Dosaggi Sierologici e Molecolari nelle Epatiti B e C METODI MOLECOLARI. Ombretta Turriziani Dipartimento di Medicina Molecolare

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1 Dosaggi Sierologici e Molecolari nelle Epatiti B e C METODI MOLECOLARI Ombretta Turriziani Dipartimento di Medicina Molecolare

2 Dosaggi Sierologici e Molecolari nelle Epatiti B e C Molecular Methods Key role in diagnosis and monitoring HBV and HCV infection

3 Hepatitis B and C Molecular Methods HBV HBV-DNA Qualitative assay Quantitative assay Genotype Identification Drug resistance assay HCV HCV-RNA Qualitative assay Quantitative assay Genotype Identification IL28 polymorphism Drug resistance assay

4 Molecular tests for HBV/HCV quantification Hybridization Methods Hybrid capture Technology Branced DNA assay Target amplification Methods Quantitative competitive PCR Real Time PCR

5 Dynamic ranges of commercial assays for HBV-DNA quantification Abbott Molecular Diagnostics LOQ (IU/mL) Abbott RealTime PCR 10 Artus-Biotech Real Art HBV Assay 31.6 Roche Molecular Systems Cobas Amplicor HBV Monitor 400 Cobas TaqMan HBV Siemens Healthcare Versant 3.0 HBV DNA b(dna) 350 Versant HBV DNA 1.0 kpcr HBV DNA copie/ml

6 Real time-pcr assay: possible outputs A quantitative result within the dynamic range of the assay >LLoQ Qualitative result < LLoQ Target not detected < LLoQ(detected)

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8 Correlation evaluation of RT and Comparator CTM 1.0

9 VERSANT HBV DNA kpcr

10 Correlation of HBV DNA quantification results between the Versant assay and the Cobas assay Lachaud et al 2012

11 VERSANT HBV DNA 1.0 Assay (kpcr) Quantitation of HBV genotypes Wong CL et al. 2012

12 HBV DNA analysis performed by VERSANT HBV DNA 1.0 Assay (kpcr) 537 serum samples (330 Pts) n. Samples (%) HBV DNA (IU/ml) 253 (47) Target not detected 60(11) <13 213( 46) Detected ( ,332,000) 11 (2) Invalid result Target not detected Genotype A, B, C, D, E

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14 Minimal residual viremia and viral breakthrough in cases with adefovir and lamivudine resistance

15 Dynamic ranges of commercial assays for HCV-RNA quantification LOD (IU/mL) Abbot Diagnostics LCx HCV-RNA QuantitativeAssay 25 Abbott Real time 12 Qiagen Artus HCV QS-RGQ assay 67 Roche Molecular Systems Amplicor HCV Monitor 600 Cobas TaqMan 2.0HCV Test 15 Siemens Healthcare Versant 3.0 Assay (bdna) 615 Versant HCV RNA 1.0 kpcr HCV RNA IU/mL

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17 Correlation of HCV RNA levels determined by CAP/CTM and ART E. Ogawa et al. / Antiviral Research 99 (2013)

18 Overall concordance between the CAP/CTM and ART assays E. Ogawa et al. / Antiviral Research 99 (2013)

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20 VERSANT HCV RNA kpcr

21 Intra-assay evaluation (n= 60) Versant HCV RNA 1.0 Assay (kpcr) HCV RNA (Log IU/mL) 2,50 2,40 2,30 2,20 2,10 2,00 1,90 1,80 1,70 1,60 1,50 1,40 1,30 1,20 1,10 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 0,20 0,10 0, Replicate 1 Replicate 2 Replicate 3 CV mean = 0.09

22 Correlation of HCV RNA levels determined by Versant kpcr and Cobas TaqMan N. 78 R = Significance level P<

23 Bland Altman plot of differences in HCV RNA quantification results between the VERSANT kpcr Assay and the COBAS Assay among 78 serum specimens.

24 Overall concordance between the CAP/CTM and ART assays Cobas Taqman assay TND <15 IU/mL (detected) 15 IU/mL Versant kpcr assay TND <15 IU/mL (detected) IU/mL

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26 Methods for HBVgenotyping. Chakravarty Expert Opin. Med. Diagn. 2012

27 VERSANT HCV GENOTYPE ASSAY (LIPA)

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32 HBV genotype analysis methods comparison Basaras Intervirology 2013

33 Methods for HCV genotyping Chakravarty Expert Opin. Med. Diagn. 2012

34 HCV GENOME 5 UTR strutturale non strutturale C E1 E2 NS2 NS3 NS4 NS5 3 Untraslated region core Envelope protein elicase/protease RNA polimerase 5 - UTR Region CORE 5 - UTR Region NS5B INNO LIPA RealTime HCV genotype

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38 Maximum likelihood phylogenetic tree of NS5B-sequences

39 Molecular methods : HBV DNA/HCV RNA quantification Because of their sensitivity, specificity, accuracy and broad dynamic range the use of real-time PCR quantification assay allow to adequately monitor viral kinetic and predict the risk of relapse In HCV treatment SVR is tantamount to cure Further studies are needed to evaluate the clinical significant of viremia levels below LOD

40 Molecular methods : HBV/HCV genotyping Genotype determination is needed in clinical practice to decide treatment The choice of the target genome region is crucial for HCV/HBV genotyping/subtyping Viral evolution represents a continuous challenge for scientists involved in molecular diagnosis of viral diseases. Comparative studies are useful means to improve the knowledge of genomic regions involved in genotype assignment and contribute to a better primer/probe. design