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Human Cytomegalovirus (HCMV)
Proposed 1st International Standard
WHO/BS/08.2099
Jacqueline Fryer
National Institute for Biological Standards and Control
Assuring the quality of biological medicines
Rationale 1
• Ubiquitous and persistent infection, causes disease in
immunologically naïve (foetus and newborns) and suppressed
(transplant recipients, AIDS patients).
• Leading infectious cause of deafness and brain damage in newborns,
most significant viral pathogen after solid-organ transplantation.
• High viral load is most important risk factor for CMV disease in
transplant recipients; HCMV DNA quantification assays are used to
guide pre-emptive antiviral therapy to prevent viral load rising above
critical disease threshold.
• Viral load measurements increasingly being used to predict
sensorineural hearing loss congenitally-infected infants.
Rationale 2
• Viral load measurements performed using NAT, particularly real-time
PCR. Many assays developed in-house, although a number of new
commercial assays have been developed.
• High level of inter-laboratory variability in viral load measurements
(AST/CST study, EQA proficiency programmes).
• Cut-off thresholds for initiation of pre-emptive therapy are site-specific
and vary significantly, therefore, difficult to compare clinical practice
and standardise patient management.
• IHMF* recommendations (2004) called for ‘international quantification
standard to compare studies using different PCR-based systems and
facilitate patient management at multiple care centres’.
* International Herpes Management Forum
Rationale 3
Variability in performance of HCMV viral load assays
Log10 variation in reported results
(relative to expected)
Plasma spiked with HCMV Merlin
Pang et al, Am J Transplant. 2009;9:258-68
Clinical samples
Source material for HCMV candidate
• Whole virus preparation of prototype clinical HCMV
strain Merlin
• Produced in cell culture, formulated in universal buffer
and freeze dried
• Concentration of ~1x107 copies/mL (IU when
established)
• ~5000 vials to be filled (August 2009)
Collaborative study protocol
• Candidate standard to be evaluated alongside frozen
liquid preparations:
– Merlin liquid bulk
– Prototype laboratory HCMV strain AD169 (whole
virus)
– Purified BAC-cloned Merlin DNA
• ~30 participants (clinical and research labs, assay
manufacturers) performing range of NAT-based assays
• To ECBS 2010
Intended use
• Calibration of secondary references used in routine
HCMV viral load assays
• Calibration/validation of commercial NAT assays
• Evaluation of HCMV-positive materials distributed in
molecular quality assurance programmes
Epstein-Barr Virus (EBV)
Proposed 1st International Standard
WHO/BS/08.2099
National Institute for Biological Standards and Control
Assuring the quality of biological medicines
Rationale 1
• EBV-associated Post Transplant Lymphoproliferative Disease
(PTLD) affects 1-20% of allografts.
• Viral load measurements by NAT used to guide pre-emptive
therapy in transplant recipients.
• High level of inter-laboratory variability in viral load measurements
(AST/CST study, EQA proficiency programmes).
• Cut-off thresholds for initiation of pre-emptive therapy are sitespecific and vary significantly, therefore, difficult to compare clinical
practice and develop standardised treatment models.
• EBV Viral Load Standardisation Workshop (Third European
Congress of Virology, Nürnberg, 2007) called for the development
of an International Standard for EBV DNA.
Rationale 2
Variability in performance of EBV viral load assays
Clinical samples
Log10 variation in reported results
(relative to expected)
Plasma spiked with Namalwa cells
Preiksaitis et al, Am J Transplant. 2009;9:269-79
Source material for EBV candidate
• Whole virus preparation of prototype laboratory EBV strain
B95-8
• Produced in cell culture, formulated in universal buffer and
freeze dried
• Concentration of ~1x107 copies/mL (IU when established)
• ~5000 vials to be filled (August 2009)
Collaborative study protocol
• Candidate standard to be evaluated alongside frozen liquid
preparations:
– B95-8 liquid bulk
– EBV-positive Namalwa cells
– EBV-positive Raji cells
• ~30 participants (clinical and research labs, assay
manufacturers) performing range of NAT-based assays
• To ECBS 2010
Intended use
• Calibration of secondary references used in routine EBV
viral load assays
• Calibration/validation of commercial NAT assays
• Evaluation of EBV-positive materials distributed in
molecular quality assurance programmes