Viral Hepatitis Serology

GUASA, JT (adapted from BANSUELA, JC)

 VIRAL HEPATITIS SEROLOGY
Laboratory testing for viral infections
Two disciplines of laboratory science involved in the
detection of viral infections to date:
1. Serological Assays
2. Molecular Assays
Both have become some of the most important and the most
frequently performed tests in the clinical laboratory today.
Can be easily and rapidly performed; thus playing an
essential role in aiding the prompt management of most
cases.
 VIRAL HEPATITIS SEROLOGY
Serological assays for viral infections
Roles in laboratory medicine:
Detecting active infections
Monitoring the course of infection
Detecting past infections
Assessing immune status

Two main techniques (based on target analyte):

Detection of virus-specific antibodies
Detection of viral antigens
 VIRAL HEPATITIS SEROLOGY
Hepatitis in general
Hepatitis – “Inflammation of the liver”
Causes:
1. Viruses (Viral Hepatitis)
2. Noninfectious Agents
o Ionizing Radiation
o Chemicals
o Drugs
o Autoimmune Processes
o Other Disease States
 VIRAL HEPATITIS SEROLOGY
Etiology of viral hepatitis
“Most common liver disease worldwide.” (Turgeon, 2014)
Primary Hepatitis Viruses:
o Hepatitis A, B, C, D, E, & G Viruses
Secondary Hepatitis Viruses:
o Epstein-Barr Virus (EBV)
o Cytomegalovirus (CMV)
o Herpes Simplex Virus (HSV)
o Others
 VIRAL HEPATITIS SEROLOGY
Signs and symptoms of viral hepatitis
Disease can occur in four acute or chronic forms:
1. Acute
2. Fulminant acute
3. Subclinical without jaundice
4. Chronic
Signs and symptoms are extremely variable (i. e. can be
completely asymptomatic, mild, transient, severe, prolonged,
or ultimately fatal).
May progress to hepatocellular carcinoma
 VIRAL HEPATITIS SEROLOGY
Forms of hepatitis
Acute hepatitis Typical form associated with jaundice
Four phases – incubation, preicteric,
icteric, & convalescence
Incubation period – few days to many
months
Average length of time – 75 days (40-180
days) in HBV infection
Fulminant acute Rare form of hepatitis associated with
hepatitis hepatic failure
 VIRAL HEPATITIS SEROLOGY
Forms of hepatitis
Subclinical Probably accounts for persons with
hepatitis without demonstrable antibodies in their serum
jaundice but no reported history of hepatitis

Chronic hepatitis Accompanied by hepatic inflammation and

necrosis that lasts for at least 6 months
VIRAL HEPATITIS SEROLOGY
Primary hepatitis viruses

Hepatitis A Virus (HAV)

Hepatitis B Virus (HBV)
Hepatitis C Virus (HCV)
Hepatitis D Virus (HDV)
Hepatitis E Virus (HEV)
Hepatitis G Virus (HGV)
 VIRAL HEPATITIS SEROLOGY
Hepatitis A
ETIOLOGY
Agent: Hepatitis A Virus (HAV)
Nonenveloped, single-stranded RNA virus of Hepatovirus
genus of the Picornaviridae family
Replication occurs in the cytoplasm of the hepatocyte
Only hepatitis virus that has been successfully grown in
culture
High titers detected in acute-phase stool samples
 VIRAL HEPATITIS SEROLOGY
Hepatitis A
EPIDEMIOLOGY
Formerly called infectious hepatitis or short-incubation
hepatitis
Primarily a disease of young children in developing
countries
Transmitted primarily by the fecal-oral route, by close
person-to-person contact, or by ingestion of contaminated
food or water
 VIRAL HEPATITIS SEROLOGY
Hepatitis A
SIGNS & SYMPTOMS
Incubation period: 15-45 days (average: 28 days)
Virus produces symptoms of acute hepatitis in the majority
of infected adults 2 to 6 weeks after exposure
Most infections in children are asymptomatic
Infection does not progress to a chronic state and is usually
self-limiting, with symptoms typically resolving within 2
months
 VIRAL HEPATITIS SEROLOGY
Hepatitis A
IMMUNOLOGIC MANIFESTATIONS
HAV antigens are shed in the feces of infected individuals
during the incubation period and the early acute stage of
infection
Shortly after the onset of fecal shedding, an IgM Ab is
detectable in serum, followed within a few days by the
appearance of an IgG Ab
 VIRAL HEPATITIS SEROLOGY
Hepatitis A
IMMUNOLOGIC MANIFESTATIONS
IgM anti-HAV: almost always detectable in patients with
acute HAV
IgG anti-HAV: manifestation of immunity; peaks after the
acute illness and remains detectable indefinitely (perhaps
lifelong)
 VIRAL HEPATITIS SEROLOGY
Hepatitis A
LABORATORY DIAGNOSTIC EVALUATION
Main approach: serologic tests (RIA and ELISA) to detect the
presence of specific HAV antibodies
o IgM Anti-HAV: Routinely detected by solid-phase
antibody-capture ELISA
o IgG Anti-HAV: Detected by Competitive Inhibition ELISA
HAV antigens are not clinically useful indicators of the
disease
Other available tests: Molecular tests – RT-PCR (HAV RNA)
Reverse Transcriptase - PCR
 VIRAL HEPATITIS SEROLOGY
Hepatitis E
Water-borne hepatitis
Agent: Hepatitis E Virus (HEV)
o Nonenveloped, single-stranded, RNA virus
o Formerly, Caliciviridae
o Reclassified to Hepeviridae
Mode of transmission: Fecal-oral
Incubation period: 2-9 weeks (average 6 weeks)
 VIRAL HEPATITIS SEROLOGY
Hepatitis E
Signs and Symptoms:
o Fulminant hepatitis with high mortality rate in pregnant
women
o No progression to chronic forms
Laboratory Diagnosis:
1. Serologic: ELISA
o Anti-HEV (IgM/IgA/IgG)
2. Molecular: RT-PCR (HEV RNA)
3. Others: Immunoelectron Microscopy (stool)
 VIRAL HEPATITIS SEROLOGY
Hepatitis C
ETIOLOGY
Agent: Hepatitis C Virus (HCV)
Nonenveloped, single-stranded RNA virus of Hepacivirus
genus of the Flaviviridae family.
Six discovered genotypes (1 through 6)
Over 50 different subtypes
 VIRAL HEPATITIS SEROLOGY
Hepatitis C
EPIDEMIOLOGY
Formerly called non-A, non-B (NANB) hepatitis prior to the
discovery of the hepatitis c virus
Transmitted primarily by exposure to contaminated blood
(especially blood transfusion)
Other modes of transmission: parenteral, sexual, vertical
 VIRAL HEPATITIS SEROLOGY
Hepatitis C
SIGNS AND SYMPTOMS
Incubation period: 2 to 30 weeks (average 7 weeks)
Virus produces symptoms of acute hepatitis in only 20% of
cases
Majority of infections are asymptomatic
Progress to a chronic state in about 85% of cases and
usually progresses slowly to cirrhosis and even
hepatocellular carcinoma
 VIRAL HEPATITIS SEROLOGY
Hepatitis C
DIAGNOSTIC EVALUATION
Viral culture is extremely difficult; viral detection is made
solely by other laboratory methods
Serological testing
o Used for screening blood and organ donors and initial
diagnosis of symptomatic patients
o Detection of IgG anti-HCV against recombinant and
synthetic antigens (Core, NS3, NS4, NS5 proteins) by third
generation EIAs or CIA methods
 VIRAL HEPATITIS SEROLOGY
Hepatitis C
DIAGNOSTIC EVALUATION
Traditional confirmatory method: Recombinant Immunoblot
Assay (RIBA) to detect specific HCV antibodies
Recently, confirmatory testing has been replaced by
molecular testing methods:
o Qualitative and quantitative test to detect HCV RNA
o Reverse transcriptase (RT) -PCR (most common)
o Trancription-mediated Amplification (TMA) (highly
sensitive)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
ETIOLOGY
Agent: Hepatitis B Virus (HBV; Dane particle)
Enveloped, partly double-stranded DNA virus of the
Hepadnaviridae family
Replication occurs in the cytoplasm of the hepatocyte
Eight distinct genotypes (designated A to H) with different
clinical outcomes
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
EPIDEMIOLOGY
Known as serum hepatitis or long-incubation hepatitis
A major cause of morbidity and mortality throughout the
world as a blood-borne pathogen
Transmitted through the parenteral route, by intimate
contact with HBV-contaminated blood or other body fluids,
via the (vertically) perinatal route, or via sexual contact
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
SIGNS AND SYMPTOMS
Incubation period: 45-90 days
Symptoms that vary among different age groups:
o 90% of newborns – asymptomatic
o 5-15% of children (1-5 years) – acute hepatitis
o 33-50% of older children, adolescents, and adults –
acute hepatitis
“Development of chronic HBV infection, in which the virus persists in
the body for 6 months or more, is independently related to age”
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
SIGNS AND SYMPTOMS
chronic hepatitis B develops in about
o 10% of infected adults
o 25-50% of young children
o 80-90% of infected infants
Chronic infection is also more likely to develop in persons
who are immunosuppressed and those who have HIV
Most HBV-infected adults recover within 6 months and
develop immunity but about 1% develop fulminant liver
disease (high fatality rates)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
HBV VIRAL STRUCTURE
Important viral proteins:
1. Envelope protein – HBsAg (Surface antigen)
2. Structural nucleocapsid core protein – HBcAg (Core
antigen)
3. Soluble nucleocapsid protein – HBeAg (Related antigen)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
IMMUNOLOGIC MANIFESTATIONS
HBV ANTIGENS
1. Hepatitis B Surface Antigen (HBsAg)
2. Hepatitis B-Related Antigen (HBeAg)
3. Hepatitis B Core Antigen (HBcAg)

HBV ANTIBODIES
1. Antibodies to Hepatitis B Core Antigen (Anti-HBc)
2. Antibodies to Hepatitis B-Related Antigen (Anti-HBe)
3. Antibodies to Hepatitis B Surface Antigen (Anti-HBs)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
HBV ANTIGENS
Hepatitis B Surface Antigen (HBsAg) – Australia antigen
o Earliest marker of infection
o Indicates active infection
Hepatitis B-Related Antigen (HBeAg)
o Present during active replication
o Indicates high degree of infectivity
Hepatitis B Core Antigen (HBcAg)
o Not detectable in serum
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
Genomic DNA
(double- HBV ANTIGENS
stranded with
partial single
strand)
Hepatitis Envelope
B surface
antigen
(HBsAg)

Nucleocapsid
(viral capsid)
Spherical
Envelope lipid
(from host cell)

Hepatitis B e antigen Hepatitis B core antigen

(HBeAg) (HBcAg) Elongated

Complete infectious virion Viral envelope particles containing HBsAg

 VIRAL HEPATITIS SEROLOGY
Hepatitis B
HBV ANTIGENS

Elongated form Spherical form

Dane particle
(complete virion)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
HBV ANTIBODIES
1. Antibodies to Hepatitis B Core Antigen (Anti-HBc)
IgM
o first to appear
o indicates current or recent infection
o useful for detecting infection in cases in which HBsAg is
undetectable (window period)
IgG
o indicates past infection and immunity
o lifetime persistence
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
HBV ANTIBODIES
2. Antibodies to Hepatitis B-Related Antigen (Anti-HBe)
Indicates convalescence and recovery

3. Antibodies to Hepatitis B Surface Antigen (Anti-HBs)

Appears also during recovery period of acute hepatitis, a few
weeks after HBsAg disappears
Persists for years, providing protective immunity
Produced after immunization with Hepatitis B vaccine
(Recombinant HBsAg)
VIRAL HEPATITIS SEROLOGY
Hepatitis B
TYPICAL SEROLOGIC MARKERS

ACUTE HBV INFECTION

VIRAL HEPATITIS SEROLOGY
Hepatitis B
INTERPRETATION OF HEPATITIS B PANEL (TURGEON)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
LABORATORY DIAGNOSTIC EVALUATION
Serological tests for Hepatitis B markers
o Enzyme Immunoassays
o Chemiluminescent Immunoassays
Molecular methods
o Real-Time PCR (HBV DNA)
 VIRAL HEPATITIS SEROLOGY
Hepatitis B
LABORATORY DIAGNOSTIC EVALUATION
 VIRAL HEPATITIS SEROLOGY
Hepatitis D
Delta hepatitis
Agent: Hepatitis D Virus (HDV; Delta agent)
o Unclassified, single-stranded, defective/incomplete RNA
virus
o Requires the presence of HBV for replication
o Coinfection or superinfection with HBV
Coinfection: HBV and HDV infections occurring simultaneously
Superinfection: HDV infection in people with existing HBV infections
Mode of transmission: parenteral; sexual
Signs and Symptoms: acute or chronic hepatitis
 VIRAL HEPATITIS SEROLOGY
Hepatitis D
1-3% of cases of co-infections progress to a chronic state
Superinfections result in a greater risk of developing
fulminant hepatitis or chronic liver disease with an
accelerated progression toward cirrhosis, liver
decompensation, and hepatocellular carcinoma
 VIRAL HEPATITIS SEROLOGY
Hepatitis D
Laboratory Diagnosis:
1. Serologic: ELISA
o Anti-HDV (IgG/IgM)
o Anti-HBc IgM – Differentiates coinfections from
superinfections
2. Molecular: PCR (HDV RNA)
3. Histopathologic: Liver biopsy
o HDV antigen
 VIRAL HEPATITIS SEROLOGY
Hepatitis G
Agent: Hepatitis G Virus (HGV)
o Enveloped, single-stranded, RNA virus
o Flaviviridae
o 96% identical to a viral agent known as GB Virus Type C
(GBV-C)
o Frequently occurs in coinfections with HCV
Mode of Transmission: blood-borne, parenteral, sexual
 VIRAL HEPATITIS SEROLOGY
Hepatitis G
Signs and Symptoms:
o Association with hepatitis is uncertain
Laboratory Diagnosis:
1. Serologic: Only available mainly for research
2. Molecular: RT-PCR (HGV RNA)
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