DEVELOPMENT OF AN INTERNATIONAL STANDARD FOR HEPATITIS D VIRUS RNA - AN UPDATE - AREVIR-GenaFor Meeting 11-12 April 2013, Cologne Michael Chudy Paul-Ehrlich-Institut Federal Institute for Vaccines and Biomedicines WHO Collaborating Centre for Quality Assurance of Blood Products and in vitro Diagnostic Devices
Hepatitis D Virus (HDV) Member of the genus Deltavirus 36 nm defective viral particle Virion is encapsidated by HBsAg Circular ss (-) RNA genome of ~ 1.7 kb which is encased in 60 molecules HDAg 8 major clades (HDV-1 to HDV-8) Genetic variability ranges from 20 to 35% Gudima et al., J Virol 81:3608-3617 (2007) Transmitted via infected blood or blood products; sex contact Individuals at risk are HBV carriers, IDUs, hemodialysis patients Worldwide ~ 5% of HBV carriers are anti-HDV positive (10 - 15 million people) Mortality rate lies between 2 and 20% (ten times higher than for HBV) 1
Global Epidemiology of HDV Infection Wedemeyer & Manns, Nature Reviews, Gastroenterol & Hepatol 7, 2010.
Clinical Utility of HDV RNA Quantification Serological tests often lacks on sensitivity The most sensitive method is NAT* Identify individuals with active HDV infection Decide to initiate treatment Monitor antiviral treatment efficacy _______________________________________________________________ *Nucleic acid amplification technique
HDV NAT and Standardization Despite the high sequence diversity, primers and probe of NAT assays has to be selected from highly conserved regions to cover all 8 HDV clades Majority of NAT assays are in-house developed No standardization No proficiency testing program in place HDV RNA quantification currently is unreliable HDV RNA standard is particularly important for… − assay comparison − development and calibration of diagnostic assays − calibration of secondary references and working standards − evaluation of standardized preparations used in quality control and quality assurance
HDV NAT – Calibration / Quantitation HDV cDNA or plasmid Synthetic HDV RNA (transcripts) Armored RNA* No reference method for quantification HDV RNA Reference Preparation (whole virus in human plasma) IU (arbitrary unitage ) ≠ copies IU vs copies (assay related conversion factor) _______________________________________________________________ *Armored RNA, complex of MS2 bacteriophage coat protein and RNA; RNA sequences are completely protected from RNase digestion; developed by former Ambion company, now Asuragen. 5
Development of the 1 st International Standard for HDV RNA (1) Adoption of proposal by WHO ECBS* in October 2009 EASL** Monothematic Conference on Delta Hepatitis (September 24 – 26, 2010, Istanbul, Turkey) Standardized HDV RNA testing (IS) - HDV RNA quantification is a crucial tool to diagnose, treat and manage HDV infections _______________________________________________________________ *Expert Committee on Biological Standardization **European Association for the Study of the Liver 6
Development of the 1 st International Standard for HDV RNA (2) Collaboration − Institute of Hepatology, Ankara University, Turkey (Prof. Bozdayi) Seven HDV-positive plasma samples are available − Volume 250 – 300 mL − Viral load 10 5 – 10 7 cps/mL (pre-analyzed) − All samples represent genotype HDV-1 (sequenced) Further extensive characterization (e. g. HDV RNA, HBV DNA, HBsAg quant, HBe/antiHBe …) 7
Preparation of the HDV RNA standard Material N6357 (HDV-positive plasma, clinical specimen, gt 1) dilution of 1:50 in negative plasma pool Filling and freeze-drying − Preparation of 4,010 vials − 5 mL screw-cap glass vial − Filling volume 500 µl (SD ±3.9 µl / CV 0.8%) − PEI code 7657/12 HDV RNA concentration (log 10 copies/ml)* − Pre-Lyo 7.03 (SD±0.07) − Post-Lyo 6.75 (SD±0.09) Residual moisture content: Requirement <1% − 0.89% (SD±0.07) 8 * estimated by the RoboGene HDV RNA Quantification Kit
Proposed WHO IS HDV RNA – stability testing Accelerating degradation studies 35,00 30,00 Pre-Lyo (1:10) 25,00 -20°C (1:10) Ct values +4°C (1:10) 20,00 +20°C (1:10) +37°C (1:10) 15,00 No evidence for degradation at Pre-Lyo (1:100) temperatures up to +37°C for 6 months -20°C (1:100) 10,00 +4°C (1:100) +20°C (1:100) 5,00 +37°C (1:100) 0,00 wk 1 wk 2 wk 3 wk 4 wk 8 wk 12 6 mo Time * estimated by the RoboGene HDV RNA Quantification Kit 9
Collaborate study to evaluate the candidate material for 1 st International Standard for HDV RNA A total of 20 laboratories in 10 countries have been invited 19 laboratories agreed to participate 17 laboratories sent results from Phase 1 15 laboratories from 9 countries sent complete results (Phase 1 and 2) • Europe Belgium, France, Germany (4), Italy (2), UK (3) • Eurasia Russia, Turkey • North America USA • Australia Australia 10
WHO collaborate study – study design 4 study samples S1 = S2, HDV gt 1, candidate material; freeze dried preparation S3; HDV gt 1, frozen liquid bulk S4, HDV gt 1 pos plasma, clinical specimen, frozen liquid material Study protocol Phase 1; S1–S3, one run with 10-fold dilutions; S4, undiluted; report results as +/- or copies/ml and Ct values (all qual and quant assays), Phase 2; proposal based on results of Phase 1, End-point dilution (ED): minimum of 5 dilutions around the assay end-point for S1-S3, S4 undiluted and at least one further 10-fold dilution, 3 separate occasions, report results as +/- and Ct values (selected qual and quant assays) Quantitation (quant): minimum of 2 dilutions of S1-S4, S4 should start with undiluted testing, 3 separate occasions, report results in copies/mL and Ct values (selected quant assays) 11
WHO collaborative study – participants* Scientist Affiliation Bowden S VIDRL, Victoria, Australia Bozdayi M Dept. of Gastroenterology, Ankara University, Turkey Chudy, M Dept. of Virology, Paul-Ehrlich-Institut, Langen, Germany Chulanov V Reference Center for Viral Hepatitis, Moscow, Russia Garson J Clin Microbiol. & Virology, UCLH NHS Foundation Trust, London, UK Gordien E Lab. de Virologie, Hopital Avicenne, Laboratoire associé au Centre National de Référence des Hépatites B, C et delta, Université Paris, Bobigny, France Luciani, F Instituto Superiore di Sanita, Rome, Italy Miller B MVZ Labor Prof. Seelig GbR, Karlsruhe, Germany Mixson-Hayden T Division of Viral Hepatitis, Centers for Disease Control and Prevention, Atlanta, GA, USA Olivero A Dept. of Internal Medicine, Hospital-University St. Giovanni Battista, Torino, Italy Padalko E Clinical Virology, University, Ghent, Belgium Protzer U Institute of Virology, TU Munich, Munich, Germany Tettmar K Blood Borne Virus Unit, Health Protection Agency, London, UK Tilston P Dept. of Clinical Virology, Manchester Royal Infirmary, Manchester, UK Von Witzendorff D Dept. of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany *In alphabetic order 12
WHO collaborative study – HDV NAT assays Sample Lab Assay Target Dilution Code Assay Sample Prep QS Equival. Type factor Region (µl) NTR upstream 1 In-house TaqMan m2000sp quant cDNA 35.71 28 HD gene Ribozyme region 2 In-house TaqMan QIAamp Vrial RNA quant RNA transcript 35 28.6 3 In-house real-time easyMAG quant HD gene RNA transcript 11.67 86 4 In-house real-time Manual GuSCN quant HD gene RNA transcript 40 25 5 In-house TaqMan Cobas AmpliPrep quant HD gene plasmid 26.67 37.5 quant within ribozymes Synthetic DNA 27.8 36 6 In-house real-time m2000sp qual HD gene n.a. 7 In-house real-time EZ1 Advanced quant HD gene cDNA 20 50 8 commercial real-time* Manual Instant quant HD gene RNA transcript 16,67 60 9 In-house real-time MagnaPure quant HD gene RNA transcript 20 50 10 In-house TaqMan QIAamp MiniElute qual HD gene n.a. 70 14.3 11 In-house TaqMan MagnaPure qual HD gene n.a. 40 25 12 commercial real-time** RIBO-prep manual quant HD gene Amored RNA 50 20 13 In-house TaqMan Automat. Qiagen qual HD gene n.a. 83.33 12 between quant cDNA 11.67 86 14 In-house real-time Manual Qiagen autocatalytic qual n.a. cleavage sites 15 In-house real-time HPS Viral RNA quant HD gene Amored RNA 20 50 ED; end point dilution; n.a.; not available; *RoboGene HDV RNA Quantification Kit, aj Roboscreen, Germany, quant mod.; 13 **AmpliSens, Russia.
WHO collaborative study – Phase 1 (S1, S2, S3) Assay Dilution Study Protocol Lab Code S1 S2 S3 Type factor Phase 2 1 quant 28 ED 1,E-05 1,E-05 1,E-05 2 quant 28.6 ED 1,E-04 1,E-04 1,E-04 3 quant 86 quant 1,E-02 1,E-02 1,E-02 4 quant 25 ED 1,E-05 1,E-05 1,E-05 5 quant 37.5 quant 1,E-02 1,E-03 1,E-02 quant +ED quant 36 1,E-03 1,E-03 1,E-03 6 ( ED )* qual 1,E-04 1,E-04 1,E-05 7 quant 50 ED 1,E-05 1,E-05 1,E-05 8 quant 60 ED 1,E-04 1,E-04 1,E-04 9 quant 50 quant 1,E-03 1,E-03 1,E-04 10 qual 14.3 ED 1,E-04 1,E-05 1,E-05 11 qual 25 ED 1,E-05 1,E-03 1,E-05 Phase 2 12 quant 20 ED 1,E-05 1,E-06 1,E-05 13 qual 12 ED 1,E-03 1,E-03 1,E-03 11 data sets: ED protocol quant 86 quant 1,E-01 1,E-02 1,E-02 14 ─ qual 1,E-02 1,E-03 1,E-03 5 data sets: quant protocol 15 quant 50 ED 1,E-04 1,E-04 1,E-04 ED; end point dilution; *Proposal 14
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