DEVELOPMENT OF AN INTERNATIONAL STANDARD FOR HEPATITIS D VIRUS RNA - - PowerPoint PPT Presentation

DEVELOPMENT OF AN INTERNATIONAL STANDARD FOR HEPATITIS D VIRUS RNA

• AN UPDATE -

WHO Collaborating Centre for Quality Assurance of Blood Products and in vitro Diagnostic Devices

Michael Chudy Paul-Ehrlich-Institut Federal Institute for Vaccines and Biomedicines AREVIR-GenaFor Meeting 11-12 April 2013, Cologne

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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%
• 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)

Gudima et al., J Virol 81:3608-3617 (2007)

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

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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.

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Development of the 1st 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

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Development of the 1st 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 105 – 107 cps/mL (pre-analyzed) − All samples represent genotype HDV-1 (sequenced)

• Further extensive characterization (e. g. HDV RNA, HBV DNA,

HBsAg quant, HBe/antiHBe …)

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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 (log10 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)

*estimated by the RoboGene HDV RNA Quantification Kit

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Proposed WHO IS HDV RNA – stability testing

Accelerating degradation studies

*estimated by the RoboGene HDV RNA Quantification Kit

0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 wk 1 wk 2 wk 3 wk 4 wk 8 wk 12 6 mo

Ct values Time

Pre-Lyo (1:10)

• 20°C (1:10)

+4°C (1:10) +20°C (1:10) +37°C (1:10) Pre-Lyo (1:100)

• 20°C (1:100)

+4°C (1:100) +20°C (1:100) +37°C (1:100)

No evidence for degradation at temperatures up to +37°C for 6 months

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Collaborate study to evaluate the candidate material for 1st 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

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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)

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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

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WHO collaborative study – HDV NAT assays

Lab Code Assay Sample Prep Assay Type Target Region QS Sample Equival. (µl) Dilution factor 1 In-house TaqMan m2000sp quant

NTR upstream HD gene

cDNA 35.71 28 2 In-house TaqMan QIAamp Vrial RNA quant

Ribozyme region

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 6 In-house real-time m2000sp quant

within ribozymes Synthetic DNA

27.8 36 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 14 In-house real-time Manual Qiagen quant

between autocatalytic cleavage sites

cDNA 11.67 86 qual n.a. 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.; **AmpliSens, Russia.

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WHO collaborative study – Phase 1 (S1, S2, S3)

Lab Code Assay Type S1 S2 S3 Dilution factor Study Protocol Phase 2 1 quant 1,E-05 1,E-05 1,E-05 28 ED 2 quant 1,E-04 1,E-04 1,E-04 28.6 ED 3 quant 1,E-02 1,E-02 1,E-02 86 quant 4 quant 1,E-05 1,E-05 1,E-05 25 ED 5 quant 1,E-02 1,E-03 1,E-02 37.5 quant 6 quant 1,E-03 1,E-03 1,E-03 36 quant +ED qual 1,E-04 1,E-04 1,E-05 (ED)* 7 quant 1,E-05 1,E-05 1,E-05 50 ED 8 quant 1,E-04 1,E-04 1,E-04 60 ED 9 quant 1,E-03 1,E-03 1,E-04 50 quant 10 qual 1,E-04 1,E-05 1,E-05 14.3 ED 11 qual 1,E-05 1,E-03 1,E-05 25 ED 12 quant 1,E-05 1,E-06 1,E-05 20 ED 13 qual 1,E-03 1,E-03 1,E-03 12 ED 14 quant 1,E-01 1,E-02 1,E-02 86 quant qual 1,E-02 1,E-03 1,E-03 ─ 15 quant 1,E-04 1,E-04 1,E-04 50 ED

ED; end point dilution; *Proposal

Phase 2 11 data sets: ED protocol 5 data sets: quant protocol

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S1/S2 - linear relationship between log dilution and Ct values

10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 1,00E-03 1,00E-02 1,00E-01 1,00E+00

Mean Ct value S1 / S2 Dilution S1 / S2

Lab 1 Lab 2 Lab 3 Lab 4 Lab 5 Lab 6A Lab 7 Lab 8 Lab 9 Lab 10 Lab 11 Lab 12 Lab 13 Lab 14 Lab 15

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S1/2 - linear relationship - slope

Lab code Slope (-3.32) Slope (-3.32—x) 1

• 3.78

+0.46 2

• 3.54

+0.22 3

• 3.36

+0.04 4

• 3.92

+0.60 5

• 3.23
• 0.09

6A

• 4.03

+0.68 7

• 3.78

+0.46 8

• 2.86
• 0.46

9

• 3.54

+0.22 10 (qual)

• 2.10
• 1.22

11 (qual)

• 3.19
• 0.13

12

• 3.22
• 0.10

13 (qual)

• 3.79

+0.47 14

• 3.01
• 0.31

15

• 2.80
• 0.52

Similar results with S3

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WHO collaborative study

0,000 1,000 2,000 3,000 4,000 5,000 6,000 7,000 1 2 3 4 5 6 7 8 9 12 14 15

HDV RNA (log10 copies/ml) Lab Code No

Sample 4* / WHO Study Phase 1+2

Neat 1:10 (x10)

Mean

*Quantitation based on internal QS

Max-Min 2.4 log10

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WHO collaborative study – quant protocol

3,0 4,0 5,0 6,0 7,0 3 5 6 9 14

HDV RNA (log10 copies/ml) Lab Code No S1/S2 S3 S4

*Quantitation based on internal QS

S1/S2 mean 6.1 log10 / max-min 2.5 log10 S3 mean 6.2 log10 / max-min 2.6 log10 S4 mean 5.6 log10 / max-min 1.6 log10

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WHO collaborative study – all assays

relationship S1/S2 to S3 and S1/S2 to S4

• 5
• 4
• 3
• 2
• 1

1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Delta Ct values Lab Code No

S1/2 - S3 (neat) S1/2 - S3 (1:10) S1/2 - S4 (neat) S1/2 - S4 (1:10)

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WHO collaborate study – proposed outcome

• Proposed standard #7657/12 (S1/S2)
• Lyophilised material S1 = S2
• Comparable to frozen-liquid bulk material
• Commutable (S4; clinical specimen)
• Will have a concentration of 100,000 – 1,000,000 IU/ml
• Results from the ED protocol will evaluated by Probit analysis
• Combination of results from ED and quant protocol
• Standard is stabile over a long period

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WHO International Standard for HDV RNA – next steps

• Statistical analysis completed by end of April 2013
• Draft report for circulating by end of May 2013
• Submission of final report to WHO ECBS in July 2013
• (hopefully) Establishment by ECBS in October 2013
• Available from PEI (custodian)

Development of WHO Biological Reference Preparations

PEI Projects

Establishment IVD PEI-Code WHO BRP Marker ECBS 2009 NAT 5086/08 1st Ref Panel HBV genotype panel ECBS 2011 HBsAg 6100/09 1st Ref Panel HBV genotype panel ECBS 2011 NAT 1st IS HEV RNA Proposed end of 2013 NAT 7657/12 1st IS HDV RNA ECBS 2011 Bacterial detection methods PEI-B-06 1st Ref Repository Transfusion relevant bacterial strain panel

For more information: www.pei.de/who-reference-material

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− Mithat Bozdayi, Institute of Hepatology, Ankara University, Turkey − Colleagues from PEI

• Section of Molecular Virology
• PEI-IVD
• Section of Statistics
• Administration staff

Thanks to all cooperators in the projects, in particular to…

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Thank you for your attention!