Hepatitis A, B, C, D, E, G,…diagnostic tools and their use

Geert Leroux-RoelsLaboratorium voor Klinische Biologie

UZ Gent

Human Hepatitis VirusesDiscovery and characteristics

Virus Discovered Genome Envelope Classification

HAV 1975 ss-RNA Picorna

HBV 1970 ds-DNA HBsAg HepaDNA

HCV 1989 ss-RNA E1-E2 Flaviviridae

HDV 1978 ds-RNA HBsAg viroid

HEV 1990 ss-RNA Caliciviridae

HGV 1995 ss-RNA E1-E2 Flaviviridae

TTV 1997 ss-DNA Circoviridae

Human Hepatitis VirusesDiagnostic tools

Virus Serology Antibodies Antigens

Molecular det/quant

Advanced molec anal

HAV IgM, IgG

HBV HBsAl HBeAl HBcAl

HBsAg HBeAg

det/quant genotype resistance

HCV IgG det/quant genotype

HDV Anti-delta Delta-Ag

HEV IgM, IgG

HGV IgG

TTV IgG det

Epidemiology of HAV

Hepatitis A virus – diagnostic tools• Serology

– Anti-HAV IgM acute HAV infection– Anti-HAV Totaal immunity

- natural- vaccine-induced protective level 10-30 IU/L

• HAV detection in blood and blood productsin faeces, saliva, …in shelfish, food productsin water,sewage,

HAV vaccines• Children (2-15)

Havrix Junior (720 U/dose), 0.5 ml ml/dscheme : 0 and 6-12 mo (2 doses, IM)

• Adults (>15)Havrix 1440 (1440 U/dose, 1 ml/dscheme : 0 and 6-12 mo (2 doses, IM)

Epidemiology of HEVepidemische en sporadische

gevallen

Rare cases in western countries after recent travel in endemic area

Hepatitis E virus

Serology anti-HEV IgM anti-HEV IgG

HEV-RNA

Hepatitis B virus Diagnostic tools and

interpretation

HBV: genome and gene products

3200 baseparen4 open reading frames7 proteins• large (preS1-preS2-S)• Middle (preS2-S)• Small or HBsAg• nucleocapsid or HBcAg• secreted HBe-Ag• Polymerase• X protein

Diagnostic markers of HBV infection

Antigens Antibodies

HBsAg anti-HBsHBcAg anti-HBc-Tot

anti-HBc-IgM

HBeAg anti-HBe

Inflammation and liver cell damageTransaminases ALT/ASTen other biochemical markers

Detection/quantific. of HBV DNACommercial assays- Branched DNA (Bayer)- PCR (Roche Amplicor)In-house nested PCRIn-house real-time PCR

Usefulnessof HBV-DNA quantification

• Diagnosis– Acute HBV infection : HBV DNA is not useful– Chronic HBV infection – Is HBV replicating ?

• HBeAgpos : not useful• HBeAgneg/anti-HBepos : useful, “threshold value” ?

• Prognosis• Therapy

– Decision to treat : ALT, biopsy, HBeAgpos

– Selection treatment– Monitoring : HBV DNA, ALT, HBeAg, anti-HBe,

Evolution of an acute, self-limited HBV infection

Serologic profile of a chronic HBV infection,with a late seroconversion to anti-HBe

Case 1

• 41 years old Afghan male• political refugee• In training for assistant-cook• Medical screening exam for ‘hepatitis’• No symptoms• No history of hepatitis

Results - blood chemistryTest Result Unit Ref. intervalBili Tot 0.66 mg/dL 0.00-1.00Bili Dir 0.10 mg/dL 0.00-0.20Bili Ind 0.55 mg/dL 0.20-0.80ALT 16 U/L 0-41AST 21 U/L 0-38LDH 351 U/L 0-480Alk Fosf 106 U/L 0-128GT 20 U/L 8-61

Results - serology

Test Result

HBsAg Positive

Anti-HBs 46 IU/L

Anti-HBc-Tot Positive

Anti-HAV-IgM Negative

Anti-HCV Negative

Question 1Can HBsAg and anti-HBsoccur concomitantly ?

[HBsAg-anti-HBs] immune complexes

• Are present during the clearance phase of an acute HBV infection in the “window phase” and in some chronic HBV patients

• Routine tests for HBsAg/anti-HBs do not detect immune complexes (IC)

• IC dissociatie (ICD) by treatment of serum (100µl) with HCl (50 µl, 0.5 N, 1h at 37°C) and neutralisation with NaOH (50 µl, 0.5N) (Rabenau et al. 1996)

• Research tests can detect IC’s

HAV serostatus• anti-HAV-IgM antibodies are only

present in the acute phase of HAV infections

• ALT/AST activities are normal• Anti-HAV status (IgG antibodies) would

have been useful to see whether this person still needs HAV vaccination– Food handling– Chronic HBV infection

Results – additional HBV tests

Test Resultaat

HBsAg Positive

Anti-HBs 46 IU/L

Anti-HBc-Tot Positief

HBeAg Negative

Anti-HBe Positive

HBV DNA 8 400 gEq/ml

Question 2Is this person infectious ???

Question 3Does he need treatment ?

• Infectivity is LOW– Spouse and daughter show no signs or

markers of HBV infection (HBsAlneg)– Vaccination of household (sexual contact) !– Twinrix is an alternative for Engerix-B

• Therapy is not indicated– Normal transaminases, low DNA– Follow-up : annually ?

Case 2• Man, born in 1947• 1986 – Ulcerative colitis • 1992 - liver enzymes slightly elevated, no

further investigations • 1998 – abnormal liver tests, alcohol

consumption,• June 1998 – exacerbation of colitis• Nov 2001 - exacerbation of colitis

Evolution of laboratory testsTest May 1998 June 1998 June 1999 Sep 2003

ALT 129 35 24 42AST 94 30 17 33-GT 120 56 28 58HBsAg Pos Pos Pos NegAnti-HBs Neg Neg Neg NegAnti-HBc Pos Pos Pos PosHBeAg Neg NegAnti-HBe Pos Pos HBV DNA <0.7 mEq/ml

Spontaneous seroconversions in chronic HBV

• HBeAg to anti-HBe seroconversion– Inflammation (ALT/AST) = 8-15% per year– Normal ALT <2% in children < 3 years

4-5% in patients > 3 years

• HBsAg to anti-HBs seroconversion– Active HBeAg- hepatitis 0.5% /jaar– Asymptomatic HBeAg- carrier

• In a western population 1-2% /jaar• Post perinatal infection 0.05-0.8% /jaar

HBeAg/anti-HBe seroconversion

• Transition to ‘inactive carrier’– Normalisation of transaminases– Low viral replication and HBV DNA (<105 gEq/ml)

• Active hepatitis with HBeAg-/anti-HBe+

– Elevated transaminases– High(er) HBV DNA (> 105 gEq/ml)– Precore mutation (G1896A: stop codon)– Core/precore promotor mutations

Hepatitis B core and e antigen

-29 aa 183aa 1

HBeAg precursor

HBcAg

aa 1 aa 183

mature HBeAg

aa -10 aa 149 aa 1

AU

G

AU

GP

G18

96A

Interpretation of the serology and its evolution in this patient (case 2)• Spontaneous seroconversion of HBsAg

to anti-HBs• No detectable [HBsAg-anti-HBs] IC’s• HBV DNA detection, quantification and

sequence analysis are needed for a correct diagnosis and prognosis

Case 3

• Man, 45 years• Traffic accident => brain death• Possible organ donor (liver ?)• Serology :• HBsAgneg, anti-HBsneg,anti-HBcpos, anti-

HCVneg,anti-HIVneg, CMVneg

• Biochemistry : no abnormalities

Prevalence of “anti-HBc alone” in low seroprevalence populations

Country (year)

Study group

Population studied

Prevalence number (%)

HBV DNA+ number (%)

Germany 1998

Unselected 18-70 yrs

5300 65 (1.4) 5/65 (7.7)

Germany 1997

IVD users 377 94 (25)

Switserland 1996

Pregnant 9000 104 (1.2) 0/104 (0)

Germany 1998

Blood donors

15,000 27 (0.2) 2/27 (7.4)

Switserland 1999

Blood donors

9751 51 (0.5) 2/51 (3.9)

Prevalence of “anti-HBc alone” in high seroprevalence

populationsCountry (year)

Study group

Population studied

Total HBV(%)

Anti-HBc alone (%)

USA (1984) Male homosexuals

1461 55.2 3.3

USA (1984) Prison inmates

685 18.8 5.0

Senegal (1987)

Africans 3033 88.7 20.8

Hong-Kong (1988)

Chinese 1801 68 11.9

Hong-Kong (1992)

Chinese 4001 40 1.3

Possible causes of “Anti-HBc alone” serology

1

1 = window phase

2

2 = late immunity, only anti-HBc persists

3 = chronic infection with low replication/production of HBV or with HBsAg mutant

3

“Occult hepatitis”

• Typical serology

HBsAgneg, anti-HBsneg, anti-HBcpos

• HBV DNA < detection limit of routine PCR test (e.g. < 200 gEq/mL)

• HBV DNA in the liver

Influence of HCV infection

on HBV replication• HCV core protein suppresses HBV

replication with a factor 2 to 4

• HCV infection reduces the expression of HBsAg in the liver

• Treatment of HCV with IFN also has an effect on HBV

“anti-HBc alone” can be :

1. Window phase2. Late immunity – only sign of past infection3. Chronic infection – “occult infection”4. HBsAg mutant5. “False” positive test result

• really “false positive” – low signal, 2nd EIA

• Core-binding antibodies (IgM vs IgG)

-Nested

PCRLo PCRHi PCRHi PCRHi PCRHBV DNA

+ or -+ or -++--Anti-HBe

--- -++HBeAg

++++++Anti-HBc

+-+++++++HBsAg

== to Hi= to HiHiHi=ALT

HBVReplication

HostImm Resp

LiverDisease

ToleranceActive Hepatitis

HBeAg+ HBeAg-

mutant serocon

Occult Inactivecarrier

Lab Tools IgM anti-HDV HDV-Aganti-HDV HDV-RNA

Hepatitis C virus Diagnostic tools and interpretation

The HCV genome and expressed polyprotein

• Indirect markers – anti-HCV

• ELISA 3-4th generation• Confirmation tests RIBA, LIA

• Direct markers = HCV genome • RT-PCR qualitative and quantitative• Branched-DNA (quantitative) • Genotyping• HCV core antigen

Markers of HCV infection

Anti-HCV assays: generations 1 to 4

1989 1993 2000

Generation 1 2 3 4

Antigens NS3/4 C,NS3,NS4 C,NS3,NS4A/B,NS5A

Sensitivity 95-98% >99%Specificity <95% 99.8%Lag time (wks) 16-24 4-12 4-8

Confirmation of anti-HCV+

• Repeat ELISA on the same sample• Another ELISA on the same sample• Same ELISA on a new sample• Confirmation assays

– RIBA (recombinant immunoblot assay)– LIA (Line immuno assay)

• Confirmation by PCR

Molecular tests for HCV

• Molecular detection – qualitative

• Molecular quantification

• Genotyping

HCV-RNA detection – qualitative

Assay Manufacturer Method Lower limit of detection

Amplicor HCV v2.0

Roche Molec Systems

Manual, qualit PCR

50 IU/ml

Cobas Amplicor HCVV2.0

Roche Molec Systems

Semi-automated, qual PCR

50 IU/ml

Versant HCV RNA qualitative assay

Bayer Corpor, Diagnostics Div

Manual qualit TMA

10 IU/ml

Qualitative HCV-RNA tests

• Confirms diagnosis of HCV infection• Useful for the early diagnosis of acute

hepatitis C• Demonstrates the presence of active

infection• « Gold standard » for documenting

response to therapy

Interpretation of combined anti-HCV and HCV-RNA

resultsAnti-HCV HCV-RNA Interpretation

Neg Neg No infectionEarly post exposure (<1 week)

Neg Pos Acute infection (no Ab’s yet)Chronic in immune deficient p.

Pos Neg Past, recovered infection

Pos Pos Acute HCV infection with Ab’sChronic HCV infection

Treatment of HCV• Clinical studies evaluating the

efficacy of different treatment protocols : drugs, doses, duration, … have revealed the importance of 1) HCV-RNA quantification 2) genotyping

Quantitative HCV RNA tests

• Generally less sensitive than qualitative HCV-RNA test => Taqman !

• Positive in >95% of untreated patients with chronic hepatitis C

• Useful in predicting response to therapy and determination of early virological response (EVR)

Range of HCV-RNA assays

Amplicor Monitor v2.0(PCR)

VersantHCV RNA v3.0(b-DNA)

LCx 25HCV RNA

SuperQuant

20 200 2,000 20,000 200,000 2,000,000 IU/ml

600

615

500,000

7,700,000

2,630,000

30 1,470,000

Molecular Genotyping

• Direct Sequencing– ‘Home-made’ methods: NS5B-, E1-based– 5’-noncoding: Trugene - Visible Genetics

• ‘Reverse Hybridization’– Inno LiPA (Innogenetics)

Analysis of the viral genome sequenceReverse hybridisation of PCR

amplicons

Treatment of HCV

• Interferon-

• Interferon- + Ribavirine

• Pegylated IFN- + Ribavirine

Early stopping rules

• 1997 Consensus conference– IFN monotherapy : stop therapy when

HCV-RNA (sensitive qualitative test) remains POSITIVE after 12 weeks

• 1998 McHutchison – NEJM 339:1485 – IFN + ribavirin : stop therapy when

HCV-RNA (sensitive qualitative test) remains POSITIVE after 24 weeks

n = 390 n = 390 (86%)(86%)

n = 63n = 63(14%)(14%)

2 log reduction or 2 log reduction or HCV RNA (-) HCV RNA (-)

YESYES

NONO

Week 12 (N = 453)Week 12 (N = 453)n = 253n = 253(65%)(65%)SVRSVR

n = 137 n = 137 (35%)(35%)No SVRNo SVR

n = 2n = 2(3%)(3%)

n = 61 n = 61 (97%)(97%)Fried et al. NEJM 2002;347:975-982Fried et al. NEJM 2002;347:975-982

SVRSVR

No SVRNo SVR

PEG-IFN-2a QW + ribavirin QDAll genotypes

2 log drop of HCV-RNA at week 12

• in patients treated with PEG-IFN + ribavirin• undetectable HCV-RNA or log 2 drop at

week 12, is predictive for sustained response (>60%)

• absence of 2 log drop is extremely (>99%) predictive for non-sustained response

• should lead to early stop of treatment• leads to significant cost reduction • avoids inconvenience and side effects

GENOTYPE 2 OR 3GENOTYPE 2 OR 3 GENOTYPE 1 (and 4, 5 or 6)GENOTYPE 1 (and 4, 5 or 6)==

PEG-IFN-PEG-IFN- + 800 mg ribavirin + 800 mg ribavirin24 weeks24 weeks

End-of-treatment virological responseEnd-of-treatment virological responseSustained virological responseSustained virological response

> 2 log decrease> 2 log decreaseor HCV RNA (-)or HCV RNA (-)

at week 12at week 12<2 log decrease<2 log decrease

at week 12at week 12

End-of-treatment virological responseEnd-of-treatment virological responseSustained virological responseSustained virological response

== Stop treatmentStop treatment

or continue in order or continue in order to slow evolutionto slow evolutionof liver diseaseof liver disease

HCV Genotype determinationHCV Genotype determination

Viral load quantification Viral load quantification at baseline and week 12at baseline and week 12

HCV RNA detection HCV RNA detection (sensitive qualitative assay) (sensitive qualitative assay)

at the end of treatment and 24 weeks laterat the end of treatment and 24 weeks later

Liver biopsyLiver biopsy

Bad prognosisBad prognosis==

PEG-IFN-PEG-IFN- + + 1000-1200 mgribavirin1000-1200 mgribavirin

48 weeks48 weeks

Good prognosisGood prognosis==

No treatmentNo treatment

CHRONIC HEPATITIS CCHRONIC HEPATITIS C

HCV RNA detection HCV RNA detection (sensitive qualitative assay) (sensitive qualitative assay)

at the end of treatment and 24 weeks laterat the end of treatment and 24 weeks later

Cost-benefit of monitoring early viral response (EVR) a simulation for Belgium

Cost-benefit of log2 at week 12 Assumptions and data

• Number of new cases per year = 1000• Number of treated cases = 600• Fraction of HCV genotype 1 = 70 %• Cost of quantitative HCV-RNA = 150 Euro per

test, x 2 (w0 and w12) = 300 Euro• Cost of PEG-IFN + Riba = 400 Euro per week• Diagnostic sensitivity (detection of NR) = 33%• NPV of EVR = 100%

Cost-benefit of log2 at week 12

1000 new HCV

600 treated

180 non-gt1 420 gt1

350 EVR

HCV-RNA at w12 63000

HCV-RNA at w0 63000

Cost Benefit

210 SR 140 NSR

70 No EVR

STOP 126000 1008000

w0

w12

W48 End of treatment Net savings 882000

Molecular diagnostic tools for detection and monitoring of HCV

infections• Correct diagnosis• Selection of treatment and duration• Decision to “Stop treatment”

– Reduce costs (medication, doctor visits, ..)– Reduce the discomfort and suffering of patients– Reduce the loss of labour time

– The impact on costs (direct, indirect) will increase if the diagnostic sensitivity of the EVR algorythm can be improved (>33%)

Additional Hepatitis Agents

• 12% o post-transfusion hepatitis unrelated to A-E

• 18% of acute hepatitis unrelated to A-E• Up to 40% of fulminant hepatitis no

etiology is present• Cases of acute hepatitis followed by

aplastic anemia

New viruses not proven to cause human hepatitis

• HGV• TTV• TLMV• TTV-like minivirus• Sanban• Yonban• Sen virus

HGV – GBV-C• Related to HCV - Flaviviridae• Parenteral transmission• Replicates in lymphocytes and not in

hepatocytes• HGV infection prolongs survival in HIV• Does not cause hepatitis and not even

co-morbidity in (frequent) association with HBV or HCV

Prevalence in Belgium Sheng - Doctoral Thesis - KUL 1998

HGV-RNA (%) Anti-E2 Ab (%)

Blood donors 1.8 6

Clotting disord 14.8 25.7

Haemodialysis 17 14.2

Chronic HBV 5.6

Chronic HCV 50.5

Fulminant hep 8.3

TT virus infection in acute and chronic liver diseases and in patients regularly receiving blood products in BelgiumAli S, van Pelt JF, Verslype C, Nevens F, Fevery J, Yap SH – Acta Gastroenterol Belg 2004,67:161

TTV-DNA was present in • 49% of patients with chronic HCV• 54% in patients with chronic HBV• 47% in patients receiving clotting factors• 64% in patients in chronic haemodialysis• 29.7% in (340) healthy blood donors

Analysis of samples for less common forms of hepatitis Dr. Robert Vranckx HEV-AlWetenschappelijk Instituut VolksgezondheidHDV-Ag ?Juliette Wytmansstraat 14 HGV-RNA ?1050 Brussel

Prof. Dr. Patrick Goubau HDV-AlAIDS Reference Laboratory, UCLAvenue Hippocrate 54/92B-1200 Brussels