2. HBV viral load 측정 및 임상적 의의 진단검사의학과이희주

3. Content v 임상적 의의 v 측정법

4. Scheme of Hepatitis B Virus Outer lipid envelop containing HBsAg HBV DNA DNA Polymerase Inner protein core (HBcAg) Secreted HBeAg HBsAg

5. Hepatitis B Virus Genome HBcAg HBeAg DNA Polymerase HBsAg

6. HBV DNA v Reliable marker of active HBV replication v Detectable within a few days after infection v Not stable over time and depend on the infection phase –Immunotolerance after acute infection: high levels of viral replication –Immuno-elimination phase: generally lower, often fluctuating HBV DNA –‘Clinical latency’ phase: very low or undetectable levels of viral replication –Reactivation phase: high levels v Not affected by the severity of liver lesions

7. Practical use of HBV quantification v Dx of HBV infection –Acute hepatitis B : not necessary –Chr. HBV infection : necessary whether or not virus replicating –Inactive carrier < clinically active chronic Active Hepatitis –<10 5 HBV DNA copies/mL : inactive carrier v Assessment of disease severity &prognosis –Valuable prognostic information v Treatment of HBV infection –Decision to treat –Selection of optimal therapy –Treatment monitoring : u Non responder : no change during therapy u Responder : significant decrease

8. Algorithm for use & interpretation of laboratory tests

9. Molecular methods for HBV DNA v v Hybridization – – Hybrid capture (Digene): Antibody capture – – Liquid hybridization (Abbott): Radioactive – – branched DNA (Bayer): Signal detection system v v PCR – – PCR, Nested PCR: 정성검사법 – – Amplicor-HBV (Roche), Real-time PCR: 정량검사법

10. Hybridisation assays vs Amplicor Weeks of therapy HBV DNA Copies/ml – Predict relapse – Predict escape mutants – Predict risk of post OLT recurrence Amplicor sensitivity Hybrid sensitivity

11. Characteristics of each tests Assay Vol. of Sample sensitivity Linearity (copies/ml) GenotypeIndependent pg/m l copies/ml bDNA (Bayer) 10 ㎕ 2.1 7×10 5 7×10 5 ∼ 5×10 9 A,B,C,D,E,F Hybrid Capure (Digene) (Digene) 30 ㎕ 1 ㎖ 0.50.02 1.4×10 5 5×10 3 2×10 5 ∼ 1×10 9 5×10 3 ∼ 3×10 6 A,B,C,D Liquid hybridization (Abbott) (Abbott) 100 ㎕ 1.6 4.5×10 5 5×10 5 ∼ 1×10 10 D>A PCR-Amplicor (Roche) (Roche) 50 ㎕ 0.00 1 4×10 2 4×10 2 ∼ 1×10 7 (A),B,C,D,E Molecular Beacon 10- 50 ㎕ - 〈 50 50 ∼ 1×10 9 A-F

13. Available HBV DNA tests 10 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 Digene Corp. Roche Molecular Systems Bayer Corp. HBV Digene Hybrid-Capture I HBV Digene Hybrid-Capture II Ultra-sensitive Digene Hybrid-Capture II Amplicor HBV Monitor Cobas Amplicor HBV Monitor Versant HBV DNA 1.0 Versant HBV DNA 3.0 Cobas Taqman HBV DNA Abbott Molecular Diagnostics Abbott HBV DNA Kit Graph adapted from J. Hepatol., 39, S3-S25, 2003 Graph adapted from J. Hepatol., 39, S3-S25, 2003

14. Microwell Target RNA Target Probe* Pre-amplifier* Amplifier* with hybridized Label Probes* Capture Probe (solution) Capture Probe (microwell) * components containing Iso-C/Iso-G bDNA 3.0 Technology

15. Digene Hybrid Capture ® II System

17. RNA probe with target nucleic acid Hybridization

18. DenatureHybridizeCapture Detec t Read

19. Cobas amplicor (PCR hybridization)

20. Geometric Plateau Linear Variability High precision Classic PCR quantification

21. log (F2/F1) Target cycles log (F2/F1) Crossing Point (Cycles) log (copy number) Unknown Sample Standard Curve cycles Real time PCR

23. 원리 : SYBER Green Real time PCR 원리 : SYBER Green

24. 원리 : TaqMan ® ( 5' nuclease assay) Real time PCR 원리 : TaqMan ® ( 5' nuclease assay) R Q forward primer reverse primer 3' 5' 3' 5' 1. polymerisation probe R = reporter dye Q = quencher dye 3' 5' 3' 5' 4. polymerisation completed Q R 3' R Q 5' 3' 5' 2. strand displacement Q 3' 5' 3' 5' 3. cleavage R

25. Real time PCR 원리 :

26. HBV DNA 정량 방법 v Digene Hybrid Capture 15 기관 (54%) v Bayer bDNA 6 기관 (21%) v Roche Cobas Amplicor 6 기관 (21%) v Arthus RealArt 1 기관 (4%)

29. HBV DNA by Amplicor in KHMC

30. HBV DNA by Digene in KHMC

31. Comparison between Cobas vs Digene assay in KHMC

32. Summary v HBV DNA : Reliable marker of DNA replication v Use : Dx, assessment of dis severity & Px v The limited range of linearity with conventional molecular methods v Real-time PCR methods showed the widest detection ranges

33. 감사합니다.

34. HBV DNA 정량검사의 의의 v HBV 증식을 측정하는 가장 정확한 검사법 – 건강보균자와 만성간염환자를 분류 u 10 5 HBV DNA copies/mL (NIH consensus conference) – 간 손상 정도 파악 u 10 4 HBV DNA copies/mL 이하는 간손상정도가 경미 v 항바이러스 약제 치료시 치료효과의 판정 v 항바이러스 약제 치료 방침 결정 –HBV DNA > 200 pg/mL( 약 6×10 7 copies/mL) u 인터페론 치료가 효과적이지 않은 경우 많음