www.pei.de

Progress with HBV Genotype Panels

SoGAT XXII

14th-15th April 2011, Rome, Italy

M. Chudy, Division of Virology,

Paul-Ehrlich-Institut, Langen, Germany

Virology Division

Worldwide Distribution of HBV Genotypes

Virology Division

Background

Current WHO ISs for HBsAg or HBV-DNA represent HBV

genotype A2

HBV gt A2 in <1% of HBV-infected persons globally

Commutability of gt A2 for other HBV genotypes?

Virology Division

Background

WHO Consultation on Global Measurement Standards and their

use in the in vitro Diagnostic Field (June 2004)

“HBsAg and HBV NAT test kits might be less sensitive for some

HBV genotypes other than A2…”

ECBS 2005

Endorsement of PEI proposal to establish WHO International

Reference Panels representing different HBV genotypes /

HBsAg subtypes

These projects were assigned high priority by ECBS

Virology Division

Design of HBV Genotype Panels

Collection of plasma units from different regions

- HBV DNA/HBsAg high titre plasma samples with sufficient volume

Characterization of 215 potential candidate materials

- quant HBV DNA, quant HBsAg

- Sequencing of entire S ORF

- Analysis for genotype, escape mutations; HBsAg subtyping; HBV-

markers

- HIV-1 RNA, HCV-RNA

HBV genotypes A – G (H)

- One genotype H sample received at late stage, only for HBsAg

Virology Division

Design of HBV Genotype Panels

Project (part 1) ECBS 2009

HBV genotype panel for HBV-DNA tests 15 panel members

Target concentrations: 106 IU HBV-DNA/ml (12)

105 IU HBV-DNA/ml (1)

104 IU HBV-DNA/ml (2)

Project (part 2) proposed for ECBS 2011

HBV genotype panel for HBsAg tests 15 panel members

Reduction of potential infectivity (ultracentrifugation)

Target concentration: 100 IU HBsAg/ml

12 members of each panel have common origin (same donor)

Virology Division

HBV Genotype Panel (HBV DNA)

Collaborative study

17 labs from 12 different countries

Total of 19 data sets

16 different assays (13 quant, 3 qual)

One set on sequence analysis and phylogenetic tree

Virology Division

Results of collaborative study

Mean absolute estimates for the WHO IS (97/750); gt A2

Assigned 6.00 log10 IU/ml / Study 6.01 0.17 log10 IU/ml

Arithmetic mean values from 3 independent runs (replicate testing)

Virology Division

Results of collaborative study

Mean estimates relative to WHO IS

Sample 14: gt F2 Sample 15: gt G

6

8

3.78 0.50 log10 IU/ml4.76 0.45 log10 IU/ml

4.86 0.20 log10 IU/ml (excl. Lab 2) 3.85 0.21 log10 IU/ml (excl. Lab 4,6,7B,8)

Virology Division

Box Plot

The box-and-whisker-plots show the distribution of the data.

The boxes contain the middle 50 per cent of the results (interquartile range, IQR)

and the median as horizontal line.

The end of the whiskers denote the minimum and maximum values.

Virology Division

Results of HBV Genotype Panel (HBV-DNA)

Box-and-whisker-plots: Distribution of dataA1 A1 A2 B2 B2 B4 C2 C2 C2 D1 D3 D1 E F2 G IS

Virology Division

HBV Genotype Panel (HBV DNA)

Collaborative study - outcome

Overall good correlation in detecting most of the genotypes

Small number of assays detected genotypes F and G less efficiently

Few genotype B, C, and E samples were underquantified by two

methods

Need of a genotype panel confirmed

Virology Division

International Reference Panel for HBV genotypes

- NAT assay -

Panel for NAT tests established by ECBS of WHO in October 2009

Hepatitis B virus DNA; PEI 5086/08; 1st International Reference

Panel 2009

More information about the panel and ordering under:

WHO/BS/09.2121

www.pei.de

www.who.int/biologicals/en/

For future: Proposal to ECBS/WHO for extension (gt H …)

Thanks to the participants of the collaborative study

Virology Division

Design of HBV Genotype Panels

Project (part 2):

HBV genotype panel for HBsAg tests

15 panel members

Reduction of potential infectivity (ultracentrifugation)

Target concentration: 100 IU HBsAg/ml (EIA Architect, Abbott)

Virology Division

Flowchart Sample Processing

*Schüttler et al. (2010), J Clin Virol 47:238-242

Panel Samples

Infectivity-

reduced Samples

Samples Pre-Lyo

Samples Post-Lyo

Dilution

UC

Lyophilization

QIE - HBsAg

(PEI-U/ml)

HBsAg

(ng/ml)

EIA - HBsAg

(IU/ml)

EIA - HBsAg

(IU/ml)

EIA - HBsAg

(IU/ml)

Collaborative

Study

Native HBsAg Pos

Plasma Samples

Sample Characterization

EIA - HBsAg

(IU/ml)

QIE - HBsAg

(PEI-U/ml)

HBsAg

(ng/ml)

HBsAg

(ng/ml)

QIE - HBsAg

(PEI-U/ml)

QIE - HBsAg

(PEI-U/ml)

EIA HBsAg, quant enzyme immunoassay (Architect, Abbott)

QIE HBsAg, quant immunoelectrophoresis (Laurell)*

HBsAg, biochemical purification of native HBsAg*

Virology Division

HBV Genotype Panel (HBsAg) – Ultracentrifugation

Panel

Member OriginHBsAg

subtype1

HBV Sub-

genotype1HBV DNA (log10 ge/ml)2

Before UC After UC Reduction factor

1/A South Africa adw2 A1 9,52 7,98 1,54

2/A Brazil adw2 A1 9,55 6,04 3,31

3/A Germany adw2 A2 9,60 5,56 4,04

4/B Japan adw2 B1 8,90 7,41 1,49

5/B Japan adw2 B2 9,19 7,59 1,60

6/C Japan adr C2 9,33 5,91 3,42

7/C Japan adr C2 8,83 7,30 1,53

8/C Russia adr C2 9,20 6,85 2,35

9/D Germany ayw2 D1 9,80 6,17 3,63

10/D3 Russia ayw3 D2 4,79

11/D South Africa ayw2 D3 8,75 6,71 2,04

12/E West Africa ayw4 E 9,71 7,84 1,87

13/F Brazil adw4 F2 7,78 5,97 1,81

14/F Brazil adw4 F2 3,70 3,01 0,69

15/H Germany adw4 H 9,78 8,15 1,63

1Sequencing; 2In-house LC HBV NAT (ge/ml), Cobas AmpliPrep/CobasTaqMan HBV Test, v1.0

(Roche Diagnostics; conversion factor 1IU correspond to 5,82 ge); 3no UC.

Virology Division

HBV Genotype Panel (HBsAg)Dilution to 100 IU/ml1 (Pre- and Post-Lyo)

Pre-Lyo Post-Lyo

Panel

Member

HBsAg2

(IU/ml)

HBsAg3

(PEI U/ml)

HBsAg2

(IU/ml)

Loss by

Lyo (%)

HBsAg3,4

(PEI U/ml)

HBsAg5

(ng/ml)

1/A 107,9 60,8 95,4 11,6 53,7 96,68

2/A 100,6 70,0 87,7 12,8 61,0 92,66

3/A 94,5 66,6 80,3 15,0 56,6 75,03

4/B 74,9 45,3 63,3 15,5 38,3 33,94

5/B 88,7 43,0 74,7 15,8 36,2 69,06

6/C 98,5 136,0 87,7 11,0 121,0 180,48

7/C 101,0 85,6 84,2 16,6 71,4 101,36

8/C 100,6 73,9 90,0 10,5 66,1 92,23

9/D 100,9 57,0 83,3 17,4 47,1 78,20

10/D 91,1 105,8 88,2 3,2 102,4 n.a.

11/D 88,4 66,7 72,4 18,1 54,6 60,61

12/E 96,1 71,0 85,5 11,0 63,2 81,26

13/F 100,8 51,2 80,4 20,2 40,9 n.a.

14/F 113,1 56,0 95,0 16,0 47,0 51,36

15/H 102,7 43,0 88,5 13,8 37,1 60,03

1based on EIA (mean Architect values); 2EIA Architect; 3QIE Laurell

immunoelectrophoresis; 4assumed loss (%) as measured by the Architect; 5purified HBsAg;

n.a., not available.

Virology Division

HBV Genotype Panel (HBsAg)

- Collaborative Study -

15 Participants

Japan, USA, UK, NL, Italy, France, Germany

24 qual data sets from 21 different tests

2 data sets (one lab) were not used for the analysis

one data set: no sample cutoff

6 quant data sets from 2 different tests

Serial Dilutions of the 15 panel members and the WHO IS

Qual tests: S/CO

Quant tests: IU/ml

Virology Division

HBV Genotype Panel (HBsAg)

- Collaborative Study -

Virology Division

1/A

11/D10/D9/D6/C 7/C 8/C

15/H14/F13/F

4/B 5/B 12/E2/A 3/A

HBsAg Test (qual)

Sample dilution / IS dilution

at which the S/CO=1.00

Virology Division

Box-Plot showing the potency (relative to the Reference IS 00/588) in IU/ml for all samples (quantitative and qualitative assays)

A1 A1 A2 B1 B2 C2 C2 C2 D1 D2 D3 E F2 F2 H

HBV Genotype Panel (HBsAg)

- Collaborative Study -

Virology Division

Data analysis in progress

Stability studies are on-going

Submission of the report to ECBS June 2011

HBV Genotype Panel (HBsAg)

- Collaborative Study -

Virology Division

Thanks to the co-operators in the projects

- Prof Gerlich, Institute of Medical Virology, Justus Liebig University Giessen, Germany

- Profs Yoshizawa & Tanaka, Hiroshima University, Japan

- Dr Jochum, Biotest AG, Dreieich, Germany

- Prof Zhibourt, Federal Blood Center, Moscow, Russia

- Drs Sabino & Otani, Fundação Pró-Sangue Homocentro de São Paulo, Brazil

- Dr Schmidt, German Red Cross Blood Donation Center, Frankfurt/Main, Germany

- Drs Cheraghali & Abolghasemi, Iranian Blood Transfusion Organization, Tehran, Iran

- Mrs Sykes & Mr Watts, South African National Blood Service, Weltevreden Park, South Africa

- Dr Phil Minor, NIBSC, UK

- Dr Ana Padilla, WHO, HSS / EMP / QSM, Geneva

- Colleagues from PEI- Section of Molecular Virology

- PEI-IVD

- Section of Statistics

- Administration

Virology Division

Virology Division

HBV Genotype Panel (HBsAg)

Panel

Member

Origin HBsAg

subtype

HBV Sub-

genotype1

HBsAg2

(IU/ml)

HBsAg3

(PEI U/ml)

HBsAg4

(ng/ml)

1/A South Africa adw2 A1 80.130 48.680 77.470

2/A Brazil adw2 A1 57.737 40.400 53.500

3/A Germany adw2 A2 50.710 33.770 38.050

4/B Japan adw2 B1 45.280 20.510 15.370

5/B Japan adw2 B2 63.120 27.140 43.590

6/C Japan adr C2 34.585 47.030 62.420

7/C Japan adr C2 27.446 23.500 27.820

8/C Russia adr C2 29.384 21.700 27.100

9/D Germany ayw2 D1 94.090 53.660 73.580

10/D5 Russia ayw3 D2 8.413 8.900 n.a.

11/D South Africa ayw2 D3 42.949 28.650 26.030

12/E West Africa ayw4 E 66.204 47.030 53.800

13/F Brazil adw4 F2 22.258 11.400 n.a.

14/F Brazil adw4 F2 23.928 13.400 12.290

15/H Germany adw4 H 139.508 59.930 83.740

1INNO-LiPA; 2 EIA Architect; 3QIE Laurell immunoelectrophoresis; 4 purified HBsAg ; 5no ultracentrifugation; n.a., not available.