Progress in the Diagnosis of CMV

1
Progress in the Diagnosis of CMV

• V Emery (UK)

2
Progress in the diagnosis of CMV

• Professor Vincent Emery
• Royal Free and University College Medical School
• London

3
Overview of presentation

• CMV as a pathogen
• Importance of suppression of CMV replication in
  reducing disease
• Improved diagnostics can facilitate
• Understanding of replication dynamics
• Rapid deployment of pre-emptive therapy to reduce
  disease
• Understanding of factors affecting response to
  therapy

4
Direct and indirect effects of CMV

• In the immunocompetent
• Virus in harmony with the host
• In the immunocompromised
• Virus has the upper hand
• Active CMV infection associated with
• Direct effects such as fever, hepatitis,
  pneumonitis, gastrointestinal disease
• Indirect effects such as organ rejection,
  coronary artery disease

Rubin RH et al. JAMA 1989 2613607-9. Rubin RH.
Clinical approaches to infection in the
compromised host. 2000. pp. 573-9.
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CMV diseases in the immunocompromised
Tx transplant
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CMV load, dynamics and disease

• CMV replicates rapidly in vivo
• Doubling time of 1 day
• Several studies in patient groups at risk of CMV
  disease have shown
• Viral loads are highest in patients with symptoms
  
• Patients with high viral loads
• need a longer duration of antiviral therapy
• are more likely to have a second episode of
  viraemia

Emery et al (1999) J Exp Med 190177-82 Sia et
al (2000) J Infect Dis 181717-20 Humar et al
(2002) J Infect Dis 186829-33
Razonable et al
(2003) J Infect Dis 1871801-8
7
Antiviral therapy for CMV

• Product UL97 kinase activation
• Ganciclovir (GCV) YESValganciclovir
• Aciclovir YESValaciclovir
• Foscarnet NO
• Cidofovir NO

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Suppression of CMV reduces disease probability
1.0
0.8
0.6
Probability of disease
0.4
0.2
0.0
3
4
5
6
7
Log viral load (genomes/ml)
Cope et al (1997) J Infect Dis 176 1484-90
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Therapeutic approaches to control CMV replication

• Prophylaxis
• Universal or targeted
• Eliminates direct and indirect effects of CMV
• Subset of patients remain at risk of late CMV
  infection/disease after cessation of prophylaxis

• Pre-emptive therapy
• Targets individuals based on their virologic
  markers
• Minimises drug exposure
• Patients may require more than one treatment
• May not eliminate the indirect effects of CMV

Forum debate (2001) Rev Med Virol 1173-86
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Laboratory monitoring of CMV

• Qualitative presence of virus
• Rapid molecular methods
• PCR, NASBA
• allows identification of patients at risk of
  disease
• Semi-quantitative measures of virus
• Rapid methods such as antigenaemia
• allows identification of patients at risk of
  disease

PCR Polymerase chain reaction NASBA Nucleic
acid sequence-based amplification
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Quantitative assessment of CMV load

• In-house assays
• QC PCR systems
• Real time PCR assays
• Commercial assays
• Roche COBAS Amplicor monitor
• Murex hybrid capture version 2
• Real time assays

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Which samples can be used for diagnosis?

• Plasma, whole blood, PBL, PBMC
• All samples can provide prognostic information
• All samples can be used to initiate pre-emptive
  therapy
• All samples can be used to assess response to
  therapy
• PCR of other clinical samples
• Gastrointestinal disease
• Hepatitis
• Pneumonitis

PBMC Peripheral blood mononuclear cells PBL
Peripheral blood leukocytes
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Comparison of blood compartments

• Automated quantitative assay used on whole blood,
  plasma, PBL and PBMC
• CMV load in whole blood 0.67 log higher than
  plasma (p0.0009)
• CMV load in PBL and PBMC comparable
• Higher quantity of CMV DNA in whole blood offers
  advantages for diagnosis

Razonable et al (2002) Transplantation 73968-73
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Trials of PCR pre-emptive therapy in bone marrow
transplant recipients

• Patients randomised to PCR or cell culture
  surveillance
• Initiate treatment based on
• 2 consecutive PCR of blood
• 1 culture, any site
• treat until PCR-negative
• Median day treatment initiated
• 44 days (PCR) vs 54 days (cell culture)
• Total GCV treatment shorter
• Duration of neutropenia reduced
• 1 day (PCR) vs 5 days (cell culture)

Significant difference

Einsele et al (1995) Blood 862815
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Oral GCV pre-emptive therapysolid organ
recipients

• Randomised, double-blind, placebo-controlled
  study of pre-emptive oral GCV (8 weeks) in liver
  transplant recipients
• Treatment initiated on a positive PCR result in
  PBL
• In all patients, CMV disease incidence
• 12 placebo vs 0 GCV (p0.01)
• In DR- patients incidence of shell vial
  viraemia
• 55 placebo vs 11 GCV (plt0.01)
• Oral GCV was less able to control replication in
  patients with high CMV loads

Paya et al (2002) J Infect Dis 185854-60
Razonable et al (2003) J Infect Dis 1871801-8
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Effective pre-emptive monitoring of CMV
CMV load
Time
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Viral load kinetics and disease risk

• Early markers of replication are associated with
  progression to high level viraemia and disease
• Viral load in early samples
• Rate of increase in viral load
• Cut-off values for initiating pre-emptive therapy
  available but difficult to compare between studies

Emery et al (2000) Lancet 3552032-36 Norris et
al (2002) Transplantation 74 527-31 Razonable
et al (2003) J Infect Dis 1871801-8 Mattes et al
(2004) J Infect Dis in press
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Factors associated with therapeutic response of
CMV

• Study of replication kinetics prior to and post
  therapy in 48 transplant recipients randomised to
  receive iv GCV or half dose GCV half dose
  foscarnet
• Frequent samples available (minimum 2/week)
• No difference in decay rate between study groups
• Combination not superior
• Viral doubling time, viral decay rate and viral
  load at initiation of therapy investigated as
  factors affecting response time
• Primary end-point was CMV PCR negative by day 14

Mattes et al (2004) J Infect Dis. in press
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Univariable risk factors for CMV response to
therapy

• CI95 Odds for oddsRisk factors ratio ratio S
  ignificance
• Viral load1(per log10 higher) 2.39 1.055.44 0.03
  8
• Doubling time(per day decrease) 2.95 1286.82 0.0
  1
• Half life of decline(per day increase) 3.01 1.45
  6.25 0.003

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Risk factors for recurrence after treatment

• GCV therapy of 52 SOT patients with CMV disease
• Recurrent disease occurred in 12 patients
• Risk factors for recurrent disease were
• Time to clear CMV infection (33 vs 17 days
  p0.002)
• Half life of decline in virus load (8.8 vs 3.2
  days p0.001)
• CMV load kinetics useful in identifying patients
  who are more likely to have a recurrence of
  disease

Humar et al (2002) J Infect Dis 186829-33
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Conclusions

• Continued improvements in CMV diagnosis have
  occurred in the past 3 years
• Fully quantitative diagnostic methods becoming
  widely used
• The ability to accurately monitor and quantify
  CMV in a timely way has enabled
• A greater understanding of CMV replication
  kinetics
• Refinement of treatment algorithms
• Minimisation of CMV disease through targeted use
  of antivirals

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Amendments to IHMF guidelines

• Diagnostic testing
• CMV DNA levels in whole blood are significantly
  higher than those present in plasma, so whole
  blood should become the sample of choice
  (Category 1 recommendation)
• An international quantitation standard is
  required to compare studies using different
  PCR-based systems and facilitate patient
  management at multiple care centres (Research
  need)

• Pre-emptive therapy (SOT)
• Pre-emptive therapy with oral ganciclovir (1g tid
  for 8 weeks), upon the detection of CMV DNA,
  reduces the incidence of CMV disease and viraemia
  in liver transplant patients (Category 1
  recommendation)

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Acknowledgements

• Virology, UCL Hampstead site
• Prof Paul Griffiths
• Dr Aycan Hassan-Walker
• Dr Lea Cope
• Dr Dee Gor
• Dr E Frances Bowen
• Dr Frank Mattes
• Dr A-M Geretti

• Primary Care and Population SciencesDr Caroline
  Sabin
• Liver transplantationProf Andrew Burroughs
• Renal TransplantationDr Paul Sweny
• Bone Marrow TransplantationProf H Grant
  PrenticeDr Mike Potter

Wellcome Trust UK Medical Research Council
National Institutes of Health