PhD THESIS - Virology · neuroinvasive capacity (enterovirus D68) (Calleri et al., 2017; Mailles et...
Transcript of PhD THESIS - Virology · neuroinvasive capacity (enterovirus D68) (Calleri et al., 2017; Mailles et...
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IOD Romania Academy
ICA Institute of Virology „Ştefan S. Nicolau” Bucharest
PhD THESIS
Correlations between etiology and prognosis of viral
infections of the central nervous system
– SUMMARY –
Scientific coordinator,
Prof. Dr. Simona Ruţă
PhD student,
Dr. Corneliu Petru Popescu
2018
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CONTENTS
1. Introduction and objectives 3
2. Material and Methods 4
2.1 General data 4
2.2 Working methods 4
2.2.1 General patient’s data and case definitions 4
2.2.2 Collected data and statistical analysis 5
2.2.3. Laboratory diagnosis 5
3. Results 7
3.1 General characteristics of CNS acute viral infections 7
3.2 Neurological manifestations of West-Nile virus infection 9
3.3 Neurological complications of influenza virus infection 10
3.4 Neurological manifestations of enterovirus infection 11
3.5 Neurological manifestations of herpesvirus infection 12
4. Conclusions 15
References 17
Papers elaborated on the topic of PhD thesis 18
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1. Introduction and Objectives
The epidemiology of acute viral infections of the CNS changes periodically, some viruses
being present in seasonal outbreaks (influenza viruses, enteroviruses, West Nile virus), others
with a certain geographical spread (Japanese encephalitis virus, tick-borne encephalitis virus,
South American arboviruses, Toscana virus) and the others suffer changes in virulence or
neuroinvasive capacity (enterovirus D68) (Calleri et al., 2017; Mailles et al., 2017). There are
also a number of viruses that spread to new regions (parechoviruses, West Nile virus, Zika
virus), some affecting new species, and becoming pathogenic for humans (Powassan virus,
variegated squirrel bornavirus 1).
Although there are multiple viruses that can cause neurological manifestations (enteroviruses,
herpesviruses, paramyxoviruses, adenoviruses, flaviviruses, choriomeningitis lymphocyte
virus, and rhabdovirus), the literature shows the failure of etiological agent identification in
over 40-50% of cases. Thus, in a study in Spain that sought to identify a wide range of
neurotrophic viruses, viral etiology was highlighted in only 46.5% of cases (by Ory et al.,
2013); a US Encephalitis Surveillance Project, 1997-2010 period, managed to establish the
etiology of encephalitis in only one third of patients (Bloch et al., 2015); in France a
prospective study determined the etiology of encephalitis in 52% of cases, of which only
68.7% had a viral etiology (Mailles and Stahl., 2009), and in the United Kingdom, the
etiological diagnosis, not exclusively viral, of encephalitis could only be established in 42%
of cases (Granerod et al., 2010). This failure to establish viral etiology can be caused by low-
level use of molecular or serological diagnostics, partly justified by the often self-limiting and
benign evolution of viral infections, and by the lack of antiviral therapeutics (Vanichanan et
al, 2016; et al., 2017).
In this context, this PhD thesis aims to identify viruses frequently involved in acute CNS
infections in Romania and to highlight the correlations between etiology and prognosis of
CNS viral infections.
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2. Material and Methods
2.1 General data
Initially, a retrospective study was conducted at the “Dr. Victor Babeş” Hospital of Infectious
and Tropical Diseases in Bucharest (SVB) between January 2012 and December 2013,
involving 208 patients with acute viral infections of the CNS, where the etiologic agent was
identified clinically or by laboratory methods in only 13.5% of cases (Popescu et al., 2015),
and prospectively patients who were admitted to SVB from January 2014 to December 2016,
with the diagnosis of acute infection viral CNS, by type of presentation and clinical
development.
2.2 Working methods
2.2.1 General patient’s data and case definitions
All patients with clinical diagnosis of viral meningitis and viral encephalitis, as
defined below, were included in the study.
Case definitions:
For encephalitis, we followed the 2013 recommendations of the Consensus Statement
of the International Encephalitis Consortium:
- major criteria: alteration of mental status (defined as decrease or alteration of consciousness,
lethargy or personality changes) of duration ≥ 24 hours and no alternative cause
- minor criteria: fever ≥38 °C, generalized or partial seizures, newly installed focal
neurological findings, leukocytes in the CSF ≥5 / mmc, changes in cerebral parenchyma
visible in cerebral imaging and suggestive of encephalitis.
The original definition included minor criteria and electroencephalogram
modifications, but this was not available and the case definition was adapted.
Confirmation involves identification of the viral etiological agent by laboratory
methods or a clinical diagnosis of viral etiology (Venkatesan et al., 2013).
For meningitis, the definition involves an acute onset of meningeal manifestations
(neck stiffness, headache, fever) and pleocytosis at the CSF level without laboratory
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identification of fungi or bacteria. Confirmation involves the identification of the viral
etiological agent by laboratory methods or a clinical diagnosis of a viral etiology (CDC,
1997).
2.2.2 Data collected and statistical analysis
Patient’s data was recorded in a Microsoft Excel 2010 program and then processed
statistically in IBM SPSS statistics v.20. following parameters: age at admission, gender
(male / female), urban / rural environment, date of admission, period from onset to admission
(days), clinical form of diagnosis, duration of hospitalization (days), patient evolution (death /
survivor), treatment (antibiotic / antiviral / antibiotic + antiviral), etiology, signs and
symptoms (fever, chills, rash, diarrhea, headache, vomiting, dysarthria, coma, neck stiffness,
sign Kernig or Brudzinski, general altered status / good, nystagmus, paresthesia, psychomotor
agitation, myalgia, sleepiness, cough, focal neurologic findings, aphasia, obesity, diminished
osteo-tendinous reflex, abolition of skin reflexes, not to maintain orthostatism at admission,
photophobia, ataxia, confusion, abdominal pain, arthralgia, vertigo, hallucinations, dysmetry),
CSF characteristics (albumin, glucose, cell type - lymphomonocytes or PMN), blood tests
(hemogram, ESR, C reactive protein, glucose, creatinine, ALT). The following etiologies
have been sought: influenza viruses, herpesviruses (HSV1, HSV2, VVZ, EBV, CMV,
HVV6), enteroviruses, West Nile virus, rubella virus, mumps virus.
2.2.3. Laboratory diagnosis
I Classical PCR for the detection of 6 herpesviruses (HSV1, HSV2, VVZ, EBV,
HHV6, CMV) in the CSF using the Seeplex MeningitisV2 Diagnostic Test Kit (v2.0),
Seegene (Seoul, South Korea). The test was conducted in the virology laboratory at “dr.
Victor Babeş” Hospital with the support of Dr Gratiela Ţardei.
II RT-PCR, for the qualitative detection of enterovirus infection. The test was
conducted in the Microbiology Laboratory at “dr. Victor Babeş” Hospital with the support of
Dr Maria Nica.
For enteroviral RNA detection in cerebrospinal fluid, the GeneXpert system was used
the Cepheid Xpert EV kit. 60 types of enteroviruses (polio viruses 1, 2 and 3), Coxsackie A
(3, 5-7, 9-22, 24), Coxsackie B 1-6, ECHO viruses (1-9, 11-21, 24-27, 29-33) and
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enteroviruses 68-71. The primers and sequences used for the detection of enteroviral RNA
have no cross-reactivity with the nucleic acid of other viruses or bacteria known to be
involved in infectious neurological pathology (EBV, HSV-1, HSV-2, HHV-6, HHV-7, AdV-
2, Measles, Mumps, Parainfluenza 1-3, Influenza A, Influenza B, VZV, CMV, Group B
Streptococcus, Haemophilus influenzae B, H. influenzae non-B, Escherichia coli, Neisseria
meningitides, Citrobacter freundii, and Citrobacter koserii).
III Genotyping in order to identify the type of enterovirus. The test was conducted
in the reference laboratory at the Cantacuzino Institute with the support of Dr Gabriela
Oprisan.
IV For the virological diagnosis of influenza, nasopharyngeal swabs were collected
from all influenza-like illness (ILI) patients and sent for antigenic and genetic characterization
to the National Reference Center for Influenza at the Cantacuzino Institute with the support of
Dr Emilia Lupulescu. Samples were screened for the identification of influenza A or B
nucleic acid using real-time quantitative RT-PCR assays, the Invitrogen Superscript III
Platinum One-Step Quantitative RT-PCR System (Carlsbad, CA, USA). Positive samples for
influenza A were subsequently tested by a second rRT-PCR to differentiate between the
H1N1 subtypes pdm09 and H3N2. In positive samples for influenza B, a second rRT-PCR
assay with specific binding probes was performed to determine the line of origin. Then,
positive specimens were inoculated on MDCK cell lines, and viral isolates were characterized
antigenically by hemagglutinin inhibition (RHAI) with specific antisera and guinea pig turkey
/ guinea pig indicator system.
The viral hemagglutinin (HA) gene was characterized entirely by Sanger sequencing
technique.
V For the diagnosis of neuro-invasive infection with West Nile virus. According to
the National Surveillance Program, blood pair and CSF samples were collected and then were
transported for the laboratory diagnosis of the Cantacuzino Institute's National Reference
Center. Laboratory criteria adapted after the European Commission Decision included: the
presence of WNV (IgM) specific antibodies in the CSF for confirmed cases and the presence
of serum-specific WNV antibodies for probable cases.
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3. Results
3.1 General characteristics of CNS acute viral infections
296 patients with neurological manifestations in SVB, in the 3 years of the prospective
study, with a mean age of 31 years, 55% of male, were identified. Most 73.6% (218/296) were
urban, out of which 69.3% (151/218) in Bucharest.
Clinical diagnosis was meningitis in 144 cases (48.6%), encephalitis - 89 (30%),
meningoencephalitis - 60 (20.3%), cerebellar ataxia - 2 and meningoencephalomyelitis in one
case.
Etiological diagnosis was established in 88 (29.7%) patients: enterovirus - 26 (all
under 16 years old age), West Nile virus (WNV) - 21 (all adults), influenza viruses - 15,
herpesviruses - 21 [herpes simplex virus 1 (HSV1) in 9 cases, varicella-zoster virus (VVZ) -6,
Epstein-Barr virus (EBV) - 6], mumps virus - 5 cases. Cases distribution after the months of
hospitalization highlights most cases in August (21.3%), followed by July (16.2%),
September (13.2%) and June (10.1%), with the least cases registered in April (2.7%).
Comparative analysis based on age at admission, comparative analysis of clinical
forms of acute CNS infections, comparative etiology analysis and comparative analysis
according to evolution were performed. The percentage of cases of acute viral infections of
the CNS with confirmed etiology significantly increased by an epidemiologically close
surveillance doubled by the constant use of molecular diagnostic methods such as GeneXpert
or PCR multiplex, from 13.5% in 2012/2013 reaching 38.5% in 2016. The most common
etiology of meningitis is enteroviruses and mumps virus, whereas encephalitis is caused by
influenza viruses, herpes viruses and WNV. The mortality rate is significantly increased in
patients with encephalitis or meningoencephalitis (especially for WNV infections, herpes
viruses or influenza), those with viral meningitis generally having benign development,
regardless of etiology.
The lowest age was in patients with enteroviral etiology with a median of 6 years,
followed by those with mumps virus – 19 years, old influenza virus – 31 years, herpes viruses
40 years and the highest WNV - 66 years. The mortality rate was significantly higher at
16.8% in adults compared to 1.4% in children, p <0.001. The tendency to administer
antibiotic alone is statistically significantly higher in children 73.5% vs. 33.6% in adults, p
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<0.001. Period disease from onset to admission was shorter for children 1 day vs. 3 days in
adults, p <0.001.
The most common etiology of meningitis was enteroviruses and mumps viruses,
whereas encephalitis is caused by influenza viruses, herpes viruses and WNV. The mortality
rate is significantly increased in patients with encephalitis or meningoencephalitis (especially
for WNV infections, herpes viruses or influenza viruses), those with viral meningitis
generally having benign development, regardless of etiology.
No deaths were recorded for cases of meningitis, but deaths were reported in 15.7% of
encephalitis and 23.3% in meningoencephalitis (p <0.001). In meningitis, 86.8% of patients
received only antiviral, without antibiotic treatment, while in the case of encephalitis (64%)
and meningoencephalitis (63.3%) received a combination of antibiotic and antiviral treatment
(p <0.001).
There were no deaths for enteroviral etiology and mumps virus, but the mortality rate
was 14.3% for influenza viruses, 28.6% for herpes and 33.3% for WNV. The shortest
hospitalization duration was a median of 10 days for influenza virus infection, and the longest
for herpesviruses and WNV with 14 days.
In a multivariate analysis using logistic regression, coma (Wald test = 7.127, p =
0.004), elevated CSF albumin (Wald test = 5.109, p = 0.024), creatinine (Wald test = 5.935, p
= 0.015) and serum glucose (Wald test = 3.943, p = 0.047) were maintained as independent
predictive factors for progression to death.
There are statistically significant differences between acute infections of the CNS of
children and adults in terms of disease forms (adult encephalitis and meningoencephalitis),
prognosis (adult mortality rate) and the tendency to use antibiotics in treatment (more
common in children). Old age is the main risk factor for death, and encephalitis (coma,
sleepiness, focal neurologic findings, aphasia, obesity, and confusion) are predictive of severe
progression. CSF analysis shows an association between elevated albumin and glucose values
as well as decreased cellularity with the progression to death of patients with acute viral
infections of the CNS.
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3.2 Neurological manifestations of West-Nile virus infection
A prospective analysis of cases hospitalized during the 2014-2016 period with clinical
suspicion of WNV infection, according to the case definition of the surveillance and control
system: fever and neurological manifestations, with clear CSF, age ≥ 16 years. As a result of
this analysis, observing an increase in the number of patients hospitalized with WNV
infection in 2016, we prospectively followed the patients from May to October 2017 and also
for the period 2012-2013 a retrospective analysis was carried out by extracting the data from
the hospitalized patient’s records with WNV neuro-invasive disease diagnosis.
Thus, a total of 534 patients with acute viral infections of the CNS were identified, of
which 47 cases had West Nile virus infection. WNV-infected patients were predominantly
male (55.3%), most of the urban (78.7%), median age 64 (IQR 47.0-76.0), statistically
significantly higher than the rest of acute viral infections of CNS, 14 years (IQR 6.0-44.0).
Following the analysis of the studied group, there was an increase in the number of
cases registered in 2016 and 2017, as well as the increase in the mortality rate, suggesting a
change in the pattern of neuro-invasive WNV infection in Romania. Considering the presence
in Romania of two different strains of viruses from the lineage 2 - Volgograd 2007 and Nea
Santa 2010, favorable conditions can be created for genetic recombination and emergence of
strains with increased virulence and new characteristics that can influence the prognosis, but
also the growth morbidity.
Of the 47 patients, 33 (70.2%) had one or more co-morbidities; 22 (46.8%) had
multiple co-morbidities. The most common patients were associated with hypertension 26
cases (55.3%), prior neuropsychiatric disease in 10 (21.3%) cases (dementia - 5, stroke - 3,
schizophrenia - 2) diabetes mellitus - 8 cases (17%), neoplasm - 5 (10.6%), obesity - 4, kidney
transplantation - 3, chronic hepatitis - 3, cardiac failure - 3, malnutrition 2, pulmonary TB,
alcoholism, chronic kidney failure and autoimmune thyroiditis - one case.
The median of age is significantly higher in the deceased group: 77.5 years (IQR,
73.75 years versus 57.5 years (IQR 40-69.25 years) in the survivors group, p <0.001. Coma,
confusion and obtundation are also considered to be negative prognostic factors. In addition to
this, with a higher but statistically significant p, confusion syndrome, sleepiness, aphasia are
considered negative prognostic factors. From the group of co-morbidities, only the association
of the neoplasm is significant statistically as prognostic factor to death, p = 0.013.
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Older age (over 75 years) is the only independent prognostic factor in the death of
patients infected with WNV. Although even if co-morbidities are frequently associated with
neuro-invasive WNV infection, they have not been shown to be risk factors for death, maybe
with exception of the neoplasms association. Clinical forms of WNV infection include
especially encephalitis (encephalitis or meningoencephalitis), comparative with other acute
viral CNS infections, and from laboratory data only low chloride in CSF was associated with
the progression to death of patients.
3.3 Neurological complications of influenza virus infection
A prospective study was conducted in the influenza seasons 2014/2015, 2015/2016
and 2016/2017, which included patients admitted to SVB that presented neurological
manifestations associated with influenza symptomatology.
In the 3 influenza seasons, 15 patients were diagnosed with neurological complications
associated with influenza virus infection:
- 9 influenza virus B,
- 4 influenza virus A/H1pdm09 and
- 2 with A/H3N2.
Thus, in the 2014/2015 season there were 9 cases (8 influenza virus B and one
influenza virus A/H1N1), another 2 cases that only had febrile seizures without other
neurological manifestations were excluded from this analysis (one child with a history of
febrile seizures and an adult with epilepsy), 4 cases were identified in the 2015/2016 season
(3 influenza virus A/H1N1 and one influenza B virus) and 2016/2017 were 2 cases, both with
A/H3N2 influenza virus.
This study signals for the first time in the literature the neurotropic potential of
influenza B virus in adults, neurological manifestations being previously reported only in
pediatric cases.
Genetic analysis by hemagglutinin (HA) sequencing for influenza B isolate from
nasopharyngeal swab was successful in 4 cases. The phylogenetic analysis of HA showed that
all strains belong to Yamagata-lineage clade 3, the isolates being representative of strain
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B/Phuket/3073/2013, different from the strain included in the influenza vaccine for the 2014-
2015 season (B/Massachusetts/2/2012-like virus). The analyzed B influenza virus sequences
were found in the B-lineage Yamagata, clade 3, and presented mutations: S150I, N165Y,
G229D, N116K, N202S, K298E and E312K characteristic of the B/ Phuket/3073/2013
reference strain.
The continuous evolution of influenza viruses can lead to the emergence of
neurovirulence strains that escape the immune response, causing severe neurological
complications, even in young, immunocompetent individuals, especially when a large mass of
the population remains unvaccinated.
In the literature, a number of genetic mutations, mostly at the level of innate immune
signaling pathways (TLRs), or dominant autosomal transmitted mutations in Ran-binding
protein 2 (RANBP2) have been reported (Wang et al, 2010; Goenka et al , 2014), which
appear to predispose to recurrent encephalitis associated with influenza virus infection. In the
present study, one patient had an episode of influenza virus encephalitis with clinical
manifestations, similar to the current episode, in 2012, being admitted to the same clinic, the
viral isolate was A/H3N2; there was no possibility of genetic testing.
Cerebral imaging can support a controversial diagnosis and can guide treatment, as
evidenced by viral nucleic acid in the CSF is rare. In one of the cases diagnosed in 2016/2017,
which showed co-infection with influenza A and HSV1, MRI images highlight specific
changes in HSV 1 infection (asymmetric damage of temporal lobes, hippocampus, insular
cortex, limbic system), but also rare changes for the herpetic etiology, but possible in the
influenza etiology (thalamic impairment).
Treatment with neuraminidase inhibitors is particularly useful if given within the first
48 hours of onset, and acyclovir combination is necessary until a possible etiology with
herpesviruses is avoided.
3.4 Neurological manifestations of enterovirus infection
During the study, 25 patients with neurological manifestations were identified in the
context of a possible viral infection that was tested for enteroviruses infection by GeneXpert
method. Of the 25 patients, 17 were male and 8 female, with an average age of 10 years
(between 1 and 39 years, only 2 were over 16 years of age). 21 patients were from the urban
area and only 4 from the rural area, 16 from Bucharest. Onset of affection was on average
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1.84 days prior to admission (1-7 days). The average hospital length of stay was 12 days (9 to
17 days).
The GeneXpert method has identified enterovirus RNA in 15 cases, all patients under
15 years (average 8 years), 13 in the urban area (11 in Bucharest). Distribution by age group
was equal to 5 patients in the 2-5, 6-10 and 11-16 years age groups; no case was under 2 years
of age. Most patients were diagnosed in August and October (Figure 1). The average duration
of hospitalization was 11 days, and the average of the period elapsed between the onset of the
illness and hospitalization was 1.6 days.
The use of GeneXpert is a modern method of identifying the enterovirus etiology of
viral meningitis that could lead to better patient management in terms of treatment and
duration of hospitalization.
The features of CSF in meningitis with enteroviruses (pleiocytosis, sometimes
predominantly PMN) pose difficulties in establishing etiology and therapeutic attitudes.
Although epidemics of meningitis with enteroviruses occur periodically, their circulation in
the population is permanent, with simultaneous presence of several serotypes, some with
epidemiological potential - in our case ECHO 6 and 30 and Coxsackie B5 are identified.
3.5 Neurological manifestations of herpesvirus infection
This chapter consists of 2 subchapters:
- complications of varicella in unvaccinated children admitted to SVB for the period 2002-
2013
- neurological manifestations of herpes virus infection in SVB patients with acute viral CNS
infections during 2014-2016
I. In the first subchapter we carried out a retrospective study and thus the records of all
patients admitted to the SVB with varicella diagnosis from January 2002 to December 2013.
There were included 1302 patients (average 12.36 years, median 7 years) diagnosed with
varicella. 674 patients (51.8%) experienced one or more varicella complications. Neurological
complications were recorded in 60 patients (4.6%) and had the highest length of stay (median
of 10.1 days and an average of 12.2 days): in order for patients with meningoencephalitis
(mean 15.5 days), then for those with cerebellar ataxia (mean 12.58 days) and those with
encephalitis (11.6 days). They had the following types of syndromes: 39 (65%) - cerebellar
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ataxia, 11 (18.3%) - meningitis, 5 (8.3%) - encephalitis, 4 (6.7%) - meningo-encephalitis and
one (1.7%) of meningoencephalomyelitis. In the absence of vaccination, varicella can lead to
severe complications with long hospitalization.
It has been observed that younger age is an independent predictor for the occurrence of
varicella-associated complications. In the retrospective analysis of the 13 years there is an
increase in the cases of chickenpox that associate complications and require hospitalization,
especially during the 2007-2013 period. The rate of neurological complications has a constant
distribution over time, although in the literature are described peaks of their occurrence,
possibly due to changes in virulence of the varicella-zoster virus. Cerebellar ataxia is the main
neurological complication of varicella, followed by meningitis, encephalitis and
meningoencephalitis. The type of neurological complications is correlated with the age of the
patient, cerebellar ataxia and meningitis occurring at significantly older age, compared to
encephalitis and meningoencephalitis.
The occurrence of seizures in neurological complications is rare if febrile seizures are
excluded from the definition of fever without other neurological manifestations. The length of
hospitalization of complicated cases of CNS-related varicella was significantly longer than
that of other complications, varicella meningoencephalitis requiring the longest
hospitalization of neurological complications. In our study, the progression was favorable in
all cases of neurological complications under acyclovir treatment, although the literature
describes cases of neurological sequelae and even deaths from CNS depression in the context
of varicella.
II. In the second subchapter, a prospective study was conducted between July 2014
and December 2016, involving 86 patients with neurological manifestations and suspicion of
viral infection, admitted to the SVB. In 23 cases, a PCR positive result was obtained in the
CSF: 10 for HSV1, 7 for VVZ and 6 for EBV. To be specified, one of the cases of HSV1 and
one of VVZ infection was co-infection with influenza viruses.
Statistically significant differences were observed between the three types of herpes
viruses identified by PCR in CSF in terms of year of hospitalization (no EBV cases in 2014
and VVZ in 2015), length of stay (median of 23 days for HSV 1 vs. 13.5 days for EBV and 12
days for VVZ, p = 0.036), the period from onset to hospitalization (median of 3 days for VVZ
versus 5 days for HSV 1 and 8 days for EBV, p = 0.025).
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Also statistically significant differences were found between the three viruses and in
terms of clinical manifestations: rash (85.7% of VVZ cases and only 20% for HSV 1 and
16.7% for EBV, respectively, p = 0.01), aphasia (presence only in EBV infection, p = 0.008),
abolished cutaneous reflexes (also for EBV infection, p = 0.008). Patients with HSV 1 and
VVZ have received antiviral treatment (Acyclovir) in 100% and those with EBV in only
33.3% (2 patients), p = 0.015.
There were no statistically significant differences in age, gender, type of clinical
diagnosis, other clinical manifestations, laboratory data (CSF, blood), and prognosis among
the three viruses. Although not statistically significant, the mortality rate was 50% for EBV,
20% for HSV 1 and 14.3% for VVZ and the lowest age for patients infected with VVZ
(median of 29 years), while patients with EBV had the highest age (median of 43 years).
An increased mortality rate of 50% for EBV infection has been observed, which should be
interpreted with caution in view of the small number of patients and the existence of
important co-morbidities in these patients - two being co-infected with HIV in the AIDS
stage, one of them being simultaneously infected with Rhodococcus equi.
On the other hand, one of the cases whose healthy survival without sequelae is unusual
may suggest a protective effect of EBV against Bacillus anthracis infection. Some studies
suggest that herpesvirus latency provides symbiotic protection against bacterial infection
(Barton et al, 2007).
Encephalitis is predominant in HSV1 and EBV infections, with severe progression, in
a significant number of cases with evolution to death or neurological sequelae, these being
influenced by age or comorbidities of the patient as well as by the time of initiation of
Acyclovir injectable therapy in HSV1 infection.
Cerebral imaging, especially MRI, is useful in acute HSV1 CNS infections, the
presence of pathological changes, especially in temporal lobes, supporting the rapid initiation
of Acyclovir treatment. The use of modern molecular multiplex PCR methods for the
detection of herpes viruses in the CSF is necessary in the management of all acute viral
infections of the CNS. The presence of EBV DNA in the CSF should be interpreted with
caution and correlated with the presence of other pathogens as well as with the patient's
immune status.
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4. Conclusions
• A careful epidemiological surveillance, doubled by the constant use of molecular diagnostic
methods such as GeneXpert or PCR multiplex, allows significant improvement in etiologic
diagnosis in acute viral infections of the CNS.
• The most common etiology of meningitis is enteroviruses and mumps virus, while
encephalitis is caused by influenza viruses, herpesviruses and WNV.
• West Nile virus is re-emerging in Romania, 2016/2017, with significant changes in the
pattern of neuro-invasive infection, by increasing the number of cases and especially the
mortality rate. Older age (over 75 years) is the only independent prognostic factor in neuro-
invasive WNV infection. The association of co-morbidities is common, but with the exception
of neoplasms, they are not risk factors for death.
• Influenza viruses are an important etiology in acute viral infections of the CNS. During the
season of influenza, severe neurological complications are reported annually, even in young,
immunocompetent people, especially when a large mass of the population remains
unvaccinated.
• This study signals for the first time the neurotropic potential of influenza B in adults. The
continuous evolution of influenza viruses can lead to the emergence of neurovirulence strains
that escape the immune response and cause severe, even lethal, forms of encephalitis.
Cerebral imaging may support a controversial diagnosis when evidence of viral nucleic acid
in the CSF occurs only rarely and can guide treatment, with early administration of
neuraminidase inhibitors during the first 48 hours of onset.
• Identifying viral nucleic acid in the CSF is a precise method of identifying the enterovirus
etiology of viral meningitis, which could lead to better patient management in terms of
treatment and duration of hospitalization. The characteristics of CSF in meninges with
enteroviruses (pleiocytosis, sometimes with PMN predominance) pose difficulties in
establishing etiology and therapeutic attitudes. Although periodic meningitis epidemics occur
with enteroviruses, their circulation in the population is permanent, with simultaneous
presence of several serotypes, some with epidemic potential - in our case ECHO 6 and 30 and
Coxsackie B5 are identified.
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• In the absence of vaccination, varicella can lead to severe complications, with long
hospitalization. Younger age is an independent predictor for the occurrence of varicella-
associated complications. The rate of neurological complications has a constant distribution
over time, although in the literature are described peaks of their occurrence, possibly due to
changes in virulence of the varicella-zoster virus. Cerebellar ataxia is the main neurological
complication of varicella, followed by meningitis, encephalitis and meningoencephalitis. The
type of neurological complications is correlated with the age of the patient, cerebellar ataxia
and meningitis occurring at significantly older age, compared to encephalitis and
meningoencephalitis.
• The use of modern molecular multiplex PCR methods for the detection of herpes viruses in
the CSF is necessary in the management of all acute viral infections of the CNS. Encephalitis
is predominant in HSV1 and EBV infections, with a significant number of cases leading to
death or healing with sequelae. The prognosis is influenced by the age or co-morbidity of the
patient as well as the time of initiation of the Acyclovir injectable treatment for HSV1
infection. Cerebral imaging, especially MRI, is useful in acute HSV1 CNS infections, the
presence of pathological changes, especially in the temporal lobes, which supports the rapid
initiation of Acyclovir treatment. The presence of EBV in the CSF should be interpreted with
caution and correlated with the presence of other pathogens as well as with the patient's
immune status.
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References
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Bloch KC, Glaser CA. Encephalitis surveillance through the Emerging Infections Program,
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Calleri G, Libanore V, Corcione S. et al. Infection 2017;45:227.
https://doi.org/10.1007/s15010-017-0993-4.
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MMWR, 46(RR-10), 1-55. https://www.cdc.gov/mmwr/preview/mmwrhtml/00047449.htm.
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Infect Dis. 2010 doi: 10.1016/S1473-3099(10)70222-X.
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manifestations of influenza infection in children and adults: results of a national British
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microbiology and infection: the official publication of the European Society of Clinical
Microbiology and Infectious Diseases, 2017; 23:607-613. doi:10.1016/j.cmi.2017.05.002.
de Ory F, Avellón A, Echevarría JE, et al. Viral infections of the central nervous system in
Spain: A prospective study. J Med Virol, 2013, vol.85 (pg. 554-62). doi: 10.1002/jmv.23470.
Popescu CP, Ruta S, Lazar S, Rusu R, Homos M, Simion V, Nica M, Tardei T, Purice P,
Gutan A, Florescu S, Ceausu E. Difficulties in the etiological diagnosis of viral
meningoencephalitis. Journal of Translational Medicine and Research, 2015;Vol.20,Supl2,
51-52.https://docs.google.com/viewerng/viewer?url=http://www.jtmr.ro/pdfs/supplements/
2015-supplement-2.pdf.
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Papers elaborated on the topic of PhD thesis
Articles titles
1. Complications of Varicella in Unvaccinated Children From Romania, 2002-2013: A
Retrospective Study - Popescu CP, Ceausu E, Florescu SA, Chirita D, Ruta S.,
Pediatr Infect Dis J. 2016 Feb;35(2):211-2. doi: 10.1097/INF.0000000000000969,
revista indexata PubMed PMID: 26544989, Factor de Impact 2.723.
2. Neurologic Complications of Influenza B Virus Infection, Romania. - Corneliu P.
Popescu, Simin A. Florescu, Emilia Lupulescu, Mihaela Zaharia, Gratiela Tardei,
Mihaela Lazar, Emanoil Ceausu, and Simona M. Ruta. Emerg Infect Dis.
2017;23(4):574-581. https://dx.doi.org/10.3201/eid2304.161317, revista indexata
PubMed, PMC 5367398, Factor de Impact 8.22.
3. Etiological profile of infectious meningitis at “Dr. V. Babes” Clinical Hospital of
Infectious and Tropical Diseases, Bucharest - Ceausu Emanoil, Florescu Simin-Aysel,
Nica Maria, Smadu Sebastian, Codreanu Daniel, Oprisan Corina, Dascalu Ana Maria,
Oprea Cristiana, Lazar Stefan, Popescu Corneliu, Kosa Alma, Calistru Petre Iacob,
Revista Romana de Boli Infectioase, vol XX, Nr.3, pag 135-139, 2017, revista cotata
B+.
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4. Mortality causes in infectious diseases- Simin Aysel Florescu, Petre Iacob Calistru,
Sebastian Smadu, Daniel Codreanu, Ana Maria Popescu (Veja), Corneliu Petru
Popescu, Emanoil Ceausu, Rom J Leg Med , vol 25, issue 1, March 2017.
doi:10.4323/rjlm.2017.20, Factor de Impact 0.1444.
5. Re-emergence of severe West Nile virus neuroinvasive disease in humans in Romania,
2012 to 2017 – implications for travel medicine - Corneliu Petru Popescu, Simin
Aysel Florescu, Ani Ioana Cotar, Daniela Badescu, Cornelia Svetlana Ceianu, Mihaela
Zaharia, Gratiela Tardei, Daniel Codreanu, Emanoil Ceausu, Simona Maria Ruta;
Travel Medicine and Infectious Diseases Journal, Volume 22 , 30 – 35,
DOI: https://doi.org/10.1016/j.tmaid.2018.03.001
Oral and poster communications at national and international scientific event
1. Neurological manifestations in varicella-zoster virus infections – C.P.Popescu,
S.Ruta, S.Florescu, E.Ceausu, P.Calistru, IXth Simpozon „Nicolae Cajal”, Bucuresti,
12-14 Mai 2014 (poster)
2. Dificultati in diagnosticul etiologic al meningoencefalitei virale – C.P.Popescu, S.
Ruta, S. Lazar, R. Rusu, M. Homos, V. Simion, M. Nica, G. Tardei, S. Purice, A.
Gutan, S. Florescu, E.Ceausu, Simpozion Academician Nicolae Cajal editia a X-a,
Bucuresti, 01-04.04.2015 (poster)
3. Complications and epidemiological aspects of varicella – a 9-year retrospective study -
C.P.Popescu, S. Ruta, S. Florescu, E. Ceausu, P. Calistru, 25th
European Congress of
Clinical Microbiology and Infectious Diseases, Copenhagen, 25-28.04.2015 (poster)
4. Acute central nervous system infections in children – a 2 years retrospective study -
C.P. Popescu, S. Ruta, M. Nica, S. Lazar, R. Rusu, A. Gutan, S. Purice, E. Nedu, S.A.
Florescu, E. Ceausu, 33th
Annual ESPID Meeting, Leipzig, 12-16.05.2015 (poster)
5. Encefalitele virale – prezentari de caz – Corneliu Popescu, Conferinta de Imunitate
Clinic Aplicata, Bucuresti, 13-14.11.2015 (prezentare orala)
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6. Severe cases of influenza during the 2014/2015 season - C. P. Popescu, S. Ruta, S. A.
Florescu, C. Ciobanu, E. Lupulescu, M. Lazar, G.Tardei, E. Ceausu; Simpozion
Academician Nicolae Cajal editia a XI-a, Bucuresti, 17-19.03.2016 (prezentare orala)
7. Encephalitis and meningitis unexpected etiologies - Corneliu Petru Popescu, 4th
edition National Conference of infectious diseases with international participation;
Cluj, 29.09-01.10.2016 (prezentare orala)
8. West Nile meningoencephalitis - Filofteia Cojanu Banicioiu, Dana Serbanescu,
Corneliu Popescu, Olivia Burcos, 34th Balkan Medical Week, Bucharest, 7-8
October 2016 (poster) Archives de lÚnion Medicale Balkanique, Supplement I (2016)
http://www.balkanmedicalunion.com/en/supplements/34-balkan-medical-week/
9. Complicatii neurologice ale infecțiilor cu arbovirusuri – Corneliu Petru Popescu,
Simin Aysel Florescu, Graţiela Tardei, Emanoil Ceauşu, Simona Ruţă, A IX-a
Conferinta Nationala de Microbiologie si Epidemiologie, Brasov, 20-22.10.2016
(prezentare orala)
10. Neurologic manifestation of viral infections in children – Corneliu Petru Popescu,
Simona Ruta, 9th ESPID suported teaching course; 8th East-European and
Mediterranean Teaching Course on Pediatric Infectious Diseases, 25-26.11.2016
Brasov, Romania (prezentare orala)
11. Infectii acute ale sistemului nervos central la copii: abordare clinica, diagnostic si
tratament – Corneliu Popescu, Scoala de iarna a SNRBIP, 16-18.03.2017, Brasov,
Romania (prezentare orala)
12. Complicatii neurologice severe ale infectiei cu virus gripal B – Simona Ruta,
Corneliu Popescu, E Lupulescu, Conferinta Nationala RoVaccin 2017 – Vaccinarea –
urgenta de sanatate publica, 25.03.2017, Bucuresti, Romania (prezentare orala)
13. Virusurile gripale si sistemul nervos central - C. P. Popescu, E. Ceaușu, E. Lupulescu,
S. Ruta, Simpozion Academician Nicolae Cajal editia a XII-a, Bucuresti, 30.03-
01.04.2017 (prezentare orala)
14. Encephalitis by co-infection with A/H3N2 influenza virus and Herpes simplex virus 1
in a teenager girl - Popescu Corneliu Petru, Florescu Simin Aysel, Zaharia Mihaela,
Marcu Carmen, Vasile Teodor, Tardei Gratiela, Lupulescu Emilia, Ceausu Emanoil,
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Ruta Simona. 35th Annual ESPID Meeting, Madrid, Spania, 23-27.05.2017.
(prezentare orala)
15. Infectii virale acute ale sistemului nervos central - diferente intre copii si adulti/Acute
viral infections of the central nervous system – differences between children and
adults - Corneliu Petru Popescu, Simin Aysel Florescu, Mihaela Zaharia,
Gratiela Tardei, Maria Nica, Daniel Codreanu, Emanoil Ceausu, Simona Ruta. A X-
a Conferinta Nationala de Microbiologie si Epidemiologie, Bucuresti, 2-4.11.2017
(poster)
16. Risk factors for fatal evolution in acute viral infections of the central nervous system -
three years prospective study in a tertiary facility – C. P. Popescu, S. A. Florescu, M.
Zaharia, G. Tardei, M. Nica, D. Codreanu, E. Ceausu, S. Ruta. 18th International
Congress in Infectious Diseases, Buenos Aires, Argentina, 1-4 Martie 2018. (poster)
17. Characteristics of the patients in an Infectious Diseases Intensive Care Unit -
S.A.Florescu, C. P. Popescu, D. Ciortea, D. Stanciu, M. Zaharia, E. Nedu, M. Nica,
D. Codreanu, A. G. Kosa, S. Ruta, P. Calistru, E. Ceausu. 18th International Congress
in Infectious Diseases, Buenos Aires, Argentina, 1-4 Martie 2018. (poster)
18. Re-emergence of West Nile virus neuroinvasive disease in Romania, 2016-2017,
changes in evolution and prognosis - Corneliu Petru Popescu, Simin Aysel Florescu,
Ani Ioana Cotar, Daniela Badescu, Cornelia Svetlana Ceianu, Mihaela Zaharia, Delia
Stanciu, Violeta Melinte, Voinescu Bianca, Olivia Burcos, Cristiana Cristea, Stela
Mereuta, Simona Tetradov, Valentina Simion, Sebastian Smadu, Gratiela Tardei,
Daniel Codreanu, Petre Calistru, Emanoil Ceausu, Simona Maria Ruta. Simpozion
Academician Nicolae Cajal editia a XIII-a, Bucuresti, 17-19.03.2016 (prezentare
orala)
19. Risk factors for fatal evolution in acute viral infections of the central nervous system -
two-year prospective study in a tertiary facility - Corneliu Petru Popescu, Simin
Aysel Florescu, Mihaela Zaharia, Gratiela Tardei, Maria Nica, Daniel Codreanu,
Emanoil Ceausu, Simona Ruta 28th
European Congress of Clinical Microbiology and
Infectious Diseases, Madrid, 21-24.04.2018 (poster).
20. Infectia neuroinvaziva cu virus West Nile in Romania/ Neuroinvasive West Nile virus
disease in Romania – Popescu CP, Florescu Simin Aysel, Ruta Simona Maria,
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Ceausu E. Conferinta Nationala de Boli Infectioase „Patologia infectioasa la
frontiere”, Galati, 7-9.06.2018 (oral). ISBN 978-606-696-117-2.
21. Re-emergent WNV encephalitis in Romania - Corneliu Popescu. International
Symposium on Innate Immunity and Inflammation in conjunction with The 5th
National Conference of Infectious Disease Infection nowadays. Therapy, where to?,
Cluj, 5-8.09.2018.