National Institute for Biological Standards and Control Assuring the quality of biological medicines Human Cytomegalovirus (HCMV) Proposed 1 st International Standard WHO/BS/ 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: Log 10 variation in reported results (relative to expected) 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 ~1x10 7 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 Control Assuring the quality of biological medicines Epstein-Barr Virus (EBV) Proposed 1 st International Standard WHO/BS/

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 2 Log 10 variation in reported results (relative to expected) Variability in performance of EBV viral load assays Preiksaitis et al, Am J Transplant. 2009;9: 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 ~1x10 7 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