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Immuno-Mycologics, Inc.
Serodiagnosis of Mycoses Sean K. Bauman, Ph.D. V.P. Operations Immuno-Mycologics, Inc.
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Outline Why use serology? Mechanisms of different tests
Serology of specific mycoses
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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.
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Immunodiffusion Immunobiology, 4th Edition
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Immunodiffusion Concentration Immunobiology, 4th Edition
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Immunodiffusion Concentration Immunobiology, 4th Edition
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Immunodiffusion Kuby Immunology, 4th Edition
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Immunodiffusion Kuby Immunology, 4th Edition
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Immunodiffusion Kuby Immunology, 4th Edition
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Immunodiffusion Types of Immunodiffusion Reactions: Identity
Partial Identity Non-Identity
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Immunodiffusion Identity Anti-A Antigen with epitope A A
Adapted from Kuby Immunology, 4th Edition
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Immunodiffusion Partial Identity Anti-B Anti-A Anti-A
Antigen with epitopes A and B A B Adapted from Kuby Immunology, 4th Edition
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Immunodiffusion Non-Identity Anti-B Anti-A
Antigen with epitopes A and B A B Adapted from Kuby Immunology, 4th Edition
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Immunodiffusion
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Qualitative vs Quantitative Immunodiffusion
Ag CS Pt 1 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 Positive Undiluted 1:4 Identity & Partial Identity bands are considered POSITIVE
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Complement Fixation Serum without antibodies Serum with Antigen binds
to antibodies Unbound antigen Day 1 Complement binds to Ag/Ab complex Unbound complement Hemolysin sensitized red blood cells serve as an indicator Day 2 No lysis Positive Lysis Negative
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Complement Fixation
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Latex Agglutination + + Antigen Test Antibody-Coated Specimen Latex
Containing Antigen Antibody Test Antigen-Coated Latex Specimen Containing Antibody +
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Latex Agglutination
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Enzyme-Linked Immunosorbent Assay
Sandwich ELISA Antigen Test Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Sandwich ELISA Antigen Test Add Specimen Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Sandwich ELISA Antigen Test Add Specimen Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Sandwich ELISA Antigen Test Add Specimen Containing Ag Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Indirect ELISA Antibody Test Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Indirect ELISA Antibody Test (Specimen) Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Indirect ELISA Antibody Test (Specimen) Adapted from Kuby Immunology, 4th Edition
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Enzyme-Linked Immunosorbent Assay
Indirect ELISA Antibody Test (Specimen) Adapted from Kuby Immunology, 4th Edition
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Mycotic Diseases Organism, Disease, Diagnosis Aspergillosis
Blastomycosis Candidiasis Coccidioidomycosis Cryptococcosis Histoplasmosis Paracoccidioidomycosis Organism, Disease, Diagnosis
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Aspergillosis The Organism Primary etiologic agents A. fumigatus
A. flavus A. niger A. terreus Aspergillus spp. have a global distribution
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Aspergillosis The Disease
Infection initiates by inhalation of Aspergillus spores A. niger fruiting body in a lung cavity (Aspergilloma) Forms of the Disease Allergic Bronchopulmonary Aspergillosis (ABPA) Pulmonary Aspergilloma (fungus ball) Invasive Aspergillosis (IA) (usually immunocompromised)
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Aspergillosis Antibody Test
Diagnosis Immunodiffusion 1 band indicates current/recent Aspergillus infection 3 bands is indicative of Aspergilloma &/or IA Species # Bands Sensitivity Specificity A. fumigatus 2 90% 99.6% A. flavus 1 100% 100% A. niger 1 100% 100% A. terreus 1 n.d. n.d. Disease Sensitivity Specificity Aspergilloma 97% 100% ABPA 70% 100% IA 79% 100% Sabouraudia (1982) 20:63
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Aspergillosis Antigen Tests
Diagnosis Latex Agglutination Sensitivity Specificity Pastorex LA % 91-95% ELISA Platelia ELISA % 82-99% Clin Micro Rev (2002) 15:465
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Blastomycosis The Organism Etiologic agent: Blastomyces dermatitidis
B. dermatitidis is endemic to the Ohio and Mississippi river valleys l
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Blastomycosis The Disease Infection initiates by inhalation
of the organism
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Blastomycosis Antibody Test
Diagnosis Immunodiffusion ‘A’ antigen (a.k.a. WI-1) 120-kDa glycoprotein 25-amino acid tandem repeat is immunodominant epitope Precipitin band indicates current/recent B. dermatitidis infection Sensitivity 100% Specificity 100% J. Med. Vet. Mycol. (1995) 33:123 Complement Fixation Sensitivity 40% Specificity 100% J. Clin. Micro. (1986) 23:294
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Candidiasis The Organism Primary etiologic agent: Candida albicans
C. albicans is part of the normal human flora ~80% have positive DTH reactions to Candida antigens by 1 year of age
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Candidiasis The Disease
Immunocompromised states susceptible to Candidiasis Neutropenia Corticosteroids Diabetes mellitus HIV infection Cancer Oropharyngeal (thrush) Antibiotic therapy Systemic/Invasive Infection types: Vaginal
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Candidiasis Antibody Test
Diagnosis Immunodiffusion 1 band indicates current/recent infection Systemic candidiasis should be suspected when Number of precipitins increases on serial specimens -or- Seroconversion (i.e. negative patient becomes positive) Sensitivity 80% in immunocompetent individuals * Manual of Clinical Laboratory Immunology, 6th Edition Sensitivity is less in immunodeficient individuals
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Candidiasis Antigen/Metabolite Tests
Diagnosis Latex Agglutination Sensitivity Specificity Cand-Tec assay LA 77% 88% Pastorex LA % 100% ELISA Platelia ELISA 42% 98% b-Glucan b-Glucan Limulus 84% 88% Clin Micro Rev (2002) 15:465
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Coccidioidomycosis The Organism
C. immitis is a soil fungus endemic to: Southwestern United States Northern Mexico Central America Central South America Etiologic agent: Coccidioides immitis
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Coccidioidomycosis The Disease Infection initiates by inhalation
of arthroconidia Pulmonary and/or meningeal infection
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Coccidioidomycosis Antibody Test
Diagnosis Latex Agglutination Sensitivity 51% Specificity 89% Complement Fixation Sensitivity 89% Specificity 89% ELISA Sensitivity 94% Specificity 92% J. Clin. Micro. (1995) 33:940
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Coccidioidomycosis Antibody Test
Diagnosis Immunodiffusion IDTP 120-kDa glycoprotein (BGL2) 3-O-methylmannose (3-O-MM) is immunodominant epitope Heat stable antigen (60°C, 30 min) Predominantly IgM reactivity in early primary cases IDCF 47-kDa chitinase enzyme Heat labile antigen Predominately IgG reactivity Closest to the antigen well Sensitivity 100% Specificity 100% J. Clin. Micro. (1995) 33:618
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Cryptococcus neoformans
Cryptococcosis The Organism Etiologic agent: Cryptococcus neoformans C. neoformans is encapsulated yeast with a global distribution often found in soil and bird droppings
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Clinical microbiology made by M. Gladwin and B. Trattler
Cryptococcosis The Disease Immunocompromised (e.g. AIDS) are most susceptible Pigeon Meningitis Cryptococcus neoformans AIDS Adapted from Clinical microbiology made ridiculously simple by M. Gladwin and B. Trattler
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Cryptococcosis Antigen Tests
Diagnosis Latex Agglutination Sensitivity 100% Specificity 100% ELISA Sensitivity 85% Specificity 97% Mycoses (1993) 36:31
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Histoplasmosis The Organism H. capsulatum is endemic to the
Etiologic agent: Histoplasma capsulatum H. capsulatum is endemic to the Ohio and Mississippi river valleys
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Histoplasmosis The Disease Infection initiates by inhalation
of the organism Intracellular proliferation
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Histoplasmosis Antibody Test
Diagnosis Immunodiffusion M Antigen 81-kDa glycoprotein (catalase) M precipitins first to appear in acute disease Closest to the antigen well H Antigen 93-kDa glycoprotein (b-glucosidase) H precipitins occur later in infection (likely extrapulmonary dissemination) Closest to the serum well Sensitivity 90% Specificity 94% J. Clin. Micro. (1976) 2:77
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Histoplasmosis Antibody Test
Diagnosis Complement Fixation Sensitivity 94% Specificity 95% J. Clin. Micro. (1976) 2:77
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Histoplasmosis Antigen Tests
Diagnosis EIA/RIA Sensitivity Specificity Urine RIA 89% 90% Urine EIA 89% 90% Serum RIA 82% 77% Serum EIA 68% 81% Clin Micro Rev (2002) 15:465
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Paracoccidioidomycosis
The Organism P. brasiliensis is endemic to Mexico Central America South America Doctorfungus Corporation ? Etiologic agent: Paracoccidioides brasiliensis
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Paracoccidioidomycosis
The Disease Infection initiates by inhalation of the organism Ulcerative lesions are common Doctorfungus Corporation
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Paracoccidioidomycosis Antibody Test
Diagnosis Immunodiffusion Up to 3 precipitin bands are observed gp43 Antigen 43-kDa glycoprotein gp43 is the immunodominant antigen (closest to Ag well) (a.k.a. E2 or A) Most prevalent and longest lasting of the major precipitins Sensitivity = >90% * Manual of Clinical Laboratory Immunology, 6th Edition
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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.
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The Fungal Diagnostic Specialists
Do one thing, do it well!
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