Sean K' Bauman, Ph'D'

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Serodiagnosis of Mycoses
Sean K. Bauman, Ph.D. V.P. Operations Immuno-Mycol
ogics, 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.

4
Immunodiffusion
Immunobiology, 4th Edition
5
Immunodiffusion
Concentration
Immunobiology, 4th Edition
6
Immunodiffusion
Concentration
Immunobiology, 4th Edition
7
Immunodiffusion
Kuby Immunology, 4th Edition
8
Immunodiffusion
Kuby Immunology, 4th Edition
9
Immunodiffusion
Kuby Immunology, 4th Edition
10
Immunodiffusion
Types of Immunodiffusion Reactions

• Identity
• Partial Identity
• Non-Identity

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Immunodiffusion
Identity
Adapted from Kuby Immunology, 4th Edition
12
Immunodiffusion
Partial Identity
Adapted from Kuby Immunology, 4th Edition
13
Immunodiffusion
Non-Identity
Adapted from Kuby Immunology, 4th Edition
14
Immunodiffusion
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Qualitative vs Quantitative Immunodiffusion
Positive Undiluted
Positive _at_ 14
Identity Partial Identity bands are considered
POSITIVE
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Complement Fixation
Day 1
Day 2
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Complement Fixation
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Latex Agglutination
Antigen Test
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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
27
Enzyme-Linked Immunosorbent Assay
Indirect ELISA
Antibody Test
(Specimen)
Adapted from Kuby Immunology, 4th Edition
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Mycotic Diseases

• Aspergillosis
• Blastomycosis
• Candidiasis
• Coccidioidomycosis
• Cryptococcosis
• Histoplasmosis
• Paracoccidioidomycosis

Organism, Disease, Diagnosis
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Aspergillosis
The Organism
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Aspergillosis
The Disease

• Infection initiates by inhalation of
  Aspergillus spores

• Forms of the Disease

• Allergic Bronchopulmonary Aspergillosis (ABPA)

• Pulmonary Aspergilloma (fungus ball)

• Invasive Aspergillosis (IA) (usually
  immunocompromised)

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Aspergillosis Antibody Test
Immunodiffusion
Diagnosis

• ? 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.

Sabouraudia (1982) 2063
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Aspergillosis Antigen Tests
Diagnosis
Latex Agglutination
Sensitivity Specificity
Pastorex LA 31-95 91-95
ELISA
Platelia ELISA 60-93 82-99
Clin Micro Rev (2002) 15465
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Blastomycosis
The Organism
Etiologic agent Blastomyces dermatitidis
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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

Complement Fixation
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Candidiasis
The Organism

• Primary etiologic agent Candida albicans

• C. albicans is part of the normal human flora

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Candidiasis
The Disease

• Immunocompromised states susceptible to
  Candidiasis
• Neutropenia
• Corticosteroids
• Diabetes mellitus
• HIV infection
• Cancer

• Antibiotic therapy

• Infection types

• Vaginal

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Candidiasis Antibody Test
Immunodiffusion
Diagnosis

• ? 1 band indicates current/recent infection

• Systemic candidiasis should be suspected when

• Seroconversion (i.e. negative patient becomes
  positive)

• 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 25-28 100
ELISA
Platelia ELISA 42 98
b-Glucan
b-Glucan Limulus 84 88
Clin Micro Rev (2002) 15465
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Coccidioidomycosis
The Organism
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Coccidioidomycosis
The Disease
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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) 33940
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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 (60C, 30 min)

• Predominantly IgM reactivity in early primary
  cases

• Heat labile antigen

• Predominately IgG reactivity

• Closest to the antigen well

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Cryptococcosis
The Organism
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Cryptococcosis
The Disease
Immunocompromised (e.g. AIDS) are most susceptible
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) 3631
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Histoplasmosis
The Organism
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Histoplasmosis
The Disease

• Infection initiates by inhalation
• of the organism

• Intracellular proliferation

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Histoplasmosis Antibody Test
Immunodiffusion
Diagnosis

• 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)

• Closest to the serum well

J. Clin. Micro. (1976) 277
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Histoplasmosis Antibody Test
Diagnosis
Complement Fixation
Sensitivity 94 Specificity 95
J. Clin. Micro. (1976) 277
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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) 15465
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Paracoccidioidomycosis
The Organism
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Paracoccidioidomycosis
The Disease
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Paracoccidioidomycosis Antibody Test
Immunodiffusion
Diagnosis

• Up to 3 precipitin bands are observed

• Most prevalent and longest lasting of the major
  precipitins

• Sensitivity gt90

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.

56
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