Laboratory Diagnostics in Hepatitis

T. Mazzulli, MD, FRCPC

Department of Microbiology

UHN/Mount Sinai Hospital

Objectives

Review the serologic diagnosis of viral hepatitis

Review the methodologies available for molecular testing and describe some of the advantages and disadvantages

Discuss the currently available commercial assays that are available and those which are in use in Toronto

Discuss the use of molecular methods for genotyping and resistance testing

Hepatitis A - Diagnosis

Three serologic markers available:1. Hepatitis A Total (IgG and IgM) antibody

2. Hepatitis A IgM

3. Hepatitis A IgG

First tests available since 1978

No antigen test

Antibody response is similar following vaccination or wild type infection

Incubation time is 7 to 28 days

FecalHAV

Symptoms

ALT

IgM anti-HAV

Total anti-Total anti-HAVHAV

Months after Exposure

Tit

erT

iter

Typical Serologic Course

0 1 2 3 4 5 6 12

24

Hepatitis A Virus Infection

Laboratory Tests for HBV

Serology:– Many tests available – most common tests are

Enzyme Immunoassays (EIAs, MEIAs)– First tests available in 1972– For every rule, there is an exception/caveat– No single test tells you everything

Molecular:– HBV DNA (quantitative)– HBV genotyping– HBV resistance testing

Hepatitis B – Laboratory Tests

1) HBsAg (Hepatitis B surface antigen):

• if positive, person is infectious• Sensitivity = 0.15 ng/ml• Specificity = 99.5%

2) Anti-HBs (Antibody to HBV surface antigen):

• indicates immunity to HBV and protection from disease

• Protective level is >10 IU/ml

Serologic markers:

Hepatitis B – Laboratory Tests

3) Anti - HBc (Antibody to HBV core antigen):

• Total - indicates past or active infection; present whether person is immune or chronic carrier

• Specificity = 99.8% to 99.9%• IgM - early indicator of acute infection• No antigen test

Serologic markers:

Hepatitis B – Laboratory Tests

4) HBeAg (Hepatitis Be antigen):

• indicates person is highly infectious

• Selecting patients for therapy

5) Anti-HBe (Antibody to HBVe antigen):

• prognostic for resolution of infection; less infectious; spontaneous seroconversion in 10 to 20% of healthy adults per year

Serologic markers:

Acute Hepatitis B Virus Infection with RecoveryTypical Serologic Course

Weeks after Exposure

Titer

Symptoms

HBeAg anti-HBe

Total anti-HBc

IgM anti-HBc anti-HBsHBsAg

0 4 8 12 16 20 24 28 32 36 52 100

Progression to Chronic Hepatitis B VirusTypical Serologic Course

Weeks after Exposure

Titer

IgM anti-HBc

Total anti-HBc

HBsAg

Acute(6 months)

HBeAg

Chronic(Years)

anti-HBe

0 4 8 12 16 20 24 28 32 36 52 Years

Virological and Biochemical Course of Chronic Hepatitis B

Disease Phases in Chronic HBV Infection

Phase HBsAg HBeAg Anti-HBe

ALT HBV DNA range

Immune Tolerant

+ + - Normal >8 log IU/mL

Immune Clearance

+ + - Normal or elevated

3-8 log IU/mL

Inactive Disease

+ - + Normal <3 log IU/mL

HBeAg-negative Chronic HBV

+ - + Normal or elevated

3-8 log IU/mL

Interpretation of Serologic Tests in Hepatitis B

Hepatitis B – Laboratory Tests

Serologic markers – caveats:Persistent HBsAg for >6 mos = chronic infectionHBsAg and anti-HBs may co-exist in up to 24% of chronically infected individuals; likely due to mutations in the “a” determinant of the S gene– Surface antigen escape mutants described in infants

infected with HBV after HBIG + vaccination and in Liver transplants after prolonged HBIG

Anti-HBc IgM may persist for up to 2 years in 20%; chronically infected individuals may have low titres which rise during acute flares

Hepatitis B – Laboratory Tests

Serologic markers – caveats:Precore or HBeAg negative mutants:– Due to mutation in precore (abolishes HBeAg

production) or core promoter region (down-regulates HBeAg production)

– No effect on viral replication (may be enhanced)– More difficult to treat; greater risk of cirrhosis

Co-infection with HCV may suppress both HBeAg and HBsAg

HBV Viral Genome Organization

HBsAg

HBcAg

HBeAg

3200 Base Pair Genome

Hepatocyte receptor bindng site

HBV DNA Polymerase

Protein that transactivates transcriptional promotors

Hepatitis B – Laboratory Tests

Serologic markers – caveats:

Isolated HBcAb may be due to:– Remote infection (immune or chronic carrier)– “Window” period between HBsAg and HBsAb– Co-infection with HCV– False positive test result – HBcAb is marker

most prone to false positives

HBV DNA may help sort this out

Laboratory Tests for HCV

Serology:Detection of anti-HCV antibodiesSerologic test available since 1990

Molecular:HCV RNA detectionDetermination of HCV genotype Viral load determination

Laboratory Tests for HCV

Serology:Screening:– 3rd generation EIAs measure antibodies directed

against recombinant peptides NS4, core, NS3, and NS5 proteins

– Sensitivity = 97%– Detects antibodies within 6 to 8 weeks– No HCV IgM test available

Confirmatory/supplementary:– RIBA, LiPA, Second EIA, HCV RNA

Serologic Pattern of Acute HCV Infection with Progression to Chronic Infection

Symptoms +/-

Time after Exposure

Tit

eranti-HCV

ALT

Normal

0 1 2 3 4 5 6 1 2 3 4YearsMonths

HCV RNA

TREATMENT

DiagnosisDiagnosis

Serological assays

Qual HCV RNA

PrognosisPrognosis

Liverhistology

DecisionDecisionto treatto treat

ALTLiver histologyQual HCV RNA

TreatmentTreatmentdurationduration

Genotyping Viral load

ResponseResponseand resistanceand resistance

assessmentassessment

Qual HCV RNAViral load

Rational Use of HCV Diagnostic Tests

Hepatitis D Virus - Diagnosis

• Anti-HDV Total (IgG & IgM) available• Incubation time – similar to Hepatitis B• High titres of HDV antibodies indicate ongoing

chronic infection• Available only at National Microbiology Lab in

Winnipeg

Hepatitis E Virus - Diagnosis

• Both IgG and IgM antibody tests are available

• Incubation period – 7 to 28 days• No domestically acquired cases in Canada• Available only at the National Microbiology

Lab in Winnipeg

Molecular Tests for Hepatitis

Hepatitis Virus – Molecular Tests

Molecular assays available as follows:– Commercial assays for HBV DNA and HCV RNA– In-house assays for HAV RNA & HDV RNA– No molecular assay for HEV RNA

HCV RNA & HBV DNA, plasma or serum must be separated from cells within 6 hrs and plasma can be stored at 4oC for several days or -70oC for long-termNo licensed tests for diagnostic purposes; all tests are for monitoring or donor screening– HCV RNA will be done in HIV or other

immunocompromised patients if requested

Hepatitis Virus – Molecular Tests

Lower limit of Detection (LLD) does not equal dynamic (linear) range of quantitative assays– Determined by PROBIT analysis to determine

the value that is consistently detected 95% of the time

Results of different assays may (HBV) or may not (HCV) be interchangeable

Nucleic Acid Amplification Tests (NAAT) for Detection of RNA/DNA

Quantitation of RNA or DNA may be reported as copies/ml or IU/ml

Conversion factor for copies/ml to IU/ml is not the same for different assays measuring the same target or different targets– HBV DNA: 5.82 copies/IU– HCV RNA: PCR - 2.4 copies/IU; bDNA: 5.2 copies/IU

Coefficient of variation (COV) may range from 15 to 50%

HBV DNA Quantification Assays

Assay Sensitivity (pg/ml)*

LLD (copies/ml)*

Linearity (copies/ml)

Coefficient of Variation

Versant bDNA v3.0 (Siemens)

2.1 2 x 103 2 x 103 to 1 x 108

15 - 37%

Hybrid Capture II (Digene)

0.02 to 0.5 5 x 103 5 x 103 to 6 x 107

10 – 15%

Liquid Hybridization (Abbott)

1.6 6 x 105 5 x 105 to 1 x 1010

12 – 22%

Cobas Amplicor Monitor (Roche)

0.001 2 x 102 2 x 102 to 2 x 105

14 – 44%

Cobas Taqman (Roche)

35 (Manual)

70 (Automated)

2 x 102 to 1 x 1010

16 – 54%

RealArt HBV PCR (artus/Qiagen)

10 1 to 4 x 108

A. Lok et al. Hepatology 2001;34; J. Servoss et al. Infect Dis Clin N Am 2006;20; B. Weber. Future Drugs 2005*283,000 copies/pg; 5.26 copies/IU

Measuring HBV DNA

Gish and Locarnini, Clin Gastro Hep 2006

Comparison of Quantitative HBV DNA Assays

Yao J et al. J Clin Microbiol 2004:42(2)

Versant vs. Digene:

R2 = 0.9849

Versant vs. Cobas:

R2 = 0.7711

Versant 3.0 vs. Versant 1.0:

R2 = 0.9001

HCV RNA Detection AssaysAssay Method LLD*

(IU/ml)a

Linearity (IU/ml)

Versant Qualitative (Siemens) TMA 5 - 10 NA

Amplicor Qualitative v2.0 (Roche) RT-PCR 50 NA

Ampliscreen (Roche) RT-PCR 50 NA

Amplicor Monitor v2.0 (Roche) RT-PCR 600 600-800,000

Cobas Taqman (Roche) RT-PCR 15 15 – 1 x 108

Abbott RealTime (Abbott) RT-PCR 12 - 30 10 – 1 x 107

Versant Quantitative v3.0 (Siemens)

bDNA 615 615 -7,700,000

S. Chevaliez et al. World J Gastro 2007;13; J Scott et al. JAMA 2007;297; A. Caliendo et al. J Clin Microbiol 2006;44

*LLD = Lower Limit of Detection;

aConversion factor IU/ml to copies/ml varies with each assay (e.g. PCR: 1 IU/ml = 2.4 copies/ml; bDNA: 1IU/ml = 5.2 copies/ml)

HBV DNA in Clinical Practice

Routine monitoring on therapy to assess response to treatment– Every 3 months X years on oral agents– Every 1 month X 6-12 on PEG/IFN

Routine monitoring off therapy to estimate prognosis and to evaluate need for treatment– Every 6 –12 months normally– Diagnosis of occult HBV infection

Laboratory Tests for HCV

Molecular:Both qualitative and quantitative HCV RNA assays available

Used for treatment monitoring (and in some circumstances for confirmation of positive or indeterminate serology)

HCV RNA is detectable 2 to 14 days after an exposure

Ministry of Health Ministère de la Santé and Long-Term Care et des Soins de longue durée

Laboratories Branch Direction des Laboratoires

P.O. Box 9000, Terminal “A” C.P. 9000, Terminal “A” (81 Resources Road, Etobicoke) (81 Chemin Resources, Etobicoke) Toronto, Ontario M5W 1R5 Toronto (Ontario) M5W 1R5

Telephone: (416) 235-5737 Téléphone: (416) 235-5737

Facsimile: (416) 235-6197 Télécopieur: (416) 235-6197

PHL LAB INFORMATION FORM FOR HEPATITIS C RNA AND HEPATITIS B VIRAL LOAD

THIS IS NOT A DATA SHEET, please attach to PHL test requisition Minimum volume 2.5 mL serum, removed from clot within 4 hours and submitted frozen to the lab.

PATIENT'S NAME: ___________________________________ PHL LAB#: _________________ DR’s NAME ______________________________

HEPATITIS C (HCV) RNA - QUANTITATIVE

Baseline and Genotyping (Pre-Treatment)

Quantitative Viral Load - Week 12 of treatment Quantitative Viral Load – due to change in treatment/dose ______ weeks HEPATITIS C (HCV) RNA - QUALITATIVE

Query the presence of active HCV infection

(HIV immunocompromised, infant of HCV positive mother, patient with anti HCV indeterminate result, 8-10 weeks post exposure, etc).

Confirm successful treatment at 4 weeks (genotypes 2 and 3) On Treatment _________ weeks Post Treatment _________ weeks (2 samples less than the detection limit (<50 IU/mL) and 6 months apart are required to confirm successful treatment. No follow up required unless there is a new exposure).

HEPATITIS B (HBV) VIRAL LOAD (NOTE: Test is not useful for diagnosis. Research use only)

Pre-Treatment for HBV On Treatment _________ weeks Post Treatment

LFTs/Clinical Information: ALT ___________ AST __________

Genotyping

Used for:– Detection of mutations that confer resistance to

antiviral agents– Genotyping of isolates for epidemiological purposes;

categorizes patient isolates into 8 different HBV genotypes (A to H) and 6 different HCV genotypes (1 to 6 with 24 subtypes)

Methods include:– Sequencing– Hybridization (Line Probe Assay, Trugene Assay)

Laboratory Diagnosis of Resistance

ProsPros ConsCons

SequencingDiscovers Labor-intensive

new mutations Low sensitivity

(15-20% pop.)

Line Probe High throughput Detects known

High sensitivity mutations only

(5-10% pop.)

Marker lineConj.cont.Amp.cont.

L180

M180

M204

V204

I204

V207L207

M207

I207

Amplified Amplified targettarget

StreptavidinStreptavidin

BiotinBiotin

Alkaline Alkaline PhosphatasePhosphatase

Purple Purple precipitateprecipitate

ChromogenChromogen(NBT/BCIP)(NBT/BCIP)

DNA-probeDNA-probeNitrocellulose Nitrocellulose

stripstrip

InnoLiPA Principle

InnoLIPA HBV Drug Resistance

M arke r line

C onj. C on tro lA m p.C ontro lL80 W TV 80 M u tant

80 M u tantV 173 W T G 173 W TL173 M utantL180 W TM 180 M u tantA 181 W TT181 M utan tV 181 M utan tM 204 W TV 204 M utan t

204 M utan tS 204 M utan tN 236 W TT236 M utan t

I

I

1 - 2 - 3 -4 - 5 - 6 - 7 - 8 - 9 -

10 - 11 - 12 - 13 - 14 - 15 - 16 - 17 - 18 - 19 -

HBV Resistance Testing

HBV Resistance Testing

InnoLiPA vs. Sequencing

Hussein et al, J Clin Micro 2006

Drug Resistance Report

  

Present Mixed Absent

A181V    X

A181T   X  

N236T   X  

LAMIVUDINE Present Mixed Absent

L80V    X

V173L X    

L180M  X  

M204V  X

M204I   X

M204S   X  

ADEFOVIR

Interpreting HBV DR Reports

 

Resistance Mutation

  Lamivudine Resistance

L180M +M204V

Adefovir Resistance

N236T

Adefovir Resistance

A181V/T

Sensitive AdefovirTenofovir

LamivudineEntecavir

TelbivudineEmtricitabine

Lamivudine Tenofovir Entecavir

Emtricitabine

Resistant or Reduced Susceptibility

LamivudineEntecavir

TelbivudineEmtricitabine

Clevudine 

AdefovirTenofovirClevudine

Adefovir 

 L180M + M204V/I are the key lamivudine-resistant mutations

 A181V/T leads to 4-fold increase in IC50

  N236T leads to 7-fold increase in IC50

Diagnostics in Viral Hepatitis: Summary

Serology remains the cornerstone for diagnosis and screeningNAAT is critical to patient managementOf the many NAAT tests available, PCR, bDNA and TMA remain most popular– Sensitivity and dynamic range varies between assays– Standardization allows (to some degree)

interchangeability of the results with different assaysResistance/Genotyping requires amplification first– Increasing role in making treatment decisions as more

drugs become available for HBV