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Presentation Transcript

Slide1: 

Sean K. Bauman, Ph.D. V.P. Operations Immuno-Mycologics, Inc. Serodiagnosis of Mycoses

Slide2: 

Outline Why use serology? Mechanisms of different tests Serology of specific mycoses

Slide3: 

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.

Slide4: 

Immunobiology, 4th Edition Immunodiffusion

Slide5: 

Immunobiology, 4th Edition Immunodiffusion Concentration

Slide6: 

Immunobiology, 4th Edition Immunodiffusion Concentration

Slide7: 

Kuby Immunology, 4th Edition Immunodiffusion

Slide8: 

Kuby Immunology, 4th Edition Immunodiffusion

Slide9: 

Kuby Immunology, 4th Edition Immunodiffusion

Slide10: 

Immunodiffusion Types of Immunodiffusion Reactions: Identity Partial Identity Non-Identity

Slide11: 

Immunodiffusion Identity Adapted from Kuby Immunology, 4th Edition

Slide12: 

Immunodiffusion Partial Identity Adapted from Kuby Immunology, 4th Edition

Slide13: 

Immunodiffusion Non-Identity Adapted from Kuby Immunology, 4th Edition

Slide14: 

Immunodiffusion

Slide15: 

Qualitative vs Quantitative Immunodiffusion Positive Undiluted Positive @ 1:4 Identity andamp; Partial Identity bands are considered POSITIVE

Slide16: 

Complement Fixation Day 1 Day 2

Slide17: 

Complement Fixation

Slide18: 

Latex Agglutination Antigen Test

Slide19: 

Latex Agglutination

Slide20: 

Sandwich ELISA Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay Antigen Test

Slide21: 

Sandwich ELISA Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay Add Specimen Antigen Test

Slide22: 

Sandwich ELISA Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay Add Specimen Antigen Test

Slide23: 

Sandwich ELISA Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay Add Specimen Containing Ag Antigen Test

Slide24: 

Indirect ELISA Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay Antibody Test

Slide25: 

Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay (Specimen) Indirect ELISA Antibody Test

Slide26: 

Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay (Specimen) Indirect ELISA Antibody Test

Slide27: 

Adapted from Kuby Immunology, 4th Edition Enzyme-Linked Immunosorbent Assay (Specimen) Indirect ELISA Antibody Test

Slide28: 

Mycotic Diseases Aspergillosis Blastomycosis Candidiasis Coccidioidomycosis Cryptococcosis Histoplasmosis Paracoccidioidomycosis Organism, Disease, Diagnosis

Slide29: 

Aspergillosis The Organism

Slide30: 

Infection initiates by inhalation of Aspergillus spores Aspergillosis The Disease Allergic Bronchopulmonary Aspergillosis (ABPA) Pulmonary Aspergilloma (fungus ball) Invasive Aspergillosis (IA) (usually immunocompromised) Forms of the Disease

Slide31: 

Aspergillosis Antibody Test  1 band indicates current/recent Aspergillus infection Immunodiffusion 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.  3 bands is indicative of Aspergilloma andamp;/or IA Sabouraudia (1982) 20:63 Diagnosis

Slide32: 

Aspergillosis Antigen Tests Latex Agglutination ELISA Sensitivity Specificity Pastorex LA 31-95% 91-95% Platelia ELISA 60-93% 82-99% Clin Micro Rev (2002) 15:465 Diagnosis

Slide33: 

Blastomycosis Etiologic agent: Blastomyces dermatitidis The Organism

Slide34: 

Blastomycosis Infection initiates by inhalation of the organism The Disease

Slide35: 

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 Diagnosis Complement Fixation

Slide36: 

Candidiasis Primary etiologic agent: Candida albicans C. albicans is part of the normal human flora The Organism

Slide37: 

Candidiasis Immunocompromised states susceptible to Candidiasis Neutropenia Corticosteroids Diabetes mellitus HIV infection Cancer Infection types: The Disease Vaginal Antibiotic therapy

Slide38: 

Candidiasis Antibody Test Systemic candidiasis should be suspected when  1 band indicates current/recent infection Seroconversion (i.e. negative patient becomes positive) Sensitivity is less in immunodeficient individuals Immunodiffusion Diagnosis

Slide39: 

Candidiasis Antigen/Metabolite Tests Latex Agglutination ELISA b-Glucan Cand-Tec assay LA 77% 88% Clin Micro Rev (2002) 15:465 Sensitivity Specificity Pastorex LA 25-28% 100% Platelia ELISA 42% 98% b-Glucan Limulus 84% 88% Diagnosis

Slide40: 

Coccidioidomycosis The Organism

Slide41: 

Coccidioidomycosis The Disease

Slide42: 

Coccidioidomycosis Antibody Test Sensitivity 51% Specificity 89% J. Clin. Micro. (1995) 33:940 Diagnosis Sensitivity 89% Specificity 89% Sensitivity 94% Specificity 92% Latex Agglutination Complement Fixation ELISA

Slide43: 

Heat labile antigen Predominately IgG reactivity Heat stable antigen (60°C, 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 Coccidioidomycosis Antibody Test Diagnosis

Slide44: 

Cryptococcosis The Organism

Slide45: 

Cryptococcosis The Disease Immunocompromised (e.g. AIDS) are most susceptible Adapted from Clinical microbiology made ridiculously simple by M. Gladwin and B. Trattler

Slide46: 

Cryptococcosis Antigen Tests Sensitivity 100% Specificity 100% Mycoses (1993) 36:31 Sensitivity 85% Specificity 97% Diagnosis Latex Agglutination ELISA

Slide47: 

Histoplasmosis The Organism

Slide48: 

Histoplasmosis The Disease Infection initiates by inhalation of the organism Intracellular proliferation

Slide49: 

Histoplasmosis Antibody Test M Antigen 81-kDa glycoprotein (catalase) M precipitins first to appear in acute disease H Antigen 93-kDa glycoprotein (b-glucosidase) Closest to the antigen well Closest to the serum well Immunodiffusion J. Clin. Micro. (1976) 2:77 Diagnosis

Slide50: 

Complement Fixation Sensitivity 94% Specificity 95% J. Clin. Micro. (1976) 2:77 Histoplasmosis Antibody Test Diagnosis

Slide51: 

Histoplasmosis Antigen Tests EIA/RIA Urine RIA 89% 90% Sensitivity Specificity Serum RIA 82% 77% Serum EIA 68% 81% Urine EIA 89% 90% Clin Micro Rev (2002) 15:465 Diagnosis

Slide52: 

Paracoccidioidomycosis The Organism

Slide53: 

The Disease Paracoccidioidomycosis

Slide54: 

Paracoccidioidomycosis Antibody Test Sensitivity = andgt;90% * Manual of Clinical Laboratory Immunology, 6th Edition Up to 3 precipitin bands are observed Most prevalent and longest lasting of the major precipitins Immunodiffusion Diagnosis

Slide55: 

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.

Slide56: 

Do one thing, do it well! The Fungal Diagnostic Specialists www.immy.com

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