster a,b , Fábio L. Onuki-Castro a , Vanda A. Souza c ,
José E. Levi c , Cláudio S. Pannuti c
a
Department of Pathology, Escola Superior de Ciências da Saúde de Vitória (EMESCAM), Vitória, Brazil
b
Centro Integrado de Neurologia, Hospital Meridional, Espírito Santo, Brazil
c
Laboratório de Virologia, Instituto de Medicina Tropical, Departamento de Moléstias Infecciosas e Parasitárias da Faculdade de Medicina,
Universidade de São Paulo, São Paulo, Brazil
Received 4 April 2007; received in revised form 12 September 2007; accepted 24 September 2007
Available online 23 October 2007

Abstract

The findings of a neurological evaluation in 85 patients with confirmed, acute, dengue virus infection are described. Signs of central
nervous system involvement were present in 18 patients (21.2%). The most frequent neurological symptom was mental confusion. The
frequency of neurological involvement did not differ between patients with primary and secondary dengue infection, and the prevalence of
central nervous system involvement in dengue fever and dengue hemorrhagic fever also did not differ significantly. The presence of CNS
involvement did not influence the prognosis of dengue infection. Dengue viral CSF RNA was found in 7 of 13 patients submitted to a spinal
tap, the CSF viral load being less than 1000 copies/ml. PCR was negative in serum samples obtained from three patients on the same day as
the CSF samples, suggesting that the dengue virus actively enters the CNS and that the presence of the virus in the CNS does not result from
passive crossing of the blood–brain barrier.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Dengue virus; Neurological manifestations; CSF; PCR

1. Introduction ability, thrombocytopenia, and hemorrhagic manifestations

Dengue fever (DF) is the most frequent human arboviral
infection, with nearly 50 million cases occurring annually
worldwide. The most common clinical manifestations are
febrile state with severe frontal headache, retro-ocular pain,
muscle and joint pain, rash, nausea and vomiting, abdominal
pain, and other less frequent symptoms. The more severe
manifestation of the disease, dengue hemorrhagic ever
(DHF) is associated with fever, increased vascular perme-
⁎ Corresponding author. Av. Nossa Senhora da Penha, 699, sala 709,
Vitória ES 29055-131, Brazil. Tel.: +55 27 33457056.
E-mail address: renan-domingues@uol.com.br (R.B. Domingues).
0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jns.2007.09.040
[1,2].
Involvement of the central nervous system (CNS) in
dengue infection is well known [3–16]. The most frequent
manifestation of CNS involvement is altered consciousness,
although it is still unclear whether this state results from
metabolic imbalance, immune-mediated tissue damage, or
direct viral invasion of the CNS. Some workers have isolated
dengue virus antigens from brain in autopsy of dengue
patients [17,18], while others have demonstrated viral RNA
amplification in cerebrospinal fluid samples obtained during
the acute phase of the disease [5]. However, whether the
virus passively crosses the blood–brain barrier during the
course of systemic infection or whether it actively invades
the CNS is not known.
 R.B. Domingues et al. / Journal of the Neurological Sciences 267 (2008) 36–40
The aim of this study was to investigate the frequency and
the spectrum of neurological symptoms in acute dengue
infection, to evaluate the influence of CNS involvement on
disease prognosis, to assess whether neurological manifesta-
tions are more frequent in secondary infection, and to assess
whether the frequency of CNS involvement differs between
DF and DHF. The findings of serum and CSF PCR analyses
are described.
2. Patients and methods
All patients with suspected dengue infection admitted to
the Santa Casa de Misericórdia de Vitória, Espírito Santo
State, Brazil, between October 2002 and February 2003,
were submitted to serum sample collection and a standard-
ized, clinical evaluation protocol, including physical and
neurological examinations, tourniquet test, blood cell counts,
creatinine level assessment, and cerebrospinal fluid analysis.
A diagnosis of dengue fever was established when the
patient presented a positive serum IgM or a four-fold increase in
serum IgG titers or detection of dengue RNA by multiplex RT-
PCR or real-time PCR. Serum multiplex PCR was performed to
detect dengue virus RNA and to establish the dengue virus
serotype. Real-time PCR was also applied to all available serum
samples. Patients were classified as exhibiting primary or
secondary infections according to an IgG affinity test as
described below. Diagnoses of DF and DHF were established
according to the WHO criteria [19]. CSF was examined for
protein, cells, sugar, bacteria, and dengue RNA by PCR.
This study was approved by the Ethics Committee of the
Santa Casa de Misericórdia de Vitória and informed consent
was obtained from each patient.
2.1. Dengue serology
Sera were tested for the presence of IgM and IgG
antibodies against dengue virus by enzyme-linked immuno-
sorbent assay (ELISA) (Focus Technologies, Cypress, CA).
An IgG affinity test was used to differentiate between
primary and secondary dengue infection and was performed
as described previously [20].
2.2. Polymerase Chain Reaction
From each patient, 200 μl of serum, and, CSF samples
from those patients submitted to spinal tap, were used for
RNA extraction employing the Qiagen Viral RNA kit
(Qiagen, Valencia, CA). Nucleic acids were eluted in 60 μl
of elution buffer and processed using one of two PCR
methods: (1) Multiplex RT-PCR (mx-PCR); cDNA was
synthesized with random primers and M-MLV reverse
transcriptase (Invitrogen, Carisbad, CA) and amplified in
an Eppendorf MasterCycle Gradient thermocycler (Eppen-
dorf, Hamburg, Germany) as described [21], or (2) directly
applied to a single-step, real-Time PCR kit (Real-Time PCR
Artus/Qiagen Real-Art Dengue Kit, Qiagen, Hamburg,
37
Germany) for the Light-Cycler device (Roche Applied
Sciences, São Paulo, Brazil) [22].
2.3. Neurological evaluation
A neurological examination was performed on all patients.
Based on their neurological findings, the patients were
classified into one of the following categories: (1) encephalitis:
a febrile patient with any sign of focal involvement of the CNS
(motor deficit, sensory abnormality, cranial nerve deficit,
visual field disorder, aphasia, and ataxia) and/or seizures with
or without CSF examination abnormalities [23]; (2) enceph-
alopathy: a patient with non-localizing neurological symptoms
and signs of only non-focal involvement of the CNS such as
mental confusion, a reduced level of consciousness, and
abnormal behavior without CSF abnormality; (3) meningitis:
no symptoms and signs of CNS involvement but with signs of
irritation of the meninges and pleocytosis.
2.4. Data analysis
A descriptive analysis of the clinical and laboratory data
for the patients with dengue and neurological involvement
was performed. Statistical comparisons of the frequency of
neurological involvement in patients with primary or secon-
dary dengue as well as in those with DF or DHF were
performed using a t-test or chi-squared test.
3. Results
From October 2002 to February 2003, during a dengue
epidemic, 101 patients with suspected dengue virus infection
were admitted to our hospital. The diagnosis of dengue was
established in 85 (84.1%). A diagnosis of acute dengue
infection was confirmed by a positive IgM in 32 cases; by a
four-fold increase in IgG titer in 16, and by both criteria in
37. Of the 85 patients, 18 (21.2%) manifested some form of
neurological involvement, according to the criteria men-
tioned above. The clinical and laboratory data for the patients
with neurological involvement are given below. Thirty
patients were male and 52 were female. Their ages ranged
from 14 to 72 years, with a mean of 33.5 ± 13.9 years. The
mean duration of the disease was 7 ± 3 days. The clinical
findings of the patients with and without neurological
involvement are listed in Table 1. Creatinine levels were
normal in all patients.
3.1. Neurological findings
Nine patients with neurological manifestations were male,
and nine were female. Eleven of these patients were classified
as having encephalitis, six were classified as having
encephalopathy, and one had meningitis. The neurological
findings were: altered consciousness (17 patients, 94.4%); a
reduced level of consciousness (8 patients, 44.4%); abnormal
coordination (5 patients, 22.2%, four with bilateral and one
38 R.B. Domingues et al. / Journal of the Neurological Sciences 267 (2008) 36–40

Table 1
Clinical findings in patients with and without neurological involvement
Clinical finding Patients with neurological findings Patients without neurological findings P value
Fever (%) 18 (100) 67 (100) 1
Muscle pain (%) 18 (100) 67 (100) 1
Headache (%) 18 (100) 65 (97) 1
Anorexia (%) 17 (94.4) 66 (98.5) 0.38
Nausea and/or vomiting (%) 17 (94.4) 56 (83.6) 0.45
Gastrointestinal and/or urinary bleeding (%) 6 (33.3) 25 (37.3) 0.79
Joint pain (%) 6 (33.3) 25 (37.3) 0.79
Diarrhea (%) 4 (22.2) 14 (20.1) 1
Positive tourniquet test (%) 3/15 (20) 22/52 (42.3) 0.14
Abdominal tenderness (%) 7 (38.9) 33 (51.6) 0.59
Skin rash (%) 2 (11.1) 15 (23.4) 0.5
Mean systolic blood pressure (mm Hg, ±SD) 120 ± 20 120 ± 19.4 0.56
Mean diastolic blood pressure (mm Hg, ±SD) 80 ± 16.8 80 ± 11.35 0.64
Pulse rate/min (± SD) 71 ± 13.86 70 ± 11.3 0.85
Hematocrit (%, ±SD) 40.4 ± 7.3 36.5 ± 5.4 0.12
Leukocytes/mm3 (±SD) 3425 ± 1972 3450 ± 2722 0.73
Platelets/mm3 (± SD) 72500 ± 46232 78000 ± 56255 0.62

with unilateral dysmetria); nuchal rigidity (2 patients,
11.1%); altered behavior (2 patients, 11.1%); mild hemipar-
esis with ipsilateral Babinski sign (1 patient, 5.5%), syncope
(1 patient, 5.5%), and meningitis (1 patient, 5.5%). In this last
patient CSF examination showed 130 white blood cells/mm3
(104 lymphomononuclear and 26 polymorphonuclear cells);
CSF glucose measured 37 mg/dl, and CSF protein 35 mg/dl.
According to the IgG affinity test, nine of the patients
with neurological involvement had primary dengue infec-
tions, and nine had secondary dengue virus infections. The
risk of neurological manifestation was unaltered between the
primary and secondary dengue virus infections (P = 0.27).
According to the WHO criteria, 13 of the patients with
neurological manifestations had classic dengue fever, and 5
had dengue hemorrhagic fever (DHF). The frequency of
neurological manifestation was unaltered in the patients with
dengue hemorrhagic fever compared to those with classic
dengue fever (P = 0.32).
3.2. PCR findings
Forty-eight of the 85 patients (56.5%) exhibited positive
serum multiplex-PCR results. One patient was positive for
dengue type 1, one for dengue type 2, 45 for dengue type 3,

Table 2
Results of PCR and characteristics of the disease in patients with dengue and neurological manifestations
Days of Age Neurological Disease CSF Serum
disease a (years) findings b type
mx-PCR rt-PCR: (copies/ml) mx-PCR rt-PCR: (copies/ml)
29 16 E DHF type 3 0 NEG 0
18 56 E DF NEG 0 NEG 0
4 34 E DHF NEG 0 NEG 0
7 15 E DF type 2 417 NEG 0
3 27 E DF NEG 282 type 3 100,375
3 43 E DF type 3 307 type 3 191,3753
29 12 E DHF type 3 0 type 3 2832
1 64 E DF NEG 0 type 3 1385
6 37 E DHF type 3 0 NEG 0
5 44 M DF NEG 0 type 1 342
4 67 E DF NEG 0 type 3 314
6 8 E DHF NEG 265 type 3 571
8 23 E DF NEG 0 type 3 705
c c c
3 47 E DF type 3
c c
6 55 E DF NEG NEG
c c
6 66 E DF NEG NEG
c c
8 73 E DF NEG NEG
c c
1 70 E DF NEG NEG
mx-PCR: multiplex PCR; rt-PCR: Real-time PCR.
a
Time elapsed after the beginning of fever.
b
E—encephalopathy/encephalitis, M—meningits.
c
No CSF or serum available.
 R.B. Domingues et al. / Journal of the Neurological Sciences 267 (2008) 36–40
and one patient was positive for both dengue type 1 and
dengue type 3. The sera of 70 of these patients were also
tested by real-time PCR. One patient was excluded from the
analysis since amplification inhibition of the internal control
was detected in this case. In 60 of the 69 sera (87%), the mx-
PCR and real-time PCR (rt-PCR) results were in agreement.
Only one serum was mx-PCR positive for dengue type 3 but
tested negative with real-time PCR. Positive results with
real-time PCR were found in 8 samples that were negative
for mx-PCR (Table 2), some of which presented high viral
loads (i.e. N10,000 copies/ml).
Dengue RNA was detected in seven (54%) of the 13 CSF
samples. Four (31%) of the 13 CSF samples were positive by
real-time PCR, all showing a viral load of less than 1000
copies/ml. Five of the 13 CSF samples were positive by mx-
PCR, and in three of these, real-time PCR was negative. In
four of the seven PCR positive CSF samples, concomitant
viremia was detected in the serum. Conversely, discordant
results between CSF PCR and serum PCR were found in
seven patients, four of whom had serum-positive and CSF-
negative samples, while three had positive CSF PCR and
serum negative samples (Table 2).
4. Discussion
Dengue virus infection was confirmed by serology in
84% of the suspected cases. Although antibodies against
other flaviviruses (e.g., yellow fever virus and Japanese
encephalitis virus) may cross-react with the dengue virus
leading to false positive reactions, there are no other
flavivirus infections known in this region of Brazil [24,25],
so that the serology can be considered highly reliable.
Involvement of the central nervous system in patients
with acute dengue infection has been described. Solomon et
al. reported dengue infection in 4% of all cases of CNS
infection [5]. Pancharoen and Thisyakorn studied 1400
children with dengue, and found neurological manifestations
in 5.4% [7]. We found that 21.2% of patients with dengue
showed CNS involvement. This prevalence is higher than
that reported in previous studies. However, the high pre-
valence of neurological manifestations noted in our study
may not represent the true prevalence of CNS involvement in
dengue patients since we only evaluated patients admitted to
a tertiary care hospital where it is expected that patients have
a severe condition. In a population study including patients
with mild disease, the prevalence of neurological manifesta-
tions of dengue would probably be lower. The most common
neurological finding also differed in our study. In previous
studies of children with dengue and CNS involvement, a
reduced level of consciousness was the most frequent
clinical finding [7,10]. In our study, which included adult
patients almost exclusively, mental confusion without a
reduced level of consciousness was the most frequent neu-
rological finding. Other authors have described spinal cord,
nerve, and muscle abnormalities in patients with dengue,
however, none of our patients have presented with symptoms
39
or signs of myelitis or peripheral nervous system involve-
ment [15,26].
In another series of patients with dengue and neurological
involvement, encephalopathy was associated with a more
severe illness [15]. In our study the prognosis was not in-
fluenced by the presence of neurological symptoms. Second-
ary dengue infections may be associated with increasing
severity of the disease [1,27]. Data comparing neurological
involvement in primary and secondary dengue infections are
scarce although a recent report has shown that headache
features do not differ between primary and secondary infec-
tions [28]. Our data suggest that the frequency of neurological
symptoms does not increase with secondary dengue virus
infection. In previous studies, encephalopathy has been more
frequently associated with DF, suggesting that the pathogen-
esis of neurological involvement in classic DF and DHF may
differ [29]. Although our data do not allow comparison of the
pathogenesis of CNS involvement in DF and DHF, the
prevalence of neurological involvement did not differ between
these two forms of the disease.
Neurological involvement can be due to the infection
itself or secondary to metabolic or hematological distur-
bances. In this study the neurological findings could not be
attributed to hypotension, hematological abnormalities,
kidney or coagulation dysfunction, suggesting that the neu-
rological symptoms were related to the dengue virus
infection itself. A few reports have demonstrated dengue
virus antigen in brain specimens or dengue viral RNA in
CSF samples [5,17,18]. However, it is not clear whether the
virus passively crosses the blood–brain barrier or whether
active viral invasion of the CNS takes place. In our study, we
used two assays for dengue RNA detection. Although both
methods gave very similar results for the serum samples,
there was some divergence in the CSF samples. This finding
may derive from the low viral loads found in CSF in contrast
to the sera. However, we are confident that the data represent
the true detection rates of dengue RNA in the CSF, since in
two cases both methods were positive, and each amplifies a
distinct genomic region. The possibility that dengue RNA in
the CSF is a consequence of blood leakage to the CSF cannot
be ruled out. However, the fact that three of the seven
patients with dengue RNA in the CSF had negative serum
samples obtained on the same day as the CSF sample, and
that four patients exhibited serum-positive and CSF-negative
PCRs, suggests that the presence of viral RNA does not
reflect a passive crossing of the blood–brain barrier by the
virus during active viremia.
Several unanswered questions remain to be investigated
in future studies. Studies with magnetic resonance imaging
in patients with dengue and central nervous system
involvement may provide important information. It is still
unclear why some patients infected with the dengue virus
develop neurological manifestations while most do not. The
pathogenesis of neurological involvement and the exact role
of the active viral penetration into the CNS in patients with
dengue remain to be established. It is unclear why different
40 R.B. Domingues et al. / Journal of the Neurological Sciences 267 (2008) 36–40
neurological syndromes can be found in patients with
dengue. We recommend that patients with dengue undergo
a detailed neurological investigation and follow up.
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