Diagnostics tests for viral hepatitis

1. Diagnostic Evaluation of
Viral Hepatitis
Dr Ashok Rattan,
Chief Scientific Officer, RAK Hospital
&
COO & Medical Director,
Star Metropolis Clinical Laboratories,

2. Hepatitis
• Hepatitis is a general term referring to
inflammation of the liver
• Causes:
– Infectious
•
•
•
•
Viral
Bacterial
Fungal
Parasitic
– Non infectious
•
•
•
•
Alcohol
Drugs
Autoimmune
Metabolic diseases

3. Viral Hepatitis
• Hepato-tropic viruses
–
–
–
–
–
Hepatitis A Virus (HAV)
Hepatitis B Virus (HBV)
Hepatitis C Virus (HCV)
Hepatitis D Virus (HDV)
Hepatitis E Virus (HEV)
• Other viruses
–
–
–
–
–
Adenovirus
Cytomegalovirus (CMV)
Epstein Barr virus (EBV)
Herpes simplex virus (HSV)
Yellow fever virus (YFV)

4. Case Study
• A 27-year-old African female presented to the
emergency room with a five day history of malaise,
fatigue, low-grade fever and nausea. Yesterday, she
noted that her urine was very dark and her stools were
very light in color. She also complained of joint pain.
Her liver was noted to be enlarged on physical exam and
the whites of her eyes were yellow. She had a history of
three sex partners in the past few months, denied illegal
drug use and otherwise was in good health.

5. What viral hepatitis serologic tests would be
appropriate to order?
1. HBsAg, Ig M anti HBc, Ig M anti HAV, anti HCV
2. Total anti HBc, Total anti HAV, anti HCV
3. Ig M anti HBc, Ig M anti HAV

6. Correct answer: 1
• This patient has signs and symptoms of acute
viral hepatitis. Using serologic testing for
acute viral hepatitis is the correct approach
for this patient.
– HBsAg
– IgM anti HBc
– IgM anti HAV
– Anti HCV

7. Results of Serological tests
•
•
•
•
HBsAg positive
IgM anti-HBc - positive
IgM anti-HAV - negative
Anti-HCV –
negative
• Based on the serological results, Your diagnosis is:
1. Acute Hepatitis A, Acute Hepatitis B and Acute Hepatitis C
2. Acute Hepatitis B
3. Acute Hepatitis C

8. Correct Answer: 2
• A positive IgM-anti-HBc test indicates recent
HBV infection.
• The IgM anti-HAV test is negative, indicating
that she does not have recent HAV infection.
• The anti-HCV test is negative, indicating she
does not have HCV infection

9. What else should the treating physician do ?
1. Report the case of acute hepatitis B to public health
department
2. Counsel the patient that any sex contact within the
past 6 months are at risk of infection & should
contact their Health care provider
3. Counsel patient about importance of using barrier
precaution to prevent transmission of infection to
sex partners
4. All of the above

10. Correct answer: 4
• Acute hepatitis B is a reportable disease.
• Hepatitis B is a sexually transmitted disease and
patients should be counseled on how to prevent
transmission.

11. Diagnostic Considerations
Overview
• Non icteteric patient
– Simple screening test for
• Urine for bilirubin
• Serum for Liver enzyme penal
• Blood glucose by finger prick for pts with altered
sensorium
– High bilirubin patients
•
•
•
•
Alkaline phosphatase
Prothrombine time
BUN & serum creatinine
Serum ammonia (in altered mental status)

12. Viral markers
overview
• IgM HAV is standard for diagnosing HAV acute
infection
• IgM HBc Ag for acute HBV infection
• HBsAg may be present in acute as well as in
chronic carriers, its presence in symptomatic
pts suggests acute HBV infection
• Anti HCV or NAAT positive for HCV infection,
pt is normally asymptomatic

13. Hepatitis B Virus
• Hepa DNA viridae
family, 3.2 kp partially
ds DNA
• Four overlapping genes
– Gene S for HBsAg
– Gene C for HBcAg
– Gene P for DNA
polymerase (RT activity)
– Gene X codes for X
protein (regulatory)

14. Routes of Transmission
• Vertically,
– between mother with chronic infection & her
baby
• Horizontally,
– through close person to person contact (through
cuts or sores)
– Parenterally, via injections
– Sexually

15. World wide distribution of HBV

16. HBV infections
• Parameters used to define & characterize HBV
infection include
– HBV antigens & host antibodies
– HBV DNA & genotype
– Biochemical markers
– Degree of hepatic fibrosis & inflammation

17. HBs Ag
• Forms part of the
envelope
• Also exists in large
quantity within serum
• Outnumber viron 102 to
105 times
• HBs Ag positive persons
have overt HBV
infection, but not
necessarily liver disease

19. • Serum anti HBc is most useful & inexpensive marker
for identification of occult HBV infection in HBs Ag
negative individuals

20. Protection
• In HBs Ag negative persons
• protective immunity
– anti HBs antibodies +
• anti HBc antibodies – ve (following vaccination)
• Anti HBc antibodies + ve (following natural infection)

21. Occult HBV infection
• Presence of HBV DNA in the liver
– with detectable (< 200 IU/ml)
– or undetectable HBV DNA in serum
– HBs Ag negative
– Anti HBc antibodies
Can persist throughout lifespan of the individual

22. Chronic HBV infection
• American Association of the Study of Liver
Diseases (AASLD) defined chronic hepatitis B
as HBsAg positivity for more than six months

23. Immune
status
Im. tolerant
Im. clearance
Im. control
Im. escape
(healthy carrier)

24. HBe Ag
• Secretory form of HBc Ag, released into serum
• HBcAg is assembled within nucleocapsid
• Detection of HBe Ag is hall mark of 1st phase of
infection with wild HBV
• HBe Ag is a marker of replicative HBV infection
• HBe Ag clearance & anti HBe seroconversion
indicates a switch from e+ CBH to inactive
HBV carrier state
• HBe Ag seroconvertion is an important
therapeutic milestone & goal

25. HBV DNA
• Direct product & hallmark of HBV infection
• REVEAL study:
– Liver disease progression was intrinsically linked
to extent of viral replication
– Quantification of serum HBV DNA has become a
pivotal tool
•
•
•
•
in management of HBV carriers
Identify of disease progression
Select candidates for antiviral therapy
Guide treatment with nucleoside/nucleotide analogues

27. Non invasive liver disease markers
• Combination of serum markers have been
shown to predict liver fibrosis.
• FibroTest
– Aminotransferases
– Α 2 macroglobulin
– Apolipoprotein A1
– Haploglobin
– γ glutamyl transpeptidase
– Total bilirubin

28. Clinical significance of HBV markers
HBV marker
Diagnostic category
Anti HBs antibodies
Immunity
Anti HBc antibodies
Exposure
HBs Ag &/or HBV DNA
Infection
HBe Ag &/or HBV DNA
Replication
IgM anti HBc &/or HBV DNA
Disease

29. End points of therapy for
chronic hepatitis B infection
End point
Criteria
Biochemical
Normal ALT levels
Serologic
HBeAg loss & sero conversion to anti HBe
HBsAg loss , with/or without sero conversion to
anti HBs
Virologic
Sustained decrease in serum HBV DNA to
undetectable level
Histologic
Reduction in fibrosis stage
No worsening of fibrosis
Reduction of inflammatory activity

31. Hepatitis B Panel interpretation
HBsAg anti IgM
HBc
Anti
HBc
Anti
HBs
---
---
---
-+
--
--
+
+
+
+
--
+
---
+
+
+
----
Four possibilities : 1. Resolving infection
3. Low level chronic infection
Interpretation
Susceptible
Immune due to
vaccination
Immune due to natural
infection
Acute infection
Chronic Infection
Interpretation unclear
2. False positive anti HBc
4. Resolving acute infection

32. HCW
• A 43-year-old registered nurse was hired to work in
the emergency room at a large tertiary care center.
She was given the 3-dose hepatitis B vaccine series
followed by post vaccination testing two months
after the last dose for antibody to hepatitis B surface
antigen (anti-HBs). Her anti-HBs concentration was 5
mIU/mL.

33. What is your interpretation
• 1. She is infected with HBV
• 2. She is protected from infection with HBV
• 3. She is not protected against HBV

34. Correct answer: 3
• Anti-HBs is the marker that indicates
immunity to HBV infection.
• An anti-HBs result less than 10 mIU/mL within
1-2 months after completion of the hepatitis B
vaccine series indicates that she is not
protected against HBV infection.

35. What should be done ?
1. She should be revaccinated with three doses
of HBV vaccine
2. She should have anti HBs retested.
3. Nothing needs to be done.

36. Correct answer is: 1
• She should be revaccinated with a 3-dose
hepatitis B vaccine series followed by
postvaccination testing for anti-HBs (1-2
months after the last dose). 50-75% of people
develop seroprotection after an additional
series.

37. • She was revaccinated. Her postvaccination antiHBs test result was 150 mIU/mL. She is now
protected from HBV infection. The result was
placed in her occupational health record.
• Six years later, she had a needlestick. The source
patient was HBsAg positive and anti-HCV
positive.
• What should be done for Hepatitis B:
– 1. She should have a booster dose of the vaccine
– 2. She should be retested for HBsAg
– 3. Nothing needs to be done

38. Correct answer is: 3
•No postexposure prophylaxis is recommended for
persons who have ever had a documented anti-HBs
result of at least 10 mIU/mL after hepatitis B
vaccination, even if this result was many years in the
past. Immunocompetent persons who respond to
hepatitis B vaccination remain protected even if the
anti-HBs concentration falls below measurable levels.
•What needs to be done for the exposure to blood
from an anti-HCV positive source patient?
– 1. Nothing needs to be done
– 2. Test for ALT only
– 3. Baseline Test for ALT and anti HCV

39. Correct answer is: 3
• Baseline testing for anti-HCV and ALT activity is
recommended. (If an earlier diagnosis of HCV
infection is needed, testing for HCV RNA by PCR may
be performed at 4-6 weeks.) All positive anti-HCV
results by enzyme immunoassay should be verified
by supplemental testing with a recombinant
immunoblot assay or PCR for HCV RNA.

40. • Baseline testing for HCV is NEGATIVE
• What additional follow-up should be done
regarding her exposure to HCV-positive
blood?
– 1. Follow up testing for anti HCV and ALT at 4 to 6
months
– 2. Counseling about infection control practices at
work
– 3. Counseling about not to donate blood for 4 to 6
months
– 4. All of the above

41. Correct answer is: 4
•Follow-up testing for anti-HCV and ALT testing at 4-6 months
after the needlestick should be done. Persons who are anti-HCV
negative at 4-6 months can be assured that they did not become
infected from the exposure.
•Persons who are exposed to HCV-infected blood should refrain
from donating blood, plasma, organs, tissue, or semen during
the follow-up period.
•No modifications to an exposed person's patient care
responsibilities are necessary to prevent transmission. All health
care professionals should follow recommended infection control
practices to prevent blood exposures, including standard
precautions and appropriate use of hand washing, protective
barriers, and care in the use and disposal of needles and other
sharp instruments.

42. Hepatitis C Infection
•
•
•
•
•
RNA virus
9.4 kb
55 nm diameter
One serotype
6 major genotypes &
80 subtypes

43. HCV Prevalence
Prevalence of HCV Infection

44. Genotypes of HCV

45. Risk Factors

47. Optimal approach to detection of
HCV infection
• Screen persons for a history of risk of exposure to
the virus
• Test selected individuals who have identifiable risk
factor
– IV drug abuser
– Received blood component transfusion
– Haemodialysis
– Children born to HCV positive mothers
– Exposure to an infected sexual partner
– Needle stick injury in HCW

51. ALT

58. 85%

61. Interpretation of HCV assays
Anti HCV HCV RNA
interpretation
+
+
Acute or chronic HCV depending
upon clinical context
+
-Resolution of HCV
-+
Early acute HCV, chronic HCV in
immunosuppressed states
--Absence of HCV

64. Hepatitis A Abs. (Anti- HAV) IgG, IgM
Hepatitis A Diagnostic Panel (Anti HAV IgG, IgM)
Hepatitis Acute Diagnostic Panel (Anti HAV IgM, HBsAG, ANTI HBc IgM)
Hepatitis Acute Virus Screen (Anti HAV IgM, HBsAG, ANTI HBc IgM, ANTI HCV IgM,
ANTI HEV IgM)
Hepatitis Acute Virus Confirmation (anti HAV IgM, HBsAG, HBeAG, ANTI HBe, ANTI
HBc IgM, ANTI HCV IgM, ANTI HEV IgM)
Hepatitis B Viral DNA (HBV DNA ) Quantitative, RT PCR
Hepatitis B Viral DNA (HBV DNA ) Qualitative PCR
Hepatitis B Chronic Panel (HBsAG, HBeAG, ANTI HBe)
Hepatitis B Immunity Screen (ANTI HBc TOTAL, ANTI HBs, HBsAG)
Hepatitis B Profile (HBsAG, ANTI HBs, HBeAG, ANTI HBe, ANTI HBc IgM, ANTI HBc
TOTAL)

65. Hepatitis C Abs (Anti HCV)
Hepatitis C Abs (Anti HCV) IgM
Hepatitis C Viral Combo (HCV RNA QUANTATIVE RT PCR, HCV GENOTYPE)
Hepatitis C Viral RNA, Genotype 1,2,3,4
Hepatitis E Abs (Anti HEV) IgG, IgM
Hepatitis Viral Comp. Panel (ANTI HAV IGG, ANTI HAV IGM, ANTI HBC TOTAL,
ANTI HBC IGM, ANTI HBS , HBASG, ANTI HBE, HBE AG, ANTI HCV , ANTI HCV IGM,
ANTI HEV IGG, ANTI HEV IGM )

66. Patient history:
• A 25 year old Asian male had been feeling very tired,
was jaundiced and had vague "flu-like" symptoms.
He went to see his primary care physician who did a
history and physical and ordered blood tests that
included serology for acute viral hepatitis.
• What serological tests were ordered ?
1. HBsAg, IgM anti HAV, IgM anti HBc and anti HCV
2. IgM anti HAV
3. IgM anti HBc

67. Correct answer: 1
• Testing for hepatitis A, B, and C using IgM anti-HAV,
IgM anti-HBc, HBsAg, and anti-HCV are the most
appropriate serologic tests for the diagnosis of acute
viral hepatitis.
• The test results are as follows:
–
–
–
–
HBsAg - negative
IgM anti-HBc - negative
IgM anti-HAV - negative
Anti-HCV - positive by enzyme immunoassay (EIA)
•
- positive by recombinant immunoblot assay (RIBA)
• ALT - 1500 IU (upper limit of normal - 45 IU).

68. What is your diagnosis ?
1. Acute hepatitis B
2. Acute hepatitis C
3. Chronic hepatitis C

69. Correct answer is : B
• The diagnosis is acute hepatitis C.
• In patients with acute hepatitis C, alanine
aminotransferase (ALT) levels are usually at least
seven times the upper limit of normal.
• ALT levels in patients with chronic HCV infection are
elevated, but are usually lower than in patients with
recently acquired infection.

70. What additional steps need to be
carried out ?
•
•
•
•
1. An interview to identify risk factors
2. Counseling for prevention of transmission
3 Follow up to evaluate outcome of infection
4. All of the above

71. Correct answer is : 4
• All patients with acute hepatitis C should be interviewed
to identify a risk factor(s) for infection in the 2 weeks to
6 months before illness onset. If the patient has
received blood or blood products, has been hospitalized,
or has had hemodialysis, surgery, or other medical or
dental procedures, further investigation is needed to
determine if additional cases are associated with a
common source of exposure.
• All patients with acute hepatitis C should receive
counseling about how to prevent transmission of HCV to
others and should receive followup to evaluate the
outcome of infection and possible need for treatment.