An outbreak of echovirus meningitis in children.
ABSTRACT An outbreak of aseptic meningitis in children as evidenced by increase in the number of admissions in a tertiary care hospital is described. Clinical data and stool samples were collected from 25 hospitalized infants and young children. The stool samples were subjected to virological investigations. Fever and vomiting were the commonest symptoms. Cerebrospinal fluid (CSF) showed lymphocytic pleocytosis in majority of cases. Of the 25 stool samples, 14 showed an enterovirus specific cytopathogenic effect (CPE) in rhabdomyosarcoma (RD) cell line. All the 14 samples were positive for enterovirus RNA by reverse transcription-polymerase chain reaction (RT-PCR). Partial sequencing of the Virion protein 1 (VPI) region of the enterovirus genome carried out on the first 7 isolates revealed 5 isolates to be echovirus serotype 4 and one each to be echovirus serotypes 3 and 30. All children showed a rapid recovery and were discharged within 3 days of admission.
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ABSTRACT: Human enteroviruses (HEVs) are a rare cause of encephalitis, presenting in endemic or epidemic form. The aim of the study is to identify and characterise the causative agent of the encephalitis epidemic, which occurred in Uttar Pradesh, India during the summer of 2008. A total of 90 cerebrospinal fluid (CSF) specimens were collected between June and October 2008 from children with symptoms of encephalitis admitted to Chhatrapati Shahuji Maharaj Medical University and Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India. Conventional and molecular methods were used to identify and characterise the viral agent associated with the epidemic. Enterovirus RNA was detected in 37 (41.11%) of 90 CSF samples by real-time polymerase chain reaction (PCR). Seroneutralisation, amplification and sequencing of the 3'-end of the VP1 region of EV isolates revealed coxsackievirus B5 (CBV) and echovirus 19 (ECV) as the main serotypes causing this epidemic. Phylogenetic analysis showed that sequence divergence among the same serotypes was 0-4% at the nucleotide level. This is the first report suggesting that CBV 5 and ECV 19 may be responsible for an epidemic of encephalitis in India. These serotypes were variant and evolved within the studied area.Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 03/2011; 51(2):142-5. · 3.12 Impact Factor
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ABSTRACT: Human enteroviruses (HEVs) are the most common viral pathogen associated with paediatric aseptic meningitis. From October 2010 to February 2011 a cluster of HEV-associated meningitis cases was identified in paediatric patients who had presented at two large tertiary hospitals in Pretoria in the Tshwane Metropolitan Area, Gauteng, South Africa (SA).Journal of Clinical Virology. 01/2014;
Conference Paper: A novel blind diversity combining receiver for CDMA systems[Show abstract] [Hide abstract]
ABSTRACT: This paper addresses the problem of blind multiple access interference (MAI) and intersymbol interference (ISI) suppression in direct sequence code division multiple access (DS-CDMA) systems. In order to take advantage of the diversity inherent to multipath channels, we introduce a blind RAKE multiuser receiver that proceeds in two steps. First, soft estimates of the desired user transmitted symbols are obtained from each propagation path using a bank of appropriate linearly constrained maximum likelihood linear (LCMLL) receivers. Afterwards, these estimates are adequately combined to enhance the signal to interference and noise ratio (SINR)Signal Processing and its Applications, Sixth International, Symposium on. 2001; 02/2001
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here an outbreak of echovirus meningitis in
Vellore and surrounding areas as evidenced by
a sudden increase in the number of infants and
children admitted to the pediatric wards of our
Subjects and Methods
An outbreak of enterovirus meningitis was
suspected when there was a sudden increase in
number of patients admitted with a diagnosis
due to vitamin A deficiency in selected states of
India. Ministry of Health & Family Welfare,
Government of India, New Delhi 1991.
7.Ching P, Birmingham M, Goodman T, Sutter
Enteroviruses are the commonest cause of
aseptic meningitis with majority of cases
attributable to Coxsackie and Echo viruses(1-
3). Enteroviral meningitis occurs sporadically
or as epidemics(1), with some of the largest
outbreaks caused by echoviruses(4).
meningitis are commonly reported from
industrialized countries there are few
documented reports from India. We report
outbreaks of enterovirus
An Outbreak of Echovirus Meningitis in Children
Narayanan Sathish, Julius Xavier Scott*, Shaji RV+, Gopalan Sridharan, Theophilus S.
Vijayakumar, Antony Raj and Thomas Cherian*
From the Departments of Clinical Virology, Child Health* and Clinical Hematology+, Christian
Medical College and Hospital, Vellore, India.
Correspondence to: Dr. Thomas Cherian, 67 Rue des Lattes, 1217 Meyrin, Switzerland.
Manuscript received: April 9, 2003, Initial review completed: June 24, 2003;
Revision accepted: October 27, 2003.
An outbreak of aseptic meningitis in children as evidenced by increase in the number of admissions
in a tertiary care hospital is described. Clinical data and stool samples were collected from 25
hospitalized infants and young children. The stool samples were subjected to virological
investigations. Fever and vomiting were the commonest symptoms. Cerebrospinal fluid (CSF)
showed lymphocytic pleocytosis in majority of cases. Of the 25 stool samples, 14 showed an
enterovirus specific cytopathogenic effect (CPE) in rhabdomyosarcoma (RD) cell line. All the 14
samples were positive for enterovirus RNA by reverse transcription-polymerase chain reaction
(RT-PCR). Partial sequencing of the Virion protein 1 (VPI) region of the enterovirus genome
carried out on the first 7 isolates revealed 5 isolates to be echovirus serotype 4 and one each to be
echovirus serotypes 3 and 30. All children showed a rapid recovery and were discharged within 3
days of admission.
Key words: Aseptic meningitis, Enterovirus, Outbreak.
R, Loevinsohn B. Childhood mortality impact
and costs of integrating vitamin A supple-
mentation into immunization campaigns. Am J
Pub Health 2000; 90: 1526-1529.
VOLUME 4 1_ _APRIL 1 7 , 2 0 0 4
of aseptic meningitis in the pediatric wards of
our hospital. Data were retrospectively
analyzed for 25 such children (age 0-12 years)
admitted from July through September 2002.
Clinical and laboratory data were abstracted
from the clinical records of the patients into a
standardized proforma. Cerebrospinal fluid
(CSF) examination for cytology, glucose and
protein content, Gram stain and bacterial
culture was done in all cases. In addition, stool
samples were subjected to virological
Stool samples were collected in a tube with
viral transport medium (VTM) and trans-
ported to the laboratory on wet ice at +4ºC
immediately. Samples were processed by
centrifugation at 2000 rpm for 10 minutes at
+ 4ºC and 0.2 ml of the supernatant obtained
was inoculated (by enhanced adsorption
method)(5) onto susceptible cell culture
monolayers (Vero and rhabdomyosarcoma
[RD]) and incubated at 37ºC. The tubes were
observed daily for cytopathogenic effect
(CPE) characteristic of enterovirus, up to
7 days for RD and upto 10 days for Vero.
Cell cultures showing enterovirus specific
CPE were passed again into the cell line
for confirmation. Supernatants of cell
lines showing CPE upon the second passage
were stored at –60ºC for confirmation by
RNA was extracted from the cell culture
supernatants showing enterovirus-specific
CPE using a commercial kit (QIAGEN
QIAamp Viral RNA Mini kit). A random
primed two-step nested RT-PCR was carried
out as previously described(6). Amplified
products were visualized by agarose gel
electrophoresis with the expected product size
being 304 base pairs.
Partial Sequencing of Virion Protein 1 (VP1)
From the RNA extracts, synthesis of
cDNA and PCR amplification were carried
out separately. Following cDNA synthesis(6)
PCR amplification using 3 deoxyinosine
containing degenerate primers (012, 040 and
011)(7) was performed to generate an approxi-
mately 450 base pair product (spanning the
VPI-2A region of the enterovirus genome) that
was detected by ethidium bromide stained
agarose gel electrophoresis. Cycle sequencing
of the amplified product and identification of
the serotype was done as described(8).
Twenty five children (18 males and
7 females) admitted to the hospital from the
29th of June 2002 to 4th of September 2002
were investigated. Agewise, 4 were infants,
ten aged between 1 to 5 years, 9 aged between
6 to 10 years and 2 were older than 10 years of
age. Most of the subjects were residents of
Vellore and Tiruvannamalai districts in
Tamilnadu or the adjoining Chittoor district in
The presenting signs and symptoms in the
subjects are shown in Table I. Vomiting was
the commonest symptom seen in 88% of the
subjects followed by fever in 80%. Meningitis
was suspected because of the presence of
meningeal signs (neck stiffness or kernig
sign), headache or persistent vomiting in older
children and a bulging anterior fontanelle,
irritability and incessant crying in children less
than two years of age. All the patients were
treated with intravenous antibiotics pending
the results of the CSF culture. All children
showed rapid improvement in sympto-
matology and 23 children were discharged
from hospital within 3 days of admission and
the remaining 2 within 5 days.
The CSF was clear in appearance in all
VOLUME 4 1_ _APRIL 1 7 , 2 0 0 4
children. Total leukocyte counts ranged from
20 to 760 per cu mm (median count 130 per cu
mm). There was lymphocytic predominance
in 20 subjects, whereas 5 had neutrophilic pre-
dominance. The glucose concentration was
normal in all subjects and the protein
concentration was less than 45 mg/dL in all but
one patient, in whom it was 108 mg/dL. Gram
stain of the CSF smears showed occasional
pus cells with no bacteria. CSF cultures were
negative for any bacterial growth.
Of the 25 stool samples collected, 14
samples showed enterovirus-specific cyto-
pathogenic effect in only RD cell line, which
were confirmed by a second passage. None of
these showed any cytopathogenic effect in
Vero cell line cultures. Random-primed two
step RT-PCR carried out on the RD cell line
supernatants showing enterovirus-specific
CPE, detected the presence of the 304 base
pair enterovirus-specific product in all 14
The first seven of the 14 isolates amplified
using primers (011, 012 and 040) directed
against the VPI-2A region generated an
approximately 450 base pair product. All these
isolates sequenced using 2 (011, 012) of the
above-mentioned 3 primers could be assigned
to a particular serotype by sequencing. Of the
7 isolates, 5 were identified as echovirus
serotype 4 and one each was identified as
echovirus serotypes 3 and 30 respectively.
The pairwise identity score for the
enterovirus isolates from this study with the
respective homologous GenBank sequences
ranged from 83 % to 89%.
The etiological diagnosis of aseptic
meningitis is seldom attempted in most
sporadic cases due to cost constraints and lack
of laboratory facilities. This outbreak provided
us with the opportunity to conduct an
investigation including virological studies.
The similarity in clinical presentation and the
high rates of detection of enteroviruses from
our cases indicate that we did encounter an
outbreak of meningitis due to echovirus. Data
from the previous year also showed an
increase in cases of aseptic meningitis during
June and July. This suggests that there is a
seasonal increase in cases of aseptic
meningitis during these months in our area as
also reported in North America and Europe.
The majority of cases were due to echovirus
type 4. The two cases with detection of
echovirus 3 and 30 might either represent the
sporadic cases or could be contributory to the
outbreak as epidemics due to multiple sero-
types of echovirus have been described
previously(4). The study documents the
occurrence of an outbreak of aseptic
meningitis due to echovirus serotype 4.
However, the study has limitations as it was
hospital-based and virus typing was done in
only small number of cases. Thus it does not
document the extent of the outbreak or
accurately describe all the serotypes involved
and their proportional contribution to the
TABLE I–Presenting Signs and Symptoms in the
Study Subjects (n =25).
Sign or symptomNo.(%)
* Abdominal pain (1), blurring of vision(1).
VOLUME 4 1_ _APRIL 1 7 , 2 0 0 4
Though excretion of enteroviruses may be
prolonged in the stool of children with
asymptomatic infection(9), isolation in a child
with aseptic meningitis makes it very likely
that the enterovirus is the etiological agent,
especially when it is isolated from multiple
cases in an outbreak situation. Stool samples
are useful for the detection of nonpolio
enteroviral infections(10-12) and sometimes
serve as the only clue to diagnosis(l).
Enterovirus specific cytopathogenic effect in
cell cultures in a majority of samples, presence
of an enterovirus specific PCR signal in all the
culture positive stool samples and detection of
potentially neurotropic enterovirus serotypes
serotypes 4 and 30, which are known to be
associated with outbreaks, substantiates that
the viruses obtained from stool samples did not
represent asymptomatic excretion but the
agents involved in the outbreak of aseptic
The use of more than a single cell line for
virus culture as in our study, increases the
sensitivity of detection of enteroviruses(13).
RD cell line is more sensitive for isolation of
echoviruses(14) which can explain echovirus
isolates growth only in RD in our study. Use of
RD cell line also increases the speed of virus
isolation(14). Sequencing using primers as
done in our study is useful for the rapid
determination of epidemiological relatedness
of viruses isolated during an outbreak(7).
In conclusion, our study documented an
outbreak of echovirus serotype 4 in Vellore
and surrounding areas and also suggested the
benign self-limiting course of the illness.
Contributors: NS was responsible for all the
virological investigations and drafted the manuscript;
JXS contributed to clinical data and specimen
collection and reviewed the manuscript; SRV
contributed towards DNA sequencing of virus
isolates and reviewed the paper; GS contributed to
critical review of the manuscript and coordinated the
concept and design of the study; TSV contributed to
concept design and to reviewing of the manuscript;
AR contributed to sample processing and culture
work; TC initiated and coordinated the study and was
responsible for manuscript preparation.
Competing interests: None stated.
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