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Sean K. Bauman, Ph.D. V.P. Operations Immuno-Mycologics, Inc. Serodiagnosis of Mycoses Slide 2 Outline Why use serology? Mechanisms of different tests Serology of specific mycoses Slide 3 Why Use Serology For Case Finding? Antigens and antibodies are easier to detect than finding the organism directly. Antibodies are produced in large quantities and circulate in the blood even though they are not usually protective against mycotic agents. Soluble antigens frequently enter the circulation of the host and are sometimes excreted in the urine. They are produced in discrete infected lesions. Culture is relatively Insensitive due to the low concentration of the agents in tissues. Multiple cultures are usually required. Slide 4 Immunobiology, 4 th Edition Immunodiffusion Slide 5 Immunobiology, 4 th Edition Immunodiffusion Concentration Slide 6 Immunobiology, 4 th Edition Immunodiffusion Concentration Slide 7 Kuby Immunology, 4 th Edition Immunodiffusion Slide 8 Kuby Immunology, 4 th Edition Immunodiffusion Slide 9 Kuby Immunology, 4 th Edition Immunodiffusion Slide 10 Types of Immunodiffusion Reactions: Identity Partial Identity Non-Identity Slide 11 Immunodiffusion Identity Antigen with epitope A A A Anti-A Adapted from Kuby Immunology, 4 th Edition Slide 12 Immunodiffusion Partial Identity Adapted from Kuby Immunology, 4 th Edition Antigen with epitopes A and B A A B B Anti-A Anti-B Anti-A Slide 13 Immunodiffusion Non-Identity Anti-A Anti-B Adapted from Kuby Immunology, 4 th Edition Antigen with epitopes A and B A A B B Slide 14 Immunodiffusion Slide 15 Qualitative vs Quantitative Immunodiffusion Positive UndilutedPositive @ 1:4 Identity & Partial Identity bands are considered POSITIVE Ag CS Pt1 2 4 3 Screening Test QUALitative Undil. 1:2 1:8 1:4 Ag CS Patient 1 QUANTitative Undil. 1:2 1:8 1:4 Ag CS Patient 3 QUANTitative Slide 16 Serum without antibodies Serum with antibodies Complement Fixation Antigen binds to antibodies Unbound antigen Complement binds to Ag/Ab complex Unbound complement No lysis Positive Lysis Negative Hemolysin sensitized red blood cells serve as an indicator Hemolysin sensitized red blood cells serve as an indicator Day 1 Day 2 Slide 17 Complement Fixation Slide 18 Latex Agglutination Antigen Test + Antibody-Coated Latex Specimen Containing Antigen Antibody Test Antigen-Coated Latex Specimen Containing Antibody + Slide 19 Latex Agglutination Slide 20 Sandwich ELISA Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay Antigen Test Slide 21 Sandwich ELISA Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay Add Specimen Antigen Test Slide 22 Sandwich ELISA Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay Add Specimen Antigen Test Slide 23 Sandwich ELISA Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay Add Specimen Containing Ag Antigen Test Slide 24 Indirect ELISA Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay Antibody Test Slide 25 Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay (Specimen) Indirect ELISA Antibody Test Slide 26 Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay (Specimen) Indirect ELISA Antibody Test Slide 27 Adapted from Kuby Immunology, 4 th Edition Enzyme-Linked Immunosorbent Assay (Specimen) Indirect ELISA Antibody Test Slide 28 Mycotic Diseases Aspergillosis Blastomycosis Candidiasis Coccidioidomycosis Cryptococcosis Histoplasmosis Paracoccidioidomycosis Organism, Disease, Diagnosis Slide 29 Primary etiologic agents A. fumigatus A. flavus A. niger A. terreus Aspergillosis Aspergillus spp. have a global distribution The Organism Slide 30 Infection initiates by inhalation of Aspergillus spores Aspergillosis A. niger fruiting body in a lung cavity (Aspergilloma) The Disease Allergic Bronchopulmonary Aspergillosis (ABPA) Pulmonary Aspergilloma (fungus ball) Invasive Aspergillosis (IA) (usually immunocompromised) Forms of the Disease Slide 31 Aspergillosis Antibody Test 1 band indicates current/recent Aspergillus infection Immunodiffusion Species # BandsSensitivitySpecificity A. fumigatus 290%99.6% A. flavus 1100%100% A. niger 1100%100% A. terreus 1n.d.n.d. 3 bands is indicative of Aspergilloma &/or IA Disease Sensitivity Specificity Aspergilloma 97%100% ABPA 70%100% IA 79%100% Sabouraudia (1982) 20:63 Diagnosis Slide 32 Aspergillosis Antigen Tests Latex Agglutination ELISA SensitivitySpecificity PastorexLA31-95%91-95% PlateliaELISA60-93%82-99% Clin Micro Rev (2002) 15:465 Diagnosis Slide 33 Blastomycosis Etiologic agent: Blastomyces dermatitidis B. dermatitidis is endemic to the Ohio and Mississippi river valleys The Organism Slide 34 Blastomycosis Infection initiates by inhalation of the organism The Disease Slide 35 Blastomycosis Antibody Test A antigen (a.k.a. WI-1) 25-amino acid tandem repeat is immunodominant epitope Precipitin band indicates current/recent B. dermatitidis infection 120-kDa glycoprotein Immunodiffusion Sensitivity 100% Specificity 100% J. Med. Vet. Mycol. (1995) 33:123 Diagnosis Complement Fixation Sensitivity 40% Specificity 100% J. Clin. Micro. (1986) 23:294 Slide 36 Candidiasis Primary etiologic agent: Candida albicans C. albicans is part of the normal human flora The Organism ~80% have positive DTH reactions to Candida antigens by 1 year of age Slide 37 Candidiasis Immunocompromised states susceptible to Candidiasis Neutropenia Corticosteroids Diabetes mellitus HIV infection Cancer Infection types: The Disease Oropharyngeal (thrush) Vaginal Systemic/Invasive Antibiotic therapy Slide 38 Candidiasis Antibody Test Systemic candidiasis should be suspected when Sensitivity 80% in immunocompetent individuals * Manual of Clinical Laboratory Immunology, 6 th Edition 1 band indicates current/recent infection Seroconversion (i.e. negative patient becomes positive) Number of precipitins increases on serial specimens -or- Sensitivity is less in immunodeficient individuals Immunodiffusion Diagnosis Slide 39 Candidiasis Antigen/Metabolite Tests Latex Agglutination ELISA -Glucan Cand-Tec assayLA77%88% Clin Micro Rev (2002) 15:465 SensitivitySpecificity PastorexLA25-28%100% PlateliaELISA42%98% -GlucanLimulus84%88% Diagnosis Slide 40 Coccidioidomycosis Etiologic agent: Coccidioides immitis C. immitis is a soil fungus endemic to: Southwestern United States Northern Mexico Central America Central South America The Organism Slide 41 Coccidioidomycosis Infection initiates by inhalation of arthroconidia Pulmonary and/or meningeal infection The Disease Slide 42 Coccidioidomycosis Antibody Test Sensitivity51% Specificity89% J. Clin. Micro. (1995) 33:940 Diagnosis Sensitivity89% Specificity89% Sensitivity94% Specificity92% Latex Agglutination Complement Fixation ELISA Slide 43 Heat labile antigen Predominately IgG reactivity IDCF 47-kDa chitinase enzyme Heat stable antigen (60C, 30 min) 3-O-methylmannose (3-O-MM) is immunodominant epitope IDTP 120-kDa glycoprotein (BGL2) Predominantly IgM reactivity in early primary cases Closest to the antigen well Immunodiffusion Sensitivity100% Specificity100% J. Clin. Micro. (1995) 33:618 Coccidioidomycosis Antibody Test Diagnosis Slide 44 Cryptococcosis The Organism Etiologic agent: Cryptococcus neoformans C. neoformans is encapsulated yeast with a global distribution often found in soil and bird droppings Slide 45 Cryptococcosis The Disease Immunocompromised (e.g. AIDS) are most susceptible Pigeon Meningitis Adapted from Clinical microbiology made ridiculously simple by M. Gladwin and B. Trattler Slide 46 Cryptococcosis Antigen Tests Sensitivity100% Specificity100% Mycoses (1993) 36:31 Sensitivity85% Specificity97% Diagnosis Latex Agglutination ELISA Slide 47 Histoplasmosis The Organism Etiologic agent: Histoplasma capsulatum H. capsulatum is endemic to the Ohio and Mississippi river valleys Slide 48 Histoplasmosis The Disease Infection initiates by inhalation of the organism Intracellular proliferation Slide 49 Histoplasmosis Antibody Test M Antigen 81-kDa glycoprotein (catalase) M precipitins first to appear in acute disease H Antigen 93-kDa glycoprotein ( -glucosidase) H precipitins occur later in infection (likely extrapulmonary dissemination) Closest to the antigen well Closest to the serum well Immunodiffusion Sensitivity90% Specificity94% J. Clin. Micro. (1976) 2:77 Diagnosis Slide 50 Complement Fixation Sensitivity94% Specificity95% J. Clin. Micro. (1976) 2:77 Histoplasmosis Antibody Test Diagnosis Slide 51 Histoplasmosis Antigen Tests EIA/RIA UrineRIA89%90% SensitivitySpecificity SerumRIA82%77% SerumEIA68%81% UrineEIA89%90% Clin Micro Rev (2002) 15:465 Diagnosis Slide 52 Paracoccidioidomycosis The Organism Etiologic agent: Paracoccidioides brasiliensis P. brasiliensis is endemic to Mexico Central America South America Doctorfungus Corporation ? Slide 53 The Disease Infection initiates by inhalation of the organism Paracoccidioidomycosis Ulcerative lesions are common Doctorfungus Corporation Slide 54 Paracoccidioidomycosis Antibody Test Sensitivity = >90% * Manual of Clinical Laboratory Immunology, 6 th Edition Up to 3 precipitin bands are observed gp43 is the immunodominant antigen (closest to Ag well) (a.k.a. E 2 or A) Most prevalent and longest lasting of the major precipitins gp43 Antigen 43-kDa glycoprotein Immunodiffusion Diagnosis Slide 55 Why Use Serology For Case Finding? Antigens and antibodies are easier to detect than finding the organism directly. Antibodies are produced in large quantities and circulate in the blood even though they are not usually protective against mycotic agents. Soluble antigens frequently enter the circulation of the host and are sometimes excreted in the urine. They are produced in discrete infected lesions. Culture is relatively Insensitive due to the low concentration of the agents in tissues. Multiple cultures are usually required. Slide 56 Do one thing, do it well! The Fungal Diagnostic Specialists www.immy.com