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Virus Infections in the Central Nervous System 2 main causes • Herpes simplex: more 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/yr Skoldenberg, Lancet 1984; ii: 707 • Viral Encephalitis: Incidence • Sweden: 2. 3/106 people/yr Skoldenberg, Lancet 1984; ii: 707 • Denmark: 1. 8/106 people/yr Fonsgaard, CDU 1998; 9: 45 • Finland: 1. 4/105 adults/yr: 16% due to HSV Rantalaiho, J. Neurol. Sci 2001; 184: 169 • Vienna: 1/105 adults/yr: incidence of herpes encephalitis Puchhammer-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 • Structure of the virus - core, caspid, tegument and envelope

Herpes simplex Virus Infections in the Central Nervous System • Neonatal HSV - mostly 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: 2127 Tookey et al. Pediatr Perinat Epid 1996; 10: 432

HSV Encephalitis • Leading cause of fatal encephalitis • 10 - 20 % of 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 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 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 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 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 falsepositive results.

Molecular Diagnosis of HSV Encephalitis • 1 st Molecular diagnosis: in situ hibridization with Molecular Diagnosis of HSV Encephalitis • 1 st Molecular diagnosis: in situ hibridization with biotinylated c. DNA 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 54/54: (controls) sens. 92% NEGATIVE spec. 100% Bamborschke S et al. J Neurol 1990; 237: 73

PCR for Diagnosis of HSV Encephalitis • Brain biopsy ± CSF in patients suspected 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 Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS Test Ease of Turnaround performancea time Antigen detection 1 Cell culture 2 -3 Serology 2 PCR 3 -4 Result interpretation 1 -3 h May indicate infection if correlated with symptoms 2 -7 days Indicates active infection 4 -6 h Indirect; probably indicates active infection 1 -2 days Indicates active infection 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. a Tang YW et al. J Clin Microbiol 1999; 37: 2127 -36

Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS Test Antigen detection Laboratory Techniques for Specific Diagnosis of HSV Infection in the CNS Test Antigen detection Cell culture Serology PCR Advantage(s) Rapid Isolate available for phenotypic antiviral susceptibility testing Potential for automation High sensitivity and specificity Disadvantage(s) Poor sensitivity and specificity Very poor sensitivity; timing of early specimen collection critical Results generally retrospective Facility requirement; false positive due to carryover 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 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 CSF Preparation Prior to Nucleic Acid Amplification Principle Method (examples) CSF cell lysis Heating to 95°C, freezing thawing CSF cell lysis-protein digestion Detergents (SDS), proteases (protease K), 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 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, HHV 6 • PCR with consensus primers • Real time PCR Cinque P et al. J Clin Virol 2003; 26: 1 -28.

Example of Multiplex PCR M HSV-1 (138 bp) HSV-2 (101 bp) VZV (266 bp) Example of Multiplex PCR M HSV-1 (138 bp) HSV-2 (101 bp) VZV (266 bp) Cinque P et al. J Clin Virol 2003; 26: 1 -28.

Example of PCR Assay with Consensus Primers Cinque P et al. J Clin Virol 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 (HSV 1 Indications for molecular amplification techniques for the detection of Herpes Simplex Virus (HSV 1 -HSV 2) 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 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

Types of NATs in use in 2002 Types of NATs in use in 2003 Types of NATs in use in 2002 Types of NATs in use in 2003 Forde C. ECCMID 2004, P 831

Molecular Diagnostic Tests : Proficiency Testing • To confirm skill of lab in test 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 MM 3 -A. , 1995

Quality Control for Molecular Diagnostics Past, present trends……. Program *No Participants QCCA **No Part Quality Control for Molecular Diagnostics Past, present trends……. Program *No Participants QCCA **No Part 3 cipants 2003 *QCCA % false Positive **2003 % false Positive QCCA % false Negative **2003 % false Negative *QCCA % Commercial Assays **2003 % commercial Assays CMV 79 95 2. 4 1. 9 21. 0 18. 0 28. 0 16. 3 CT 96 122 0. 7 1. 3 45. 0 16. 4 91. 0 88. 8 EBV N/A 84 N/A 2. 3 N/A 17. 7 N/A 22. 7 EV 59 101 4. 0 6. 7 31. 0 18. 9 5. 0 11. 2 HBV 42 79 5. 3 1. 1 18. 0 9. 7 56. 0 48. 3 HCV 55 92 1. 3 0. 0 11. 0 7. 1 76. 0 74. 6 HIV 50 90 2. 3 0. 0 15. 7 89. 0 86. 1 HSV 71 30% 103 4. 8 5. 6 16. 0 15. 7 6. 0 11. 2 TB N/A 74 N/A 22. 9 9% N/A ~18% 65. 9 <3% 7. 0 Improved specificity Source: *E Valentine-Thon JCV 25 (2002) S 13 -S 21 Sensitivity Issues Rates variable Program dependant ** Forde C, ECCMID 2004, P 831

Quality Control for the Detection of HSV • Techniques for NA extraction, amplification and 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 for given test : Se = 99%, Sp Influence of Prevalence on Predictive Values for given test : Se = 99%, Sp = 98% Prevalence PPV NPV 1°/°°° 1 °/°° 1% 2% 3% 4% 5% 10 % 20 % 30 % 4. 9 % 4. 7 % 33. 3 % 50. 0 % 67. 0 % 72. 0 % 84. 0 % 92. 0 % 95. 0 % 99. 99 % 99. 98 % 99. 97 % 99. 96 % 99. 95 % 99. 89 % 99. 75 % 99. 56 % Goldberg M, 1990; “L’epidémiologie sans peine”

Algorithm for Specimen Processing and Reporting Results Specimen type / volume adequate Yes No Algorithm for Specimen Processing and Reporting Results Specimen type / volume adequate Yes No specimen preparation Control amplification - + dilute, reamplify Control amplification - Target amplification - Product analysis Qiagen extraction + Repeat testing - Report : “unable to process” Reject Report as - + Report as -

Utility of Amplification Methods for Virus Detection in CSF • HSV: PCR was shown Utility of Amplification Methods for Virus Detection in CSF • HSV: PCR was shown to be the reference method Lakeman 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 • 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

Molecular Diagnostics for Meningo-encephalitis pos HSV neg VZV M. pneumoniae pos Repeat to confirm Molecular Diagnostics for Meningo-encephalitis pos HSV neg VZV M. pneumoniae pos Repeat to confirm neg pos CMV T. gondii neg Report result

Detection of HSV DNA from CSF Specimens Collected at the Mayo Clinic from August Detection of HSV DNA from CSF Specimens Collected at the Mayo Clinic from August 1993 through December 1997 Yr No. of specimens % No. of subjects positive Male HSV-positive Female Unknown tested specimens 1993 80 3 3. 8 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 male/female gender ratio 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 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 protein level leukocyte count abnormal count normal abnormal Total Herpesvirus* 0/209 (0) 24/732 (3. 3) 1/33 (3. 0) 5/317 (1. 6) 18/173 (10. 4) 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) Tang et al. Clin Infect Dis 1999; 29: 805 -06 * Including HSV, EBV, VZV, and CMV

Restriction Rules for HSV Detection in CSF Reference N° cases / specimens Criterium Tang Restriction Rules for HSV Detection in CSF Reference N° cases / specimens Criterium Tang (1999) 24 / 723 WBC > 5 cells / mm 3 and / or > 45 mg/d. L protein workload reduction 29% Simko (2002) 10 / 406 WBC > 5 cells / mm 3 and / or > 55 mg/d. L protein workload reduction 38% increase of positivity rate: 1. 9% 4% 2 -fold Tang et al. Clin Infect Dis 1999; 29: 803 Simko et al. Clin Infect Dis 2002; 35: 414

Results of HSV DNA Detection in CSF by PCR and of HSVspecific Antibody Measurement Results of HSV DNA Detection in CSF by PCR and of HSVspecific Antibody Measurement in CSF and Sera in Patients with Clinical Suspicion of Encephalitis Method/detection Number of patients results PCR/HSV-1 DNA in CSF 631 PCR/HSV-2 DNA in CSF 631 IFAT/intrathecal HSV Ig. M 624 IFAT/intrathecal HSV Ig. G 624 IFAT/HSV Ig. M, 4 fold 2367 increase in Ig. G titres, seroconversion Positive Negative Interpretation 8 (1. 3%) 623 Direct confirmation of CNS infection by PCR 7 (1. 1%) 624 13 (2. 1%) 611 Serological evidence of CNS infection 12 (1. 9%) 612 268 (11. 3%) 2099 Serological evidence of active infection Sauerbrei A et al. J Clin Virol 2000; 17: 31

Virological Diagnosis of Herpes simplex Encephalitis PCR versus serological diagnosis Study design: • 624 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: 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 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/mm 3 De Tiege X et al. Clin Infect Dis 2003; 36: 1335

Quantitative PCR for Diagnosis of HSE • No correlation between quantity of virus genomes 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 copies Dominguez R. J Clin Microbiol 1998; 36: 2229

Quantitative Real-Time PCR for the Detection of VZV in CSF Methods • Quantitative PCR Quantitative Real-Time PCR for the Detection of VZV in CSF Methods • Quantitative PCR on Light. Cycler 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: 3 x 106 copies/ml correlation between viral load and severity of disease remains uncertain ! Zampachova E et al. ECCMID 2004, P 840

Example of NA Quantification in the CSF Virus Quantitative techniques Significance of NA quantitation Example of NA Quantification in the CSF Virus Quantitative techniques Significance of NA quantitation in CSF HSV-1 Competitive PCR, real-time PCR Wide range of level variation (up to 107 copies/ml). 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, real-time PCR Higher levels in patients with herpes zoster compli- cations than in those with varicella Cinque P et al. J Clin Virol 2003; 26: 1 -28.

PCR Results following Completion of Antiviral Therapy Infant characteristic value PCR Negativea Positiveb P PCR Results following Completion of Antiviral Therapy Infant characteristic value PCR Negativea Positiveb P 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%) 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 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 Enterovirus

Types and Characteristics of Human Enterovirus 66 serotypes known Group Virus types CPE in Types and Characteristics of Human Enterovirus 66 serotypes known Group Virus types CPE in cell cultures Monkey Pathology in Human newborn Kidney cells Poliovirus 3 types: 1 -3 + Coxsacke A 23 types/ A 1 -22, A 24 Coxsackie B 6 types : B 1 -B 6 Echovirus 31 types (1 -9, 11 -27, mice + - or ± + + + ± 29 -33) Enteroviruses 4 types (68 -71) + + + - -

Types and Characteristics of Human Enterovirus Group Virus types Poliovirus Major disease associations 3 Types and Characteristics of Human Enterovirus Group Virus types Poliovirus Major disease associations 3 types (poliovirus 1 -3) Paralytic poliomyelitis; aseptic meningitis; febrile illness Coxsackie virus 23 types (A 1 -A 22, A 24) Aseptic meningitis; herpangina; febrile group A illness; conjunctivitus (A 24); hand, foot and mouth disease Coxsackie virus 6 types (B 1 -B 6) Aseptic meningitis; severe generalised group B neonatal disease; myopericarditis; : encephalitis; pleurodynia (Bornholm disease); fibrile illness Echovirus 31 types (types 1 -9, Aseptic meningitis, rash, febrile illness 11 -27, 29 -33) conjunctivitis; severe generalised neonatal disease Enterovirus 4 types (types 68 -71) Polio-like illness (E 71): aseptic meningitis (E 71); hand, foot and mouth disease (E 71); epidemic conjunctivitis (E 70)

Enterovirus: Epidemiology Distribution of the 15 most commonly reported nonpolio enterovirus, serotypes, by rank Enterovirus: Epidemiology Distribution of the 15 most commonly reported nonpolio enterovirus, serotypes, by rank - National Enterovirus Surveillance System, United States, 2000 -2001 Rank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total 2000 (n=577) Serotype Coxsackie B 5 echo 6 coxsackie A 9 Coxsackie B 4 echo 11 echo 9 coxsackie B 2 echo 25 echo 18 enterovirus 71 echo 16 echo 30 echo 13 echo 21 parecho 1* % 34. 4 8. 8 8. 7 8. 3 6. 9 6. 2 3. 5 2. 6 2. 3 2. 1 1. 9 1. 7 1. 6 1. 4 92. 2 2001 (n=1, 285) Serotype % 2000 -2001 (n=1, 862) Serotype % echo 18 echo 13 coxsackie B 2 echo 6 echo 4 echo 11 coxsackie B 3 coxsackie B 1 echo 9 coxsackie A 9 coxsackie B 5 echo 30 coxsackie B 4 echo 25 enterovirus 71 echo 18 echo 13 coxsackie B 5 coxsackie B 2 echo 6 echo 11 coxsackie A 9 echo 9 coxsackie B 4 echo 4 coxsackie B 3 coxsackie B 1 echo 30 echo 25 enterovirus 71 30. 8 29. 3 7. 6 4. 8 4. 1 3. 4 3. 0 2. 7 2. 0 1. 7 0. 9 0. 6 95. 3 22. 0 20. 8 11. 9 6. 3 6. 1 4. 5 4. 0 3. 3 3. 2 3. 1 2. 4 2. 0 1. 8 1. 2 1. 1 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. Belgie 2000: echo 30; echo 6, coxsackie B 5 (M. Van Ranst) MMWR 2002; 51: 1047 -49

Enteroviral Meningitis • Incidence: 219/105 children < 1 yr of age 19/105 children 1 Enteroviral Meningitis • Incidence: 219/105 children < 1 yr of age 19/105 children 1 -4 yrs of age Rantakallia 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: 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 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 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. Diagnosis of Enteroviral Meningitis by Culture Total number of isolates: 73 Number RD cells. MRC 5 Vero 33 + + o o 25 + o o o 5 o o + + 5 o o + o 4 o + o o 1 o o o Hep 2 + Verstrepen et al. , 2002

Diagnosis of Enteroviral Meningitis by Culture Interpretation of results • CSF: very specific but 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 faeces in pharynx : 1 -2 weeks : 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 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 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 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 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 Organization of the enterovirus RNA genome. Amplification Methods for the Diagnosis of Enteroviral Meningitis Organization of the enterovirus RNA genome. NTR inidicates nontranslated region. 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

Selection Criteria for PCR on EV in CSF • WBC: increase with increasing age Selection Criteria for PCR on EV in CSF • WBC: increase with increasing age • children: 15% < 10 WBC / mm 3 Henquell et al. J Clin Virol 2001; 21: 29 -35 Adults: - 29/30 (97%) pleocytosis : > 5/mm 3 - 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 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+ (N=95) Additional laboratory tests IV antibiotics hospitalization time EV-PCR(N=92) P values 26% 2 d 42 hours 72% 3. 5 d 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 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 : 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 5 d/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 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.