The Diagnosis of

Hepatitis A, B and C Virus

 The phases of the Acute Viral
Hepatitis
The classical phases of the Acute viral hepatitis

Pre-icteric Icteric Convalescense

Flu-like syndromes malaise

Appetite
History taking Dyspepsia +
Tea-colour urine

jaundice
Physical exam hepatomegaly jaundice
<

Laboratorium Alt/ASTALP/ AST/ALT/AP/

GGT/BIL., GGT,BIl./N
Blood ALT/AST seromarker + Seroconversion

Urine Uro/bili + Uro/bili + Uro/bili. .

Hepatitis A Virus
 Hepatitis A Virus

Naked RNA virus

Related to enteroviruses, formerly known as
enterovirus 72, now put in its own family:
heptovirus
One stable serotype only
Difficult to grow in cell culture: primary marmoset
cell culture and also in vivo in chimpanzees and
marmosets
4 genotypes exist, but in practice most of them are
group 1
 Hepatitis A - Clinical
Features
Incubation period: Average 30 days
Range 15-50 days
Jaundice by <6 yrs, <10%
age group: 6-14 yrs, 40%-50%
>14 yrs, 70%-80%
Complications: Fulminant hepatitis
Cholestatic hepatitis
Relapsing hepatitis
Chronic sequelae: None
 Hepatitis A Virus Transmission

Close personal contact

(e.g., household contact, sex contact,
child day care centers)
Contaminated food, water
(e.g., infected food handlers, raw shellfish)
Blood exposure (rare)
(e.g., injecting drug use, transfusion)
 Laboratory Diagnosis

Acute infection is diagnosed by the detection of HAV-IgM

in serum by EIA.
Past Infection i.e. immunity is determined by the detection
of HAV-IgG by EIA.
Cell culture difficult and take up to 4 weeks, not
routinely performed
Direct Detection EM, RT-PCR of faeces. Can
detect illness earlier than serology but rarely
performed.
Hepatitis B Virus
 Hepatitis B Virus - Virology
Double stranded DNA virus,the + strand not complete
Replication involves a reverse transcriptase.
Complete Dane particle 42 nm, 28 nm electron dense core,
containing HBcAg and HBeAg. The coat and the 22 nm
free particles contain HBsAg
At least 4 phenotypes of HBsAg are recognized;
adw, adr, ayw and ayr.
The HBcAg is of a single serotype
Hepatitis B virus (HBV) has been classified into 8
genotypes (A-H).
It has not yet been possible to propagate the virus
in cell culture.
 Prevalence of HBsAg Carrier State

HBV - Epidemiology

>8%
2-8%
<2%

WHO
 Transmission of HBV
Horizontal transmission Vertical transmission

Host Recipient Mother

Child to child Perinatal

Contaminated needles
Sexuals
Health care workers Infant
Transfusions
6% infected after age 5 years
become chronically infected 90% infected infants become
Chronically infected
No clear risk factors in 20-30% patients
 Concentration of HBV
in Various Body Fluids
High Moderate Low/Not
detectable
Blood Semen Urine
Serum Vaginal fluid Feces
Wound exudates Saliva Sweat
Tears
Breast milk
CDC
 Hepatitis B disease Progression
Liver
Cancer (HCC)
5-10% of CHB
individuals

>30% of CHB
individuals
Acute Chronic Liver Liver Death
Infection Infection Cirrhosis Transplantation

>90% of infected
children progress to
chronic disease
<5% of infected
Immunocompetent
LiverFailure
adults progress to
(decompen-
chronic disease.
sation))

23% of patients decompensate

within 5 years of developing
cirrhosis
 Early Childhood HBV Infection

Risk of Chronicity
Age at infection (years) Proportion who become carriers(%)
<1 70 90

23 40 70

46 10 40

7 6 - 10
Immunopathology of HBV Infection
apoptosis
HBV

APC

HBV

CTL CD8 HLA class I Macro

TH CD4 HLA class II
b phage

Cytokines

Viral Peptide

a Lyse hepatocyte directly. Clearance

b Release IFN-activates
Ag-nonspecific mononuclear
cells in the liverdestruction
hepatocytes.
Natural History From Exposure to Acute Infection

Blood(parenteral)
HBV
Body fluid Mucosal } Patient (acute/chronic hepatitis) New Host (adult)

Vigorous imune response Acute infection

Viral clearance(ALT ,icterus )

Convalescence 6m
Simple Infection Fulminant
HBsAg - 1-2%
Mortality rate
63-93%
Icteric
Icteric Pre-icteric Incubation
1-3 w
1-3 w 3-10 d 6w-6m

Chronic
(HBV DNA >6m
>105copies/ml
ALT / HBsAg +
HBV - Natural History

Clinical Outcome of Chronic

Hepatitis B
Chronic HBV Infection

Inactive Carrier
State Chronic Hepatitis

Cirrhosis

HCC
 Hepatitis B Virus

Gene Products and Functions

Polymerase Terminal protein (priming)
Reverse transcriptase, RNAse H
Surface Envelope proteins
Pre - S1 Receptor binding,
Regulation of cccDNA, viral assembly
Pre - S2 Viral assembly, fusion sequence
S Primary structural component,
major antigenic determinants
Core
HBeAg Secreted, immunomodulatory function
Core Nucleocapsid component
HBX Pleiotropic effects
 Diagnosis
A battery of serological tests are used for the diagnosis of acute and
chronic hepatitis B infection.
HBsAg - used as a general marker of infection.
HBsAb - used to document recovery and/or immunity to HBV
infection.
anti-HBc IgM - marker of acute infection.
anti-HBcIgG - past or chronic infection.
HBeAg - indicates active replication of virus and therefore
infectiveness.
Anti-Hbe - virus no longer replicating. However, the patient can still
be positive for HBsAg which is made by integrated HBV.
HBV-DNA - indicates active replication of virus, more accurate than
HBeAg especially in cases of escape mutants. Used mainly for
monitoring response to therapy.
 HBV - Diagnosis

Serological Markers of Acute HBV Infection

HBV DNA

HBeAg Anti-HBe
Anti-HBs
Anti-HBc

HBsAg Anti-HBc
IgM

0 2 4 6
Months Year
Years
Serologic Profiles in patients with
Hepatitis B Infection
Serological Acute HBV Recovered Chronic Inactive Occult
tests from HBV Hepatitis B carrier Hepatitis B

HBsAg + - + + -

Anti HBs - + -/+ - -

Anti HBc + + + + -
Total

HBeAg + - +/- - -

Anti HBe - + -/+ + -

HBV DN A + - +/>105 +/<104 +/<103

copies copies copies
 The Variables to define Chronic
Hepatitis B (CHB)
Chronic Hepatitis B

ALT HBeAg Anti HBe HBV DNA

To measure the
Necroinflammatory +active replica- Positive,not always Levels of viral
of hepatocytes tion mean nonactive/ Replication,
Noninfectious-> Branched/PCR
Precore/core pro DNA
Active hepatitis moter Mutant
Negative->not indicating
Nonreplicative/
Normal, Noninfectious-> -decision to initiate Tx
not always mean Pre-core/core promo - initial evaluation of Tx
Inactive. ter MUTANT - forecasting the pro-
gression of the disease
>2UNL,meaningful
 Chronic Hepatitis B
HBsAg + (>6m)

Active Inactive

N/ N N
ALT

+ HBeAg

+ Anti-HBeAg +

105 104 HBV DNA <3x102

Copies/ml

Wild Mutant
Common Mutant Type of B hepatitis virus

1. Escape mutant
Mutant on S Ag
Detected in Chronic hepatitis or as a novel acute hepatitis.
The marker is HBsAg + and anti-HBs +,in a patient.

2. Pre-core or core promoter mutant

Mutant on pre-core/E Ag
In chronic active B hepatitis ,HBeAg - ,anti-HBe +,but
HBV DNA +

3. YMDD mutant
Mutant that develops during lamivudine therapy.
This mutant type resistant to Lamivudine.
 Relative Risk of HCC for various serum HBsAg-HBeAg

10-
HBsAg+,HBeAg+

8-

% 6-
cumulative
incidence

4-

HBsAg+,HBeAg -
2-

HBsAg-,HBeAg-
0
1 2 3 4 5 6 7 8 9 10 Years

Relative risk of HCC versus uninfected individuals:

60,2 in patients HBsAg+and HBeAg +
9,6 in patients HBsAg +,but HBeAg-

Yang HI et al,New Engl J Med 2002;347:168-174

 Adjusted Relative Risk of Liver Cancer
for various serum HBV DNA and ALT levels
Number of Number of liver Adjusted relative
HBV DNA level subjects cancer cases risk 95% CI
ALT < 1 x ULN

Undetectable(<102) 908 13 1,0

<105 1825 37 1,6(0,9-3,0)

>105 868 93 10,2(5,7-18,4)

ALT 1 x ULN

Undetectable (<102) 43 3 4,3(1,2-15,9)#

<105 61 3 1,6(1,2-15,1)

>105 146 27 16,9(8,1-33,7)*

# p<0,05. *p>0,01
Ilouje UH et al J.Hepatol 2005;42:S179(abst 495)
 Hepatitis C Infection

Transmission is mainly related to contact with blood

and blood product.

The main routes of spread are blood transfusion and the use
of shared,non-sterilized needles and syringes.

Since 1991 ,transfusion-related hepatitis C has virtually dis-

appeared;now through needles/syringesIDU/tattouage etc.

The rest the transmission is undetermined (50%?)

HCV - Epidemiology

Prevalence

Worldwide 170 million (3%)

United States
Anti-HCV positive 3.9 million (1.8%)
HCV RNA positive 2.7 million (1.4%)

Alter MJ et al., New Engl J Med 1999; 341:556

Lavanchy D & McMahon B, In: Liang TJ & Hoofnagle JH (eds.)
Hepatitis C. New York: Academic Press, 2000:185
 Risk Groups for HCV Infection

High Risk Groups Moderate Risk Groups

*Blood transfusion (before 1991) *High risk sexual behaviour,multiple

*Frequent exposure to blood products:
haemophilia,transplants,haemodyalisis
chronic renal failure,cancer chemothera-
py.
partners,history of herpes simplex
type 2 infection
*Cocaine use,with sharing of intranasal
administration equipment.

*Injecting drug users, even if use was

brief and/or many years ago.

*Healthcare workers with needlestick

injuries.

*Infants born to HCV-infected mothers,

particularly those coinfected with HIV.
 HCV - Epidemiology

Current Likelihood of Transmission

Transfusion ~ 1 in 1,000,000
Maternal-Infant
Mother HIV-negative ~ 5%
Mother HIV-positive 15 - 20%

Heterosexual partner ~1 in 1,000 per yr

Needlestick injury
HCV-positive source ~ 5%
HCV status unknown ~ 1%

Terrault NA, Hepatology 2002 ;36(Suppl 1):S99

Roberts EA, Yeung L. Hepatology 2002 ;36(Suppl 1):S106
HCV - Natural History

Outcome Following Hepatitis C

Infection
Acute hepatitis C

55 - 85%

Chronic infection

70%

Chronic hepatitis
1 - 4%/yr
20% HCC

Cirrhosis

Decompensation
4 - 5%/yr
Time
(yr)
10 20 30
HCV - Diagnosis

Diagnostic Tests

Hepatitis C antibody tests (EIA-RIBA)

Qualitative HCV RNA tests

Quantitative HCV RNA tests

Genotyping
HCV - Diagnosis

Acute hepatitis C infection

1000
HCV RNA positive
800
Anti-HCV
600
ALT
(IU/L)
400
Symptoms

200

Normal
0
ALT
0 2 4 6 8 10 12 24 1 2 3 4 5 6 7
Weeks Months
Time After Exposure
Hoofnagle JH, Hepatology 1997; 26:15S
HCV Diagnosis

Low prevalence popu Indicate

lationpoor positive past/present/resolve
predictive value infection

Antibody test
for
Hepatitis C

Immunocompromized *EIA +(low risk) Inexpensive

low sensitivity *RIBA(confirmatory) sensitive-spesific

ALT + risk factor+,anti HCV +

95% have Hep C
HCV Diagnosis

Gold standard Confirm Dx

for documenting of HCV infection
Respons to Tx

Qualitative test
for HCV RNA

Demonstrate Early Dx
the presence of of
Active Infection Acute Hepatitis C
 Prognostic Tests

Genotyping genotype 1 and 4 have a worse prognosis overall and

respond poorly to interferon therapy. A number of commercial and in-
house assays are available.
Genotypic methods DNA sequencing, PCR-hybridization e.g. INNO-
LIPA.
Serotyping particularly useful when the patient does not have detectable
RNA.
Viral Load patients with high viral load are thought to have a poorer
prognosis. Viral load is also used for monitoring response to IFN
therapy. A number of commercial and in-house tests are available.
Conclusion
 Acute infection is diagnosed by the detection of
HAV-IgM in serum by EIA.
Past Infection i.e. immunity is determined by the
detection of HAV-IgG by EIA.
Cell culture difficult and take up to 4 weeks,
not routinely performed
Clinical Features of Hepatitis B
Transmission
Oral Not likely
Percutaneous Common
Sexual Common
Perinatal Common
Incubation period 60-180 (days)
Clinical Illness at 10 - 15%
presentation
Clinical Features of Hepatitis B
Jaundice 5 20%
Fulminant <1%
Diagnostic tests
Acute infection HBsAg, IgM anti- HBc
Chronic infection HBsAg, IgG anti-HBc
Immunity IgG anti-HBc, anti-HBs
Case-fatality rate 1 3%
Chronic infection >90% infants
<5% adults
HBV - Diagnosis

Clinical Significance of Serological

Markers for HBV Infection
HBsAg Acute/Chronic
infection
Anti-HBc IgM Acute infection
HBeAg High infectivity
Anti-HBe Low infectivity
Anti-HBs Immunity
Anti-HBc IgG and HBsAg Chronic infection
Anti-HBc IgG and anti-HBs Resolved infection
Clinical Features of Hepatitis C

Jaundice 5 10%
Fulminant Rare
Diagnostic tests
Acute infection HCV RNA (anti-HCV)
Chronic infection HCV RNA (anti-HCV), >6 months
Immunity Unknown
Case-fatality rate 1 2%
Chronic infection 60 85%
 Clinical Features of Hepatitis C
Transmission
Oral No
Percutaneous Common
Sexual Yes, rare
Perinatal Yes, low frequency
Incubation period 14 160 (days)
Clinical Illness at 5 - 10%
presentation
HCc Ag Dx and monitoring Tx
Indirect marker of HCV
replication;predicting
EVR <sensitive to replace
HCV RNA to demonstrate
eradication
 Diagnosis of Hepatitis B Infection
Is HBsAg present?

Yes
No

Is IgM anti-HBc present?

No
Yes

Chronic Hepatitis
Acute Hepatitis

Is HBeAg/anti HBe or HBV DNA present? Is anti-HBs present?

HBeAg +,antiHBe HBeAg -,antiHBe + HBeAg -,antiHBe + Yes No

HBV DNA + HBV DNA + HBV DNA -

Recovered or No HBV
Replicative Non-Replicative Vaccination Infection
Replicative
HBV Infection HBV Infection HBV Infection
 HCV Infection Testing Algorithm for
Diagnosis of Asymptomatic Persons
Negative
EIA for anti-HCV Stop

Positive

Negative
RIBA for anti-HCV RT-PCR for HCV-RNA

Positive
Negative Indeterminate Positive

Stop Additional Lab. Evaluation Medical

(eg.PCR,ALT) evaluation

Negative PCR Positive PCR

Normal ALT Abnormal ALT