laboratory diagnosis of viral hepatitis (B & C)


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Diagnostics tests for viral hepatitis

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laboratory diagnosis of viral hepatitis (B & C)

  1. 1. Diagnostic Evaluation of Viral Hepatitis Dr Ashok Rattan, Chief Scientific Officer, RAK Hospital & COO & Medical Director, Star Metropolis Clinical Laboratories,
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 15. World wide distribution of HBV
  16. 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. 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
  18. 18. • Serum anti HBc is most useful & inexpensive marker for identification of occult HBV infection in HBs Ag negative individuals
  19. 19. Protection • In HBs Ag negative persons • protective immunity – anti HBs antibodies + • anti HBc antibodies – ve (following vaccination) • Anti HBc antibodies + ve (following natural infection)
  20. 20. 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
  21. 21. Chronic HBV infection • American Association of the Study of Liver Diseases (AASLD) defined chronic hepatitis B as HBsAg positivity for more than six months
  22. 22. Immune status Im. tolerant Im. clearance Im. control Im. escape (healthy carrier)
  23. 23. 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
  24. 24. 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
  25. 25. 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
  26. 26. 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
  27. 27. 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
  28. 28. 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
  29. 29. 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.
  30. 30. 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
  31. 31. 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.
  32. 32. 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.
  33. 33. 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.
  34. 34. • 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
  35. 35. 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
  36. 36. 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.
  37. 37. • 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
  38. 38. 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.
  39. 39. Hepatitis C Infection • • • • • RNA virus 9.4 kb 55 nm diameter One serotype 6 major genotypes & 80 subtypes
  40. 40. HCV Prevalence Prevalence of HCV Infection
  41. 41. Genotypes of HCV
  42. 42. Risk Factors
  43. 43. 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
  44. 44. ALT
  45. 45. 85%
  46. 46. 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
  47. 47. 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)
  48. 48. 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 )
  49. 49. 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
  50. 50. 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).
  51. 51. What is your diagnosis ? 1. Acute hepatitis B 2. Acute hepatitis C 3. Chronic hepatitis C
  52. 52. 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.
  53. 53. 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
  54. 54. 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.