Laboratory diagnosis of herpesvirus infections of the CNS

Giorgio Palù, MD Padova University, Italy

Herpesvirus Infections of the CNSVirus Clinical diagnosis

• HSV-1 & 2 Encephalitis, meningitis, Mollaret’s (benign recurrent lymphocytic) meningitis, neonatal

meningoencephalitis and disseminated disease

• VZV Zoster sine herpete, aseptic meningitis, encephalitis, transverse myelitis, CNS vasculitis, cerebellitis

• CMV Encephalitis, polymyeloradiculitis, ventriculitis, myelitis, inflammatory polyneuropathy (predominantly in

AIDS/HIV), congenital CMV

• HHV-6 & 7 Meningoencephalitis, recurrent febrile seizures of childhood, possible association with multiple

sclerosis

• EBV Meningoencephalitis, acute cerebellar ataxia, asepticmeningitis, transverse myelitis, autonomic neuropathy,

primary CNS lymphoma in AIDS

• 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 Sensitivity and specificity

HSV-1 and 2 >95% sensitivity and specificity; quantitative PCR available; potential use in determining course of iv therapy (especially in neonatal disease)

VZV Sensitivity and specificity >95%

CMV 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

HHV-6 Excellent sensitivity, but poor positive predictive value in clinical disease (30–40% of asymptomatic controls positive)

EBV 98.5% sensitive and 100% specific as a tumour marker

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%)

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anti-HSV-2 prevalence

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(Palù et al. Scand.J.Infect.Dis. 2001)

Positive predictive values at different anti-HSV-2 prevalence in the

population

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 PCRconcordance kappa sensitivity specificity OR P

pp65 antigen 0.72 0.45 0.65 0.91 19.50 0.0000pp67 RNA 0.41 0.11 0.18 1.00 10.92 0.0137

Gold standard: pp65concordance kappa sensitivity specificity OR P

real-time PCR 0.72 0.45 0.95 0.50 19.50 0.0000pp67 RNA 0.57 0.12 0.20 0.93 3.15 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-7CSF-PCR is the technique of choice for the diagnosis of the CNS infection

Brain biopsy recommended to confirm diagnosis in conflicting cases

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

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39.0 38.5 38.0 37.5 37.0

M. pneumoniae: 1:5,120

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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)

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EBV Infection of the CNS

• EBV is rarely cultured from CSF during CNS infection

• Quantitative PCR - EBV DNA copy numbers are significantly higher in patients with active EBV infection

• Analysis by RT-PCR of specific viral mRNA

Discrimination between lytic and latent infection is important

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EBV DECAY, RAPID (EARLY) AND SLOW (LATE) COMPONENT

t1/2 early = 29.6 hr

t1/2 late = 111.6 hr

(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