Laboratory diagnosis of herpesvirus infections of the cns

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  • Jubelt B. Neuro Clin 2:187, 1984.
  • because : -Mortality in untreated patients is in excess of 70%. Only 2.5% of all patients return to normal neurologic function. -The therapy to be successful has to be started as soon as possible Cerebrospinal fluid examination elevated levels of cells and protein red blood cells are found in most CSF obtained from patients EEG) spike and slow-wave activity to the temporal lobe (Burst suppression pattern) Scanning procedures technetium, CT, MRI
  • Laboratory diagnosis of herpesvirus infections of the cns

    1. 1. Laboratory diagnosis of herpesvirus infections of the CNS Giorgio Palù, MD Padova University, Italy
    2. 2. Herpesvirus Infections of the CNS <ul><li>Virus Clinical diagnosis </li></ul><ul><li>HSV-1 & 2 Encephalitis, meningitis, Mollaret’s (benign recurrent lymphocytic) meningitis, neonatal meningoencephalitis and disseminated disease </li></ul><ul><li>VZV Zoster sine herpete, aseptic meningitis, encephalitis, transverse myelitis, CNS vasculitis, cerebellitis </li></ul><ul><li>CMV Encephalitis, polymyeloradiculitis, ventriculitis, myelitis, inflammatory polyneuropathy (predominantly in AIDS/HIV), congenital CMV </li></ul><ul><li>HHV-6 & 7 Meningoencephalitis, recurrent febrile seizures of childhood, possible association with multiple sclerosis </li></ul><ul><li>EBV Meningoencephalitis, acute cerebellar ataxia, aseptic meningitis, transverse myelitis, autonomic neuropathy, primary CNS lymphoma in AIDS </li></ul><ul><li>HHV-8 ??? </li></ul>
    3. 3. Diagnosis of CNS Infection <ul><li>Standard neurodiagnostic procedures include: </li></ul><ul><ul><ul><ul><li>CSF examination </li></ul></ul></ul></ul><ul><ul><ul><ul><li>EEG </li></ul></ul></ul></ul><ul><ul><ul><ul><li>scanning </li></ul></ul></ul></ul><ul><li>These can be normal in early stages of the disease </li></ul><ul><li>Other diagnostic evaluations should be initiated immediately </li></ul>
    4. 4. Role of PCR of CSF <ul><li>PCR is the standard method of laboratory diagnosis for many viral CNS infections </li></ul><ul><li>CSF PCR testing may antedate clinically recognizable disease </li></ul><ul><li>Quantitative CSF-PCR may also be useful for monitoring therapy. </li></ul><ul><li>Must be performed by a reliable laboratory </li></ul>
    5. 5. Sensitivity and Specificity of PCR 98.5% sensitive and 100% specific as a tumour marker EBV Excellent sensitivity, but poor positive predictive value in clinical disease (30–40% of asymptomatic controls positive) HHV-6 Sensitivity nearly 100% in immunosuppressed patients with neurological symptoms; can be quantitated (range:10–10 4 copies/ml); possible use to monitor therapy. Positive results in 60% of affected infants; correlates with poor neurological outcome CMV Sensitivity and specificity >95% VZV >95% sensitivity and specificity; quantitative PCR available; potential use in determining course of iv therapy (especially in neonatal disease) HSV-1 and 2 Sensitivity and specificity Virus
    6. 6. HSV-1/-2 Infection of the CNS <ul><li>Serological procedures performed on serum or CSF are not helpful early in the disease course when therapeutic decisions are needed </li></ul><ul><li>Detection of viral CSF-PCR is the diagnostic method of choice for confirmation of HSV involvement in CNS disease </li></ul><ul><li>The use of CSF-PCR instead of brain biopsy has expanded awareness of mild or atypical cases (16%-25%) </li></ul>
    7. 7. 0% 20% 40% 60% 80% 100% 0% 10% 20% 30% anti-HSV-2 prevalence Positive Predictive Value Gull MRL POCkit (Palù et al. Scand.J.Infect.Dis. 2001) Positive predictive values at different anti-HSV-2 prevalence in the population
    8. 8. VZV Infection of the CNS <ul><li>Serum anti-VZV antibody is of no value since VZV antibodies persist in the serum of nearly all adults </li></ul><ul><li>BUT </li></ul><ul><li>Testing of CSF for VZV antibodies helps to confirm the role of VZV in producing clinical syndromes of the CNS. </li></ul><ul><li>Diagnosis of VZV infection of the CNS is supported by the detection of VZV antibody in the CSF, even in the absence of PCR-amplifiable VZV DNA </li></ul><ul><li>Clinicians should request both PCR and antibody analysis </li></ul>
    9. 9. CMV Infection of the CNS <ul><li>Diagnosis of CMV-related CNS disease is based upon clinical presentation, neuroradiological studies, CSF chemistries, serological testing, and culture and PCR of CSF </li></ul><ul><li>Clinical presentations of CMV-related CNS disease can be nonspecific </li></ul><ul><li>CSF viral culture can be insensitive </li></ul><ul><li>Qualitative DNA PCR can detect both latent and replicating virus </li></ul><ul><li>RT- PCR for specific viral transcripts and quantitative PCR are useful </li></ul>
    10. 10. Measuring HCMV viral load <ul><li>High systemic CMV load is generally correlated with CMV disease </li></ul><ul><li>Measuring the viral load at specific sites may help diagnosis when systemic viral load correlates poorly with disease activity </li></ul><ul><li>Quantitation of DNA in both CSF and brain tissue sensitively diagnoses and monitors antiviral treatment, e.g. </li></ul><ul><ul><li>AIDS patients with HCMV-related CNS disease have high quantities of HCMV DNA in their CSF </li></ul></ul><ul><ul><li>Copies of HCMV DNA in CSF are higher in persons with HCMV-related polyradiculopathy than encephalitis </li></ul></ul><ul><li>More data are required on the correlation between changes in viral load, development of resistance, and clinical outcome </li></ul>
    11. 11. HCMV quantitation (methods) <ul><li>CMV quantitation can be performed in different fractions of the blood (i.e., cellular fractions and plasma) and organ fluids (e.g., CSF, urine, throat wash, and semen) </li></ul><ul><li>Methods available: </li></ul><ul><ul><li>Quantitative viral cultures: plaque assay, determination of TCID50, shell vial centrifugation cultures </li></ul></ul><ul><ul><li>Quantitative pp65 antigenemia </li></ul></ul><ul><ul><li>Quantitative PCR </li></ul></ul><ul><ul><li>Branched-DNA (bDNA) signal amplification assay </li></ul></ul><ul><ul><li>Hybrid capture CMV DNA assay </li></ul></ul><ul><li>The pp65 antigenemia assay appears to be useful as well, especially for patients with polyradiculopathy </li></ul>
    12. 12. Diagnostic accuracy indexes Mengoli et al., 2003
    13. 13. HHV-6/-7 Infection of the CNS <ul><li>Virus Isolation and Assay </li></ul><ul><li>Serological Assays </li></ul><ul><li>Genomic Detection by PCR </li></ul><ul><ul><li>Numerous PCR primer sets available for HHV-6 </li></ul></ul><ul><ul><li>Reverse transcription–PCR (RT-PCR) assay - latent or replicating virus? </li></ul></ul><ul><ul><li>Quantitative PCR assay - persistence of a high HHV-6 load in the absence of apparent disease </li></ul></ul><ul><ul><li>Multiplex PCR method - simultaneous detection of HHV-6 and HHV-7 </li></ul></ul>CSF-PCR is the technique of choice for the diagnosis of the CNS infection Brain biopsy recommended to confirm diagnosis in conflicting cases
    14. 14. mannitol ampicillin, acyclovir doxycycline ceftizoxime, netilmicin EEG: diffuse irritation chest x-ray: lung consolidation CT: normal LP: bacterial / viral cultures, PCR CT: diffuse edema LP extubation EEG: fewer signs chest x-ray: normal CT: normal LP 1 2 3 4 5 6 12 Days 225 100 75 CSF cells/  l 39.0 38.5 38.0 37.5 37.0 M. pneumoniae : 1:5,120 °C HHV-6/7: DNA+ M. pneumoniae : DNA+, mRNA - HHV-6/7: mRNA - ( Sgarabotto D. et al, Scand J Infect Dis 2000, 32(6):689-92 ) A TWO PATHOGEN CASE OF MENINGOENCEPHALITIS
    15. 15. EBV Infection of the CNS <ul><li>EBV is rarely cultured from CSF during CNS infection </li></ul><ul><li>Quantitative PCR - EBV DNA copy numbers are significantly higher in patients with active EBV infection </li></ul><ul><li>Analysis by RT-PCR of specific viral mRNA </li></ul>Discrimination between lytic and latent infection is important
    16. 16. EBV DECAY, RAPID (EARLY) AND SLOW (LATE) COMPONENT t 1/2 early = 29.6 hr t 1/2 late = 111.6 hr ( Biasolo et al, JMedVirol. 2003)
    17. 17. HHV-8??? <ul><li>The high frequency of HHV-8 in AIDS-related primary CNS non-Hodgkin’s lymphoma in patients with Kaposi's sarcoma suggests that this virus could play a role in the pathogenesis of some cerebral lymphomas. </li></ul><ul><li>This finding needs to be more extensively studied </li></ul>
    18. 18. Conclusions <ul><li>Herpesvirus infections of CNS are a difficult diagnostic problem for both clinicians and microbiologists </li></ul><ul><li>As effective antiviral drugs are available, rapid and reliable diagnosis is mandatory </li></ul><ul><li>The isolation of the etiological agent is still important </li></ul><ul><li>The introduction of the non-invasive, rapid and specific CSF-PCR revolutionized the diagnosis of these infections </li></ul><ul><li>Due to the peculiar biological characteristics of the herpesvirus infections, quantitative PCR and discrimination between lytic and latent infection are in many cases essential for the diagnosis </li></ul>

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