Original Article

Journal of Child Neurology

1-10
Respiratory and Enteric Virus Detection in The Author(s) 2016
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Children: A Prospective Study Comparing DOI: 10.1177/0883073816670820
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Children With Febrile Seizures and
Healthy Controls

Marko Pokorn, MD, MSc1, Monika Jevsnik, PhD2, Miroslav Petrovec, MD, PhD2,
Andrej Steyer, PhD2, Tatjana Mrvic, MD1, Stefan Grosek, MD, PhD3,
Lara Lusa, PhD4, and Franc Strle, MD, PhD1

Abstract
The majority of children with febrile seizures have viral infections and viruses were detected in 22% to 63% of children in
published studies. Using molecular methods, viruses were also detected in asymptomatic persons. A prospective study was
conducted to detect respiratory and enteric viruses in 192 children with febrile seizures and compare the detection rates to those
found in 156 healthy age-matched controls. A respiratory or enteric virus was detected in 72.9% of children with febrile seizures
and in 51.4% of healthy controls. The viruses most strongly associated with febrile seizures were influenza, respiratory syncytial
virus, parainfluenza, human coronavirus, and rotavirus. Compared to healthy controls, the age-adjusted odds ratios for naso-
pharynx virus positivity in febrile seizure patients were 79.4, 2.8, 7.2, and 4.9 for influenza virus, parainfluenza virus, respiratory
syncytial virus, and human coronavirus, respectively, and 22.0 for rotavirus in stool. The detected virus did not influence clinical
features of febrile seizure.

Keywords
influenza virus, parainfluenza virus, respiratory syncytial virus, human coronavirus, rotavirus

Received March 2, 2016. Received revised April 19, 2016. Accepted for publication August 29, 2016.

Introduction knowledge on the significance of these viruses in febrile sei-

Febrile seizures are a relatively benign convulsive disorder
triggered by fever that occurs in 2% to 5% of children, with
30% to 50% of them experiencing recurrences.1,2 The majority
of children with febrile seizures have signs of upper respiratory
tract infection, presumably of viral origin.3 The approaches to
demonstrate the etiology of respiratory viral infection in
patients with febrile seizures have been diverse and were suc-
cessful in 22% to 63% of cases, influenza virus and adenovirus
being found most frequently.4-9
With the use of molecular methods, the diagnosis of infec-
tious diseases has improved. In patients with respiratory infec-
tion, polymerase chain reaction (PCR) of upper respiratory
tract samples was more sensitive than antigen-detection immu-
nofluorescent methods.10 However, with the use of new sensi-
tive methods, viruses were also detected in asymptomatic
children, making interpretation of positive results difficult.11
The last 15 years have seen the discovery of human metapneu-
movirus, human coronaviruses NL63, HKU1 and SARS,
human bocavirus, new human rhinoviruses strains and the
emergence of a new H1N1 influenza virus-A variant.12-17 The
zures is rather limited.18-24 Although the detection rates of
respiratory or enteric viruses were previously compared among
patients with respiratory or gastrointestinal symptoms and
asymptomatic controls using molecular methods, no similar
study was performed in febrile seizure patients.25-28
The main aims of our study were (1) to detect the presence of
respiratory and gastrointestinal viruses using molecular methods
1
Department of Infectious Diseases, University Medical Centre Ljubljana,
Ljubljana, Slovenia
2
Institute of Microbiology and Immunology, Faculty of Medicine, University of
Ljubljana, Ljubljana, Slovenia
3
Department of Pediatric Surgery and Intensive Care, University Medical
Centre Ljubljana, Ljubljana, Slovenia
4
Institute for Biostatistics and Medical Informatics, Faculty of Medicine,
University of Ljubljana, Ljubljana, Slovenia
Corresponding Author:
Marko Pokorn, MD, MSc, Department of Infectious Diseases, University
Medical Centre Ljubljana, Japljeva 2, SI-1525 Ljubljana, Slovenia.
Email: marko.pokorn@mf.uni-lj.si

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2 Journal of Child Neurology
in children with febrile seizures; (2) to critically appraise their
role by comparison of the proportions of particular virus, the
frequencies of co-presence of other viruses, and cycle-
threshold values (viral burdens) in patients with febrile seizures
and in healthy controls; and (3) to assess whether features of
febrile seizures were specific for the detected viruses.
Methods
The clinical part of the study was performed at the Department of
Infectious Diseases, University Medical Centre Ljubljana, which
serves as a secondary center for a population of 530 000 inhabitants
covering the central region of Slovenia, 9.8% of them being <6 years
of age. All children with febrile seizures in the catchment area are
referred to the Department and are admitted for a 24-hour observation
period. The study was approved by the Republic of Slovenia National
Medical Ethics Committee (Nr.87/08/09) and registered at Clinical
Trials.gov (NCT00987519).
Study Population and Design
Febrile seizure patients. Children <6 years of age with febrile sei-
zures admitted to our Department in a 2-year period from October 1,
2009, to September 30, 2011, were included in the study. For study
purposes, a nasopharyngeal swab, stool, and blood sample were
obtained on admission; however, to fulfill the enrollment criteria,
only the result of nasopharyngeal swab was required. The subsequent
management, that is, other diagnostic procedures, antimicrobial
treatment, and discharge from the hospital, was left to the discretion
of the treating physician.
Febrile seizure was defined as a cerebral paroxysm accompanied
by fever without signs of central nervous system infection. Seizures
were classified as simple if they were generalized, lasted <15 minutes,
and occurred only once within 24 hours and as complex if they were
focal, persisted for 15 minutes, or recurred within 24 hours.29
Upper respiratory tract infection was defined as presence of nasal
discharge, inflamed pharyngeal mucosa, or conjunctivitis. Bronchio-
litis was diagnosed when clinical signs of lower airway involvement
(rales, wheezing) were present. Gastroenteritis was defined as 3 or
more liquid stools in a 24-hour period with or without vomiting.
Children with febrile seizures were classified according to the accom-
panying clinical syndrome(s) (febrile seizure with upper respiratory
tract infection, bronchiolitis, and/or gastroenteritis, respectively) or as
febrile seizure without localizing signs of infection, that is, febrile
seizure alone.
Control group. The control group consisted of healthy children <6
years of age admitted to the Department of Pediatric Surgery, Univer-
sity Medical Centre Ljubljana, for an elective surgical procedure dur-
ing the study period. A nasopharyngeal swab was obtained after
anesthesia induction and a stool sample was provided on admission.
Virology
Sample preparation and nucleic acid extraction. Nasopharyngeal
swabs were collected using flocked-tip swabs and transported in
Copan universal transport medium system (Copan Italia, Brescia,
Italy). Stool samples were diluted in sterile phosphate-buffered saline
to a 10% stool suspension. Aliquots of 180 mL of MagNA Pure bac-
teria lyses buffer (Roche Applied Science, Mannheim, Germany) and
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20 mL of proteinase K (Qiagen, Hilden, Germany) were added to 190
mL of stool suspension.
Before the extraction procedure, 5 mL of equine herpesvirus 1 and
5 mL of equine arthritis virus isolates were added to all samples for
external deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
control. Specific target sequences of these viruses were subsequently
amplified in separate real time reverse transcription PCR as nucleic
acid extraction and reverse transcription PCR inhibition control.30,31
The initial volume used for extracting total nucleic acids was 190 mL
of vigorously vortexed nasopharyngeal swab medium, 400 mL of stool
suspension, and 190 mL of whole blood samples. Nucleic acids were
extracted using total nucleic acid isolation kits on a MagNa Pure
Compact instrument (Roche Applied Science), according to the man-
ufacturers instructions.
Detection methods. In nasopharyngeal swabs, respiratory syncytial
virus, influenza viruses A and B, parainfluenza virus 1-3, human
metapneumovirus, human bocavirus, human coronaviruses (including
HKU1, NL63, 229E, and OC43), adenovirus, and human rhinovirus
were searched for using real-time reverse transcription PCR.10,32-38
For amplification of target fragments of individual virus, a one-step
real-time reverse transcription PCR assay was used in a Step One
Real-Time PCR system (Applied Biosystems, Carlsbad, CA). Briefly,
5 mL of total nucleic acid was added to 15 mL of reaction mixture
including 2 x Reaction Mix, SuperScript III RT/Platinum Taq Mix
(Invitrogen, Carlsbad, CA). Cycling conditions were as follows: 20
minutes at 50 C, 2 minutes at 95 C, and 45 cycles of 15 seconds at
95 C and 45 seconds at 60 C. Cycling conditions were universal for
all tested respiratory viruses. Stool samples were tested for the pres-
ence of human bocavirus, human coronavirus, adenovirus, astrovirus,
and norovirus genogroup I and II using molecular methods as
described previously,36,37,39,40 and for group A rotavirus by antigen
enzyme-linked immunosorbent assay (ELISA) Premier Rotaclone
(Meridian Bioscience, Cincinnati, OH). Blood samples were tested
only for viruses that were positive in nasopharyngeal swab or stool
sample as described previously.
Statistical Methods
Categorical variables were summarized with frequencies and percen-
tages, numerical variables with medians and interquartile ranges; 95%
confidence intervals for percentages and medians were also reported.
The proportions of children that tested positive for each virus (the
proportions of children with at least one virus detected) among febrile
seizure patients and controls were compared using logistic regression
with Firth correction, estimating a model for each of the viruses. The
outcome in each model was febrile seizure patients versus controls, the
covariate was viral detection (positive versus negative reverse tran-
scription PCR) and the analysis was adjusted for the age of children.
Results were reported as adjusted odds ratios for virus positivity (with
their 95% confidence intervals and P values). A similar analysis was
performed on the subgroup of febrile seizure patients and controls that
were virus-positive for the comparison of the cycle-threshold values.
Clinical features of febrile seizures (duration, simple vs complex)
were compared between specific virus-positive and virus-negative
cases using Mann-Whitney test for numerical variables and Pearson
chi-squared test using Yatess continuity correction for categorical
variables. Cycle-threshold values of virus-positive patients were com-
pared between simple and complex febrile seizures for a specific virus
using Wilcoxon rank sum test with continuity correction. Statistical
analyses were performed using R statistical language, 41 the
Pokorn et al
Figure 1. Enrollment of patients with febrile seizures and healthy controls throughout the study period.
confidence interval for the median of differences were calculated
using the R wilcox.test function.
Results
Children With Febrile Seizures and Controls
In the study period, there were 290 admissions for febrile sei-
zures in 278 children. On 68 febrile seizure admissions the
parents refused inclusion in the study, and in 30 episodes chil-
dren were not included because of unspecified reasons. In
remaining 192 febrile seizure episodes, 189 children were
included in the study; during the study period, one girl had 3
and one boy had 2 febrile seizure episodes. Of 192 episodes, 97
were in boys and 95 were in girls. The median age of children
with febrile seizures was 18.4 (interquartile range 14.6-27.4)
months. The enrollment of febrile seizure patients and healthy
controls throughout the study period is presented in Figure 1.
Family history was positive for febrile seizures in 57 (30%)
and for epilepsy in 13 (6.8%) children with febrile seizures.
There were perinatal problems in 20 (10.5%) and neurologic or
developmental problems in 21 (11.1%) children. Of 192 febrile
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3
seizure episodes, 163 (84.9%) were first, 17 (8.9%) were sec-
ond, 6 third, 3 fourth, and there were single children with a 5th,
6th, and 7th episode, respectively.
Febrile seizures occurred within the first 24 hours of fever in
172 (89.6%) cases, within 24 to 48 hours in 13 (6.8%), within
48 to 72 hours in 6 (3.1%), and in 1 episode (0.5%) after 72
hours of fever. The seizure was generalized in 183 (94.8%) and
recurrent in 22 (11.4%) episodes. Rectal diazepam was used to
stop seizures in 36 (18.7%) episodes. Altogether, 132 (68.8%)
seizures were simple and 60 (31.2%) were complex. The med-
ian seizure duration was 2.3 minutes, mean duration was 3.9
minutes; 93 (48.2%) episodes lasted up to 2 minutes, 161
(83.4%) up to 5 minutes, and 177 (91.7%) up to 10 minutes.
Of 192 febrile seizure episodes, 139 (72.4%) children had
upper respiratory tract infection, 13 (6.8%) had upper respira-
tory tract infection and gastroenteritis, 7 (3.6%) had gastroen-
teritis, 1 (0.5%) had gastroenteritis and bronchiolitis, 8 (4.2%)
had bronchiolitis, and 24 (12.5%) had febrile seizure alone.
Among these patients, 1 child also had pneumococcal bacter-
emia, 3 had urinary tract infection, 1 pneumonia, and 1 acute
mastoiditis.
4 Journal of Child Neurology

Figure 2. The co-presence of viruses in febrile seizure patients with positive nasopharyngeal swabs and/or stool samples. Black bars indicate
positive, gray negative, and white missing samples. Abbreviations: AdV, adenovirus; AstV, astrovirus; HBoV, human bocavirus; HCoV, human
coronavirus; hMPV, human metapneumovirus; hRV, human rhinovirus; InfV, influenza virus; NoV, norovirus; NP, nasopharynx; PIV, parainfluenza
virus; RoV, rotavirus; RSV, respiratory syncytial virus.

There were 156 children in the control group, 132 boys
and 24 girls. Their median age was 25.8 (interquartile range
14.635.8) months.
Virological Results
At least one virus was detected in nasopharyngeal swab or stool
in 140/192 (72.9%, 95% confidence interval: 66.0%-79.1%)
febrile seizure episodes and in 72/156 (51.4%, 95% confidence
interval: 42.9%-59.9%) healthy controls.
Nasopharyngeal swabs. At least 1 virus was detected in the
nasopharyngeal swab in 122/192 (63.5%, 95% confidence
interval: 56.3%-70.3%) febrile seizure episodes; adenovirus,
influenza virus and human rhinovirus were detected most
frequently. In 88/122 (72.1%, 95% confidence interval:
63.2%-80.0%) nasopharyngeal swabs, only 1 virus was
detected whereas in 34/122 (27.9%, 95% confidence interval:
20.1%-36.7%) samples, multiple viruses were found. The gra-
phical representation of the co-presence of viruses among
febrile seizures patients with positive nasopharyngeal swabs
is shown in Figure 2.

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Pokorn et al 5

Table 1. The Prevalence of Viruses in Nasopharyngeal Swabs and Stool in Patients With Febrile Seizures and Healthy Controls.

Febrile seizure patients, Healthy controls,

% (95% CI) % (95% CI)
Virus (n 192 for NP and 165 for stool samples) (n 156 for NP and 150 for stool samples) OR adjusted for age (95% CI) P value

Any virus 72.9 (66.0-79.1) 51.4 (42.9-59.9) 3.5 (2.2-5.6) <.001

InfV 14.6 (9.9-20.4) 0 (0-2.3) 79.4 (10.6-10163.7) <.001
AdV 15.6 (10.8-21.6) 9.6 (5.5-15.4) 1.7 (0.9-3.4) .109
hRV 13.5 (9.0-19.2) 10.9 (6.5-16.9) 1.4 (0.7-2.7) .332
PIV 10.4 (6.5-15.6) 3.9 (1.4-8.2) 2.8 (1.2-7.8) .020
hMPV 2.1 (0.6-5.3) 0 (0-2.3) 6.2 (0.7-827.4) .128
RSV 10.9 (6.9-16.2) 1.3 (0.2-4.6) 7.2 (2.2-36.7) <.001
HCoV 9.9 (6.1-15.0) 1.9 (0.4-5.5) 4.9 (1.7-19.1) .003
HBoV 10.4 (6.5-15.6) 5.8 (2.7-10.7) 1.9 (0.9-4.6) .102
RoV (stool) 4.2 (1.7-8.6) 0 (0-2.4) 22.0 (2.3-2969.3) .003
NoV (stool) 9.1 (5.2-14.6) 4.0 (1.5-8.5) 1.9 (0.8-5.4) .174
AstV (stool) 2.4 (0.7-6.1) 3.3 (1.1-7.6) 0.7 (0.2-2.4) .516
AdV (stool) 31.5 (24.5-39.2) 27.3 (20.4-35.2) 1.2 (0.7-2.0) .474
HCoV (stool) 0.6 (0-3.3) 0.7 (0-3.7) 1.4 (0.1-20.8) .780
HBoV (stool) 4.2 (1.7-8.6) 3.3 (1.1-7.6) 1.3 (0.4-4.3) .698
Abbreviations: AdV, adenovirus; AstV, astrovirus; CI, confidence interval; HBoV, human bocavirus; HCoV, human coronavirus; hMPV, human metapneumovirus;
hRV, human rhinovirus; InfV, influenza virus; NoV, norovirus; NP, nasopharynx; OR, odds ratio; PIV, parainfluenza virus; RoV, rotavirus; RSV, respiratory syncytial
virus.

Of 34 samples with multiple viruses detected, there were 28
samples with 2 viruses, one sample with 3, three with 4 and two
samples with 5 viruses. The virus most frequently detected with
other viruses was adenovirus.
Among 156 healthy controls, 42 (26.9%, 95% confidence
interval: 19.9%-33.9%) had positive nasopharyngeal samples,
with human rhinovirus and adenovirus being detected most
frequently; no nasopharyngeal samples were positive for influ-
enza virus and human metapneumovirus.
The comparison of respiratory virus detection in febrile sei-
zure patients and controls with age-adjusted odds ratios is
shown in Table 1. Respiratory viruses significantly more fre-
quently detected in febrile seizure patients were influenza
virus, parainfluenza virus, respiratory syncytial virus, and
human coronavirus. The comparison of cycle-threshold values
for respiratory viruses in febrile seizure patients and controls is
shown in Figure 3 and the results of logistic regression with
age-adjusted odds ratios are shown in Supplementary Table 1.
Cycle-threshold values were not statistically significantly dif-
ferent between febrile seizure patients and controls, but most of
the comparisons were based on few samples.
Stool samples. Stool samples were obtained in 165/192 febrile
seizure episodes and in 150/156 healthy controls. The com-
pared prevalences of enteric viruses in febrile seizure patients
and controls with age-adjusted odds ratios are presented in
Table 1.
Among febrile seizure patients, the most frequently detected
virus was adenovirus, followed by norovirus, rotavirus, and
human bocavirus. In healthy controls, 29.3% stool samples
were positive, with adenovirus being detected predominately,
followed by norovirus, human bocavirus, and astrovirus. There
were no rotavirus-positive samples in the control group, and the
odds ratio for febrile seizure was highest among rotavirus-
positive patients. A comparison of cycle-threshold values in
stool between febrile seizure patients and controls is shown
in Figure 3 and Supplementary Table 1. Only adenovirus-
positive febrile seizure patients had statistically significantly
lower cycle-threshold values than adenovirus-positive healthy
controls whereas the corresponding cycle-threshold values in
nasopharyngeal samples showed only a trend.
Blood samples. Among 140 children with at least one virus
positive in nasopharyngeal or stool sample, 15 (10.7%, 95%
confidence interval: 6.1%-17.1%) had a virus detected in
blood. Adenovirus was detected in blood in 11 children, rota-
virus and human bocavirus in 2 children, respectively. All chil-
dren with positive blood results had viruses present in stool,
one adenovirus-positive and both human bocaviruspositive
children also had positive nasopharyngeal samples for respec-
tive viruses. Data on children with febrile seizure and viruses
detected in blood are shown in Supplementary Table 2. Of 11
adenovirus-positive children, 54.5% had upper respiratory tract
infection, 36.3% had febrile seizure alone and 9% had upper
respiratory tract infection and acute gastroenteritis. Both
human bocaviruspositive children had upper respiratory tract
infection and both children with rotavirus in blood had acute
gastroenteritis.
Simple vs Complex Febrile Seizures and Specific Viruses
Seizure duration between specific virus-positive and virus-
negative patients was compared using Wilcoxon rank-sum test
with continuity correction. Apart from norovirus with shorter
median seizure duration in positive compared to negative
patients (P .065), seizure duration in virus-positive and

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6 Journal of Child Neurology

Figure 3. A comparison of cycle threshold values of respiratory and enteric viruses between children with febrile seizures and healthy controls,
positive for specific virus. Abbreviations: AdV, adenovirus; AstV, astrovirus; FS, febrile seizure; HBoV, human bocavirus; HCoV, human
coronavirus; hMPV, human metapneumovirus; hRV, human rhinovirus; InfV, influenza virus; NoV, norovirus; NP, nasopharynx; PIV, parainfluenza
virus; RoV, rotavirus; RSV, respiratory syncytial virus.

virus-negative patients was similar for all other viruses. The
observed differences in median durations were very small (usu-
ally 1 minute); however, several of the results were based on
a very small number of virus-positive patients. Also, cycle-
threshold values were compared between complex and simple
febrile seizures among specific virus-positive patients, and no
significant differences were found for all viruses tested, indi-
cating no difference in viral quantity between children with
simple and complex febrile seizures. We also compared pro-
portions of simple and complex seizures among specific virus-
positive patients and calculated odds ratios for complex febrile
seizure among virus-positive patients. Apart from norovirus
with a significantly reduced odds ratio for complex seizures
in positive patients, there were no increased odds ratios for
complex seizures in all other viruses tested. Seizure duration
between specific virus-positive and virus-negative patients,
comparison of cycle-threshold values between complex and
simple febrile seizures among specific virus-positive patients,
and odds ratios for complex febrile seizures among virus-
positive patients are shown in Supplementary Tables 3 to 5.
Discussion
In the present study, we determined the occurrences of individ-
ual viruses, their cycle-threshold values, and the frequency of
co-presence of more than 1 virus in respiratory and stool sam-
ples of children with febrile seizures, and to assess the clinical
relevance of the results, we contrast the results with the find-
ings in a control group of healthy children. Although previ-
ously published studies have compared virus detection in
patients with respiratory or gastrointestinal symptoms and
asymptomatic controls, no study focused on febrile seizure
patients and no study detected viruses simultaneously in both
respiratory and stool samples.

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Pokorn et al
With the use of molecular methods, we were able to find a
virus in nasopharyngeal and/or stool samples in 72.9% (95%
confidence interval: 66.0%-79.1%) of children with febrile sei-
zures. Moreover, in children with both nasopharyngeal and
stool sample acquired, 75% had at least one virus detected that
is higher than previously reported. However, viruses were also
found in 51.4% (95% confidence interval: 42.9%-59.9%) of
healthy children.
At least 1 respiratory virus was detected in 63.5% of febrile
seizure patients and 26.9% of healthy controls. Influenza virus,
respiratory syncytial virus, parainfluenza, and human corona-
virus were significantly more frequently present in febrile sei-
zure patients, whereas the detection rates of adenovirus,
rhinovirus, and human bocavirus in nasopharynx were higher
in patients with febrile seizures than in healthy children but the
differences were not significant. These findings go rather well
in parallel with previously published results on children with
respiratory symptoms and asymptomatic controls,11,25-27,42 and
imply that the viruses associated with clinical disease are sim-
ilar in respiratory infections and febrile seizures. A study from
Johns Hopkins Hospital demonstrated that 41.7% of infants and
toddlers hospitalized in winter months without respiratory
symptoms had a virus detected in respiratory sample and that
the detection rates of adenovirus, rhinovirus, and coronavirus
were similar in children with and without respiratory symp-
toms.42 In a study from Alaska, respiratory viruses were
detected in 90% of children <3 years of age hospitalized for
respiratory tract infection and in 52% of healthy control chil-
dren, with human rhinovirus and adenovirus being the most
prevalent in the latter group.25 A study from the Netherlands
has found that 27% of asymptomatic children tested positive
for respiratory viruses, and the viruses most frequently detected
were human rhinovirus and human coronavirus.11 Self et al26
have compared virus detection in patients with community-
acquired pneumonia and asymptomatic controls and found
that parainfluenza virus, human coronavirus, and rhinovirus
as well as adenovirus were frequently present in asympto-
matic children and that their role in the etiology of
community-acquired pneumonia required further scrutiny.
Finally, a meta-analysis of studies on respiratory viruses in
lower respiratory tract infections in children <5 years of age
that compared patients with asymptomatic controls has shown
that respiratory syncytial virus, influenza virus, parain-
fluenza, human metapneumovirus, and also rhinovirus were
associated with clinical disease, whereas detection rates of
adenovirus, human coronavirus, and human bocavirus were
similar in patients and asymptomatic controls.27
In our study, only rotavirus was more frequently present in
stool samples of febrile seizure patients compared to healthy
controls. In a study from China comparing detection rates of
enteric viruses in children with gastroenteritis and asympto-
matic controls, rotavirus was found in 68.5% of children with
gastroenteritis (vs 13.2% of asymptomatic controls), followed
by norovirus (20.4% children with gastroenteritis vs 35.9% of
asymptomatic controls) and adenovirus (5% children with gas-
troenteritis vs 9.2% of asymptomatic subjects).28
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7
To obtain a further insight into clinical relevance of the
findings, we have also compared cycle-threshold values for
specific viruses between positive febrile seizure patients and
healthy controls with the hypothesis that lower cycle-
threshold value (indicating a higher viral content) is associ-
ated with clinical relevance. The comparison revealed
significantly lower cycle-threshold values for adenovirus in
stool of febrile seizure patients compared to healthy controls,
non-significant lower cycle threshold values in febrile seizure
patients for astrovirus in stool as well as for respiratory
syncytial virus, coronavirus, parainfluenza and adenovirus
in nasopharyngeal swabs, and similar values for human
bocavirus and rhinovirus in respiratory samples. However, the
comparison was limited because for several viruses the num-
ber of positive samples was rather small.
We have shown that only influenza virus, respiratory syn-
cytial virus, parainfluenza, and human coronavirus in naso-
pharynx and rotavirus in stool were significantly more
frequently detected in febrile seizure patients compared to
healthy controls. Based on these results, we believe that
detection of one of these viruses in a patient with febrile
seizure very likely indicates that the detected virus is associ-
ated with the febrile episode. For other viruses, the causa-
tive role is less clear.
Influenza virus has emerged as the virus most frequently
associated with febrile seizures in our study. It was also most
frequently detected without co-presence of other viruses. Influ-
enza virus has been the virus most frequently associated with
the occurrence of febrile seizures.8,43 A time-series analysis
comparing the population incidence of febrile seizures with the
seasonal epidemics of influenza and respiratory syncytial virus
from 2003 to 2010 in Sidney has shown that the incidence of
febrile seizures increased with the appearance of influenza-like
illnesses in the community and was not associated with an
increase in bronchiolitis incidence.44
Respiratory syncytial virus was found in almost 11% (95%
confidence interval: 7%-16%) of febrile seizure episodes in our
study; in a study from Hong Kong, respiratory syncytial virus
was associated with 2.7% (95% confidence interval: 1.8%-
4.0%) of febrile seizure episodes, but antigen detection meth-
ods with lower sensitivity were used.8 It is interesting that more
than 75% of respiratory syncytial viruspositive febrile seizure
patients in our study had upper respiratory tract infection and
less than a quarter of them had bronchiolitis. Upper respiratory
tract infection is usually the first clinical sign of respiratory
syncytial virus infection in young children and lower airway
is involved after 3 to 5 days of illness. Fever, if present, usually
occurs at the beginning of illness and this coincides with the
occurrence of febrile seizures.
The prevalence of parainfluenza virus in our study was sim-
ilar to respiratory syncytial virus (10%, 95% confidence inter-
val: 6%-16%). In a study of 923 febrile seizure admissions in
Hong Kong, parainfluenza virus was found in 6% (95% confi-
dence interval: 4.6%-7.8%) of them; when comparing the rates
of febrile seizures in all virus-specific admissions, parain-
fluenza virus was almost as important as influenza virus
8 Journal of Child Neurology
(20.6% of all admissions for parainfluenza virus vs 20.8% of all
admissions for influenza virus were due to febrile seizures).8
The prevalence of human coronavirus in nasopharyngeal
samples of febrile seizure patients was significantly higher
than in controls. The predominant type was OC34, present
in 59% of human coronaviruspositive cases. This is in con-
trast with data from Hong Kong, where the predominant
human coronavirus associated with febrile seizures was
HKU-1, followed by NL63.19
The prevalence of rotavirus in our febrile seizure patients
(4.2%, 95% confidence interval 1.7%-8.6%) was lower than
that reported by Martin et al,24 who found rotavirus in stool
specimens in 7/78 (9%, 95% confidence interval: 4%-18%)
febrile seizure patients by using PCR. Apart from other viruses,
in our study rotavirus was detected using enzyme-linked immu-
nosorbent assay and not PCR, but the test used has shown a
85% sensitivity and 100% specificity compared to PCR in a
recent study.45 Because enzyme-linked immunosorbent assay
detection limit is close to cycle-threshold cut-off value (24-
27) estimated to be of clinical significance,46,47 using antigen
detection test for rotavirus infection instead of highly sensitive
molecular test might give a more reliable result in terms of
clinical importance.
In our study, we could not demonstrate that the clinical
features of seizures, including the median duration and the rate
of complex vs simple seizures, were different between virus-
positive and virus-negative patients for any specific virus
tested; however, the results were based on a rather small num-
ber of virus-positive patients. The only exception was signifi-
cantly shorter seizure duration in norovirus-positive patients
and the finding that majority of norovirus-associated febrile
seizures were simple. In a recent study on respiratory viral
infections in febrile seizure from China, a higher rate of com-
plex febrile seizures was observed in influenza virusassoci-
ated febrile seizures vs noninfluenza virus cases, but the
difference was not statistically significant.9 It seems that clin-
ical features of febrile seizures are not related to the virus
causing the febrile illness. Host factors seem more important:
data from studies in twins suggest that there is a strong genetic
contribution to the type of febrile seizure.48
Our study has several limitations: there was marked male
preponderance in the control group, mainly due to sex-related
differences in elective surgical procedures, that is, inguinal
hernia repair and testicular retention surgery.49 Also, control
patients were older than febrile seizure patients; however,
enrollment of patients was uniform throughout the study
period, providing a reliable background on the seasonal pres-
ence of viruses in the studied population. Moreover, our anal-
yses that compared febrile seizure patients and controls were
adjusted for the age of children.
Conclusion
This is the first study to compare respiratory and enteric virus
detection rates in children with febrile seizures and healthy
controls. With the use of molecular methods, we detected a
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respiratory or enteric virus in 75% of children with febrile
seizures and the viruses most strongly associated with febrile
seizures were influenza virus, respiratory syncytial virus, para-
influenza virus, human coronavirus, and rotavirus. Efforts to
reduce the incidence of febrile seizures should be aimed at
prevention of these infections in susceptible children.
Author Contributions
MaP, MiP, TM, MJ, and FS conceived the study. MaP, TM, and SG
were responsible for the clinical part of the study; MiP, MJ, and AS
performed microbiological procedures; and LL performed statistical
analyses. MaP, MiP, MJ, FS, and LL analyzed the data. MaP and FS
drafted the manuscript. All authors read and approved the final
manuscript.
Declaration of Conflicting Interests
The author(s) declared the following potential conflicts of interest with
respect to the research, authorship, and/or publication of this article:
Marko Pokorn received lecture fees from GSK, Pfizer and MSD.
Funding
The authors disclosed receipt of the following financial support for the
research, authorship, and/or publication of this article: This work was
supported by Slovenian Research Agency (Research Programs P3-
0083 and P3-0296) and institutional department funds.
Supplemental Material
The online [appendices/data supplements/etc] are available at http://
jcn.sagepub.com/supplemental
Ethical approval
Written informed consent was obtained prior to inclusion in the study
from the childs parents or legal guardians. This study was approved
by the Republic of Slovenia National Medical Ethics Committee (No.
87/08/09), registered at ClinicalTrials.gov (NCT00987519) and per-
formed according to the principles of the Helsinki Declaration.
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