Immunology and Serology - 07 Introduction To Serological Tests

IMMUNOLOGY AND SEROLOGY INTRODUCTION TO SEROLOGICAL TESTS OUTLINE • • Bacterial Diseases • o Syphilis o Typhoid Fever o Rickettsial Diseases o Other Bacterial Diseases Viral Diseases • o Viral Hepatitis o Infectious Mononucleosis o HIV/AIDS o Dengue o Other Viral Diseases Parasitic and Fungal Diseases o Amoebiasis o Toxoplasmosis o Fungal Diseases • BACTERIAL DISEASES SYPHILIS • • • • Sexually transmitted disease caused by Treponema pallidum Transmitted directly from one host to another o 90% is through sexual contact o 10% - non-genital transmission (blood transfusion, needle injury) Stages of Syphilis o Primary stage - chancre o Secondary stage - rashes, lymphadenopathy, condyloma latum o Latent stage - asymptomatic o Tertiary stage - gummata Comparison of Syphilis Antigens Antibodies Tests Nontreponemal/ Nonspecific Treponemal/ Specific Wasserman REAGIN ag (anti(Cardiolipin) cardiolipin) • Treponemal • Group AbsGroup anti-Reiter antigens CHON REITER • Specific CHON T.pallidum • Specific antibodiesT.pallidum antiantigens T.pallidum (pathogenic) abs VDRL RPR TREPONEMAL MTDS/SPECIFIC MTDS • • • • • TPI FTA-ABS MHA-TP TPHA • NON-TREPONEMAL MTDS • • o RPR Test o uses unheated serum as specimen and the result is read macroscopically o uses the incorporation choline-chloride to modify the basic VDRL Ag and allows testing of plasma without preliminary heating o Principle: Flocculation o Rgt Ag: colorless alcoholic soln containing cardiolipin, lecithin, cholesterol with charcoal o Rotator : 100 rpm for 8 minutes o Macroscopically read o False (+) RPR results: IM, leprosy, Rheumatoid arthritis o *not recommended for CSF Used to detect Reagin (a substance present in the serum of individuals with certain diseases including syphilis) VDRL o uses heated serum (@56ºC for 30 min.) as specimen and result is read microscopically; heating is done to deactive/destroy proteins/complement o Principle: Flocculation (aggn of colloidal particles) o Colloidal suspensions + reagin (+) flocculation of lipoids o Rgt Ag: colorless alcoholic soln containing cardiolipin, lecithin, cholesterol o Rotator: 180 rpm for 4 minutes o (+) Reactive o (-) Nonreactive o false (+) VDRL results : SLE, Rheumatic fever, IM, malaria, pregnancy • T. pallidum Immobilization Test (TPI) o Most specific for syphilis o Principle: The Ab produced against T. pallidum plus complement can IMMOBILIZE the LIVE Treponemes. o Rgt Ag: live actively motile T pallidum organisms (extracted from lesions of infected rabbits) o Ab: px syphilitic serum o Complement: guinea pig complement o (+) result : immobilization of treponemes (>50%immobile) Fluorescent Treponemal Antibody Absorption Test (FTA-ABS) o Principle: fluorescence; complement fixation o Rgt Ag: Dead T. pallidum (Nichol’s strain) dried and fixed on slide o Ab: px’s syphilitic serum o Absorbent: Reiter treponemes o *Px serum is prediluted in a heated culture filtrate of Reiter Treponemes so that group Abs are blocked and the specific Ab will be then free to bind with the treponemal Ag. o Conjugate : Fluorescent-labeled AHG o (+) result: Fluorescence OTHER BACTERIAL DISEASES LEPTOSPIROSIS • • • • • o T. pallidum Hemagglutination Test (TPHA)/ Hemagglutination Treponemal Test for Syphilis (HATTS) o Principle : Hemagglutination o Rgt. Ag: glutaraldehyde stabilized turkey RBC coated with treponemal antigen o Ab: px’s syphilitic serum o (+) result - hemeaggln Microhemagglutination T. Pallidum Test (MHA-TP) o Principle : Hemeaggln o Rgt Ag: tanned formalin sheep RBC coated with treponemal antigen o Ab: px’s syphilitic serum o (+) result - hemeaggln TYPHOID FEVER • • • • • • • • STREPTOCOCCAL INFECTIONS • • • Causative agent - Streptococcus pyogenes Untreated streptococcal pharyngitis may lead to rheumatic fever Virulence factors o M protein o Streptokinase, hyaluronidase o Streptococcal pyrogenic exotoxins VIRAL DISEASES VIRAL HEPATITIS HEPATITIS A Caused by Salmonella typhi Fecal-oral route Antigenic molecules used in serotyping o H antigen o K antigen o O antigen Widal Test for Typhoid Fever o Principle: Direct Agglutination o Interpretations: ▪ O means Typhoid Fever. ▪ O and H means Typhoid Fever. ▪ H means Past Infection, Convalescence, Exposure and Recent Immunization. ▪ O, H, and PARA A means Typhoid and Paratyphoid Fever (Mixed Infection) ▪ PARA A, B, and C means Paratyphoid Fever • • • • • RICKETTSIAL DISEASES • Causes Rocky Mountain Spotted Fever Caused by Rickettsia rickettsii Weil-Felix test o OX-19 (+) and OX-2 (+) o OX-K (-) Weil-Felix reaction – A Heterophile agglutination Test o A Weil-Felix reaction is a type of agglutination test in which patients serum is tested for agglutinins to O antigen of certain non-motile Proteus and rickettsial strains(OX19, OX2, OXk) o OX19, OX2 are strains of Proteus vulgaris. OXk is the strain of Proteus mirabilis. Caused by Leptospira interrogans Direct contact transmission via urine from infected animal Two-stage disease o Septicemic stage o Immunological stage Infectious hepatitis Caused by Hepatitis A virus (Picornaviridae) MOT: fecal-oral route Most common cause of hepatitis Laboratory Diagnosis: RIA & ELISA - detect the presence of specific HAV Abs HEPATITIS B • • • • • Serum hepatitis MOT: blood transmissible Caused by Hepatitis B virus (Hepadnaviridae) Found in blood, saliva, semen and breastmilk Hepatitis B antigens o HBsAg o HBcAg - undetected serologically o HBeAg Tests Available for HBV Detxn o 1st Generation Test ▪ Ouchterlony • Principle: Precipitation o 2nd Generation Tests ▪ Counter Immunoelectrophoresis (CIE) • Principle: Precipitation with Current ▪ Rheophoresis • Principle: Precipitation by Evaporation ▪ Complement Fixation o 3rd Generation Tests (Most Sensitive) ▪ Reverse Passive Latex Agglutination • Principle: Agglutination • AntiHBsAg artificially/passively attached to latex particles ▪ Reverse Passive Hemeagglutination • Principle: Hemeagglutination • AntiHBsAg passively attached to red cells ▪ ELISA ▪ RIA INFECTIOUS MONONUCLEOSIS (IM) • • • • Aka kissing’s diseases Caused by Epstein Barr virus Causes glandular fever EBV antigens o viral capsid antigens (VCA) o EBV nuclear proteins (EBNA) o EBV early antigens (EA) TESTS TO DETECT HIV ANTIBODY • • TESTS FOR IM • • • • Paul Bunnel Test o Principle: HEMEAGGLUTINATION o Presumptive/screening test o Incapable of determining specificity and is only indicative of presence or absence of heterophil Abs o Rgt Ag: 2% suspension of SHEEP rbcs o Ab: heterophil Abs in px’s serum o (+) result: Hemeaggln- presence of Heterophil Abs Davidson Differential Test o Principle: Absorption-Hemagglutination o Exposure of Test Serum to both Beef cells and Guinea pig cells which causes absorption of either one or both of these antibodies. o Ag: Guinea Pig Kidney cells and Beef rbcs o Ab: heterophil Abs in px’s serum o Indicator cells: Sheep RBCs Monospot o Principle: ABSORPTION-HEMEAGGLUTINATION o Ag: Ag: Guinea Pig Kidney cells and Beef rbcs o Ab: heterophil Abs in px’s serum o Indicator cells: Horse RBCs Rapid Differential Slide Test using Papain-Treated Sheep RBCs o Principle: Hemeagglutination o When papain is added to sheep cells, the receptors for the Abs are specifically inactivated. o Papain - inactivates abs for IM • Screening Tests o ELISA (INDIRECT) ▪ most widely used screening test for HIV antibodies o ELISA (Competitive Assay) o Slide Agglutination Tests (Passive Aggln) o RIA Confirmatory Tests o Western Blot Assay ▪ Most widely used supplementary test for confirming reactive HIV ELISA Ab test ▪ (+) result - if, gp41 band appears alone or when an envelope Ab(gp41, gp120 or gp160) appears in combination with another HIV band. ▪ (-) result - if no bands appear o Indirect Immunofluorescent Assay (IFA) Tests for Detecting HIV Genes o In situ hybridization o Filter hybridization o Southern Blot Hybridization – DNA o DNA Amplification o PCR – tests HIV RNA o Northern Blot – measures mRNA antigen DENGUE TESTS FOR DENGUE • • IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) o Qualitative detection of dengue virus IgM antibodies o Interpretation of results ▪ Positive IgM: Patients with a positive IgM test result are classified as presumptive, recent dengue virus infections. Plaque Reduction Neutralization Test (PRNT) o Detect specific neutralizing antibodies against dengue virus and other flaviviruses o Measures the titer of the neutralizing antibodies in the serum of the infected person OTHER VIRAL DISEASES CYTOMEGALOVIRUS (CMV) • • member of Herpesviridae family gold standard test is viral culture RUBELLA VIRUS HIV/AIDS • • • • Causative agent of HIV infection Family: Retroviridae Has a marked preference for T-helper/inducer lymphocytes (CD4+) which serves as the receptor sites for the virus Modes of Transmission o sexual contact o blood transfusion o parenteral injection o transplacental o breastfeeding o passage through infected birth canal • • • caused by RNA virus that belongs to Togaviridae family causative agent of german measles Three viral structural proteins o E1 o E2 o nucleocapsid protein C PARASITIC AND FUNGAL DISEASES AMOEBIASIS • • • • Entamoeba histolytica, major pathogenic parasite for humans Fecal-oral route Common complication - dissemination to the liver Serological Tests: o Gel diffusion precipitin test (GDP) ▪ Commercially available, simple to perform, and inexpensive o IHA and EIA tests ▪ • EIA for Entomoeba histolytica antibody detection ▪ • IHA for routine serodiagnosis of amebiasis o Conventional PCR ▪ Method of choice for discriminating between the E. histolytica and E. dispar o Real-Time PCR ▪ A TaqMan real-time PCR approach has been validated at CDC and is used for differential laboratory diagnosis of amebiasis TOXOPLASMOSIS • • • • Toxoplasma gondii is a protozoan parasite that infects most species of warm blooded animals, including humans, and can cause the disease toxoplasmosis. Definitive host - cat Intermediate host - man and other mammalian species Serologic test, primary mode of diagnosis o IFA and EIA tests for IgG and IgM antibodies ▪ Most commonly used o Toxoplasma-specific IgA antibodies ▪ More sensitive than IgM detection in congenitally infected babies o TORCH test • FUNGAL DISEASES • Available technologies include: o Immunodiffusion (ID) o Complement fixation (CF) o Enzyme immunoassay (EIA) REVIEW BACTERIAL DISEASES • Syphilis o Non-treponemal tests ▪ Flocculation ▪ Complement fixation ▪