Immunology & Serology MTAP 1 1 st Semester VIRAL HEPATITIS HEPATITIS A - A.K.A: Enterovirus 72 MOT: Fecal-Oral Incubation: 15 to 50 days (ave. 28) Has an abrupt onset (short incubation hepatitis) HAV Unlike HBV, it does not produce a coat protein and is not detectable in serum (only detected in stool) Extra notes: ❖ From family Picornaviridae ❖ Inflammation of the liver due to infection of viral agents (Hep. A virus) ❖ Self-limited disease, does not result in chronic infection (LESS FATAL, ACUTE TYPE OF INFECTION) ❖ The one that causes infectious hepatitis ❖ Most common cause of hepatitis Mode of Transmission: Ingestion of fecal matter, even in microscopic amounts, from: ➔ ➔ ➔ Close person-to-person contact w/an infected person Sexual contact w/ an infected person (direct, oral & anal contact) Ingestion of contaminated food and drinks Finals 2023 Serologic Aspect ❖ For the first 15 days, the Hepatitis A antigen or protein coat shed o in the small intestine. ❖ On the early stage of infection, antigen detection in the stool is recommended for px that is suspected with Hepa A ❖ In the 2nd week of infection, the antibodies start to produce IgM, followed by IgG. These antibodies are produced in the circulation or presence of the serum or plasma. HEPA A MARKERS OF INFECTION 1.) 2.) 3.) Early shedding of the virus in stool (Hepatitis A antigen is detected) Appearance of IgM Anti-HAV with the onset of symptoms (icterus and increased liver enzyme levels) ❖ ALT, SGPT significantly elevated ❖ SGOT, Nucleotidase also increase ❖ ↑ Bilirubin = Hepatic disorders Development of Anti-HAV IgG immunity on recovery Methods to detect antibodies: 1.) ELISA indirect (if detection of antigen: Direct ELISA) 2.) Radioimmunoassay (RIA) Serologic of Hepatitis A Persons at Risk: ● Travellers to regions w/ intermediate or high rates of Hepa A ● Sex contacts of infected person ● Household members or caregivers of infected persons ● Men who have sex with men ● Users of certain illegal drugs (injection and non-injection) ● Person with clotting-factor disorder Trivia: Is it possible for Hepa A to be transferred by blood? YES. If patient has severe Hepa A infection present in the blood (Viremia) IgM anti-HAV IgG anti-HAV Acute infection + - Old infection (Immune to HAV) - + Incubation or no infection - - HEPATITIS B - A.K.A: Serum Hepatitis or Dane PAGE1 Immunology & Serology MTAP 1 1 st Semester - Particle MOT: Contact with infectious body fluids Incubation: 45 to 160 days (ave. 120) Extra notes: ❖ A viral infection that attacks the Liver (hepatocyte) and can cause both acute and chronic disease ❖ Only DNA type of virus ❖ Family from Hepadnaviridae Mode of Transmission: ● Contact with infectious blood, semen and other bodily fluids primarily through ➔ Birth to an infected mother ➔ Sexual contact with an infected person ➔ Sharing of contaminated needles, syringes or other injection drug equipment ➔ Birth to an infected mother SEROLOGIC MARKERS FOR HBV 1.) 2.) HBsAg previously known as the Australia Antigen best indicator of early, acute infection presence indicates: active infection in either the acute or chronic stage HBcAg Found within the core of intact virus It is not detectable in serum because it is found only in liver tissue/cells Not routinely tested, liver biopsy is required 3.) HBeAg - Indicated chronic hepatitis and is a reliable marker for the presence of high level of virus in the sample HBsAg Positive anti-HBc Positive Finals 2023 (highdegreeofinfectivity) 4.) Anti-HBc First antibody to appear at the same time that enzyme elevations are first seen Can be found in asymptomatic carrier The only marker present during the window period of infection 5.) Anti-HBe Low-level of virus, low degree of infectivity First serologic evidence of convalescent phase (recovery period) 6.) Anti-HBs Bestows immunity to further HBV infection (long-term) Measured several months after Hepatitis B Vaccination HBsAg Negative - Susceptible to acquire infection anti -HBc Negative anti-HBs Negative HBsAg Negative Immune due to natural infection anti-HBc Positive (this confirmspx hadprevious infectionof HepaB) anti-HBs Positive HBsAg Negative anti-HBc Negative anti-HBs Positive Immunedueto hepatitisB vaccination (recoveryphase/ Windowperiod) Acutelyinfected PAGE2 Immunology & Serology MTAP 1 1 st Semester IgM anti-HBc Positive anti-HBs Negative ● Finals 2023 2ndGenerationTest I. Counter Immunoelectrophoresis (CIE) Principle: Precipitationwith current II. Rheophoresis Principle: Precipitationwith evaporation - III. HBsAg Positive ● Chronically Infected anti-HBc Positive IgM anti-HBc Negative anti-HBs Negative confirmHepatitisBinfection) DISEASES CAUSED BY HBV: ● Acute hepatitis ● Fulminant hepatitis (sudden severe onset conditions) ● Chronic (asymptomatic carrier, chronic persistent hepatitis, chronic active hepatitis) ● Co-infection with HEPATITIS DELTA VIRUS Complement Fixation 3rdGenerationTest(MostSensitive) I. Reverse Passive Latex Agglutination Principle: Agglutination II. Reverse Passive Hemagglutination Principle: Hemagglutination III. ELISA (commonlyusedto HBsAg Negative anti -HBc Positive anti-HBs Negative (Indicates personisinthe windowperiod orstage) Interpretation unclear;four possibilities: 1.)Resolved infection(most common) 2.)False-positive antiHBc,thus susceptible 3.)“Low-level” chronicinfection 4.)Resolving acuteinfection (HDV) ● If Hepatitis B infection is not manage it can progress to a more severe condition: Primary Hepatocellular Carcinoma (Hepatoma) Infectious cancer - start with an infection after which if not managed or controlled, will progress into malignancy Ex: Human Papillomavirus →it can progress to cervical cancer IV. RIA Epstein-Barr Virus (EBV) →it can progress to Nasopharyngeal cancer TESTS AVAILABLE FOR HBV DEXTIN PAGE3 Immunology & Serology MTAP 1 1 st Semester Finals 2023 HBV Vaccine (1982) - made by a recombinant strain of yeast Saccharomyces cerevisiae (common bakers’ yeast) ● 1stGenerationTest I. Ouchterlony Principle: Precipitationreaction (doubledi usiondouble dimension) Serologic Tests a.) HBsAg 1.) HBsAg- if present, there in an ongoing disease either acute or chronic in nature 2.) Anti-HBsAg- if present, there is no active disease and the person is already immune or cured b.) HBcAg 1.) IgManti-HBcAg- New infection 2.) IgGanti-HBcAg- Old infection ↳Both (+) = Mid infection c.) HBeAg 1.) HBeAg - indicates high level of infectivity (high viral load) 2.) Anti-HBeAg - indicates low level of infectivity (low viral load) ❖ ❖ ❖ HBcAg - can’t be detected in serum In viral serology, antigen testing is prioritized first than antibody test (antigen first appears in the body fluid) Late diagnosis = do serum antibody testing HEPATITIS C - A.K.A: Bloody-borne hepatitis; Posttransfusion Hepatitis Single-stranded RNA virus Incubation: 14-180 days (ave. 45) From family Flaviviridae Specialized method to confirm Hepatitis C: Recombinant Immunoblot Assay (RIBA) Serologic Tests I. Surrogate Testing for detecting NANBV/HCVin donated blood ALT Level Detection Anti-HBc Detection (by RIA or ELISA using Enzyme Inhibition Technique) II. Serologic Tests for antibody against HCV Ag(Anti-HCV) - ELISA RIA PREVENTION AND TREATMENT ● ● ● ● ● Serological test on donor blood Active immunization Antiviral agent for treatment of chronic or persistent HBV infection Anti-HBV drug Lamivudine Treatment with interferon alpha NON-A, NON-B HEPATITIS TEST OUTCOME INTERPRETATION HCV antibody nonreactive No HCV antibody detected HCV antibody reactive Presumptive HCV infection PAGE4 Immunology & Serology MTAP 1 1 st Semester Current HCV infection HCV antibody reactive, HCV RNA detected Finals 2023 No current HCV infection HCV antibody reactive, HCV RNA not detected HEPATITIS D SEROLOGIC MARKERS FOR HEV A.K.A: Delta Virus Helical ● No distinctive markers, diagnosis based Nucleocapsid, actually uses HBV’s on symptoms for exposed individuals in envelope, HBsAg (only replicates in endemic countries cells infected with HBV) ● Pregnant women with HEV may develop RNA virus transmitted parenterally fulminant liver failure and death and can replicate with the help of HBV - Defective hepatotropic virus which HEPATITIS G requires obligatory helper functions from HBV in order to MOT: contact with blood, sexually ensure its replication and infectivity transmitted transplacental From the same family as HCV 2 infections carried out by HDV Flaviviridae 1.) Co-infection with Hepatitis B HGV contains RNA and has envelope 2.) Super infection with Hepatitis B (more Common worldwide but seems to be severe, faster replication, faster transfer, non-pathogenic increased rate of infection and damage) INFECTIOUS MONONUCLEOSIS SEROLOGIC MARKERS FOR HDV - Also known as Kissing Disease 1.) HDV ag - found in the early stage of (French kissing) infection. Rapidly disappears in plasma, causative agent is the DNA virus, hence it is not very useful Epstein Barr Virus (causativeagent 2.) IgM anti-HDV and total anti-HDV (IgM ofinfectiousmononucleosis,canmix & IgG) - use for detecting acute phase withsalivacanbetransferredduring of infection mouthtomouthtomouth)which 3.) Presence of IgM anti-HDV and HBsAg infects B lymphocytes together with IgM anti-HBc - indicates - Downey cells (Atypical lymphocyte co-infection or Reactive lymphocyte) - enlarged 4.) Absence of IgM anti-HBc - indicates lymphocytes a ected by EBV with a patient has superinfection to Hepatitis B characteristic atypical nuclei 5.) Presence of anti-HBV IgG - indicates EBV infection is most common chronic infection during adolescence and early adulthood (ages 15 to 25) PAGE5 Immunology & Serology MTAP 1 1 st Semester Finals 2023 HEPATITIS E - A.K.A MOT: Fecal-Oral route, often in contaminated water From family Calicivirus HEV contains RNA Size of virus: 32-34 nm Disease resembles Hepatitis A virus Dx. Serology “PUS FILLED TONSIL” - - Reacts with sheep cells, ox cells, horse cells but not guinea pig cells Not Forssman in Nature ❖ - HETEROPHIL Antibodies of Forssman Reacts with Guinea pig cells, horse cells, sheep cells but not in ox or beef cells (-) ❖ HETEROPHIL Antibodies in Serum Sickness Reacts with all four cells Can be used as control Guinea pig cells and ox cells - useful to di erentiate these types of antibodies THE EXPERIMENT OF FORSSMAN Therefore, ➢ ➢ Guinea pig cells are injected into Rabbits → Antibody Formation Abs Formed are: Anti-Guinea Pig cells and Anti-Sheep cells Forssman Antigen - used for any substance that stimulated the formation of sheep hemolysin (Anti-sheep cells) Heterophil Antibodies - antibodies produced by unrelated species which can cross-react with the same antigen ❖ HETEROPHIL Antibodies in IM TEST WITH IM 1.) ❖ ❖ ❖ ❖ ❖ ❖ ❖ PAUL BUNNEL TEST Principle: HEMAGGLUTINATION Presumptive/screening test Incapable of determining specificity and is only indicative of the presence or absence of heterophil Abs Rgt Ag: 2% suspension of SHEEP RBCs Ab: heterophil Abs in patient’s serum (+) result: Hemagglutination presence of Heterophil Abs Does not determine the specific Heterophil antibody PAGE6 Immunology & Serology MTAP 1 1 st Semester 2.) DAVIDSON DIFFERENTIAL TEST ❖ Principle: ABSORPTIONHEMAGGLUTINATION Two steps to perform: I.) Absorption step Exposure of test serum to both beef cells and Guinea pig cell which causes absorption of either one or both of these antibodies Ag: Guinea Pig kidney cells and Beef RBCs Ab: Heterophil Abs in patient’s serum Indicator cells: Sheepcells ● Instep1 ➔ Positive absorption would result to decrease antibody titer ➔ Negative absorption would retain a high antibody titer II.) Hemagglutination step 3.) - The “Absorbed Agglutinins” (precipitates) are removed by centrifugation and the resultant fluid (supernatant) are then tested with Sheep RBCs 4.) Conclusion 1.) If the absorption is positive in Step 1 antibody titer decreases, hence MONOSPOT ❖ Principle: ABSORPTIONHEMAGGLUTINATION ❖ Ag: GuineaPigKidneycellsand BeefRBCs ❖ Ab: heterophilAbsinpx’sserum ❖ Indicator cells: Horse cells ❖ SAME PATTERNS WITH DAVIDSON RAPID DIFFERENTIAL SLIDE TEST USING PAPAIN-TREATED SHEEP RBC’S ❖ Principle: Hemagglutination ❖ When papain is added to sheep cells, the receptors for the Abs are specifically inactivated. Normal Serum&IM serum agglutination in Step 2 weakens Finals 2023 Serum Sicknessand other Heterophil Abs Native Sheep RBCs Papain-Treated Sheep RBCs Agglutination No/weak Agglutination Agglutination Agglutination PAGE7 Immunology & Serology MTAP 1 1 st Semester Finals 2023 2.) If the absorption is negative in Step 1 antibody titer is maintain high, hence agglutination is strong ABSORPTION PATTERN Heterophil Antibodies Forssman IM Serum Sickness Beef RBCs Guinea Pig Kidney Cells No Yes Yes No Yes Yes AGGLUTINATION PATTERN Heterophil Antibodies Beef RBCs Forssman ++++ + + ++++ ++ ++ IM Serum Sickness Guinea Pig Kidney Cells PAGE8 Immunology & Serology MTAP 1 1 st Semester Gene HIV AND AIDS HUMAN IMMUNODEFICIENCY VIRUS (HIV) - - - HIV-1: ❖ ❖ ❖ ❖ ❖ The causative agent of HIV infection Family: Retroviridae Subfamily: Lentivirus, Oncovirus - Has a marked preference for T-helper/inducer lymphocytes (CD4+) Enveloped, with coiled nucleocapsid, icosahedral, with single stranded RNA Has the unique enzyme “reverse transcriptase”. Makes it very hard for antiviral agent to act on the virus Replicates inside the nucleus France: was identified by the lab of Luc Montahnier U.S.A: Robert Gallo (1983) & Jay Levy (1984) More severe than HIV-2 and more predominant in transmission rate ❖ Formerly called: ➢ Human T-cell lymphotropic virus-type III (HTLVIII) ➢ Lymphadenopathy associated virus (LAV) ➢ AIDS-associated retrovirus (ARV) HIV-2: Majority occurred in West Africa Less pathogenic, lower rate of transmission Modes of Transmission 1.) By direct and specific routes 2.) Mainly through sexual intercourse (specifically anal sex) 3.) Transfer of blood or blood products 4.) Babies can be infected before or during birth 5.) Breastfeeding 6.) Parenteral drug use Viral Gene Products gag p24, p18, p15 (group antigen gene) (p stands for protein) pol (polymerase) Reverse transcriptase (for the enzymes) Finals 2023 Functions Codesfor core structural (groupAgs) proteins Transcribes ssRNAinto dsDNA RNAse Protease Integrase (Integrase is important for the viral insertion to the host DNA) env (envelope) gp160 gp41 BindstoCD4 receptorsfor infection (required for viral fusion of cell) CD4+ cells are responsible for helping the humoral response in immunity Primary e ects of HIV infection: Extreme leukopenia Formation of giant T-cells and other syncytia (when these are formed, this would allow the virus to spread directly from cell to cell making it faster for HIV to infect other CD4+ cells) PAGE9 Immunology & Serology MTAP 1 1 st Semester - Diagnosis of HIV Infection ❖ Testing based on detection of antibodies specific to the virus in serum or other fluids; done at 2 levels ❖ Initial screening ELISA (indirect because antibody detection), latex agglutination and rapid antibody tests Rapid results but may result in false positives ❖ Follow up with Western blot analysis to rule out false positives ❖ False negatives can also occur; persons who may have been exposed should be tested a second time 3-6 months later Laboratory Tests for HIV ● ELISAtests: detects Abs to HIV and HIV ag ● WesternblotorImmunofluorescenttest: confirmatory test (western blot is more widely used) ● Westernblotassay: confirmatory serological test for HIV, 3 bands must appear: p24, gp41 or gp120/160 (presence of these bands indicates confirmed case of HIV in the patient’s sample) ● IndirectImmunofluorescenceassay: is used to Finals 2023 Infected macrophages release the virus in central nervous system Secondary e ects of HIV infection: Destruction of CD4 lymphocytes WESTERN BLOT - Unbound conjugate is removed by washing Bound conjugate is detected after addition of substrate → Chromogenic reaction; Colored bands appear in the positions where Ag-specific HIV Abs are present Positive test: 2 of the 3 major bands p24, gp41 or gp120/160 detect HIV in infected cells. Also a confirmatory test ELISA test for HIV Ab: HIV-1 Western Blot ❖ Lane 1: Positive Control ❖ Lane 2: Negative Control ❖ Sample A: Negative ❖ Sample B: Intermediate PAGE10 Immunology & Serology MTAP 1 1 st Semester ❖ Sample C: Positive CLINICAL MANIFESTATIONS Finals 2023 KAPOSI SARCOMA 1.) PrimaryStage - Patient is either asymptomatic or may show lymphadenopathy (the primary stage of HIV infection resembles that of infectious mononucleosis or IM) 2.) IntermediateStage - This state is known as the ARC (Aids-Related Complex) Quantitative T cell deficiencies with When HIV replication occurs, the CD4 cell is killed. This results in a severe depletion of helper-inducer T lymphocytes. Normal CD4:CD8 ratio = 2 : 1 AIDS pxs = 0.5 : 1 inverted CD4:CD8 ratio OPPORTUNISTIC PATHOGENS ● ● ● ● ● ● ● ● Pneumocystis carinii (jirovecii) M. avium-intracellulare complex Candida albicans Cryptosporidium parvum Toxoplasma gondii Cryptococcus neoformans Herpes Simplex (type 1 and 2) Legionella spp. 3.) FinalStage - 2-10 years after initial infection Syndrome of CD4 depletion resulting in opportunistic infections and cancers suggestive of cell-mediated immunity defects. - Tumors in skin and linings of internal organs, In the Philippines, a new confirmatory procedure is being implemented to replace Western Blot Assay, the test is called rHIVda (Rapid HIV Diagnostic Algorithm). James Gordon Hospital is the referral hospital for HIV confirmatory because they administer this test. If the patient is reactive to HIV screening The patient is then tested for rHIVda (Test 1) IfnegativeforTest1, you report the result as inconclusive. Advise the patient to return to the testing center between 2-6 weeks IfpositiveTest1, you perform rHIVda (Test 2) WhennegativeforTest2, same procedure is followed as negative Test 1 WhenpositiveforTest2, you perform rHIVda (Test 3) Whennegativefor Test3, same procedure is followed as negative Test 1 Whenpositivefor rHIVdaTest3, the patient is reported as HIV+ - Take note: if the patient is negative for rHIVda, the sample must be sent to the San Lazaro Hospital for possible Western Blot examination lymphomas and cancers of rectum & lung PAGE11 Immunology & Serology MTAP 1 1 st Semester - Finals 2023 Caused by Human Herpes Simplex Virus Type 8 most frequent malignancy observed Anti-gp41 TESTS TO DETECT HIV ANTIBODY first antibody to be detected; persist throughout the infection ● Screening Tests 1.) ELISA (INDIRECT) 2.) ELISA (Competitive Assay) 3.) Slide Agglutination Tests (Passive Agglutination) 4.) RIA ● Confirmatory Tests 1.) WESTERN BLOT ASSAY most widely used supplementary test for confirming reactive HIV ELISA Ab test (+) result (-) result - Diagnosis of AIDS is made when a person meets the . criteria: 1. Positive for the virus 2. They fulfill one of the additional criteria: a. They have a CD4 count of fewer than 200 cells/mL of blood b. The CD4 cells account for fewer than 14% of all lymphocytes c. They experience one or more of a CDC-provided list of AIDS-defining illnesses Take note: HIV+ patient - CD4 count is more than 200 cells/mL in the blood. They may still look healthy, absent opportunistic pathogens. AIDS patient - CD4 count is fewer than 200 cells/mL of blood, the patient is immunodeficient. 2.) INDIRECT IMMUNOFLUORESCENT ASSAY (IFA) TESTS FOR DETECTING HIV GENES 1. 2. 3. 4. 5. 6. In situ hybridization Filter hybridization SOUTHERN BLOT Hybridization - DNA DNA Amplification PCR - tests HIV RNA NORTHERN BLOT - measures mRNA antigen RUBELLA: Highly contagious ❖ Congenital Rubella Caused by infection during pregnancy The fetus is most likely to develop anomalies if the mother becomes infected Lab Tests: 1.) HEMAGGLUTINATION INHIBITION 2.) Passive latex agglutination PAGE12 Immunology & Serology MTAP 1 1 st Semester Finals 2023 3.) Quantitative methods, such as EIA and IFA PAGE13
