epatitishral ihepatitisppt-191004120246.pptxvi

pathology

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 -32 nm 27-42 nm 50 -80 nm (approx.) 35-37 nm (with H B s A g 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 V acc i n e   Available. Available Not available Not available Not available 6/11/2024 NCDC

6. Salient features of viral hepatitis HAV HBV HCV HDV HEV Routes of transmission Fecal-oral Percutaneous Per mucosal Percutaneous Per mucosal Percutaneous Per mucosal Fecal-oral Sexual transmission no yes, common yes, uncommon yes, uncommon no V e r t i ca l (Mother to child) No Y e s R a r e 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 C a rr i e r state No Y e s Y e s ? 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, a bdominal 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 -80 ºC; avoid repeated freezing & thawing For NAT’s (RT-PCR) plasma collected, serum also acceptable plasma separated within 2 hrs. of collection storage at -80 ºC; 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 <10 3 IU/ mL = ?inactive carrier state if >10 3 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 .

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