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

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

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

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

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

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 2.0 20 Siemens Healthcare Versant 3.0 HBV DNA b(dna) 350 Versant HBV DNA 1.0 kpcr 13 1 10 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 HBV DNA copie/ml

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)

Correlation evaluation of RT and Comparator CTM 1.0

VERSANT HBV DNA kpcr

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

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

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 (13.39-570,332,000) 11 (2) Invalid result Target not detected Genotype A, B, C, D, E

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

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 15 1 10 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 HCV RNA IU/mL

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

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

VERSANT HCV RNA kpcr

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 0,00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Replicate 1 Replicate 2 Replicate 3 CV mean = 0.09

Correlation of HCV RNA levels determined by Versant kpcr and Cobas TaqMan N. 78 R = 0.6449 Significance level P< 0.0001

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

Overall concordance between the CAP/CTM and ART assays Cobas Taqman assay TND <15 IU/mL (detected) 15 IU/mL Versant kpcr assay TND 4 9 1 <15 IU/mL (detected) 3 12 2 15 IU/mL 2 19 78

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

VERSANT HCV GENOTYPE ASSAY (LIPA)

HBV genotype analysis methods comparison Basaras Intervirology 2013

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

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

Maximum likelihood phylogenetic tree of NS5B-sequences

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

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