Most cases of acute viral hepatitis are caused by five viruses - hepatitis A, B, C, D, and E. Hepatitis A and E are transmitted through the fecal-oral route while hepatitis B, C, and D can be transmitted through blood and body fluids. Laboratory diagnosis of viral hepatitis involves serological tests to detect antibodies and antigens to the hepatitis viruses as well as molecular tests to detect viral RNA. Interpretation of hepatitis serology can indicate acute or chronic infection.

1. Lab Diagnosis of Viral
Hepatitis
Dr. Dinesh Kr Jain, MD., Assistant professor,
Department of Microbiology, SMS Medical
college, Jaipur

2. Lab diagnosis of viral hepatitis
*Hepatitis D Virus co-infection/super-infection with HBV
D*

3. Most cases of acute viral hepatitis in children and
adults are caused by one of the following five
agents:
• hepatitis A virus (HAV), the etiologic agent of
viral hepatitis type A;
• hepatitis B virus (HBV), which is associated with
viral hepatitis B;
• hepatitis C virus (HCV), the agent of hepatitis C ;
• hepatitis D (HDV), a defective virus dependent
on coinfection with HBV; or
• hepatitis E virus (HEV), the agent of enterically
transmitted hepatitis.

4. • Similar clinical presentation but multiple
disease outcomes-acute hepatitis (A,E,B,C,);
Chronic infections (B&C)
• Sequelae – Fibrosis, cirrhosis and HCC.
• Route of transmission of HAV & HEV is the
feco-oral route (contaminated water and
food) and that of HBV and HCV is per
cutaneous

5. Salient features of viral hepatitis
HAV HBV HCV HDV HEV
Family Picorna-
viridae
Hepadna-
viridae
Flavi-
viridae
Unclassified Hepe-
viridae
Genus Hepatovirus Orthohepadna
virus
Hepacivirus Deltavirus Hepevirus
Virus RNA DNA RNA RNA RNA
Envelope Absent Present Present Present Absent
Nucleic acid ssRNA dsDNA ssRNA ssRNA ssRNA
Size 27 -32nm 27-42 nm 50 -80nm
(approx.)
35-37 nm (with
HBsAg coat)
32-34nm
Genome type +ssRNA Partial dsDNA +ssRNA −ssRNA +ss RNA
Genome size 7.5 kB 3.2 kB 9.4 kB 1.7 kB 7.5 kB
Genotypes 7 9 (A-I) 6 (1-6) 4 (1-4)
Treatment Supportive
care
Anti-viral
therapy
++ Hep B Rx Supportive
care
Vaccine Available. Available Not available Not available Not available
10/4/2019 NCDC

6. Salient features of viral hepatitis
HAV HBV HCV HDV HEV
Routes of
transmission
Fecal-oral Percutaneou
s
Per mucosal
Percutaneous
Per mucosal
Percutaneo
us
Per mucosal
Fecal-oral
Sexual
transmission
no yes,
common
yes,
uncommon
yes,
uncommon
no
Vertical
(Mother to
child)
No Yes Rare Occasional No
Incubation
period, days
15-45 30-180 15-160 30-180 14-60
Fulminant
hepatitis%
0.1 0.1-1 0.1 5-20 1-2, 10-20
in
pregnancy
Carrier state No Yes Yes ? No*
Chronic
hepatitis
No Yes Yes Yes Yes
(genotype
3)
(HCC) No Yes Yes - No
* Chronic hepatitis in immunosuppressed patients.

7. Clinical presentation of acute hepatitis
• Fever, malaise, fatigue, loss of appetite, nausea
Vomiting, abdominal pain, arthralgia, jaundice
• Usually resolve within 3 months
• Rarely fulminant hepatic failure
• Biochemical markers
– Elevated ALT (usually > 10 fold of normal limits)
– Elevated Total Bilirubin >2.5mg/mL

8. Chronic hepatitis
Hepatitis B
Chronic infection develops in:
• >90% of infants
• 25%–50% of children 1–5
years
• 6%–10% of older children
and adults
Hepatitis C
Chronic infection
develops in 75%-85%
of cases
Biochemical markers: Normal or mild to
moderate elevations in AST/ALT
Clinical features: mostly asymptomatic

9. Hepatitis A virus
(HAV)

10. Hepatitis A is a vaccine-preventable,
communicable disease of the liver caused by the
hepatitis A virus (HAV).
It is usually transmitted person-to-person
through the fecal-oral route or consumption of
contaminated food or water.
Hepatitis A is a self-limited disease that does not
result in chronic infection.

11. Sample collection and transport
For Antibody and Antigen detection assays
• serum collected in plain (red topped) tubes
• serum separator tube (SST’s) - gel vacutainers also used
• serum separated within 4 hrs. of collection
• storage at -80ºC; avoid repeated freezing & thawing
For NAT’s (RT-PCR)
• plasma collected, serum also acceptable
• plasma separated within 2 hrs. of collection
• storage at -80ºC; avoid repeated freezing & thawing
• EDTA preferred anticoagulant, citrate also acceptable
• Heparin contraindicated - inhibits PCR - binds Taq polymerase

12. Serology –HAV IgM
• The diagnosis of acute HAV infection is confirmed by the
presence of IgM antibodies to HAV.
• In acute hepatitis A, the presence of anti-HAV IgM is
detectable about 3 weeks after exposure, its titer increases
over 4 to 6 weeks, then declines to non detectable levels
generally within 6 months of infection
• Prolonged presence has been reported more than 11-20
months .Along with deranged Biochemical markers.
?Chronic HAV or Prolonged HAV
(J Med Virol 50;322-24, Journal of Pediatric Infectious Diseases ;2008:63–67)
• A small proportion (8 to 20%) of vaccinated persons have a
transient IgM anti-HAV response

13. IgG anti-HAV
• IgG also appears early in the course of infection,
remains detectable for the lifetime of the individual
and confers lifelong protection against infection.
• Previous (resolved) HAV infection is diagnosed by
detection of IgG anti-HAV.
• The presence of total anti-HAV and the absence of
IgM anti-HAV can be used to differentiate between
past and current infections.

14. Immunological & biological events in
hepatitis A

15. HAV: Laboratory Tests
Serology (Antibody detection)
- Anti-HAV IgM
- Anti-HAV IgG
-Anti-HAV Total Antibodies (IgM and IgG)
Molecular assays (NAT)
- RT-PCR for HAV RNA (from stool, blood)
Electron Microscopy (EM) and Immune EM (stool)
- Virions ≈27 nm
Only in late incubation
period and pre-icteric
phase; not routinely
used for diagnosis
RDTs and
Immunoassays - ELISA ;
CLIA

16. Hepatitis B virus

17. • Hepatitis B virus (HBV) infection remains a global
public health problem despite the availability of
an effective vaccine.
• Mother-to-child transmission is the predominant
mode of transmission in high-prevalence areas.
• In comparison, horizontal transmission,
particularly in early childhood, accounts for most
cases of chronic HBV infection in intermediate-
prevalence areas,
• while unprotected sexual intercourse and
injection drug use in adults are the major routes
of spread in low-prevalence areas

18. HBV : Structure
HBsAg = surface (coat) protein ( 4 phenotypes : adw, adr, ayw and ayr)
HBcAg = inner core protein (a single serotype)
HBeAg = secreted protein; function unknown

19. Diagnosis of HBV
• Serological Assays
• Biochemical Assays
• Molecular Diagnostics

20. Key Serological Markers for HBV
• Hepatitis B Surface Antigen (HBsAg)
• =INFECTION
– earliest viral marker to be detected
– appears before symptoms
– after elevated liver enzymes
– indicator of active HBV infection
– disappears in 2-6 months
– if persistent for > 6 months without generation of anti-
HBsAg antibody indicates a chronic carrier state

21. Hepatitis B e Antigen (HBeAg)
• WILD TYPE INFECTION
– appears after HBsAg but before symptoms
– limited presence (3-6 weeks)
– indicates viral replication in the liver and high
infectivity
– sera containing HBsAg and HBeAg is 3-5 times more
infectious than HBsAg alone
– lack of HBeAg does not mean there is no circulating
HBV
• pre-core or HBeAg (-) mutants

22. Hepatitis B e Antibody (anti-HBe)
• LOW HBV DNA OR CORE MUTANT
– detectable shortly after declination of HBeAg
– implies HBV no longer replicating & disease is
resolving
• indicates risk of infection is reduced
– if no anti-HBe seroconversion - increased disease
activity and progression

23. IgM Hepatitis Core Antibody
(IgM anti-HBc)
• ACUTE HBV
– before onset of symptoms
– same time as elevated liver enzymes
– after HBeAg
– persists for months to years
– marker of an acute HBV infection
– usually replaced by IgG anti-HBc
– Occasionally seen in patient with CHB (flare or very active
disease)

24. Hepatitis B Surface Antibody (anti-HBs)
– not evident until acute disease subsides
– weeks to months after HBsAg disappears
– persists for life
– used to document recovery and/or immunity to HBV
infection
– individuals who have responded to the vaccine will
have anti-HBs antibodies

25. Serological tests are the mainstay of 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 (after recovery or post immunization).
• anti-HBc IgM - marker of acute 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.

26. Clinical progression & serologic events in
acute HBV infection

27. Molecular Diagnostics
• Technologies to detect and quantify levels of HBV
DNA in sera
– for monitoring HBV during treatment
• Presence indicates active HBV replication
• HBV DNA is detectable prior to biochemical or
serological evidence of hepatitis(21 days prior to
HBsAg)
• HBV DNA persists through both acute and chronic
disease
• More accurate than HBeAg especially with escape
mutants

28. *Lok, AS (2001) Gastroenterology; 120: 1828-1853
Practical Use of HBV DNA Detection
• Screening of Blood Donations
• Prognosis of HBV Infection is driven by HBV DNA
quantification
– more useful for disease status*
• if <103 IU/mL = ?inactive carrier state
• if >103 IU/mL = clinically significant
• Assessment of Disease Severity & Prognosis
– active HBV replication = greater risk of progression to
chronic HBV complications

29. HBV: Interpretation of Common Serological Profiles
HBsAg Anti-
HBs
Anti-
HBc
HBeAg Anti-
HBe
Interpretation
+ - IgM + -
+ - IgG + -
- - IgM - -
- + IgG - +/-
- - IgG - -
- + - - -
Acute hepatitis B, infective
Chronic hepatitis B, infective
Acute HBV infection in the window period
HBV infection in the remote past, with recovery
Recovery from HBV infection
Immunization with HBV vaccine

30. HEPATITIS C VIRUS (HCV)

31. • HCV is a single-stranded RNA virus belonging to
the family Flaviviridae. HCV causes both acute
and chronic infection; however, unlike HBV, HCV
has a higher propensity to lead onto chronic
viraemia and 25% of these patients can develop
chronic hepatitis.
• HCV is transmitted primarily through parenteral
exposures of infectious blood or body fluids.
• Injection drug use/unsafe injection practices
(currently the most common means of HCV
transmission
• Needlestick injuries in health care settings
• Birth to an HCV-infected mother

32. HCV -diagnostic modalities
1) Antibody detection – Serology
- ELISA
- Chemiluminescence immunossay (CLIA)
3) Molecular methods - Nucleic Acid Tests (NAT’s)
- qualitative assays - quantitative assays
- RT-PCR - Real Time RT-PCR
- -
4) HCV Genotyping

33. 1) Serology – Antibody detection
• screening for HCV
• ELISA commonly preferred, CLIA increasingly being used
• Advantages - inexpensive
- easy to use, automated
- reproducible
- good sens. & spec.
• Disadvantages
- false negative results in - immunocompromised - HIV, transplant recipients
- hemodialysis patients

34. Clinical and serological events in HCV
infection

35. • Most patients who are acutely infected with hepatitis C
virus (HCV) are asymptomatic.
• Symptomatic patients may experience jaundice,
nausea, dark urine, and right upper quadrant pain.
Patients with acute HCV infection typically have
moderate to high serum aminotransferase elevations.
• These may go undetected in asymptomatic patients.
Among patients who are symptomatic, symptoms
typically develop 2 to 26 weeks after exposure to HCV,
with a mean onset of 7 to 8 weeks . The acute illness
usually lasts for 2 to 12 weeks.

36. • since the levels often fluctuate (sometimes quite
widely) and may even normalize, not all patients will
have elevated aminotransferase levels at the time of
presentation.
• HCV RNA is first detectable in serum by PCR within
days to eight weeks following exposure, depending, in
part, upon the size of the inoculum.
• Enzyme-linked immunosorbent assay (ELISA) tests
detecting anti-HCV antibodies become positive as early
as eight weeks after exposure, with most patients
seroconverting between two and six months after
exposure

37. HCV Tests: Interpretation
Anti-HCV HCV RNA
(plasma/serum)
Interpretation
+ +
Hepatitis C infection acute /chronic
+ - Recovered HCV infection
False positive serology result
- + Early acute HCV infection
Chronic HCV in an immuno-
compromised host
- - Absence of HCV infection

38. HEPATITIS E VIRUS (HEV)
• Hepatitis E virus, the agent that causes hepatitis E, has
small, 27–34 nm non-enveloped virions that contain a
small, 7.2-kb single-stranded RNA genome.
• The virus has at least four distinct genotypes,
numbered from 1 to 4. Genotypes 1 and 2 infect only
humans, and are responsible for the majority of human
disease caused by infection with this virus globally; in
contrast, genotypes 3 and 4 primarily circulate among
mammalian animals, including pigs, wildboar, deer, and
only occasionally infect humans.
• Genotypes 1 and 2 have been associated with large
waterborne outbreaks

39. • Laboratory findings — Laboratory findings
include elevated serum concentrations of
bilirubin, alanine aminotransferase (ALT), and
aspartate aminotransferase .
• Symptoms coincide with a sharp rise in serum
ALT levels, which may rise up into the
thousands and return to normal during
convalescence .
• Resolution of the abnormal biochemical tests
generally occurs within one to six weeks after
the onset of the illness .

40. Antibody testing —
• The timing of appearance of HEV markers is
important for interpreting results of serologic
testing in the setting of acute hepatitis
• IgM anti-HEV appears during the early phase of
clinical illness and disappears rapidly over four to
five months .
• The IgG response appears shortly after the IgM
response, and its titer increases throughout the
acute phase into the convalescent phase.
• It is unclear how long IgG anti-HEV antibodies
persist.

41. HEV RNA assay
• HEV can be detected in stool approximately
one week before the onset of illness and can
persist for as long as two weeks thereafter .
• In serum, HEV may be detected two to six
weeks after infection and can persist for two
to four weeks in those who resolve acute
infection. Although HEV viremia is short-lived
in most patients

42. HEV: Laboratory Markers
Krain LJ. Clin Microbiol Rev, 2014

43. • Acute HEV –In general, the initial test method is an anti-HEV IgM
assay. The presence of IgM anti-HEV antibodies is suggestive of
recent HEV infection.
• If the initial test is positive, confirmatory testing should be
performed when available since no single EIA method achieves high
specificity. Confirmatory testing may include an alternate anti-HEV
IgM, evidence of rising anti-HEV IgG titers (greater than fivefold
change over two weeks), or detection of HEV RNA in serum or
stool.
• If initial EIA testing is negative, and there is still a high suspicion for
HEV infection, repeat testing should be performed, preferably with
an HEV RNA assay. The use of RNA testing is particularly important
in immunocompromised hosts with suspected HEV due to a high
rate of false-negative antibody testing

44. • Chronic HEV – In patients with suspected chronic HEV
infection, detection of HEV RNA in serum is the
mainstay of diagnosis. Chronic HEV infection is defined
as detection of HEV RNA in serum or stool for longer
than six months.
• Testing for IgG anti-HEV antibodies is of limited utility
in the diagnosis of chronic HEV infection. The presence
of IgG (or total) anti-HEV antibodies is a marker of
exposure to HEV, which can be either recent or remote.
In addition, the decline in IgG anti-HEV titers with time
might adversely affect its sensitivity for detecting
remote infection .

45. Thank you