www.pei.de Proposed 1 st IS for Hepatitis E Virus RNA WHO/BS/09.2126 S. Baylis, Division of Virology, Paul-Ehrlich-Institut, Langen, Germany SoGAT XXII 14 th -15 th April 2011, Rome, Italy
Presented at: 3 rd WHO CC Meeting 7 th -8 th March 2011, NIBSC, UK
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 Hepatitis E Virus (HEV) 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
Background Project proposed at the 2 nd 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)
1 st Collaborative Study Aim & Approach 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
HEV Strains Investigated in 1 st Study Genotype Virus strain HEV RNA (copies/ml) Anti-HEV IgM/IgG ALT (IU/L) 3a HRC-HE104 1.6 x 10 7 -/- 36 3b JRC-HE3 2.5 x 10 7 +/- 398 3f RKI 1.3 x 10 6 -/- Negative 4c HRC-HE15 1.0 x 10 6 -/- 505
1 st Collaborative Study Labs & Methods 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
Example - Qualitative Analysis of HRC-HE104 (Genotype 3a) Nominal concentration (log 10 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
Quantitative Analysis of HEV Panel Virus strain HRC-HE104 Nominal concentration log 10 copies/ml N Geometric mean Median Min. Max. 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
Analysis of Titres and C T Values - HRC-HE104
1 st Collaborative Study Conclusions 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 log 10 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)
1 st Collaborative Study Outcome 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 1 st study which tested negative for HIV-1/2 RNA, HCV RNA, HBV DNA Roche TaqScreen MPX HEV RNA and anti-hev (IgM and IgG)
Candidate WHO Standard 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 log 10 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
2 nd Collaborative Study 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
Log 10 copies/ml 2 nd Collaborative Study contd. Data analysis in progress Stability studies are on-going Submission to ECBS June 2011 Candidate Mean log 10 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
Acknowledgements Keiji Matsubayashi, Japanese Red Cross Hokkaido Blood Center Collaborative study participants
NIID Candidate WHO Candidate