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Hepatitis E Virus – Progress in Standardization of NAT-Based Assays

Blood Products Advisory CommitteeRockville, 20th September 2012

Sally A. Baylis, Division of Virology,Paul-Ehrlich-Institut, Langen, Germany

Virology Division

Initial investigation of laboratory performance of NAT assays for the detection of HEV RNA

Evaluation of a candidate WHO International Standard for HEV RNA for NAT-based assays

Review of current NAT assays; commercial, in-house

Progress in development of an HEV genotype panel

Update on proposal to introduce HEV NAT for S/D plasma

Virology Division

HEV NAT standardization project proposed by the Paul-Ehrlich-Institut (PEI) at the 2nd World Health Organization Collaborating Centres Meeting (Langen, Feb. 2009)

Presented at SoGAT XX in Brussels, May 2009 and flagged for development in SoGAT survey

Project proposal endorsed by WHO Expert Committee on Biological Standardization - Oct. 2009 (WHO/BS/09.2126)

Anticipated users Clinical laboratories (hepatitis reference centres) Blood banks/plasma centres Research laboratories and vaccine developers IVD manufacturers

Standardizing HEV RNA Assays - Background

Virology Division

To investigate HEV NAT assay performance for the first time using blinded panel of samples – Q4 2009/Q1 2010

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

Analysis based upon partial ORF2 sequence

Virology Division

20 participating laboratories, from 10 countries

Participants with expertise in molecular analysis of HEV

Requested to use regular assays for HEV RNA 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 24Percentage 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 24Percentage positive 92/88 83 50/38 33/25 4/0

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

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 7.485.2 12 4.74 4.72 3.63 6.404.2 11 3.85 3.84 3.11 5.643.2 9 3.04 2.96 2.40 4.49

JRC-HE3

6.4 12 6.16 6.15 4.43 7.705.4 12 5.07 5.14 2.15 7.004.4 12 4.21 4.27 2.60 5.583.4 10 3.40 3.20 2.92 5.00

RKI

5.1 12 4.63 4.57 3.91 6.264.1 10 3.77 3.63 3.20 5.263.1 9 2.83 2.63 1.77 4.28

HRC-HE15

5.0 12 4.56 4.44 3.28 6.284.0 10 3.40 3.44 2.63 4.043.0 8 1.83 2.46 -1.00 4.20

Virology Division

Quantitative Analysis

HRC-HE104gt 3a

JRC-HE3gt 3b

RKIgt 3f

HRC-HE15gt 4c

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 directed 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 standardswell detected in studyrepresent globally distributed genotypeBaylis et al., J Clin Micro 49,1243-9

The following strains were lyophilized in September 2010HRC-HE104 (genotype 3a) – WHO International StandardJRC-HE3 (genotype 3b) – Japanese National Standard

Diluted in citrated plasma used in 1st study which tested negative for HIV-1/2 RNA, HCV RNA, HBV DNA – Roche TaqScreen MPXHEV RNA and anti-HEV (IgM and IgG)

1st Collaborative Study – Outcome

Virology Division

WHO CandidateAB630970

NIID CandidateAB630971

Virology Division

Genotype 3a strain - candidate WHO standardCoefficient of variation of fill volume 1.1%Residual moisture 0.73%4251 vials filledTitre of HEV RNA ~5.0-5.5 log10 copies/ml (no loss post-lyophilization)

Full length sequence determined

Candidate WHO standard evaluated together with the genotype 3b strain in a further collaborative study

Candidate WHO Standard

Virology Division

Study was 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

Labs tested samples in 4 separate asays runs (qual./quant.)

Data returned by 23 laboratories, all in house assays 21 qualitative data sets, 14 quantitative data sets

2nd Collaborative Study

Virology Division

Overall Mean Estimates from Qualitative Assays (log10 NAT-detectable units/ml)

Sample n mean sd lower cl upper cl median min max cv_geo

1 19 5.25 0.51 5.01 5.50 5.32 4.42 6.20 150%

2 20 5.26 0.62 4.97 5.56 5.29 4.00 6.37 179%

3 20 5.27 0.79 4.90 5.64 5.27 3.72 7.42 226%

4 20 5.31 0.64 5.02 5.61 5.30 4.42 6.87 183%

Candidate n mean sd lower cl upper cl median min max cv_geo

WHO 39 5.26 0.56 5.08 5.44 5.32 4.00 6.37 163%

NIID 40 5.29 0.71 5.07 5.52 5.30 3.72 7.42 202%

Virology Division

Overall Mean Estimates from Quantitative Assays (log10 copies/ml)

Sample n mean sd lower cl upper cl median min max cv_geo

1 123 5.58 0.29 5.32 5.85 5.46 4.36 6.85 98%

2 125 5.60 0.28 5.33 5.87 5.46 4.43 6.69 94%

3 124 5.66 0.20 5.40 5.93 5.50 4.49 6.63 77%

4 125 5.66 0.20 5.40 5.93 5.48 4.64 6.77 76%

Candidate n mean sd lower cl upper cl median min max cv_geo

WHO 248 5.59 0.30 5.33 5.86 5.46 4.36 6.85 99%

NIID 249 5.66 0.20 5.40 5.93 5.48 4.49 6.77 76%

Virology Division

Quantitative Data – Box Plots

Virology Division

Quantitative assays (blue - copies/ml); qualitative assays (white - NAT-detectable /ml).

Histograms of Participants Results

Virology Division

Quantitative assays (blue); qualitative assays (white).

Potency relative to candidate IS = difference in estimated log10 units/ml + assigned value of candidate IS (5.39 log10 IU/ml)

Potencies Expressed Relative to Sample 1

Virology Division

Potency Relative to Candidate (Sample 1)

Sample Assay No. data GeoMean95%-Confidence

Limits%GCV

S2

quantitative 19 5.46 5.35 – 5.58 3%

qualitative 13 5.42 5.38 – 5.46 1%

combined 32 5.45 5.38 – 5.51 2%

S3

quantitative 20 5.45 5.27 – 5.65 5%

qualitative 13 5.48 5.37 – 5.59 2%

combined 33 5.46 5.35 – 5.58 4%

S4

quantitative 20 5.51 5.38 – 5.64 3%

qualitative 13 5.47 5.36 – 5.59 2%

combined 33 5.49 5.41 – 5.58 3%

Virology Division

All assays were able to detect both candidate standards Combined mean estimates for the 2 candidate standards

5.60 log10 copies/ml (quant. NAT)

5.26 and 5.29 log10 NAT-detectable units (qual. NAT - end points)

Combined data - potency of preps - 5.39 log10 units/ml

Participants standards: Plasmid DNA Synthetic oligonucleotides In vitro transcribed RNA Calibrated plasma/stool samples

No standard controls - reflected in the observed variation Expressing results relative to Sample 1, as a standard, improved

agreement between different labs/methods

Conclusions 2nd Collaborative Study

Virology Division

1st WHO International Standard (IS) for Hepatitis E Virus RNA was established in October 2011 Japanese NIID – simultaneously establishing a national standard

The IS contains a blood donor-derived genotype 3a HEV strain, diluted in plasma, and lyophilized

The IS has a unitage of 250,000 International Units/ml

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

Baylis et al. WHO/BS/2011.2175

Establishment of the 1st WHO IS for HEV RNA

Virology Division

The availability of an IS for HEV will facilitate

Comparison of results of different HEV NAT assays Defining analytical sensitivity

Clinical diagnostics Blood/plasma screening

Viral load testing – chronic infection Validation of (new) assays

Conclusions 2nd Collaborative Study Contd.

Virology Division

HEV NAT - Commercial AssaysManufacturer 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/mlCorman et al., Vox, 260 IU/ml

Beijing Kinghawk Pharmaceutical Co. Ltd

HEV RNA (FQ-PCR) Real-time PCR IVDgt 1, 4

CEERAM S.A.S. 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

Hologic/Gen-Probe In development TMA 95% cut-off – 22 IU/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 TestIn development - Japan

Real-time PCR 95% cut-off – 50 cps/mlgt 1-4

*In compliance with Directive 98/79/EC on In Vitro Diagnostic Medical Devices

(Annex III, manufacturer's self-declaration)

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, in press

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 donors

NTC

Virology Division

The WHO ECBS endorsed a proposal by the PEI to prepare a genotype panel for HEV at the annual meeting in October 2011 (WHO/BS/2011.2179)

The panel is intended to contain representatives of all genotypes and important sub-genotypes

The panel will be lyophilized

Candidate samples for the preparation of the panel include materials evaluated in the original collaborative study, strains detected in blood/plasma donors & clinical isolates

HEV Genotype Panel Proposal

Virology Division

Samples are currently being sourced/characterized:

Gt 1 - strains sourced from IndiaGt 1 - China (Xinjiang epidemic strain)

Gt 3b - JRC-HE3 (Japanese)Gt 3c - European plasma donor samplesGt 3e - European and Japanese samplesGt 3f - RKI window period and s/c samples; other strainsGt 3 - strains which challenge current assays; rabbit strain

Gt 4c - HRC-HE15 (Japanese)Gt 4i - Japanese plasma donor

Gt 1/4 prospectively sourced, Sudan (1e), Bangladesh, China and clinical cases - Europe

Gt 2 – problematic to source; inclusion of cloned nucleic acid?

Sourcing of Samples

Virology Division

b

Analysis based upon partial RdRp sequence

Virology Division

The proposal is to amend monograph 1646 - Human plasma (pooled and treated for virus inactivation)

HEV detected in respective European plasma donations

Amendment would see the introduction of HEV NAT

The proposal was discussed at the group 6B (Human Blood and Blood Products) meeting at EDQM - March 2012 Report to be published in Pharmeuropa (issue 25.1) in January 2013 – 3

month consultation period Comments expected to be reviewed by group 6B in April 2013 If agreed, referral to the Ph. Eur. Commision – June 2013 If adopted, publication of revised monograph – January 2014 Implementation, July 2014

Proposal to Amend the Ph. Eur. Monograph 1646

Virology Division

The Hepatitis E virus RNA: The plasma pool is tested using a validated nucleic acid amplification technique (2.6.21). A positive control with 2.5 log10 IU of hepatitis E virus RNA per mililitre and, to test for inhibitors, an internal control prepared by addition of a suitable marker to a sample of the plasma pool are included in the test. The test is invalid if the positive control indicates the presence of inhibitors. The pool complies with the test if it is found non-reactive for hepatitis E virus RNA.

Proposed Text

Virology Division

Acknowledgments

JRCS Keiji Matsubayashi

NIID, Japan Saeko Mizusawa Yoshiaki Okada

Thomas Gärtner

WHO Ana Padilla

Collaborative study participants

PEI Johannes Blümel Kay-Martin Hanschmann Roswitha Kleiber Sigrid Nick Micha Nübling Gudrun Winskowsky

Institute of Virology, Bonn Felix Drexler Victor Corman

Virology Division

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