National Institute for Biological Standards and ControlAssuring the quality of biological medicines

Human Cytomegalovirus (HCMV) Proposed 1st International Standard

WHO/BS/08.2099

Jacqueline Fryer

• 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 1

• 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 2

Rationale 3

Pang et al, Am J Transplant. 2009;9:258-68

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Variability in performance of HCMV viral load assays

Plasma spiked with HCMV Merlin 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

National Institute for Biological Standards and ControlAssuring the quality of biological medicines

Epstein-Barr Virus (EBV) Proposed 1st International Standard

WHO/BS/08.2099

• 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 site-specific 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 1

Rationale 2Lo

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Variability in performance of EBV viral load assays

Preiksaitis et al, Am J Transplant. 2009;9:269-79

Plasma spiked with Namalwa cells Clinical samples

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