1

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

2

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

3

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.

4

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

5

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

6

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.

7

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

8

<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.

9

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.

10

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

11

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

12

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

13

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).

14

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.

15

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.

16

• 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.

17

References

Barton ES, White DW, Cathelyn JS, et al.: Herpesvirus latency confers symbiotic protection

from bacterial infection. Nature 2007;447:326–329. doi: 10.1038/nature05762.

Bloch KC, Glaser CA. Encephalitis surveillance through the Emerging Infections Program,

1997-2010. Emerg Infect Dis. 2015;21(9):1562-1567.

Calleri G, Libanore V, Corcione S. et al. Infection 2017;45:227.

https://doi.org/10.1007/s15010-017-0993-4.

CDC. (1997). Case Definitions for Infectious Conditions Under Public Health Surveillance.

MMWR, 46(RR-10), 1-55. https://www.cdc.gov/mmwr/preview/mmwrhtml/00047449.htm.

Granerod J, Ambrose HE, Davies NW, et al. Causes of encephalitis and differences in their

clinical presentations in England: a multicentre, population-based prospective study. Lancet

Infect Dis. 2010 doi: 10.1016/S1473-3099(10)70222-X.

Goenka A, Michael BD, Ledger E, Hart IJ, Absoud M, Chow G, et al. Neurological

manifestations of influenza infection in children and adults: results of a national British

surveillance study. Clin Infect Dis. 2014;58:775–784.

Mailles A, Stahl J-P. Infectious encephalitis in France in 2007: a national prospective study.

Clin Infect Dis 2009; 49, pp. 1838-1847.DOI: 10.1086/648419.

Mailles A, Stahl JP & Bloch KC. Update and new insights in encephalitis. Clinical

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.

18

Shukla B, Aguilera EA, Salazar L, Wootton SH, Kaewpoowat Q, Hasbun R. Aseptic

meningitis in adults and children: Diagnostic and management challenges. Journal of Clinical

Virology 2017; 94, 110-114.

Vanichanan, J., Salazar, L., Wootton, S.H., Aguilera, E., Garcia, M.N., Murray, K.O. et al,

Use of testing for West Nile virus and other arboviruses. Emerg. Infect. Dis. 2016;22.

Venkatesan A, Tunkel AR, Bloch KC, Lauring A. S., Sejvar J., Bitnun A. et al. Case

definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the

international encephalitis consortium. Clin Infect Dis. 2013;57:1114–1128.

Wang GF, Li W, Li K. Acute encephalopathy and encephalitis caused by influenza virus

infection.Curr Opin Neurol. 2010 Jun;23(3):305-11.

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+.

19

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)

20

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,

21

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,

22

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.