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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%)
0%
20%
40%
60%
80%
100%
0% 10% 20% 30%
anti-HSV-2 prevalence
Po
siti
ve P
red
icti
ve V
alu
e
Gull
MRL
POCkit
(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
225
100
75
CS
F c
ells
/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 T
WO
PA
TH
OG
EN
C
AS
E O
F
MEN
ING
OEN
CEP
HA
LIT
IS
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
0
2
4
6
8
10
12
14
0 20 40 60 80
time (days)
EB
V t
iter
/100
,000
PB
MC
s,
nat
ura
l lo
gar
ith
m
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