Virus Infections in the Central Nervous System

2 main causes

• Herpes simplex: more encephalitis

less meningitis

• Enterovirus: more meningitis

less encephalitis

sometimes the 2 combined

Other causes: CMV, T. gondii, Mycoplasma pneumoniae some in relation to HIV or very rare

Viral Encephalitis: Incidence

• Sweden: 2.3/106 people/yrSkoldenberg, Lancet 1984; ii:707

• Denmark: 1.8/106 people/yrFonsgaard, CDU 1998; 9: 45

• Finland: 1.4/105 adults/yr: 16% due to HSVRantalaiho, J. Neurol. Sci 2001; 184: 169

• Vienna: 1/105 adults/yr: incidence of herpes encephalitisPuchhammer-Stöckl, J Med Virol 2001: 64: 531-536

Most in children < 1 yr and adults > 65 yr, followed by 20-44 yr

Herpes simplex virus

• Structure of the virus- core, caspid, tegument and envelope

Herpes simplex Virus Infections in the Central Nervous System

• Neonatal HSV

- mostly HSV-2 by retrograde spread secondary to maternal genital infection

- incidence: 1/3500 - 1/5000 births in US

1.65/100.000 births in UK*

• HSV encephalitis

- most commonly caused by HSV-1

• Recurrent aseptic meningitis (Mollaret’s meninigitis)

- mainly associated with HSV-2

Tang YW et al. J Clin Microbiol 1999;27: 2127Tookey et al. Pediatr Perinat Epid 1996; 10: 432

HSV Encephalitis

• Leading cause of fatal encephalitis

• 10 - 20 % of all viral encephalitis cases

• Mortality up to 70 %

• 2/3 of survivors neurological defectsl

Herpes simplex Encephalitis: Diagnosis

Imaging Techniques

• Focal inflammatory loci in the temporal lobes of the cerebral cortex

- electro-encephalography (EEG)

- computerized tomography (CTscan)

- magnetic resonance imaging (MRI)

helpful clinical direction but results lack sensitivity and specificity

HSV Encephalitis : Diagnosis

• Diagnosis is difficult

- in early stage : 10-20 % of encephalitis have normal cell counts

- only 50 % elevated WBC

- only 50 % elevated protein level

- only 4 % with biopsy proven encephalitis :

culture positive in CSF

- serology is useless

- CSF antibodies : elevated late in disease

Herpes simplex Encephalitis: Diagnosis

Virus isolation

• Isolation of HSV from brain tissue

was considered as “gold standard”

high efficiency of virus isolation ± 45%

invasive procedure with serious complications including hemorrhage (2%)

false negative results (4%) due to focal nature• Isolation of HSV from CSF

low sensitivity

positive in maximum 4% of brain biopsy proven cases

Tang YW et al. J Clin Microbiol 1999; 37: 2127-36

Herpes simplex Encephalitis: Diagnosis

Serology

• by intrathecal antibodies:

- of little clinical value since immune response in only few patients early; in most patients only after 2-3 weeks.

- Standardization needed by concomitant detection of albumin to exclude passive diffusion of virus-specific antibodies into the CSF, thereby yielding false-positive results.

Molecular Diagnosis of HSV Encephalitis

• 1st Molecular diagnosis: in situ hibridization with biotinylated cDNA probe in cell preparations after cytocentrifugation

• 12 patients with HSE: 54 control cases

8/12: (67%) positive in methanol-fixed cells

3/12: (25%) in fresh acetone - fixed cells

11/12: (92%) POSITIVE sens. 92%

54/54: (controls) NEGATIVE spec. 100%

Bamborschke S et al. J Neurol 1990; 237:73

PCR for Diagnosis of HSV Encephalitis

• Brain biopsy ± CSF in patients suspected for HSE

• PCR in CSF

- 53/54 (98%): PCR positive in culture positive biopsies

- 3/46 (6%):PCR positive in culture negative biopsies

- all 18 CSF taken before brain biopsy: PCR positive

- 4/19 CSF: PCR positive after 2 weeks of antiviral therapy

PCR = “GOLD STANDARD”

Lakeman F. J Infect Dis 1995; 172: 1641

Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS

Test Ease of Turnaround Result interpretation

performancea time

Antigen detection 1 1-3 h May indicate infection if

correlated with

symptoms

Cell culture 2-3 2-7 days Indicates active

infection

Serology 2 4-6 h Indirect; probably indicates

active infection

PCR 3-4 1-2 days Indicates active

infection

Tang YW et al. J Clin Microbiol 1999; 37: 2127-36

a Performance scores: 1, could be performed in most routine clinical laboratories; 2, could be performed in reference clinical laboratories; 3, could be performed in specialized research laboratories; 4, could be performed in laboratories with skilled technologists and space and equipment dedicated to performing molecular techniques.

Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS

Test Advantage(s) Disadvantage(s)

Antigen detection Rapid Poor sensitivity and specificity

Cell culture Isolate available for Very poor sensitivity;

phenotypic timing of early

antiviral specimen

susceptibility collection critical

testing

Serology Potential for Results generally

automation retrospective

PCR High sensitivity and Facility requirement;

specificity false positive due to carry-

over contamination and false negative due to

inhibitors in specimen

Tang YW et al. J Clin Microbiol 1999; 37: 2127-36

PCR for HSV Encephalitis : Sample Transport and Processing

• Transport to lab at 4°C in sterile vial

• Stable for days up to weeks at 4°C

• Multiple freeze-thawing to be avoided

• Avoid contamination between samples (e.g. by

aliquoting)

Tang YW et al. J Clin Microbiol 1999; 37: 2127

CSF Preparation Prior to Nucleic Acid Amplification

Principle Method (examples)

CSF cell lysis Heating to 95°C, freezing thawing

CSF cell lysis-protein Detergents (SDS), proteases (protease K),

digestion chaotropioc agents (guanidiniun thiocyanate)a

Nucleic acid concentration Ultracentrifugation Ethanol precipitation of

nucleic acid

Nucleic acid extraction Phenol-chloroform, spin column, silicate

absorption, magnetic separation

Cinque P et al. J Clin Virol 2003; 26: 1-28.

PCR Methods for HSV Encephalitis

• Mono reaction with agarose gel electrophoresis or EIA detection

• Multiplex reaction:

- up to 6 viruses: HSV 1-2, VZV, CMV, EBV, HHV6

• PCR with consensus primers

• Real time PCR

Cinque P et al. J Clin Virol 2003; 26: 1-28.

Example of Multiplex PCR

Cinque P et al. J Clin Virol 2003; 26: 1-28.

HSV-1(138 bp)

HSV-2(101 bp)

VZV(266 bp)

M

Example of PCR Assay with Consensus Primers

Cinque P et al. J Clin Virol 2003; 26: 1-28.

Indications for molecular amplification techniques for the detection of

Herpes Simplex Virus (HSV1-HSV2)

1. Patients with neurological symptoms: encephalitis,

meningo-encephalitis, meningitis, myelitis

2. Patients with ophthalmological symptoms: keratitis, uveïtis,

acute retinitis

3. Neonatal herpes infections

4. Imunocompromised patients with oesophageal and

intestinal lesions

HSV Encephalitis : Diagnosis by PCR

• More sensitive than culture

- 53/54 biopsy proven : positive (Lakeman)

• No commercial kits available

• All methods are in house methods

• HSV PCR = not standardized

• PCR results may be different from different labs

0%

20%

40%

60%

80%

100%

Ct Mtb HCV HIV HSV CMV EV

Commercial In-house Conventional In-house Real-time

0%

20%

40%

60%

80%

100%

Ct Mtb HCV HIV HSV CMV EV

Commercial In-house Conventional In-house Real-time

Types of NATs in use in 2002 Types of NATs in use in 2003

Forde C. ECCMID 2004, P831

Molecular Diagnostic Tests : Proficiency Testing

• To confirm skill of lab in test performance

• To ensure reproducibility

• To validate amplification methods

• Frequency :

- 2-3 testing events / year

- 5 test samples / testing event covering full range : non reactive highly reactive

• Samples :

- whole organisms or isolated nucleic acids

- previously characterized specimens

- or duplicate, blinded specimens (internal consistency)

NCCLS MM3-A. , 1995

22.9

15.7

15.7

7.1

9.7

18.9

17.7

16.4

18.0

**2003% false Negative

65.9N/AN/A7.0N/A74N/A

11.26.016.05.64.810371HSV

TB

86.189.015.00.02.39050HIV

74.676.011.00.01.39255HCV

48.356.018.01.15.37942HBV

11.25.031.06.74.010159EV

22.7N/AN/A2.3N/A84N/AEBV

88.891.045.01.30.712296CT

16.328.021.01.92.49579CMV

**2003% commercialAssays

*QCCA% CommercialAssays

QCCA% false Negative

**2003% falsePositive

*QCCA% false Positive

**NoPart3cipants2003

*No Participants QCCA

Program

Quality Control for Molecular DiagnosticsPast, present trends…….

Source: *E Valentine-Thon JCV 25 (2002) S13-S21 ** Forde C, ECCMID 2004, P831

Sensitivity IssuesRates variableProgram dependant

Improved specificity

30%<3%

9% ~18%

Quality Control for the Detection of HSV

• Techniques for NA extraction, amplification and target sequences are heterogeneous

• All labs use in-house developed methods

• Application of real-time PCR increased from 7/12 (58%) labs in 2002 to 11/13 (85%) in 2004

• The use of inhibition control also increased from 7/12 (58%) labs in 2002 to 10/13 (77%)

• Sensitivity and specificity of all methods used were excellent

• No false positive results were reported in 2002; in 2004 6% of negative samples were reported false positive

Influence of Prevalence on Predictive Values

Prevalence PPV NPV

1°/°°° 4.9 % 99.99 %

1 °/°° 4.7 % 99.99 %

1 % 33.3 % 99.98 %

2 % 50.0 % 99.98 %

3 % 60.0 % 99.97 %

4 % 67.0 % 99.96 %

5 % 72.0 % 99.95 %

10 % 84.0 % 99.89 %

20 % 92.0 % 99.75 %

30 % 95.0 % 99.56 %

Goldberg M, 1990; “L’epidémiologie sans peine”

for given test : Se = 99%, Sp = 98%

Algorithm for Specimen Processing and Reporting ResultsSpecimen

type / volume adequate

Yes No

specimen preparation

Control amplification

dilute, reamplify

Control amplification

Reject

Qiagen extraction

Report : “unable to process”

Target amplification

Product analysis

Report as -

Repeat testing

Report as -

Report as +

+-

+ -

- +-

-

Utility of Amplification Methods for Virus Detection in CSF

• HSV: PCR was shown to be the reference methodLakeman et al, J. Infect. Dis. 1995; 171:857

• Extended to herpes virus group• Extended to enterovirus detection in cases of meningitis

Tanel et al., Arch. Pediatr. Adolesc. Med. 1996; 150: 919

Ahmed A et al, J. Pediatr. 1997; 131: 393

Van Vliet et al, J. Clin. Microbiol. 1998; 36: 2652

Enormous increase of requests for PCR on CSF

Molecular Diagnostic Methods in Meningo- encephalitis

• Variety of possible etiologic agents

• Stepwise approach, each step aimed at a combination of agents

• Multiplex approach

• Regional epidemiologic situation e.g. LCM, Coxiella burnetii, Borrelia burgdorferi : reference centers

• Clinical condition : immunocompromised patient : Toxoplasma gondii, CMV

HSV

VZVM. pneumoniae

CMVT. gondii

Repeat to confirm

Reportresult

Reportresult

pos

pos

pos

pos

neg

neg

neg

Molecular Diagnostics for Meningo-encephalitis

Detection of HSV DNA from CSF Specimens Collected at the Mayo Clinic from August 1993

through December 1997

Yr No. of No. of % No. of subjects positive HSV-positive

specimens positive positive Male Female Unknown male/female

tested specimens gender ratio

1993 80 3 3.8 1 1 1 1.0

1994 475 28 5.9 12 12 4 1.0

1995 1,019 90 8.8 37 48 5 0.77

1996 1,951 122 6.3 45 74 3 0.61

1997 3,082 166 5.4 63 99 4 0.64

Total 6,607 409 6.2 158 234 17 0.67

Tang YW et al. J Clin Microbiol 1999; 37: 2127-36

Effective Use of PCR for Diagnosis of CNS Infections

No. (%) of tests with indicated result/no.

of tests performed

Both protein Protein level Leukocyte Both protein

Organism level and normal, count normal, level and

detected leukocyte leukocyte protein level leukocyte

count count abnormal count

normal abnormal abnormal Total

Herpesvirus* 0/209 (0) 1/33 (3.0) 5/317 (1.6) 18/173 (10.4) 24/732 (3.3)

T. whippelii 0/56 (0) 0/3 (0) 1/101 (1.0) 0/30 (0) 1/190 (0.5)

B. burgdorferi 0/149 (0) 0/18 (0) 0/215 (0) 0/89 (0) 0/471 (0)

* Including HSV, EBV, VZV, and CMV

Tang et al. Clin Infect Dis 1999; 29: 805-06

Restriction Rules for HSV Detection in CSF

Reference N° cases / specimens Criterium

Tang (1999) 24 / 723 WBC > 5 cells / mm3

and / or > 45 mg/dL protein

workload reduction 29%

Simko (2002) 10 / 406 WBC > 5 cells / mm3

and / or > 55 mg/dL protein

workload reduction 38%

increase of positivity rate: 1.9% 4% 2-fold

Tang et al. Clin Infect Dis 1999; 29: 803Simko et al. Clin Infect Dis 2002; 35: 414

Results of HSV DNA Detection in CSF by PCR and of HSV-specific Antibody Measurement in CSF and Sera in Patients with

Clinical Suspicion of Encephalitis

Method/detection Number of Positive Negative Interpretation

patients results results

PCR/HSV-1 DNA in CSF 631 8 (1.3%) 623 Direct confirmation of

CNS infection by PCR

PCR/HSV-2 DNA in CSF 631 7 (1.1%) 624

IFAT/intrathecal HSV IgM 624 13 (2.1%) 611 Serological evidence of

CNS infection

IFAT/intrathecal HSV IgG 624 12 (1.9%) 612

IFAT/HSV IgM, 4 fold 2367 268 (11.3%) 2099 Serological evidence of

increase in IgG titres, active infection

seroconversion

Sauerbrei A et al. J Clin Virol 2000; 17: 31

Virological Diagnosis of Herpes simplex Encephalitis

PCR versus serological diagnosis

Study design:• 624 CSF samples: PCR + virus-specific antibodies• 2409 serum samples: virus-specific antibodies

CONCLUSIONS:• No intrathecal antibodies in patients with positive PCR• Intrathecal immune response when CSF negative for PCR

PCR: method of choice in early phase of disease

Intrathecal antibodies: in later stage of disease

Sauerbrei A et al. J Clin Virol 2000; 17: 31-36

Limits of Early Diagnosis of HSV Encephalitis in Children

Prognosis depends on early and appropriate administration of specific antiviral therapy

38 children with proven HSV

initial negative results: 8/33 CSF before day 3

associated with low level of protein < 10 WBC/mm3

De Tiege X et al. Clin Infect Dis 2003; 36: 1335

Quantitative PCR for Diagnosis of HSE

• No correlation between quantity of virus genomes and severity of disease or prognosis

Revello M. Clin Diagn Virol 1997; 7: 183

• Patients with >100 copies DNA/µl

- older

- brain lesions by CT scan

- poorer outcomes than patients with <100 copiesDominguez R. J Clin Microbiol 1998; 36: 2229

Quantitative Real-Time PCR for the Detection of VZV in CSF

Methods• Quantitative PCR on LightCycler with Real Art VZV LCkit• DNA isolation by High Pure Viral Nucleic Acid Kit

Results• CSF viral load:

- 10 x 102 copies/ml: meningitis- 5 x 104 copies/ml: facial nerve paresis- viral load in vesicular fluid: 3x106 copies/ml

correlation between viral load and severity of disease remains uncertain !

Zampachova E et al. ECCMID 2004, P840

Example of NA Quantification in the CSF

Virus Quantitative techniques Significance of NA quantitation in CSF

HSV-1 Competitive PCR, Wide range of level variation (up to 107 copies/ml). real-time PCR Association of high DNA levels with bad HSE out-

come? Decline of DNA levels following aciclovir

therapy in HSE

HSV-2 Real-time PCR Narrower range of level variation in patients with

HSV-2 meningitis than in patients with HSV-1 encephalitis. Highest levels found in children with

congenital infection (up to 106 copies/ml)

VZV Semiquantitative PCR, Higher levels in patients with herpes zoster compli-

real-time PCR cations than in those with varicella

Cinque P et al. J Clin Virol 2003; 26: 1-28.

PCR Results following Completion of Antiviral Therapy

PCR

Infant characteristic Negativea Positiveb P value

Disease classification

CNS 4 (36.4%) 14 (73.7%) <0.001

Disseminated 0 (0.0%) 5 (26.3 %)

SEM 7 (63.6%) 0 (0.0%)

Morbidity and mortality

after 12 mo

Normal 6 (54.5%) 1 (5.3%) <0.001

Mild 0 (0.0%) 0 (0.0%)

Moderate 1 (9.1%) 3 (15.8%)

Severe 2 (18.2%) 10 (52.6%)

Dead 0 (0.0%) 5 (26.3%)

a All samples were negative after treatment b one positive result

Romero JR, Kimberlin DW. Clin Lab Med 2003; 23:843-865

Etiology of Viral Meningitis

• Retrospective analysis of 43 causecutive cases of aseptic meningitis

43 cases : 19 (44%) enterovirus

1 (2%) HIV

2 (5%) VZV

5 (12%) HSV 1+2

1 (2%) CEE

15 (35%) unknown

Acyclovir initially administered to all cases

Hospitalization time : 16 - 31 days

Nowak A et al. Eur J Neurol 2003; 10: 271-8.

Enterovirus

Types and Characteristics of Human Enterovirus

66 serotypes known

Group Virus types CPE in cell cultures Pathology in

Monkey Human newborn

Kidney cells mice

Poliovirus 3 types: 1-3 + + -

Coxsacke A 23 types/ A1-22, A24 - or ± - or ± +

Coxsackie B 6 types : B1-B6 + + +

Echovirus 31 types (1-9, 11-27, + ± -

29-33)

Enteroviruses 4 types (68-71) + + -

Types and Characteristics of Human Enterovirus

Group Virus types Major disease associations

Poliovirus 3 types (poliovirus 1-3) Paralytic poliomyelitis; aseptic meningitis;febrile illness

Coxsackie virus 23 types (A1-A22, A24) Aseptic meningitis; herpangina; febrile group A illness; conjunctivitus (A24); hand,

foot and mouth diseaseCoxsackie virus 6 types (B1-B6) Aseptic meningitis; severe generalised group B neonatal disease; myopericarditis;:

encephalitis; pleurodynia (Bornholm disease); fibrile illness

Echovirus 31 types (types1-9, Aseptic meningitis, rash, febrile illness11-27, 29-33) conjunctivitis; severe generalised

neonatal diseaseEnterovirus 4 types (types 68-71) Polio-like illness (E71): aseptic

meningitis (E71); hand, foot and mouth disease (E71); epidemic conjunctivitis (E70)

Enterovirus: Epidemiology

MMWR 2002;51:1047-49 Belgie 2000: echo 30; echo 6, coxsackie B 5 (M.Van Ranst)

2000 (n=577) 2001 (n=1,285) 2000-2001 (n=1,862)Rank Serotype % Serotype % Serotype %

1 Coxsackie B5 34.4 echo 18 30.8 echo 18 22.02 echo 6 8.8 echo 13 29.3 echo 13 20.83 coxsackie A9 8.7 coxsackie B2 7.6 coxsackie B5 11.94 Coxsackie B4 8.3 echo 6 4.8 coxsackie B2 6.35 echo 11 6.9 echo 4 4.1 echo 6 6.16 echo 9 6.2 echo 11 3.4 echo 11 4.57 coxsackie B2 3.5 coxsackie B3 3.0 coxsackie A9 4.08 echo 25 2.6 coxsackie B1 2.7 echo 9 3.39 echo 18 2.3 echo 9 2.0 coxsackie B4 3.210 enterovirus 71 2.1 coxsackie A9 2.0 echo 4 3.111 echo 16 1.9 coxsackie B5 1.7 coxsackie B3 2.412 echo 30 1.9 echo 30 1.7 coxsackie B1 2.013 echo 13 1.7 coxsackie B4 0.9 echo 30 1.814 echo 21 1.6 echo 25 0.6 echo 25 1.215 parecho 1* 1.4 enterovirus 71 0.6 enterovirus 71 1.1Total 92.2 95.3 93.5

* Formerly echo 22. For all other serotypes, percentages were 7.8% in 2000, 4.7% in 2001, and 6.5% during 2000-2001.

Distribution of the 15 most commonly reported nonpolio enterovirus, serotypes,by rank - National Enterovirus Surveillance System, United States, 2000-2001

Enteroviral Meningitis

• Incidence: 219/105 children < 1 yr of age

19/105 children 1-4 yrs of ageRantakallia Sc. J Inf Dis. 1986; 18: 286

• Responsible for

- 85-95% of meningitis with known etiology in USA

- 10-20% of encephalitis cases

in USA : estimate of 30.000-75.000 cases annualy

• Underreported especially in adults

Enteroviral meningitis in Adults: Underestimated

• Retrospective analysis of 30 cases• Characteristics symptoms: inconstant• CSF showed pleocytosis in 29/30 cases but predominance of lymphocytes in only 44% of patients• Management of patient varied markedly

- CT scan : 33%- acyclovir: 20%- antibiotics: 53%

• Laboratory tests requested on admission:- PCR herpes simplex: 9/30 (30%): all negative after 4 days

PCR for enterovirus : 9/30 : alle positive- PCR enterovirus: 21/30 (70%) : all positive

Rapid PCR results may avoid considerable medical expenditure

Evidence for syndromic approach

Peigue-La feuille H et al. Pathologie Biologie 2002; 50: 516-24

Diagnosis of Enteroviral Meningitis by Virus Culture

• Insensitive: especially for coxsackie A viruses

• Serotyping necessary for identification and epidemiology

• Turnaround time : 4-8 days

• No cell type supports replication of all EV types

• Even with use of several cell types:

- 25%-35% negative specimens

- coxsackie on none of cell lines (suckling mice)

Virus : CPE of Enterovirus in Cell Culture

CPE after 4-8 days

Diagnosis of Enteroviral Meningitis by Culture

Total number of isolates: 73

Number RD cells MRC5 Vero Hep2

33 + + o o

25 + o o o

5 o o + +

5 o o + o

4 o + o o

1 o o o +

Verstrepen et al., 2002

Diagnosis of Enteroviral Meningitis by Culture

Interpretation of results

• CSF: very specific but low sensitivity

• blood: very specific but low sensitivity

• stool and pharynx : sensitive but low specificity

- excretion of virus in pharynx : 1-2 weeks

in faeces : 7-11 weeks

Chang et al. J Microbiol Infect 2001; 34: 167-70.

Diagnosis of Enteroviral Meningitis by Serology

• Neutralization tests: for seroepidemiological purposes- determining exposure and immunity of population group- responses to polio vaccination- tests are labour intensive, TAT 3-4 days, not widely availabe

• Seroconversion or significant increase in antibody titres- detected only occasionally

• Elevated titres - frequently occur in normal individuals

NOT RELEVANT FOR INDIVIDUAL DIAGNOSIS

Tissue Culture Versus RT-Polymerase Chain Reaction for the Detection of Enterovirus From Cerebrospinal Fluid

Source, Y RT-PCR assay Tissue culture* (%) RT-PCR* (%)

Rotbart, 1990 In-house 9/13 (69) 13/13 (100)

Sawyer et al, 1994 In-house 112/217 (52) 135/217 (62)

Riding et al, 1996 In-house 6/140 (4) 35/140 (25)

Rotbart et al, 1997 Amplicor 36/209 (17) 51/209 (24)

Ahmed et al, 1997 Amplicor 5/61 (8) 18/61 (30)

Kessler et al, 1997 Amplicor 27/103 (26) 34/103 (33)

Pozo et al, 1998 In-house 26/50 (52) 46/50 (92)

Amplicor 26/50 (52) 43/50 (86)

In-house 1/29 (3) 4/29 (14)

Amplicor 1/29 (3) 3/29 (10)

Gorgievski-Hrisoho et al, 1998 Amplicor 16/68 (24) 58/68 (85)

* Values presented as number positive/number tested.

Romero J. Arch Pathol Lab Med 1999; 123: 1161-69

PCR for diagnosis of Enteroviral Meningitis

Conventional PCR Real-Time PCR

• TAT : 2-3 days • TAT: 3-4 hours

• risk for contamination • single tube reaction: minimal

carry-over risk

• qualitative • quantitative results possible

Real-time tests are only technique allowing immediate impact on therapeutic decisions

Indications for PCR for Enteroviruses

• Viral meningitis or meningo-encephalitis (CSF)

• Acute pericarditis or myocarditis (pericard fluid, blood, myocardial biopsy)

• Prenatal diagnosis of congenital infections in case of echographic abnormalities (amniotic fluid, faetal blood)

Amplification Methods for the Diagnosis of Enteroviral Meningitis

5’NTR : - critical role in enteroviral life cycle- conserved regions of high nucleotide identity among EV- ideal for development of primers and probes for the detection of

enteroviruses : most serotypes detected

Romero J. Arch Pathol Lab Med 1999; 123: 1161-69

Organization of the enterovirus RNA genome. NTR inidicates nontranslated region.

Selection Criteria for PCR on EV in CSF

• WBC: increase with increasing age• children: 15% < 10WBC / mm3

Henquell et al. J Clin Virol 2001; 21: 29-35

Adults: - 29/30 (97%) pleocytosis : > 5/mm3

- 44%: predominance of lymphocytes- 37%: predominance of polymorphonuclear leucocytes

- mainly during first days after onset of symptoms- protein concentration: normal or slightly increased - glucose concentration: generally within normal limits

A NORMAL CSF DOES NOT EXCLUDE EV INFECTION.

Peigue-Lafeuille H et al. Pathologie Biologie 2002; 50: 516-24.

Impact of Enteroviral PCR on Patient Management

• Comparison of management in two groups of patients:

N=95 : positive PCR results

N=95: negative

EV-PCR+ EV-PCR- P values

(N=95) (N=92)

Additional laboratory tests 26% 72% <0.01

IV antibiotics 2 d 3.5 d <0.01

hospitalization time 42 hours 71.5 hours <0.01

Ramers C et al. JAMA 2000; 283: 2680-85

Impact of EV PCR on Adult Patient Management

• Nationwide outbreak of EV meningitis due to echo 30• Objective: evaluation of management strategy including early PCR on

hospitalization• Methods:

- N=21: before implementation of early PCR- N=27: after implementation of early PCR

• Results: significant reduction of - hospital stay: 103 hrs 80 hrs P=0.04- mean duration of antibiotic treatment: 115 hrs 69 hrs : P=0.02

• Conclusion: systematic testing of CSF in cases of aseptic meningitis by PCR may be cost-effective

Tattevin P et al. Scand J Infect Dis 2002; 34: 359-61

Impact of PCR on Management of Pediatric Patients with Enteroviral Meningitis

• Objectives : - Comparison of antibiotic use and hospital stay in children with EV meningitis after PCR results

available < 24 h or > 24 h after collection• Methods:

- EV PCR performed 5d/week- CSF from 113 patients with suspected meningitis

• Results:

50/113 (44%) of patients positive

17/50 (34%) results < 24 h 33/50 (66%) results > 24 h

difference in antibiotic use: 20 hrs less (P=0.006)

difference in hospital charges: 2798 $ less (P=0.001)

Rapid reporting of PCR resutls can have significant impact

Robinson CC et al. Pediatr Infect Dis 2002; 21: 283-6

Diagnosis of viral encephalitis: CONCLUSIONS

• Amplification methods are a major advance for the

detection of both herpes viruses and enteroviruses.

• Conventional PCR’s are gradually replaced by

real-time techniques.

• Rapid PCR results allow immediate impact on

therapeutic decisions and may be cost-effective.