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Proposed 1st IS for Hepatitis E Virus RNA

WHO/BS/09.2126

SoGAT XXII

14th-15th April 2011, Rome, Italy

S. Baylis, Division of Virology,

Paul-Ehrlich-Institut, Langen, Germany

Virology Division

3rd WHO CC Meeting

7th-8th March 2011, NIBSC, UK

Presented at:

Virology Division

HEV is a major cause of acute hepatitis

Major public health concern in endemic areas (vaccine efforts)

Emerging (more recognised) infection in industrialised countries

High mortality in certain patients

Individuals with liver disease

Pregnant women

Immunosuppressed – chronic infections increasingly recognised,

load testing important in evaluation of antiviral therapy regimes

Zoonotic virus, certain genotypes - swine and other animals

Testing important in patients where other causes of

hepatitis have been excluded (global issue)

HEV can be transmitted by transfusion, present in donors

Hepatitis E Virus (HEV)

Virology Division

Project proposed at the 2nd WHO CC Meeting in Langen,

Feb. 2009

Presented at SoGAT XX in Brussels, May 2009 and

flagged for development in SoGAT survey

Project proposal endorsed by ECBS in Oct. 2009

(WHO/BS/09.2126)

Anticipated users

Clinical laboratories (hepatitis reference centres)

Blood banks/plasma centres – some are screening

Research laboratories and vaccine developers

IVD manufacturers (single commercial assay)

Background

Virology Division

To investigate HEV NAT assay performance for the first

time using blinded panel of samples

To determine an appropriate strain to develop into a

candidate IS

The panel comprised 22 HEV positive samples (10-fold

serial dilutions) and 2 negative plasma controls

genotypes 3a, 3b, 3f, 4c (zoonotic genotypes)

Positive plasma samples obtained from blood donors

Japan and Germany

1st Collaborative Study – Aim & Approach

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

Virology Division

20 participating laboratories, from 10 countries

Participants have expertise in molecular analysis of HEV

Requested to use regular assays for HEV RNA and

report results as either positive or negative i.e. HEV RNA

detected or not detected

Data was returned from 24 different assays

10 labs returned quantitative data (optional)

All assays, except one, were developed in-house using

conventional or real-time RT-PCR methodologies

1st Collaborative Study – Labs & Methods

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

Quantitative Analysis of HEV Panel

Virus strain Nominal

concentration

log10 copies/ml

N Geometric

mean

Median Min. Max.

HRC-HE104

6.2 12 5.84 5.77 4.82 7.48

5.2 12 4.74 4.72 3.63 6.40

4.2 11 3.85 3.84 3.11 5.64

3.2 9 3.04 2.96 2.40 4.49

JRC-HE3

6.4 12 6.16 6.15 4.43 7.70

5.4 12 5.07 5.14 2.15 7.00

4.4 12 4.21 4.27 2.60 5.58

3.4 10 3.40 3.20 2.92 5.00

RKI

5.1 12 4.63 4.57 3.91 6.26

4.1 10 3.77 3.63 3.20 5.26

3.1 9 2.83 2.63 1.77 4.28

HRC-HE15

5.0 12 4.56 4.44 3.28 6.28

4.0 10 3.40 3.44 2.63 4.04

3.0 8 1.83 2.46 -1.00 4.20

Virology Division

Analysis of Titres and CT Values - HRC-HE104

Virology Division

Qualitative data

~100- to 1000-fold difference in sensitivity - majority of assays,

independent of strain

real-time RT-PCR methods were most sensitive

ORF1 assays were least sensitive

Quantitative data

at least two thirds of the data sets fell within ± 0.5 log10 copies/ml

of the geometric mean value for the different HEV strains

All negative plasma samples were correctly reported

(single equivocal result for one replicate sample)

One false positive result, genotyping by the lab in

question detected gt 1 (not included in the panel)

1st Collaborative Study – Conclusions

Virology Division

Project progress report submitted to WHO in Q2, 2010;

recommendation to take forward the high titre genotype

3 samples as candidate standards

well detected in study

represent globally distributed genotype

The following strains were lyophilised in September 2010

HRC-HE104 (genotype 3a)

JRC-HE3 (genotype 3b)

Diluted in citrated plasma used in 1st study which tested

negative for

HIV-1/2 RNA, HCV RNA, HBV DNA – Roche TaqScreen MPX

HEV RNA and anti-HEV (IgM and IgG)

1st Collaborative Study – Outcome

Virology Division

Genotype 3a strain - candidate WHO standard

Coefficient of variation of fill volume 1.1%

Residual moisture 0.73%

4251 vials filled

Titre of HEV RNA ~5-5.5 log10 copies/ml (no loss post-lyophilisation)

Full length sequence nearing completion

Candidate WHO standard being evaluated together with the

genotype 3b strain in a new collaborative study

Candidate WHO Standard

Virology Division

The study is being run in conjunction with the Japanese

National Institute for Infectious Diseases (NIID)

developing national standard (genotype 3b)

24 participating laboratories, from 10 countries

Each laboratory was sent 4 vials of each candidate

Sample 1 + Sample 2 - HRC-HE104 (genotype 3a)

Sample 3 + Sample 4 - JRC-HE3 (genotype 3b)

Samples shipped at ambient temperature

Data returned by 23 laboratories, all in house assays

21 qualitative data sets, 14 quantitative data sets

2nd Collaborative Study

Virology Division

Data analysis in progress

Stability studies are on-going

Submission to ECBS June 2011

2nd Collaborative Study contd.

Candidate Mean

log10 copies/ml

SD 95% CI %GCV

WHO 5.84 0.50 5.44-6.24 146

NIID 5.76 0.30 5.47-6.05 100

Participant

Lo

g10

co

pie

s/m

l

Virology Division

Keiji Matsubayashi, Japanese Red Cross Hokkaido Blood

Center

Collaborative study participants

Acknowledgements

Virology Division

WHO

Candidate

NIID

Candidate