Immunology and Serology Lab - 06 Serologic Tests for HBV and HIV

IMMUNOLOGY AND SEROLOGY LABORATORY STRUCTURAL GENES SEROLOGIC TESTS FOR HEPATITIS B VIRUS (HBV) AND HUMAN IMMUNODEFICIENCY VIRUS (HIV) • OUTLINE • • HIV and Hepatitis o Human Immunodeficiency Virus o Hepatitis Tests for HIV and HBV o HIV Antibody Test o Immunochromatographic Method HIV AND HEPATITIS • • • In layman’s term, Hepatitis means the inflammation of liver. Acute Viral Hepatitis caused by the Hepatitis B Virus is a common and serious infection. It is characterized by inflammation and necrosis of the liver. Apart from the chronic liver disease, it is the single most significant cause of mortality of liver cancer. Two important serologic markers in detecting Hepatitis B includes: HBs (Hepatitis B surface) Ag and Anti-HBs Antibody to Hepatitis B. The HbsAg is the first marker to appear and its level peak during acute stages of infection. The antiHbsAg provides protective immunity. The Human Immunodeficiency Virus (HIV) on the other hand is a retrovirus identified as the etiologic agent for Acquired Immunodeficiency Syndrome (AIDS). The principal cells infected by HIV are T4 lymphocytes that play a key role in the immune defense system of the organism. The progressive decrease of the T4 level during development of the disease leads to opportunistic infections with fatal consequences. The HIV virus consists of genomic RNA molecule protected by a capsid and an envelope. The HIV envelope is the major target for humoral antibody response. VIRAL REPLICATION • HUMAN IMMUNODEFICIENCY VIRUS (HIV) • • • • • • • Etiologic agent of acquired immunodeficiency syndrome (AIDS). Discovered independently by Luc Montagnier of France and Robert Gallo of the US in 1983-84. Former names of the virus include: o Human T cell lymphotrophic virus (HTLV-III) o Lymphadenopathy associated virus (LAV) o AIDS associated retrovirus (ARV) HIV-2 discovered in 1986, antigenically distinct virus endemic in West Africa. One million people infected in US, 30 million worldwide are infected. Leading cause of death of men aged 25-44 and 4th leading cause of death of women in the same age group in the US. Characteristics of the virus o Icosahedral (20 sided), enveloped virus of the lentivirus subfamily of retroviruses. o Retroviruses transcribe RNA to DNA. o Two viral strands of RNA found in core surrounded by protein outer coat. ▪ Outer envelope contains a lipid matrix within which specific viral glycoproteins are imbedded. ▪ These knob-like structures responsible for binding to target cell. Three main structural genes: o Group Specific Antigen (Gag) is p55 from which three core structural proteins (p15, p17 and p24) are formed. ▪ Located in nucelocapsid of virus. ▪ Icosahedryl capsid surrounds the internal nucleic acids made up of p24 and p15. ▪ p17 lies between protein core and envelope and is embedded in the internal portion of the envelope. ▪ Two additional p55 products, p7 and p9, are nucleic acid binding proteins closely associated with the RNA. o Envelope (Env) gene codes for envelope proteins gp160, gp120 and gp41. ▪ gp160 cleaved to form gp120 and gp41. ▪ gp120 forms the 72 knobs which protrude from outer envelope. ▪ gp41 is a transmembrane glycoprotein antigen that spans the inner and outer membranes and attaches to gp120. ▪ gp120 and gp41 both involved with fusion and attachment of HIV to CD4 antigen on host cells. o Polymerase (Pol) codes for p66 and p51 subunits of reverse transcriptase and p31 an endonuclease. ▪ Located in the core, close to nucleic acids. ▪ Responsible for conversion of viral RNA into DNA, integration of DNA into host cell DNA and cleavage of protein precursors. • • • • • • First step, HIV attaches to susceptible host cell. o Site of attachment is the CD4 antigen found on a variety of cells ▪ helper T cells (Primarily affected cell) ▪ macrophages ▪ monocytes ▪ B cells ▪ microglial brain cells ▪ intestinal cells o Macrophages predominant cell infected in primary infection, may serve as reservoir for virus. o T cells infected later on. The gp120 protein on virus binds specifically to CD4 receptor on host cell with high affinity. Gp41 causes fusion of the virus to the cell membrane. o After fusion virus particle enters cell. o Viral genome exposed by uncoating particle. Reverse transcriptase produces viral DNA from RNA. o Becomes a provirus which integrates into host DNA. o Period of latency occurs. After a period of latency lasting up to 10 years viral replication is triggered and occurs at high rate. CD4 cell may be destroyed in the process, body attempts to replace lost CD4 cells, but over the course of many years body is unable to keep the count at a safe level. Destruction of large numbers of CD4 cause symptoms of HIV to appear with increased susceptibility to opportunistic infections, disease and malignancy HIV TRANSMISSION/SYMPTOMS IMMUNOLOGIC MANIFESTATIONS • • • • • • Methods of transmission: o Sexual transmission, presence of STD increases likelihood of transmission. o Exposure to infected blood or blood products. o Use of contaminated clotting factors by hemophiliacs. o Sharing contaminated needles (IV drug users). o Transplantation of infected tissues or organs. o Mother to fetus, perinatal transmission variable, dependent on viral load and mother’s CD 4 count. Primary HIV syndrome o Mononucleosis-like, cold or flu-like symptoms may occur 6 to 12 weeks after infection. ▪ lymphadenopathy ▪ fever ▪ rash ▪ headache ▪ fatigue ▪ diarrhea ▪ sore throat ▪ neurologic manifestations. ▪ no symptoms may be present o Symptoms are relatively nonspecific, may not be severe and misdiagnosis may occur. o HIV antibody test often negative but becomes positive within 3 to 6 months. o Primary HIV can be diagnosed using viral load titer assay or other tests. o Primary HIV syndrome resolves itself and HIV infected person remains asymptomatic for a prolonged period of time, often years. Clinical latency period, HIV continues to reproduce, CD4 count gradually declines from its normal value of 500-1200. o Once CD4 count drops below 500, HIV infected person at risk for opportunistic infections. o The following diseases are predictive of the progression to AIDS: ▪ persistent herpes-zoster infection (shingles) ▪ oral candidiasis (thrush) ▪ oral hairy leukoplakia ▪ Kaposi’s sarcoma (KS) AIDS o CD4 count drops below 200 person is considered to have advanced HIV disease o If preventative medications not started the HIV infected person is now at risk for: ▪ Pneumocystis carinii pneumonia (PCP) ▪ cryptococcal meningitis ▪ toxoplasmosis o If CD4 count drops below 50: ▪ Mycobacterium avium ▪ Cytomegalovirus infections ▪ lymphoma ▪ dementia ▪ Most deaths occur with CD4 counts below 50. Infants with HIV may exhibit the following: o Failure to thrive o Persistent oral candidiasis o Hepatosplenomegaly o Lymphadenopathy o Recurrent diarrhea o Recurrent bacterial infections o Abnormal neurologic findings. • • • • Early stage slight depression of CD4 count, few symptoms, temporary. Window of up to 6 weeks before antibody is detected, by 6 months 95% positive. During window p24 antigen present, acute viremia and antigenemia. Antibodies produced to all major antigens. o First antibodies detected produced against gag proteins p24 and p55. o Followed by antibody to p51, p120 and gp41 o As disease progresses antibody levels decrease. Immune abnormalities associated with increased viral replication. o Decrease in CD4 cells due to virus budding from cells, fusion of uninfected cells with virally infected cells and apoptosis. o B cells have decreased response to antigens possibly due to blockage of T cell/B cell interaction by binding of viral proteins to CD4 site. o CD8 cells initially increase and may remain elevated. o As HIV infection progresses, CD4 T cells drop resulting in immunosuppression and susceptibility of patient to opportunistic infections. o Death comes due to immuno-incompetence LABORATORY DIAGNOSIS OF HIV INFECTION • ELISA Testing o First serological test developed to detect HIV infection. ▪ Easy to perform. ▪ Easily adapted to batch testing. ▪ Highly sensitive and specific. o ELISA tests useful for: ▪ Screening blood products. ▪ Diagnosing and monitoring patients. ▪ Determining prevalence of infection. ▪ Research investigations. o Antibodies detected in ELISA include those directed against: p24, gp120, gp160 and gp41, detected first in infection and appear in most individuals o • • Different types of ELISA techniques used: ▪ indirect ▪ competitive ▪ sandwich o ELISAs are for screening only, false positives do occur and may be due to AI disease, alcoholism, syphilis, and immunoproliferative diseases. Other Screening Tests o Agglutination tests using latex particles, gelatin particles or microbeads are coated with HIV antigen and will agglutinate in the presence of antibody. o Dot-Blot Testing utilizes paper or nitrocellulose impregnated with antigen, patient serum is filtered through, and anti-antibody is added with enzyme label, color change is positive. Western Blot Testing o Most popular confirmatory test. ▪ Utilizes a lysate prepared from HIV virus. ▪ The lysate is electrophoresed to separate out the HIV proteins (antigens). ▪ The paper is cut into strips and reacted with test sera. ▪ After incubation and washing anti-antibody tagged with radioisotope or enzyme is added. ▪ Specific bands form where antibody has reacted with different antigens. ▪ Most critical reagent of test is purest quality HIV antigen. ▪ The following antigens must be present: p17, p24, p31, gp41, p51, p55, p66, gp120 and gp160. o Antibodies to p24 and p55 appear earliest but decrease or become undetectable. o Antibodies to gp31, gp41, gp 120, and gp160 appear later but are present throughout all stages of the disease. • • • HEPATITIS B (HBV) • • • HEPATITIS • • • • • • • General term meaning inflammation of the liver, usually accompanied with fever, nausea, vomiting and jaundice.. Can be caused by radiation, chemicals, disease processes such as autoimmune disease, viruses and cancer. Focus here will be on 5 distinct hepatitis viruses, A, B, C, D and E which cause chronic and acute hepatitis. All of these are RNA viruses with the exception of hepatitis B which is a DNA virus. Initial infection may be clinically silent. Chronic carrier state may develop and may result in liver failure due to cirrhosis, hepatocellular carcinoma, or fulminant hepatitis. Fulminant hepatitis is the term used when the number of hepatocytes destroyed is so great that too few remain to maintain basic liver function, results in liver failure. • HEPATITIS A VIRUS (HAV) • • • • Formerly known as “short incubation” or “infectious hepatitis” Transmitted by fecal-oral route, usually associated with unsanitary conditions or contaminated food or water. Occurs worldwide, less common in industrialized nations. Most hepatitis epidemics are due to HAV and may show seasonal patterns. Progress of infection: o Incubation of 2-7 weeks, may be asymptomatic or may include jaundice. o clinical illness develops abruptly and include: fever, anorexia, vomiting, fatigue and malaise. o Increase in serum transaminases. o Right upper quadrant pain, dark urine and pale stool. o Recovery 2-4 weeks, no carrier state. o Mortality 0-1% Antibody and antigen markers. o First and most clinically useful is IgM antibody to HAV. o IgM indicates acute infection, appears 4-5 weeks after exposure. o IgM disappears in 3-6 months, replaced by IgG antiHAV. o IgG peaks during convalescence and may remain detectable for life. HAV vaccine is available and highly effective. • • Old term “serum hepatitis”, incubation time of 4-26 weeks. Route of infection is usual parenteral, direct inoculation o small breaks in skin/mucosa come in contact with infected blood or serum. o blood transfusions o Needles or sharps exposure ▪ IV drug abusers ▪ tattoos ▪ dental equipment ▪ accidental needle stick ▪ homosexual or heterosexual contact ▪ maternal to fetus or newborn ▪ sharing razors, toothbrushes, etc. Incidence of infection is 140-320,000 cases per year resulting in 5-6,000 deaths per year. o Duration of acute infection ranges from 4-8 weeks with symptoms similar to HAV. o Ten percent progress to chronic. o One-third of chronic at risk of developing chronic active hepatitis, liver cirrhosis, and/or hepatocellular carcinoma. Laboratory diagnosis involve the detection of three marker systems. (ELISA) o Hepatitis B surface antigen (HBsAg) is the first to appear, appears 2 to 4 weeks during late incubation, marker of choice for recent infection. o Anti-Hepatis B surface antigen (anti-HBs) is the last antibody to appear, may persist for life. o Between disappearance of HbsAg and appearance of anti-HBs is known as the core window. o IgM antibody to hepatitis B core antigen (antiHBc) may be the only detectable marker during the core window, differentiates recent infection from chronic carrier state. o Third marker is hepatitis Be antigen (HBeAg), appearance of HbeAg and anti-HBe, closely coincide with HbsAg. Confirmatory test is Western Blot. No cure or vaccine at the moment. HEPATITIS D VIRUS • • • • Requires infection with Hepatitis B (Infection can only occur if youre infected with Hepa B). Route of transmission the same as HBV Can occur as coinfection or superinfection Serological markers o HDAg found early, disappears rapidly, not very useful o IgM anti-D and total anti-HD (IgM and IgG) detected during acute phase. o Presence of IgM anti-D and HbsAg together with IgM anti-HBc indicates coinfection. o Absence of IgM anti-HBc indicates superinfection. o Persistence of IgG anti-HD indicates chronic infection. • • • HEPATITIS C VIRUS • • • • Clinically and epidemiologically similar to HBV. Sixty to 70% of HCV patients will develop chronic hepatitis, 10-20 % cirrhosis and 15% hepatocellular carcinoma. HCV and HBV may be present as coinfections. Serological markers o Serological profile not fully developed. o Present of HCV antibodies only indicates present or past infection. o Can have false negative in some patients. • HEPATITIS E VIRUS • • • • • Similar to HAV in transmission (fecal-oral route) and clinical course. Found primarily in developing countries, Africa and Asia, and related to poor sanitation and contaminated water. Results in acute hepatitis, no risk of chronic hepatitis. Pregnant women with HEV may develop fulminant liver failure and death. No distinctive markers, diagnosis based on symptoms for exposed individuals in endemic countries. HEPATITIS G VIRUS • • • • • • Independently discovered 1995-1996 by 2 separate research groups. RNA virus Transmissible by bloodborne route. Found in patients with acute or chronic liver diseases. Exact clinical significance needs to be further defined. ELISA and Western blot methods have been developed TESTS FOR HIV & HBV HIV ANTIBODY TEST (IMMUNOCOMB TEST): TEST FOR HIV/AIDS • The Human Immunodeficiency Virus (HIV) is a retrovirus identified as the etiologic agent for Acquired Immunodeficiency Syndrome (AIDS). The principal cells infected by HIV are T4 lymphocytes that play a key role in the immune defense system of the organism. The progressive decrease of the T4 level during development of the disease leads to opportunistic infections with fatal consequences. The HIV virus consists of genomic RNA molecule protected by a capsid and an envelope. The HIV envelope is the major target for humoral antibody response. The Immunocomb HIV 1 and 2 is a rapid test intended for the qualitative and differentiative detection of IgG antibodies to HIV types 1 and 2 in human serum or plasma. This test is an indirect solid-phase enzyme immunoassay (EIA). The solid phase is a comb with 12 projections. Each projection is sensitized at 3 spots: o Upper spot – goat antibodies to human immunoglobulin (internal control) o Middle spot – HIV-2 synthetic peptides o Lower spot – HIV-1 synthetic peptides The Developing plate has 6 rows (A-F) of 12 wells, each row containing a reagent solution ready for use at a different step in the assay. The test is performed by moving the comb from row to row with incubation at each step. Serum/plasma specimen is added to the diluent in the wells of row A of the Developing Plate. The comb is then inserted in the wells of row A. Anti-HIV antibodies, if present in the specimens will specifically bind to the synthetic peptides on the lower and/or middle spots on the teeth of the comb. Simultaneously, immunoglobulins present in the specimens will be captured by the antihuman immunoglobulin antibodies on the upper spot. Unbound components are washed away in row B. In row C, the IgG captured on the teeth will react with anti-human IgG antibodies labeled with alk-phosphatase. In the next rows,unbound components are removed by washing. In row F, the bound alk-phosphatase will react with chromogenic components. The results are visible as granule spots. MATERIALS • • • • • • • Immunocomb HIV 1& 2 Bispot Kit Contents Combs are provided in aluminum pouch Developing plates covered by aluminum foil Positive and Negative Control Perforator Watch / timer Micropipettes PREPARING THE COMB • 1. 2. CAUTION: To ensure proper functioning of the test, do not touch the teeth of the comb Tear the aluminum pouch of the comb at the comb at the notched edge. Remove the Comb. You may use the entire Comb and Developing Plate or only a part. To use part of a Comb: o Determine how many teeth you need for testing the specimen and controls. o Bent and break the Comb vertically or cut with scissors to detach the required number of teeth. o Return the unused portion of the Comb to the aluminum pouch. Close pouch tightly to maintain dryness. Store the Comb in the original kit box at 28’C for later use. PROCEDURE MATERIALS 1. • • • • 2. 3. Bring all reagents and specimen to room temperature and perform the test at room temperature. Dispense 50 ul. of each specimen and control into separate wells of row A of the Developing Plate and mix. Insert comb in row A and continue as described below. o Antigen-antibody reaction - Mix; incubate 10 minutes; absorb o Wash - Agitate; incubate 10 minutes; absorb o Absorb o Binding of conjugate - Mix; incubate 10 minutes; absorb o Wash - Agitate; incubate 2 minutes; o Absorb o Wash - Agitate; incubate 2 minutes; o Absorb o Colorless reaction - Mix; incubate 10 minutes o Stop reaction - Incubate 1minute; dry in air PROCEDURE • 1. 2. 3. VALIDATION • In order to confirm that the test functions properly and to demonstrate that the results are valid, the following three conditions must be fulfilled: o Positive control must produce three spots on comb tooth. o The negative control must produce an upper (Internal Control) and no other spots. o Each specimen tested must produce an upper spot (internal Control). This will also confirm that the specimen was added. INTERPRETATION • • Positive result shows a circular colored middle spot which indicates the presence of the antibodies to HIV-2 and a circular colored lower spot which indicates the presence of antibodies to HIV-1. A negative result shows the sole appearance of the upper spot (Internal Control) indicating that the specimen is nonreactive for antibodies to HIV-1 or HIV-2. • • In layman’s term, Hepatitis means the inflammation of liver. Acute Viral Hepatitis caused by the Hepatitis B Virus is a common and serious infection. It is characterized by inflammation and necrosis of the liver. Apart from the chronic liver disease, it is the single most significant cause of mortality of liver cancer. Two important serologic markers in detecting Hepatitis B includes: HBs (Hepatitis B surface) Ag and Anti-HBs Antibody to Hepatitis B. The HbsAg is the first marker to appear and its level peak during acute stages of infection. The anti-HbsAg provides protective immunity. The HbsAg test is a qualitative test based on an immunochromatographic method. HbsAg present in serum and plasma reacts with colloidal gold particles which have been coated with monoclonal antibodies against HbsAg. The resulting complexes migrate along the membrane and are bound in the test zone by a second monoclonal antiHbsAg antibody, which is fixed in the form of a horizontal line. Excessive immunocomplex and/ or unreacted colloidal gold particles migrate farther and are bound in a second line by anti- IgG antibodies, forming the control line. Test strips and samples should be brought to room temperature prior to use. Remove the appropriate number of test strips from the canister. Take care to touch the strips only at the upper end with the arrows pointing downward. Close the canister immediately after removal of the test strips. Immerse the test strips with the arrows pointing downward into a tube with .25 to 0.5 ml sample. The sample must not touch the blue foil of the test strips. Do not move the test strip while the chromatographic process is incomplete. Read the result 20 minutes after immersion into the sample. INTERPRETATION • • Positive result shows two visible purple lines, one in the control zone(C) and another one in the test zone(T) indicating the presence of HbsAg Negative result shows one visible line in the reaction zone which indicates the absence of HbsAg. PRINCIPLES • • IMMUNOCHROMATOGRAPHIC METHOD: TEST FOR HEPATITIS B VIRUS • HbsAg reagent test kit HbsAg strips Test tubes Serologic pipette or micropipette • Antigens o HBsAg = (+) infection; vaccine o HBeAg = replication; ↑ level; ↑ infectivity o HBcAg = not in blood (during infection) Antibodies o HBsAb = (+); (+) vaccination; (+) infection o Anti-HBe = active phase of immune response o HBcAb ▪ IgG = (+) past infection ▪ IgM = ↑ acute IgM; ↓ chronic infection