CLS 1113 Introduction to Clinical Laboratory Practices

1. CLS 1113Introduction to Clinical Laboratory Practices Hepatitis and HIV

2. HEPATITIS • Definition • Hepatitis A, B, C, D, E, G • HAV, HBV, HCV, HDV, HEV, HGV • EBV and CMV • Infectious Agents that pose a serious threat:

3. HAV • Enteric transmission • Contaminated food and water • Shellfish from contaminated waters (raw or steamed) • No chronic carrier state • Who is at risk? • Overcrowding • Poor sanitation • Military • Adult and Childcare facilities

5. HDV and HGV • HDV: Formerly called the delta agent • Can cause infection and serious hepatitis after parenteral exposure • However…HDV is a defective virus only found in HBV carriers, screening donors for HBV infection simultaneously eliminates the risk of HDV. • HGV – recently discovered virus • Distantly related to HCV, transfusion transmitted but a causal relationship has not been established • EBV and CMV – very mild in the absence of severe immunodepression

6. Clinical ManifestationsHCV and HBV • MOST have subclinical primary infection • SOME develop overt hepatitis with: • Acute Hepatitis C tends to be milder than Hepatitis B. • Clinical course of either HBV or HCV may progress to: • fulminant hepatitis • chronic hepatitis (relapsing hepatitis) • long-term progression through cirrhosis to hepatocellular carcinoma

7. Chronic Carriers of HBV • After initial infection some fail to clear infectious material and become chronic carriers for years or even for life. • Risk of HBsAg carrier is strongly age dependant: • 90% of perinatally infected infants • 5% of adults

8. Chronic Carriers of HCV • MOST persons infected with HCV become carriers • 85% have persistent HCV RNA in the serum and liver for years or decades • 50% of these have evidence of chronic liver disease • Most remain asymptomatic

9. Post Transfusion Hepatitis • Today: 1 in 60,000 to 200,000 risk of transfusion transmitted HBV or HCV • Decade ago it was 1% per unit risk

10. Serologic MarkersHBV • 2 to 6 weeks between exposure to HBV and circulating markers of infection (HBV DNA and HBsAg) • HBV DNA is the first marker, then HBsAg 5-10 days later. • Symptomatic patients: • can show anti-HBcAb, and either HBeAg or its Ab

11. Serologic MarkersHBV • Blood from individuals with circulating HBsAg can infect others • Transmission of HBV from HBsAg seronegative donors has been described. • PCR: detection of as few as 10 genomic copies of HBV DNA

12. HBV Test Reactivity Interpretation DNA HBsAg Anti-HBc Anti-HBs HBeAg Anti-HBe Total IgM + + +/ +/  +/  Early acute HBV infection/chronic carrier + + + +  +  Acute infection +/  + +  +/ +/ Early convalescent infection/possible early chronic carrier +/ + +   +/ +/ Chronic carrier   +  +  +/ Recovered infection     +   Vaccinated or recovered infection   +     Recovered infection? False positive? +       Window period

14. Serologic MarkersHCV • Anti-HCV antibody • approximately 10 weeks after exposure • EIA • Positive screen test • unclear without additional testing • some are chronic HCV or false positive • RIBA - recombinant immunoblot assay

15. Serologic MarkersHCV • Person who is positive with RIBA (Recombinant Immunoblot Assay) is considered to have true anti-HCV Ab • 70-90% HCV nucleic acid is detectable using PCR • In contrast: EIA-reactive patient with negative or indeterminate RIBA results are rarely infected or infectious.

16. HCV Testing • Nucleic Acid Technology (NAT) • Direct detection of viral genetic material • Very Sensitive • Detection before antibody production • Closing the “Window”

17. Surrogate Markers • Before HCV was detectable it was called NANB • ALT - • Anti-HBc antibody

18. Human Immunodeficiency VirusHIV • Etiologic agent of AIDS • Secondary Immune Deficiency • Properties of Virus • Persistent infection of CD4+ T lymphocytes • Some strains infect monocytes in addition to CD4+ cells • Preference for CD4+ cells due to high affinity between envelope protein of HIV-1 & cell surface CD4 marker

19. HIV • Transmission • Transplacental / Breast feeding • unless mother treated with anti-viral agents before delivery in ~ 30% cases • Sexual contact • Parenteral exposure to blood

21. Definition of AIDS • Enumeration of CD4+ cells • AIDS Classification from CDC • # of CD4+ cells • systemic symptoms • AIDS defining illnesses

22. HIV 2 • Very rare in the USA • West Africa • Longer incubation period • Lower incidence of progressing to AIDS

23. HIV Testing • Test for presence of antibodies to: • HIV-1 & HIV-2 proteins • Sensitivity of screening tests detects antibodies at about 22-25 days post infection • EIA • Positive test is referred to as “sero-conversion” or “sero-positive” for HIV

25. HIV Testing • Testing for p24 antigen began in ‘96 • Can detect p24 Ag ~ 6 days post infection • Both screening test for anti-HIV1/2 & for p24 Ag are designed to be very sensitive to reduce false neg’s

26. Confirmatory TestsHIV 1/2 • Western Blot • Viral antigens are separated into bands using electrophoresis • The bands are transferred to a nitrocellulose membrane • The Pt’s serum Antibodies • Layered on the membrane • React with the Viral Antigens depending on what antibodies to HIV the patient is producing

27. Confirmatory Tests • Current FDA & CDC criteria for being anti-HIV positive • if at least 2 of the following bands are present: p24, gp41, or gp120/160 • Confirmatory testing for HIV-1 Ag • For repeatedly positive EIA HIV-1 Ag • EIA neutralization test

28. More Sensitive Testing for HIV • NAT (nucleic acid amplification) • PCR • Culture • Viral load measurement • These tests tend to be both expensive & time consuming so they are not used for routine testing