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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
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
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