hepatitis e - diagnostics and standardization

www.pei.de

Hepatitis E

Diagnostics and Standardization

1st ECDC Hepatitis E Virus Expert Group Meeting

Stockholm, 9th-10th December, 2015

Sally A. Baylis, Division of Virology,

Paul-Ehrlich-Institut, Langen, Germany

Virology Division

1972 Federal Agency for Sera and Vaccines

2009 Federal Institute for Vaccines and Biomedicines Marketing authorization of human and veterinary medicines

Approval of clinical trials

Inspections

Pharmacovigilance

Official batch release testing for blood products, vaccines etc.

Testing laboratory for in vitro diagnostics devices (IVDs)

Research related to the above

2005 WHO Collaborating Centre for Quality Assurance

…… of Blood Products and In Vitro Diagnostics

2013 WHO Collaborating Centre for Vaccines

Paul-Ehrlich-Institut

Virology Division

Good diagnostics – critical

Correct diagnosis of hepatitis E

Prevalence/incidence of hepatitis E cases

Seroprevalence as a reflection of population exposure

Implementation of any screening programmes

Issues

Lack of awareness of HEV in differential diagnosis of acute

hepatitis/liver abnormalities

Hampered by assays lacking sensitivity, specificity, standardization

Diagnostics for Hepatitis E – Getting it Right

Virology Division

Clinical presentation - acute hepatitis

Jaundice, anorexia, hepatomegaly, abdominal pain/tenderness,

nausea/vomiting, fever etc.

Biochemical markers

↑ Alanine/aspartate aminotransferase, bilirubin, alkaline phosphatase

Confirmation of HEV infection

Detect virus or components

Particles (EM – stool)

RNA (blood, stool)

Ag (blood, stool)

Detect anti-HEV

IgM

IgG

IgA (?)

Diagnosis of Hepatitis E

Kamar et al., 2012 Lancet

Virology Division

Current infection - acute

HEV RNA

HEV RNA + anti-HEV IgM

HEV RNA + anti-HEV IgG

HEV RNA + anti-HEV IgM + anti-HEV IgG

Anti-HEV IgM + IgG (rising)

Current infection - chronic (immunocompromised)

HEV RNA (± anti-HEV) > 3 months

VL testing – monitoring therapy

Past infection

Anti-HEV IgG

Diagnosis of Hepatitis E – Laboratory Testing

Virology Division

WHO IS “Gold Standard” of biological reference materials

International Unit (IU) - uniform reporting system

Basis for calibration of assays, references materials

Allows comparison of different assays

WHO Standardization - Traceability

International Standard

2º Reference Materials

Calibration Standards

Working Reagents

Diagnostic Assays

EQA Materials

Virology Division

Detection of HEV RNA “gold standard” - active viraemia

PEI proposed the development of a WHO International

Standards for HEV RNA for NAT assays in 2009

Endorsed by Expert Committee on Biological Standardization

Initial study Q4, 2009 - Q1, 2010

HEV NAT assay performance - blinded panel of samples

Zoonotic strains of HEV gt 3a, 3b, 3f and gt 4c

Blood donor materials (Japan, Germany)

Determine a strain to develop into a WHO IS

20 laboratories, 10 different countries

In-house assays, with one exception

Standardizing HEV RNA Assays - Background

Virology Division

HEV Strains Investigated in 1st Study

Genotype Virus strain HEV RNA

(copies/ml)

Anti-HEV

IgM/IgG

ALT (IU/L)

3a HRC-HE104 1.6 x 107 -/- 36

3b JRC-HE3 2.5 x 107 +/- 398

3f RKI 1.3 x 106 -/- Negative

4c HRC-HE15 1.0 x 106 -/- 505

Analysis based upon partial

ORF2 sequence

Virology Division

Nominal concentration

(log10 copies/ml) 6.2 5.2 4.2 3.2 2.2 1.2

Lab no. 1 + + + +/- - -

2 a + + + + + -

2 b + + + + +/- -

3 + + + + + -

4 + + + + - +/-

5 + + + + + -

6 + + + + - -

7 + + + + - -

8 + + + - + -

9 + + + + - -

10 + + + - +/- -

11 a + + - - - -

11 b + + +/- - - -

12 + + + + + +

13† + + + + - -

14 + + + + + +

15 a + + + + - -

15 b + + + + - -

16 + + + + - -

17 + + + + - -

18 a + + + - - -

18 b + + + + - -

19 - - - - - -

20 + + + - +/- -

Total number of tests 24 24 24 24 24 24

Percentage positive 96 96 92/88 75/67 38/25 13/8

Example - Qualitative Analysis of HRC-HE104 (Genotype 3a)

Virology Division

Nominal concentration

(log10 copies/ml) 5.0 4.0 3.0 2.0 1.0

Lab no. 1 + + +/- - -

2 a + + - - -

2 b + + - - -

3 + + + + -

4 +/- + +/- - -

5 + + - - -

6 + + + +/- -

7 + + +/- - -

8 + - + - -

9 + + + + -

10 + + - - +

11 a + + - - -

11 b + + - - -

12 + + + + -

13 + + + +/- +/-

14 + + + + +

15 a - - - - -

15 b + + - - -

16 + + + + -

17 + + + + -

18 a + + - - -

18 b + + - - -

19 - - - - -

20 + - - - -

Total number of tests 24 24 24 24 24

Percentage positive 92/88 83 50/38 33/25 4/0

Example - Qualitative Analysis of HRC-HE15 (Genotype 4c)

Virology Division

Example - Analysis of Titres and CT Values - HRC-HE104

Baylis et al.,

J Clin Micro, 2011

Virology Division

Detection of HEV antigen is not as sensitive as NAT

New assays offer greatly improved sensitivity

Zhao et al., J Viral Hepatol, 11, 957-963

Wen et al., J Clin Microbiol, 53, 782-788

Detection of HEV Antigen

Genotype HEV RNA

(copies/ml)Antigen

HEV RNA

(copies/ml)Antigen

3a 1.6 x 106 pos. 1.6 x 105 neg.

3b 2.5 x 106 pos. 2.5 x 105 neg.

3f 1.3 x 105 neg. 1.3 x 104 neg.

4c 1.0 x 105 pos. 1.0 x 104 pos.

Wantai Assay

Virology Division

Issues of sensitivity (independent of strain)

~100- to 1000-fold difference - majority of assays

Issues of specificity

One false positive result, genotyping by the lab in question detected

gt 1 (not included in the panel)

Wide range of copy numbers reported

Real-time PCRs out-performed conventional (nested) PCR

Demonstrates need for assay standardization

Further details:

Baylis et al., J Clin Microbiol, 2011 49:1234-9

1st Collaborative Study – Conclusions

Virology Division

The following strains were lyophilized in September 2010

HRC-HE104 (genotype 3a) – WHO International Standard

JRC-HE3 (genotype 3b) – Japanese National Standard

Drs Okada & Mizusawa, National Institute for Infectious Diseases

Collaborative study – Q1, 2011

Blinded replicate samples; 4 assay runs

Data returned by 23 laboratories (10 countries); all in -

house assays

21 qualitative data sets (end point dilution analysis)

14 quantitative data sets (reporting in copies/ml)

Development of the WHO IS for HEV RNA

Virology Division

Potencies and Relative Potencies of Participants Results

Potency relative to candidate IS

= difference in estimated log10 units/ml + assigned

value of candidate IS (5.39 log10 IU/ml)

quant. assays (white - copies/ml)

qual. assays (blue - NAT-detectable units/ml)

Potencies

Virology Division

1st WHO IS for HEV RNA established in October 2011

Japanese NIID – simultaneously established a national standard

250,000 International Units/ml

The IS is available from the PEI (code # 6329/10)

Restriction 5 vials per lab per year

The standard has been distributed since March 2012

Uses

Preparation of secondary standards

Define analytical sensitivities

Compare assay performance, EQA reporting, define TT dose etc.

Further details - http://www.pei.de

Baylis et al., Emerg Infect Dis, 2013;19:729-35

Establishment of the 1st WHO IS for HEV RNA

Virology Division

WHO ECBS endorsed panel proposal in October 2011

All four genotypes & important sub-genotypes

Strains from plasma (n=8)

Strains from stool (n=3) diluted in pooled plasma

Lyophilized

Collaborative study, Q4, 2014- Q1/2, 2015

24 laboratories, 14 different countries

Evaluate samples concurrently with the WHO IS

Range of qual./quant. assays (in-house; commercial)

Simultaneously calibrating a secondary standard (HEV 3f)

Council of Europe/EU Commission

Implementation of HEV NAT screening for S/D plasma in EU

International Reference Panel – HEV Genotypes

Virology Division

Introduction of HEV NAT for S/D plasma – Ph. Eur.

Development of a Biological Reference Preparation

(BRP)

EDQM, Council of Europe and the EU Commission

Dr Eriko Terao

Candidate BRP for HEV RNA

Genotype 3f HEV strain - German plasma donor

6000 vials lyophilized

Calibration in IU/ml against the WHO IS in genotype panel study

Report reviewed by the EDQM BSP steering committee

Biological Standardisation Programme project

(BSP127)

Virology Division

Adlhoch et al., Vox Sang. 2009

A regular plasma donor was diagnosed with acute hepatitis and elevated levels of ALT

Negative for HAV, HBV, HCV, EBV, CMV and adenovirus

Tested for anti-HEV - IgM positive

Robert Koch-Institut was notified and a look-back study was performed

A genotype 3f virus was identified - donor worked in a slaughter house

HEV Infection in a German Plasma Donor

Virology Division

HEV Infection in a German Plasma Donor Contd.

Adlhoch et al., Vox Sang 2009

ELISA

MP Biomedicals/Genelabs

ELISA

Mikrogen

Immunoblot

Mikrogen

ELISA

Euroimmun

ELISA

Wantai

IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG

+ + - +/- +/- +/- + +/- + -

Virology Division

Genotype 2

Genotype 3

Genotype 4

Genotype 1

Origin of WHO HEV Genotype Panel Strains

Virology Division

HQ389543 3 England

AF060668 3a USA

AB089824 3a Japan

WHO IS AB630970 3a Japan

AB074918 3a Japan

AB074920 3a Japan

AB291962 3b Japan

AB246676 3b Japan

8570/13 AB630971 3b Japan

JQ034516 3c Germany

8571/13 JN995569 3c Sweden

JQ034514 3c Germany

FJ705359 3c Germany boar

8574/13s 3 France

JQ013792 3 France rabbit

GU937805 3 China rabbit

FJ906896 3 China rabbit

FJ906895 3 China rabbit

8572/13 JN995564 3e Germany

JQ953665 3e France pig

8573/13 JN995573 3f Sweden

BRP FJ956757 3f Germany

EU495148 3f France

AB369687 3f Japan

AB291961 3f Japan

FJ610232 4d China pig

AY594199 4g China pig

AB108537 4g China

8576/13 4g Japan

AB091395 4c Japan

AB097812 4c Japan

AB074915 4c Japan

8575/13 4c Japan

AB291959 4c Japan

AB161717 4c Japan

M74506 2a Mexico

8577/13s 2a Mexico

AY204877 1e Chad

8569/13 1e Sudan

AY230202 1d Morocco

L25595 1b China

M80581 1b Pakistan

L08816 1b China

D11092 1b China

X99441 1a India

AF051830 1a Nepal

M73218 1a Myanmar

AF185822 1a Pakistan

8567/13 1a India

FJ457024 1a India

8568/13s 1a India

AY535004 avian

94

100

100

100

42

89

100

61

60

99

89

88

65

77

40

99

88

71

36

53

20

93

69

92

99

99

88

61

84

81

40

60

97

94

73

50

85

48

79

35

68

48

92

90

69

84

86

49

0.02

Genotype 3

Genotype 4

Genotype 2

Genotype 1

3a

3b

3c

3

3e

3f

4g

4c

2a

1e

1a

Analysis based upon

partial RdRp

sequence

Virology Division

HEV in European Plasma Donors

Selection of strains from blood donors to reflect

clnically important genotypes

Nucleotide Substitution per 100 residues0

29.1510152025

D_N001113105171D_N001113082834P_N001213900311

P_N001212901361P_N001213900152

P_N001213900726D_N001112090844NL_Donor_12

P_N001214900744

P_N001213906705P_N001213910627

P_N001213908973P_N001213907985P_N001213908062P_N001112085481NL_Donor_15

P_N001212912417P_N001213901721P_N001213907677

P_N001213901631P_N001211907235NL_Patient_04

P_N001214903106

P_N001212902562D_N000113083892

D_N001113129349D_N001113129308

P_N001213903457P_N001214903706

P_N001213905608D_N000111204385NL_Donor_05

FJ705359_3i

P_N001213906628

P_N001213905961P_N001211903682

P_N001214901309

P_N001210910016NL_Patient_03

P_N001213908064P_N001213906323P_N001212908053

P_N001213907300Germany_6Sweden_2

P_N001214900585D_N001113091139

P_N001213980747D_N001113905084

P_N001211907392NL_Patient_07

D_N001811030409NL_Donor_04

P_N001214902034P_N001214902688

D_N000313078482P_AMC_21172902NL_Patient_05D_N000311136809

P_N001212910164French_Donor_12

P_N001212907511P_N001213905916

P_N001212910296P_N001211910745

P_N001210988310D_N001112240056D_N000311181536NL_Donor_10

Sweden_8Sweden_11

D_N000312028013NL_Donor_11

P_N001214903354D_N000313050878

Germany_20P_N001213906287P_N001213909683

D_N001211117594NL_Donor_07

P_AMC_2011P_N001212910998

P_N001212911077

P_N001212910704P_N001211915955NL_Patient_06

P_N001213907293Germany_7

D_N000313051864P_LUMC_13043210

P_N001212902862P_N001212904706

D_N000112080777NL_Donor_16

P_N001212904901

P_N001214903597D_N000113154350

D_N000313100849D_N001111236578NL_Donor_09

D_N000311079197NL_Donor_03

D_N001111001934NL_Donor_02

French_Donor_4French_Donor_15

French_Donor_5FJ600536_3c

D_N001811283203NL_Donor_08

Sweden_5French_Donor_8

French_Donor_17AF516178_3A

AF466662_3ASweden_16

Sweden_19

AB115544_3BAF296165_3D

French_Donor_10

P_N001214902194

P_N001213909588P_N001213905698

P_N001213907674

AB248521_3eAB094250_3E

AB248520_3e

Germany_3AF503512_3E

AF455784_3GAF336296_3F


French_Donor_14P_N001213906748




P_N001213900814D_9575_Spaans


French_Donor_1P_N001211904913NL_Patient_01


D_N001111062446NL_Donor_01

D_N000312096110French_Donor_9

P_N001212911317P_N001212903842

D_N000312101329NL_Donor_14

P_N001214901379P_N001212905169NL_Patient_02

French_Donor_23

P_N001212907243French_Donor_20

Czech_23Sweden_15

Sweden_12P_N001213909941

Germany_18

Genotype 1Genotype 4

Genotype 2

England&Wales

GT3, group 2

England&Wales

GT3, group 1

3c

3e

3fCourtesy of S. Ijaz

Virology Division

log1

0 I

U/m

l

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

#8567/13

#8568/13s

#8569/13

#8570/13

#8571/13

#8572/13

#8573/13

#8574/13s

#8575/13

#8576/13

#8577/13

#8577/13s

Cand

idate

BRP

Range of Quantitative Assays Results (IU/ml)

Box indicates interquartile range; line within box indicates median; whiskers indicate minimum

and maximum values observed. Circles depict expected values measured at PEI.

1a 1a 1e 3b 3c 3e 3f R 4c 4g 2a 2a 3f

Virology Division

Calibration of the Candidate BRP (Secondary Standard)

23A 6E 8 6E

6F

11

20

6B

6F

8

13

1

2

10

15

17

23C

2

10

15

17

19A

6B

6D

13

19B

21B

21B

3A

4

16

18

21A

1

3A

4

6C

16

18

3B

5

11

19B

9

12

19A

6A

9

12

21A

22

3B

14

14

20

22

7

BRP candidate

La

bo

rato

rie

s

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

log10 IU/ml

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

Overall potency of BRP

potency is:

4.61 log10 IU/ml

(95% CI: 4.51 – 4.72)

Virology Division

Established by the WHO ECBS in October 2015

Genotypes generally well detected

Inevitable genetic changes – other studies; some sub-

genotypes (3) not always so consistently detected

No unitage assigned to the panel members (WHO policy)

Details of potencies (+ range) will be reported in IFU

Wide range (especially gt 1, gt 2)

Panel will be ready for distribution early 2016

BRP being reviewed the CoE BSP steering committee

International Reference Panel – HEV Genotypes

Virology Division

Jothikumar et al., A broadly reactive one-step real-time RT-PCR

assay for rapid and sensitive detection of hepatitis E virus. J Virol

Meth 131, 65-71

Targets a conserved region in ORF2/ORF3

Probe is very short, Tm ~10°C lower than normal

Database - small number of HEV strains with polymorphisms

Widely Used HEV Real-Time PCR – Issues

Virology Division

Garson et al., Minor groove binder modification of widely used

TaqMan probe for hepatitis E virus reduces risk of false-negative

real-time PCR results. J Virol Meth, 186, 157-60

Serologically confirmed hepatitis E cases reinvestigated using the

modified probe, identified additional HEV RNA positive samples

Probe 5´-TGA TTC TCA GCC CTT CGC

UK patient 5´-TGA TTC TCA GCC CTT TGC

MGB modification ↑ Tm of probe and restored detection

Polymorphism seen in UK patients, caucasians

Widely Used HEV Real-Time PCR – Issues Contd.

Virology Division

Analysis of plasma donors by the PEI in collaboration with

Octapharma has identified a further polymorphism

Probe 5´-TGA TTC TCA GCC CTT CGC

UK patient 5´-TGA TTC TCA GCC CTT TGC

Swedish donor 5´-TGA TTC CCA GCC CTT CGC

Widely Used HEV Real-Time PCR – Issues Contd.

WHO IS

HEV RNA positive

plasma donor

NTC

Virology Division

HEV NAT - Commercial Assays

Manufacturer Assay name Technology Notes

altona DIAGNOSTICS RealStar® HEV RT-PCR kit

1.0

Real-time PCR CE-mark* 95% cut-off;

Vollmer et al., JCM, 5 IU/ml

Corman et al., Vox, 260 IU/ml

Beijing Kinghawk

Pharmaceutical Co. Ltd

HEV RNA (FQ-PCR) Real-time PCR IVD

gt 1, 4

CEERAM S.A.S. (BioMerieux) hepatitisE@ceeramTool® Real-time PCR CE-mark*

Genome Diagnostics Pvt. Ltd Geno-Sen’s HEV Real Time

PCR Kit

Real-time PCR CE-mark*

95% cut-off – 80 cps/ml

Liferiver (Gentaur) HEV Real Time RT-PCR Kit

RNA

Real-time PCR CE-mark*

gt 4

Mediagnost GmbH HEVGene®-Detection kit PCR RUO

MIKROGEN GmbH ampliCUBE HEV Real-time PCR CE-mark*

PrimerDesign Ltd Path-HEV Real-time PCR RUO, <100 cps

Roche Molecular Systems Inc. cobas® HEV Real-time PCR CE-mark*,

95% cut-off - 18.6 IU/ml, gt 1-4

Hologic, Inc./Grifols Diagnostic

Solutions, Inc.

Procleix HEV assay TMA CE-mark*

95% cut-off - 7.89 IU/ml, gt 1-4

Fast Track Diagnostics FTD Hepatitis E RNA Real time PCR CE-mark* LOD 100 IU/ml

TIB Molbiol LightMix® Modular HEV Real time PCR CE-mark* LOD ~200 IU/ml, gt 1-4

*In compliance with Directive 98/79/EC on In Vitro Diagnostic Medical Devices (Annex III, manufacturer's self-declaration)

Virology Division

Hologic/Grifols – Panther

Roche - cobas® 6800/8800

Automated Platforms

Virology Division

Antigens

Peptides

Recombinant proteins – greater sensitivity

Mainly ORF2 capsid protein (occasionally ORF3); different gts

Assay format

EIAs – plates, blots, rapid (POC) tests

IgM, IgG, total Ig

In-house and commercially available assays

Assay performance

Issues of sensitivity, specificity (cross-reactivity e.g. CMV, EBV etc.)

Often poor concordance between assay results

Lot-to-lot variability

Differences in duration of persistence of abs to various epitopes

Detection of Anti-HEV

Virology Division

Seroprevalence (IgG) of HEV significantly underestimated

Bendall et al., J Med Virol 2010

Wantai vs. MP Biomedicals

16.2% vs. 3.6%, blood donors UK

Wenzel et al., J Infect Dis 2013

Wantai vs. MP Biomedicals

Healthcare workers Germany 29.5% vs. 4.5%

Revised assay formats suggests better concordance

Avellon et al., J Med Virol 2015

Issues of Sensitivity – IgG Assays

Virology Division

Wenzel et al., J Infect Dis 2013

MP Biomedicals Axiom (Wantai) Mikrogen

ELISA ELISA Immunoblot

Issues of Sensitivity – IgG Assays

Virology Division

Sensitivity/specificity issues

False reactivity (e.g. EBV, CMV)

Drobeniuc et al., Clin Inf Dis 2010

Certain tests perform better at low concentrations of IgM

Avellon et al., 2015

IgM Assays - Issues

Assay Sensitivity Specificity

I (Sar-55 Ag/Purcell) 98% 78%

II (Antigens aa 452-617 ORF2 gt1) 98% 93%

III (International Immunodiagnostics) 82% 91%

IV (MP Biomedicals) 72% 93%

V (Diagnostic Systems) 98% 95%

VI (Mikrogen) 92% 96%

Virology Division

A WHO International Reference Reagent (95/584) for anti-

HEV established in 1997

Ferguson et al. Biologicals 30:43-8.

Lyophilized, serum pool - 5 separate bleeds over a 45 day

period (4-5 months after onset of illness) from a US patient

who developed acute hepatitis after visiting India

No confirmation of HEV genotype (Saleem Kamili, pers. comm.)

Not established as an IS because the number of

participants in the collaborative study was too limited (n=7)

Anti-HEV IgG; IgM positive (Mikrogen recomWell, Eurimmun,

Wantai, MP Biomedicals)

Serology - Standardization

Virology Division

Bendall et al., J Med Virol 2010

Determination of Analytical Sensitivity –

WHO IRR 95/584

Virology Division

48th report of the ECBS (WHO Technical Report

Series 889)

“The Committee was aware that assays for anti-hepatitis E

were at an early stage of development and recognized that

full assessment of new antibody assays requires panels of

sera. Nevertheless, it felt that the availability of a common

reference material would help evaluate inter-laboratory

variation and would aid developments in the serology of

hepatitis E virus. The Committee therefore established the

preparation coded 95/584, as an interim Reference Reagent

for Anti-hepatitis E Serum, Human, and assigned a value of

50 units per ampoule.”

Anti-HEV IRR

Virology Division

WHO ECBS endorsed a proposal in Oct. 2015 to develop an

international reference panel for anti-HEV

Acute and convalescent samples

IgM (± IgG), IgG

Samples confirmed by PCR (confirmation of gt)

Wide geographic area

Large volume samples

Lyophilized (shipment)

Evaluation in an international collaborative study

Development of an Anti-HEV Reference Panel

Virology Division

PEI

Johannes Blümel

Kay-Martin Hanschmann

Roswitha Kleiber

Sigrid Nick

Micha Nübling (PEI/WHO)

Gudrun Winskowsky

Japan

Keiji Matsubayashi (JRCS)

Saeko Mizusawa (NIID)

EDQM

Eriko Terao

WHO

Collaborative Study Participants

Acknowledgements

Germany

Thoma Gärtner (Octapharma)

Cornelia Adlhoch (RKI, ECDC)

Marco Kaiser (RKI)

Victor Corman (Bonn)

Jürgen Wenzel (Regensburg)

UK

Harry Dalton

France

Anne-Marie Roque-Afonso

India

Rakesh Aggarwal (SGPIMS)

Nirupma Trehanpati (ILBS)

USA

Saleem Kamili (CDC)

hepatitis e - diagnostics and standardization

Health & Medicine