Laboratory diagnosis of herpesvirus infections

Laboratory diagnosis of herpesvirus infections of the CNS Giorgio Palù, MD Padova University, Italy Herpesvirus Infections of the CNS Virus • HSV-1 & 2 Clinical diagnosis Encephalitis, meningitis, Mollaret’s (benign recurrent lymphocytic) meningitis, neonatal meningoencephalitis and disseminated disease Zoster sine herpete, aseptic meningitis, encephalitis, transverse myelitis, CNS vasculitis, cerebellitis Encephalitis, polymyeloradiculitis, ventriculitis, myelitis, inflammatory polyneuropathy (predominantly in AIDS/HIV), congenital CMV Meningoencephalitis, recurrent febrile seizures of childhood, possible association with multiple sclerosis Meningoencephalitis, acute cerebellar ataxia, aseptic meningitis, transverse myelitis, autonomic neuropathy, primary CNS lymphoma in AIDS ??? • VZV • CMV • HHV-6 & 7 • EBV • HHV-8 Diagnosis of CNS Infection • Standard neurodiagnostic procedures include: – CSF examination – EEG – scanning • These can be normal in early stages of the disease  Other diagnostic evaluations should be initiated immediately Role of PCR of CSF • PCR is the standard method of laboratory diagnosis for many viral CNS infections • CSF PCR testing may antedate clinically recognizable disease • Quantitative CSF-PCR may also be useful for monitoring therapy. • Must be performed by a reliable laboratory Sensitivity and Specificity of PCR Virus HSV-1 and 2 Sensitivity and specificity >95% sensitivity and specificity; quantitative PCR available; potential use in determining course of iv therapy (especially in neonatal disease) Sensitivity and specificity >95% Sensitivity nearly 100% in immunosuppressed patients with neurological symptoms; can be quantitated (range:10–104 copies/ml); possible use to monitor therapy. Positive results in 60% of affected infants; correlates with poor neurological outcome Excellent sensitivity, but poor positive predictive value in clinical disease (30–40% of asymptomatic controls positive) 98.5% sensitive and 100% specific as a tumour marker VZV CMV HHV-6 EBV HSV-1/-2 Infection of the CNS • Serological procedures performed on serum or CSF are not helpful early in the disease course when therapeutic decisions are needed • Detection of viral CSF-PCR is the diagnostic method of choice for confirmation of HSV involvement in CNS disease • The use of CSF-PCR instead of brain biopsy has expanded awareness of mild or atypical cases (16%-25%) Positive predictive values at different anti-HSV-2 prevalence in the population Positive Predictive Value 100% 80% 60% 40% 20% 0% 0% 10% 20% 30% (Palù et al. Scand.J.Infect.Dis. 2001) Gull MRL POCkit anti-HSV-2 prevalence VZV Infection of the CNS Serum anti-VZV antibody is of no value since VZV antibodies persist in the serum of nearly all adults BUT • Testing of CSF for VZV antibodies helps to confirm the role of VZV in producing clinical syndromes of the CNS. • 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 Clinicians should request both PCR and antibody analysis CMV Infection of the CNS Diagnosis of CMV-related CNS disease is based upon clinical presentation, neuroradiological studies, CSF chemistries, serological testing, and culture and PCR of CSF • Clinical presentations of CMV-related CNS disease can be nonspecific • CSF viral culture can be insensitive • Qualitative DNA PCR can detect both latent and replicating virus RT- PCR for specific viral transcripts and quantitative PCR are useful Measuring HCMV viral load • High systemic CMV load is generally correlated with CMV disease • Measuring the viral load at specific sites may help diagnosis when systemic viral load correlates poorly with disease activity • Quantitation of DNA in both CSF and brain tissue sensitively diagnoses and monitors antiviral treatment, e.g. – AIDS patients with HCMV-related CNS disease have high quantities of HCMV DNA in their CSF – Copies of HCMV DNA in CSF are higher in persons with HCMV-related polyradiculopathy than encephalitis • More data are required on the correlation between changes in viral load, development of resistance, and clinical outcome HCMV quantitation (methods) • 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) • Methods available: – Quantitative viral cultures: plaque assay, determination of TCID50, shell vial centrifugation cultures – Quantitative pp65 antigenemia – Quantitative PCR – Branched-DNA (bDNA) signal amplification assay – Hybrid capture CMV DNA assay • The pp65 antigenemia assay appears to be useful as well, especially for patients with polyradiculopathy Diagnostic accuracy indexes Gold standard: real-time PCR concordance kappa pp65 antigen 0.72 0.45 pp67 RNA 0.41 0.11 sensitivity 0.65 0.18 specificity 0.91 1.00 OR 19.50 10.92 P 0.0000 0.0137 Gold standard: pp65 concordance real-time PCR 0.72 pp67 RNA 0.57 kappa 0.45 0.12 sensitivity 0.95 0.20 specificity 0.50 0.93 OR 19.50 3.15 P 0.0000 0.0483 Mengoli et al., 2003 HHV-6/-7 Infection of the CNS • Virus Isolation and Assay • Serological Assays • Genomic Detection by PCR – Numerous PCR primer sets available for HHV-6 – Reverse transcription–PCR (RT-PCR) assay - latent or replicating virus? – Quantitative PCR assay - persistence of a high HHV-6 load in the absence of apparent disease – Multiplex PCR method - simultaneous detection of HHV-6 and HHV-7 CSF-PCR is the technique of choice for the diagnosis of the CNS infection Brain biopsy recommended to confirm diagnosis in conflicting cases 39.0 38.5 38.0 37.5 37.0 225 CSF cells/l EEG: diffuse irritation chest x-ray: lung consolidation CT: normal LP: bacterial / viral cultures, PCR extubation EEG: fewer signs chest x-ray: normal CT: normal LP CT: diffuse edema LP HHV-6/7: DNA+ M. pneumoniae: DNA+, mRNA HHV-6/7: mRNA M. pneumoniae: 1:5,120 100 75 ceftizoxime, (Sgarabotto D. et al, Scand J Infect Dis 2000, 32(6):689-92) netilmicin mannitol ampicillin, acyclovir doxycycline 1 2 3 4 5 6 12 Days A TWO PATHOGEN CASE OF MENINGOENCEPHALITIS °C EBV Infection of the CNS • EBV is rarely cultured from CSF during CNS infection Discrimination between lytic and latent infection is important • Quantitative PCR - EBV DNA copy numbers are significantly higher in patients with active EBV infection • Analysis by RT-PCR of specific viral mRNA EBV DECAY, RAPID (EARLY) AND SLOW (LATE) COMPONENT EBV titer/100,000 PBMCs, natural logarithm 14 12 10 8 6 4 2 0 t1/2 early = 29.6 hr t1/2 late = 111.6 hr 0 20 40 tim e (days) 60 80 (Biasolo et al, JMedVirol. 2003) HHV-8??? • 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. • This finding needs to be more extensively studied Conclusions • Herpesvirus infections of CNS are a difficult diagnostic problem for both clinicians and microbiologists • As effective antiviral drugs are available, rapid and reliable diagnosis is mandatory • The isolation of the etiological agent is still important • The introduction of the non-invasive, rapid and specific CSF-PCR revolutionized the diagnosis of these infections • 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