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

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Presentation on theme: "Sean K. Bauman, Ph.D. V.P. Operations Immuno-Mycologics, Inc. Serodiagnosis of Mycoses."— Presentation transcript:

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

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

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.

4 Immunobiology, 4 th Edition Immunodiffusion

5 Immunobiology, 4 th Edition Immunodiffusion Concentration

6 Immunobiology, 4 th Edition Immunodiffusion Concentration

7 Kuby Immunology, 4 th Edition Immunodiffusion

8 Kuby Immunology, 4 th Edition Immunodiffusion

9 Kuby Immunology, 4 th Edition Immunodiffusion

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

11 Immunodiffusion Identity Antigen with epitope A A A Anti-A Adapted from Kuby Immunology, 4 th Edition

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

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

14 Immunodiffusion

15 Qualitative vs Quantitative Immunodiffusion Positive 1:4 Identity & Partial Identity bands are considered POSITIVE Ag CS Pt 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

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

17 Complement Fixation

18 Latex Agglutination Antigen Test + Antibody-Coated Latex Specimen Containing Antigen Antibody Test Antigen-Coated Latex Specimen Containing Antibody +

19 Latex Agglutination

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

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

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

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

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

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

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

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

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

29 Primary etiologic agents A. fumigatus A. flavus A. niger A. terreus Aspergillosis Aspergillus spp. have a global distribution The Organism

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

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

32 Aspergillosis Antigen Tests Latex Agglutination ELISA SensitivitySpecificity PastorexLA31-95%91-95% PlateliaELISA60-93%82-99% Clin Micro Rev (2002) 15:465 Diagnosis

33 Blastomycosis Etiologic agent: Blastomyces dermatitidis B. dermatitidis is endemic to the Ohio and Mississippi river valleys The Organism

34 Blastomycosis Infection initiates by inhalation of the organism The Disease

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

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

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

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

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

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

41 Coccidioidomycosis Infection initiates by inhalation of arthroconidia Pulmonary and/or meningeal infection The Disease

42 Coccidioidomycosis Antibody Test Sensitivity51% Specificity89% J. Clin. Micro. (1995) 33:940 Diagnosis Sensitivity89% Specificity89% Sensitivity94% Specificity92%  Latex Agglutination  Complement Fixation  ELISA

43 Heat labile antigen Predominately IgG reactivity  IDCF 47-kDa chitinase enzyme 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  Sensitivity100%  Specificity100% J. Clin. Micro. (1995) 33:618 Coccidioidomycosis Antibody Test Diagnosis

44 Cryptococcosis The Organism Etiologic agent: Cryptococcus neoformans C. neoformans is encapsulated yeast with a global distribution often found in soil and bird droppings

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

46 Cryptococcosis Antigen Tests Sensitivity100% Specificity100% Mycoses (1993) 36:31 Sensitivity85% Specificity97% Diagnosis  Latex Agglutination  ELISA

47 Histoplasmosis The Organism Etiologic agent: Histoplasma capsulatum H. capsulatum is endemic to the Ohio and Mississippi river valleys

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

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

50 Complement Fixation Sensitivity94% Specificity95% J. Clin. Micro. (1976) 2:77 Histoplasmosis Antibody Test Diagnosis

51 Histoplasmosis Antigen Tests EIA/RIA UrineRIA89%90% SensitivitySpecificity SerumRIA82%77% SerumEIA68%81% UrineEIA89%90% Clin Micro Rev (2002) 15:465 Diagnosis

52 Paracoccidioidomycosis The Organism Etiologic agent: Paracoccidioides brasiliensis P. brasiliensis is endemic to Mexico Central America South America Doctorfungus Corporation ?

53 The Disease Infection initiates by inhalation of the organism Paracoccidioidomycosis Ulcerative lesions are common Doctorfungus Corporation

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

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.

56 Do one thing, do it well! The Fungal Diagnostic Specialists


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