Lab Testing in the Diagnosis and Management of Viral ... Testing in the Diagnosis and Management of Viral Hepatitis 1 ... Lab Testing in the Diagnosis and Management of Viral Hepatitis

Clearing the Confusion:

Lab Testing in the Diagnosis and Management of Viral Hepatitis

1


Lab Testing in the Diagnosis and

Management of Viral Hepatitis

April 12, 2011

Your Host: Karen Riba

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Welcome

Your Host: Karen Riba

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• PAML employees will be able to receive one hour

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Welcome

Dr. Katherine Soreng, Ph.D.

Dr. Soreng received her BSc. in Biology

from the University of Washington in

Seattle. She was awarded a Ph.D. in

Immunology and Molecular

Pathogenesis from Emory University in

Atlanta, GA, publishing a thesis on

protein synthesis and cytoskeletal

elements in the Class II restricted

processing of antigen.

Speaker Information

Speaker Image

At the end of this presentation participants will be

able to:

• Identify the appropriate tests involved in the

differential diagnosis of viral hepatitis (including

acute vs chronic)

• Discuss the algorithms useful for both hepatitis B

and C antibody test confirmation

• Discuss the clinical utility of viral load and

genotype in both hepatitis B and C infection.

Learning Objectives Disclosures

Full-time employee of Siemens

Healthcare

Sr. Manager of Clinical Education

and Scientific Publications

mailto:[email protected]



2

Stages of Liver Damage

NormalLiver injury/

inflammation

Liver

fibrosisCirrhosis

Liver

failure/

liver

cancer

© Siemens AG, 2009. All rights reserved

Physical Symptoms Of Liver Failure

Early Late

Nausea

Loss of appetite

Fatigue

Diarrhea

Clay stool, dark urine, tenderness

Jaundice

Bleeding/bruising easily

Swollen abdomen/legs

Intense skin itching

Mental disorientation

YEARS


Common Causes of Hepatitis

Metabolic

DiseaseBacteria

Viruses

Alcohol

Drugs


Introduction

“The beginning of health is to know the disease” Chinese proverb

h

Fever Jaundice

Acute Hepatitis Symptoms

Mouth & Upper GI Tract

Arm & Leg Joints & Muscles

Lower Digestive Tract

h© Siemens AG, 2009. All rights reserved

Common Tests for Identifying Liver Disease

Is there

liver injury?

What is causing

the damage?

ALT

AST

Alkaline phosphatase

LDH

Total bilirubin

Fatty

Liver

Alcohol

Viral

hepatitis




3

Common Types of Viral Hepatitis

Hepatitis A

Hepatitis E

Hepatitis CHepatitis D

Hepatitis B


Viral Hepatitis - Overview

Type of Hepatitis

A B C D E

Genome RNA DNA RNA RNA RNA

Source of virus

feces blood/blood-derived

body fluids

blood/blood-derived

body fluids

blood/blood-derived

body fluids

feces

Route of transmission

fecal-oral percutaneous

permucosal

percutaneous

permucosal

percutaneous

permucosal

fecal-oral

Chronic infection

no yes yes yes no

Prevention pre/post-exposure immunization

pre/post-exposureimmunization

blood donor

screening;

risk behavior modification

pre/post-exposure immunization; risk behaviormodification

ensure safe

drinking

water


Acute vs. Chronic Viral Hepatitis

Death

RecoveryTime

Liver damage

6 months

Acute

Chronic

Acute

Fulminant

HAV

HEV

Progression

Stable diseaseHBV

HDV

HCV


Hepatitis A

© Siemens AG, 2009. All rights reservedh

HAV: Severity

Usually mild, most recover

Frequently symptomless, especially in children

May be sick for several months

Can cause acute liver failure and death

Patient populations with the greatest risk for

significant disease include:

Persons 50 years of age or older

Persons with other liver diseases, such

as hepatitis B or C.


HAV Serological Course

0 1 2 3 4 5 6 12 24

Titer

Months after exposure

anti-HAV IgM

Total anti-HAV

ALT

Symptoms

Fecal HAV

shedding

Resolution and immunity

HAV Infection




4

HAV Serological Tests

Anti-HAV IgM

Indicates acute

infection

Can be detected in

blood about 3-4 weeks

after infection

IgM antibodies not

generally detectable

within 3-12 months

Anti-HAV-Total

Detects both IgM and

IgG

Positive in acute and

recovered hepatitis

Present in infected and

vaccinated populations

Hepatitis B


Andrew Magill, Creative Commons Attribution 2.0 License

HBV infected

maternal

blood

HBV Transmission Risk Factors

Birth (spread from an infected

mother to her baby during birth)

Sex with an infected partner

Sharing needles, syringes, or other

drug-injection equipment

Sharing items such as razors or

toothbrushes with an infected person

Direct contact with the blood or open

sores of an infected person

Exposure to blood from needlesticks

or other sharp instruments © Siemens AG, 2009. All rights reserved

HBV Infection Outcomes in Adults

Acute HBV

Subclinical

infection

HBsAg carrier

~5%

Chronic hepatitis

Liver injury

Fulminant

hepatitis

Liver cancer Death

Clinical

infection

Recovery

immunity ~95%

Asymptomatic

HBsAg carrier

Liver cirrhosis


HBV Transmission

High Viral Titer

Moderate Viral Titer

Low Viral Titer

Urine Feces Sweat TearsBreast

milk

SemenVaginal

fluidSaliva

Blood SerumWound

exudates

Vertical

transmission


HBV Infection Outcomes in Newborns

Acute HBV

Subclinical

infection

HBsAg carrier

~5%

Chronic hepatitis

Liver injury

Fulminant

hepatitis

Liver cancer Death

Clinical

infection

Recovery

immunity ~95%

Asymptomatic

HBsAg carrier

Liver cirrhosis

HBsAg carrier

~90%

HBsAg carrier

~10%


http://upload.wikimedia.org/wikipedia/commons/0/0d/Incubator-tahrir.jpg



5

Chronic HBV Correlates Inversely with Age

Age at

InfectionChronicity (%)

<6 months 90-95

>6 months 80

1-4 years 30-50

>4 years 5-10


Immunoprophylaxis to Prevent Perinatal Transmission

Recommended Immunization Schedule for Persons Aged 0 Through 6 Years—United States 2009

http://www.cdc.gov/vaccines/recs/schedules/downloads/child/2009/09_0-6yrs_schedule_pr.pdf

Vaccine Dose and HBIG* Age

Infant Born to Mother Known to be HBsAg Positive

First Vaccine Dose Birth (within 12 hours)

HBIG Birth (within 12 hours)

Second Vaccine Dose 1 – 2 months

Third Vaccine Dose 6 months

Infant Born to Mother Not Screened for HBsAg

First Vaccine Dose Birth (within 12 hours)

HBIG

Screen mother ASAP. If HBsAg

positive, give ASAP but by no

later than 1 week of age

Second Vaccine Dose 1 – 2 months

Third Vaccine Dose 6 months*Hepatitis B Immune Globulin


Hepatitis B Virus Structure

Nucleocapsid

(core antigen)

Envelope

Viral DNA

HBe AntigenSurface antigens


Hepatitis B Testing

Serology

Manual and automated methods are available to identify antibody to or antigen from the Hepatitis B virus

Molecular Tests

Detect viral load for therapeutic decision making


Serological Tests For HBV

Anti-HBc

IgM

Anti-HBc

total

HBsAg

Anti-HBs

Anti-HBe

HBeAgAntigen

detection

Antibody

detection


HBV Serological Profile:Acute Infection with Recovery

Weeks after exposure

HBsAg

HBeAg Anti-HBe

Symptoms

anti-HBc IgM Total anti-HBc

Titer

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

anti-HBs




6

HBV Serological Profile:Chronic “Non-replicative” Infection


HBsAg

HBeAg Anti-HBe

anti-HBc IgM

anti-HBs rarely seen

Titer

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

Chronic non-

replicative state

Chronic

replicative state

Chronic

6 month = chronic

Total anti-HBc



HBsAg

HBeAg

anti-HBc IgM


Titer

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

Chronic (Years)Acute (6 months)

Total anti-HBc

HBV Serological Profile:Chronic Replicative Infection


Hepatitis B Testing

Test Result

Probable Diagnosis HBsAg aHBcT aHBc IgM HBeAg aHBe aHBs

Uninfected, unvaccinated – – – – – –

Vaccinated (immune) – – – – – +

Acute infection + + + + – –

Active infection: recovering – + – – + –

Recovered (immune) – + – – + +

Chronic replicative infection + + – + – –

Chronic nonreplicative infection + + – – + –


Treating Chronic HBV

Interferon

Alpha

Pegylated

interferon

Lamivudine

Tenofovir

Entecavir

Adefovir

Telbivudine

Treatment

options

include

Treatment

is often lifelong -

few are cured


Treatment Goals for Chronic HBV


HBsAg

HBeAg Anti-HBe

IgM anti-HBc


Titer

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

Chronic non-

replicative state

Chronic

replicative state

Chronic

6 month = chronic

Total anti-HBc


Guidelines for treatment




7

Hepatitis C


HCV Prevalence

> 2.9%

2.0 – 2.9%

1.0 – 1.9%

< 1.0%

No data

US 3 – 4M

Americas 12 – 15M

Western Europe

5MEastern Europe

10M

Africa 30 – 40M

Southeast Asia

30 – 45M

Far East Asia 60M

Australia 0.2M

10,000 – 12,000 deaths per year, US

Leading cause of liver transplants, US

No vaccinewwwnc.cdc.gov/.../yellowbook/2008/ch4/hepa.aspx


6 HCV Genotypes and > 50 Subtypes

1(I)

1c(0)1(II) 1a

3c

1b

3e 3d

1c(E)

3(III)3(VI)

3a

TD3

10a

3f3b

h

9a

i

9b

j 9c

k 8b

8a

l

mNGIn

g

11ae7ac

7d

7bd

fb

a

a

d

e

f

6a4e

4g

4h4f

4d

5a

6b 7c/NGII/VII

4a(E)

4a(B)

4c

c b

2(I)

1

2

54

3

6

*

US &

Western

Europe

Japan &

Taiwan

Australia &

South Asia

Hong Kong

& Southeast

Asia

Middle

East South

Africa


HCV Infection In The US

HCV HIV

# Cases

(millions)

4

3

2

1

Source: Clin Gastroenterol Hepatol. 2006 October; 4(10): 1278–1282.

Infected for life

Up to 2% of US population infected

Estimated 35,000 new HCV cases

per year in the US

80 %


HCV: “The Silent Epidemic”

Symptoms rare (~20%) and generalizedEarly Stage

Symptoms still rare (nausea, jaundice, fatigue)Late Stage

Often 20-30 years post-exposureLiver Disease

Presentation

Elevated liver enzyme profiles (AST, ALT)Initial ID of

Damage

Infected individuals may unknowingly spread virusTransmission

Chronicity >75%-80%Disease


HCV Transmission

* Reduced after implementation of routine blood screening

Illegal drug abuse

60%

Sexual

21%

No Identified risks

10%

Transfusions*

3%

Occupational

3%

Household

3%


http://wwwnc.cdc.gov/travel/yellowbook/2008/ch4/hepa.aspx



8

Although IV Drug Abuse Is The Leading Risk, Other Causes Of HCV Include:

Tattoos

Occupational

hazards

Manicure and pedicure


Another Risk - Going to a Clinic?!

Source: CDC press release Jan. 6, 2009

60,000

patients

At risk ofHBV, HCV, HIVafter failure of

medical personnel tofollow proper

practice

Hepatitis C Virus (HCV)

Envelope Core

Envelope

glycoproteins

Viral RNA

(9400 nucleotides)


HCV RNA Genome

C E1 E2 NS5A NS5B

2nd or 3rd generation HCV IA detects antibody to 3 or more viral proteins

5’NTR Structural Proteins Non-structural Proteins 3’NTR

Nucleocapsid

p22

Metalloprotease

Serine Protease

RNA Helicase

p70

NS1 NS2 NS3NS4A NS4B

p8 p27

Interferon

Resistance

Protein

P56/58

RNA

Polymerase

p68

Cofactors

First Generation Second Generation Third Generation


IgG

Control

Level II

c100(p)

5-1-1(p) c33c c22 (p) NS5 hSOD

IgG

Control

Level I

Positive

Indeterminate

Negative

Negative

Positive

Indeterminate

Interpretation

RIBA for HCV antibody confirmation


HCV Infection Testing Algorithm

RIBA for Anti-HCV

Medical

EvaluationReport

Negative PCR

Normal ALT

Positive PCR

Elevated ALT

HCV RNA

Testing

Negative

ReportNegative Positive

Positive OR

Negative PositiveIndeterminatePositive

(but < Index or S/CO)

ReportValidated

Additional

Laboratory

Evaluation

(PCR, ALT)

≥

Index or S/CO




9

Normal

HCV Progression

Titer

0 1 2 3 4 5 6 1 2 3 4Months YearsTime after exposure

Symptoms ±

HCV RNA

Anti-HCV

ALT


Titer

0 1 2 3 4 5 6 1 2 3 4Months Years

Time after exposure

Normal

Symptoms ±

HCV RNA

Anti-HCV

ALT

HCV Progression

AbHCV/

RIBA

ALT/AST

Other

tests

Genotype

and Viral

Load

Liver

Biopsy


Anti-HCV serology

Viral Load

Genotyping Assay

Viral Load/Qualitative assay

Viral Load/Qualitative assay

Testing of the HCV Infected Patient

Has the patient ever been

infected?

How much virus is present?

What genotype is the patient

infected with?

Is the patient responding to

therapy?

Is the patient relapsing?


TMA*Add primers & enzymes

Copies(RNA)

One detection probe per copy

HCV RNA

HCV Virus

Add target probes and amplification probes

bDNA

Multiple detection probes per target

Technologies to detect HCV RNA include:

RT-PCRAdd primers & enzymes

Copies (DNA)

One detection probe per amplified copy

TMA = Transcription Mediated Amplification

HighSensitivityQualitative

Assay

TargetAmplificationQuantitative

Assay

SignalAmplificationQuantitative

Assay


Technologies to detect HCV genotype include:

• RT-PCR with sequencing

• Line Probe Assay (LiPA)

Genotype 1a, 1b

Genotype 2, 3


Molecular Testing in Treatment of HCV Patients

Pegylated

interferonRibavirin

Sensitive qualitative, quantitative viral load,

and genotyping central to therapy

Current HCV therapyTherapy response varies with genotype and compliance




10

Treatment of HCV

Strader, et al. 2004; Hepatology, Vol. 39. No. 4 page 1147

Ghany, et al. 2009, Hepatology, Vol. 49; No. 4, page 1335


NIH Guidelines


Side Effects of HCV Therapy

Grossman H et al.. 41st Interscience Conference on Antimicrobial Agents and

Chemotherapy. December 16-19, 2001. Chicago. Abstract I-254.

Pegylated interferon Ribavirin

Neuropsychiatric

Depression, anxiety, irritability, fatigueCough and dyspnea

Bone marrow depression

Neutropenia, thrombocytopeniaHemolytic anemia

Anorexia / weight loss Teratogenicity

Alopecia Insomnia

Exacerbation of autoimmune

disordersRash / Puritus

Thyroid dysfunction

Hashimoto’s thyroiditisNausea


HCV Treatment Nomenclature

“SVR”: Sustained Virologic Response

“EVR”: Early Virologic Response

“RVR”: Rapid Virologic Response

Stopping Rules


Management of Hepatitis C: 2002NIH Consensus Conference Statement

• Early viral response (EVR):

• A minimum 2 log decrease in viral load during the first 12 weeks of treatment

• Predictive of SVR and should be a routine part of monitoring patients

12 weeks

treatment


2002 NIH Consensus Conference

Baseline: Quantitative & Genotype

Week 12: Quantitative

<2 log10 drop

Consider

therapy end

End treatment: Qualitative

6 month follow-up: Qualitative

HCV RNA (+)

Non-responder

≥2 log10 drop

Genotype 1

48 weeks

Genotype non-1

24 weeks

HCV RNA (+)

Relapser

HCV RNA (-)

Sustained responder

HCV Therapy Algorithm




11

SVR vs. Non-Response (NR) to the Anti-Viral Therapies*

80/80/80 = >80% dose of peg-interferon;

>80% dose of ribavirin;

>80% of prescribed treatment duration* McHutchison JG et al. The effects of interferon alpha-2b in

combination with ribavirin on health related quality of life and work productivity. J Hepatol. 2001;34:140-147. McHutchison JG et al.

Adherence to combination therapy enhances sustained response in

Genotype-1-infected patients with chronic hepatitis C. J Gastroenterology. 2002; 123:1061-1069

63%

54%

41%

15%

37%

46%

59%

85%

0% 20% 40% 60% 80% 100%

Peg + RBV

(80/80/80)

Peg + RBV

IFN + RBV

IFN mono

SVR

NR


Predictive SVR

Therapy Response

Viral load

Therapy course (weeks)

0 2 4 6 8 10 12

Initial

load

2 log

drop

EVRNon-responder


Therapy Cessation at

12 Weeks?


n = 63

(14%)

n = 390

(86%)

2 log10 drop

or Neg HCV RNA

Week 12 (N = 453)

Outcome with PEG-IFN 2a +R:12 Week Stopping Rule

Yes

No

SVR

NR

SVR

NR

PPV

NPV

n = 253

(65%)

n = 61

(97%)

n = 137

(35%)

n = 2

(3%)

Fried et al. 2002. NEJM 347(13):975-982


n = 98

(21%)

n = 380

(79%)

2 log10 drop

or Neg HCV RNA

Week 12 (N = 478)

Outcome with PEG-IFN 2b +R:12 Week Stopping Rule

Davis et al. Hepatology (2003) 38(3):645-652

Yes

No

SVR

No SVR

SVR

No SVR

PPV

NPV

n = 273

(72%)

n = 98

(100%)

n = 107

(28%)

n = 0

(0%)


Therapeutic Response: RVR

Predictive SVR

Initial

load

2 log

drop

EVRNon-responder

RVR

Vir

al L

oad

Therapy course (weeks)

0 2 4 6 8 10 12

Rapid Viral Response


RVR in the 2009 AASLD HCVTreatment Update

Ghany, et al. 2009, Hepatology, Vol. 49; No. 4, page 1335




12

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Time to Undectability Predicts SVR

Ferenci, P, et al. J Hepatol 2005;43:425-433

Week 4 Negative≥ 2 log

< 2 log

≥ 2 log

< 2 log

Week 12 Negative Negative Negative≥ 2 log

≥ 2 log

Week

24Negative Negative Negative Negative Negative

91% 72% 66% 48% 43%

2002 Guidelines

Pat

ien

ts w

ith

SV

R (%

)


Using RVR in patient management


Baseline: Quantitative & genotype

Week 12: Quantitative and/or qualitative

<2 log10 drop

Consider

therapy end

End treatment: Qualitative

6 month follow-up: Qualitative

HCV RNA (+)

Non-responder

≥2 log10 drop

Genotype 1

48 weeks

Genotype non-1

24 weeks

HCV RNA (+)

Relapser

HCV RNA (-)

Sustained responder

HCV?Week 4: Quantitative or Qualitative


6 HCV Genotypes and > 50 Subtypes

1(I)

1c(0)1(II) 1a

3c

1b

3e 3d

1c(E)

3(III)3(VI)

3a

TD3

10a

3f3b

h

9a

i

9b

j 9c

k 8b

8a

l

mNGIn

g

11ae7ac

7d

7bd

fb

a

a

d

e

f

6a4e

4g

4h4f

4d

5a

6b 7c/NGII/VII

4a(E)

4a(B)

4c

c b

2(I)

1

2

54

3

6

*

US &

Western

Europe

Japan &

Taiwan

Australia &

South Asia

Hong Kong

& Southeast

Asia

Middle

East South

Africa

HCV genotype assessment guides therapy

and predicts likelihood of SVR


HCV Genotypes can Predict Treatment Success

Treatment Success

Treatment Success

Genotype 1

40% Effective

Genotype Non-1

80% Effective

Pegylated

InterferonRibavirin


HCV Treatment: Genotypes Make a Difference

Adapted from Ann Intern Med 2004; 140: 346-355 & Lancet

2001; 358: 956-965

Adapted from Hepatology. 2002;36:(5 suppl 1):S3-S20

Treatment Response (SVR) with Pegylated Interferon and Ribavirin

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

All Patients Genotype 1 Genotype 2&30%

10%

20%

30%

40%

50%

60%

70%

80%

90%

All Patients Genotype 1 Genotype 2&3

© Siemens AG, 2009. All rights reserved © Siemens AG, 2009. All rights reserved



13

HCV Mono-infected vs HCV-HIV Co-infected Patients

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Pa

tie

nts

wit

h S

VR

(%

)

All Patients Genotype 1 Genotype 2 & 3

HCV Only

HCV + HIV

63% 40% 52% 29% 85% 62%

Treatment with Pegylated Interferon + Ribavirin


Patient Presentation

Nausea, slight fever, hepatomegaly, jaundice


Acute Viral Hepatitis Testing Panel

HAV IgM HBsAg HBV Core

IgM

Interpretation

Pos Neg Neg Acute HAV

Neg Neg Neg

HCV-acute, chronic,

resolved?

Neg Pos NegChronic

HBV?

Anti-HCV

Neg

Pos

Neg

Neg Pos Pos Acute HBV?Neg


Resources

• Ann Robinson, Ph.D., DABMM, FAAM Director, Microbiology and Virology [email protected]

• Virginia Henderson, M,SV (ASCP)

Technical Supervisor, Virology

[email protected]

• American Association for the Study of Liver Disease (AASLD) – www.aasld.org

• Diagnosis, Management and Treatment of Hepatitis C: An Update – www.aasld.org

• P.A.C.E. credit may be obtained by submitting

your completed evaluation form. You will find the

form by clicking on the “handouts” icon in the

upper right hand corner of your screen

• CE credit may be obtained by downloading the

“Certificate of Completion” under the “handouts”

icon

• PAML employees will be able to receive one

hour of continuing education credit by submitting

your attendance through CE Manager.

Thank-you for Attending

This webinar has been recorded and will be

available by April 14th, on www.paml.com,

under the Hospital Tab/Hospital

Portal/Webinar Series heading

Thank-you for Attending



http://www.aasld.org/

http://www.paml.com/

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Lab Testing in the Diagnosis and Management of Viral ... Testing in the Diagnosis and Management of Viral Hepatitis 1 ... Lab Testing in the Diagnosis and Management of Viral Hepatitis