Laboratory diagnosis of herpesvirus infections of the central nervous system

1
Laboratory diagnosis of herpesvirus infections of
the CNS
Giorgio Palù, MD Padova
University, Italy
2
Herpesvirus Infections of the CNS

• Virus Clinical diagnosis
• HSV-1 2 Encephalitis, meningitis, Mollarets
  (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, aseptic meningitis, transverse
  myelitis, autonomic neuropathy, primary CNS
  lymphoma in AIDS
• HHV-8 ???

3
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

4
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

5
Sensitivity and Specificity of PCR
6
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)

7
Positive predictive values at different
anti-HSV-2 prevalence in the population
100
80
Gull
60
MRL
Positive Predictive Value
POCkit
40
20
0
0
10
20
30
anti-HSV-2 prevalence
(Palù et al. Scand.J.Infect.Dis. 2001)
8
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

9
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

10
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

11
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

12
Diagnostic accuracy indexes
Mengoli et al., 2003
13
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 transcriptionPCR (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
14
C
39.0 38.5 38.0 37.5 37.0
225 100 75
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

CSF cells/?l
M. pneumoniae DNA, mRNA -
HHV-6/7 mRNA -
CT diffuse edema LP
A TWO PATHOGEN CASE OF MENINGOENCEPHALITIS
HHV-6/7 DNA
M. pneumoniae 15,120
ceftizoxime, netilmicin
(Sgarabotto D. et al, Scand J Infect Dis 2000,
32(6)689-92)
mannitol
ampicillin, acyclovir
doxycycline
Days
1 2 3 4 5
6 12
15
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
16
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)
17
HHV-8???

• The high frequency of HHV-8 in AIDS-related
  primary CNS non-Hodgkins 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

18
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