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SEROLOGY OF FUNGAL INFECTIONS

SEROLOGY OF FUNGAL INFECTIONS. Diagnosis in the setting of increasing fungal burden. Biological infection. Clinical infection. Pathological changes. . INFECTION. Fungitell Aspergillus PCR Aspergillus GM. Current diagnostic methods. Targeted prophylaxis/ Pre-emptive therapy.

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SEROLOGY OF FUNGAL INFECTIONS

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  1. SEROLOGY OF FUNGAL INFECTIONS

  2. Diagnosis in the setting of increasing fungal burden Biological infection Clinical infection Pathological changes  INFECTION Fungitell Aspergillus PCR Aspergillus GM Current diagnostic methods Targeted prophylaxis/ Pre-emptive therapy Empirical/targeted therapy

  3. ANTIBODIES (Use of commercially available antigens) ANTIGENS (Use of specific antibodies) SEROLOGICAL TARGETS Serology methods utilise the reactions and properties of serum Exo-antigen Exo-antigen Antigen Antigen Antibody

  4. Antigens and antibodies are easier to detect than finding the organism directly Why use serology • Antigens and antibodies are produced in large quantities and can be found in body fluids (blood, CSF, urine, BAL) • Culture is often problematic, time consuming and insensitive due to the low concentration of the organism in tissue

  5. Available tests Antibodies • Immunodiffusion • Radioallergosorbent Test (RAST) Antigens • Latex Agglutination • Radioimmunoassay (RIA) Antibodiesandantigens • Complement fixation • Enzyme-linked immunosorbent assay (ELISA) • Enzyme Immunoassay (EIA)

  6. Measures of Accuracy in Serology Assays • Sensitivity • Quantifies the number of false negatives • 80% sensitivity = 80/100 patients with culture positive sample produce positive result in test • Specificity • Quantifies the number of false positives • 80% specificity = 20/100 healthy volunteers with no disease produce positive test result • Results are variable depending on factors such as patient group and monitoring

  7. Aspergillosis Candidiasis Cryptococcosis Mycotic diseases Antigen & antibody (?) detection Opportunistic pathogens • Histoplasmosis • Blastomycosis • Coccidioidomycosis • Paracoccidioidomycosis Antigen & antibody (?) detection True pathogens

  8. Aspergillosis Primary aetiological agents: • A. fumigatus, • A. flavus, • A. niger, • A. terreus. Aspergillus spp. have a global distribution: • airborne spores, • soil, • water supplies, • construction sites, • pillows.

  9. Treatment strategies based on the pathophysiology of aspergillosis

  10. McCormick et al. 2010

  11. Aspergillosis Serological Diagnosis • Diagnosis of invasive disease • Based on the detection of Aspergillus antigens • ELISA kits to detect Galactomannan and -Glucan • Diagnosis of allergic disease • Based on the detection of IgE by RAST/ELISA. • May also use complement fixation or Immunodiffusion.

  12. DIAGNOSTIC TOOLS 1976

  13. DIAGNOSTIC TOOLS 1979

  14. DIAGNOSTIC TOOLS 2011 • Sandwich-ELISA • galactomannan • mannan • High-resolution CT-scan • Ultrasound • Bronchoalveolar lavages • Biopsy techniques • (BLOOD)CULTURES • Fungitell (-1-3-D-glucan) • PCR

  15. Aspergillosis Diagnosis of invasive disease Galactomannan (GM) • polysaccharide component of the cell wall - highly immunogenic antigen - present in most Aspergilli - exo-antigen that can be detected in serum, BAL or CSF • monitoring of GM during antifungal therapy allows progression of treatment to be measured • several commercially available ELISA tests (Platelia, Pastorex) However... • GM presence in patient’s blood is determined by multiple factors - sensitivity of GM detection depends on the site of infection - certain antibiotics (e.g. ampicillin, amoxicillin, amoxicillin-clavulanate) may give false-positive results • Aspergillosis should also be confirmed by other diagnostic tools (CT) Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755

  16. Aspergillosis Antigen Tests: Galactomannan ELISA, LA; Cut off 0.5, except neutopenic group Wheat L.J, Transplant Infect Dis (2006), 8:128

  17. Aspergillosis (1→3)-β-D-glucan • widely distributed in nature (fungi, yeast, algae, bacteria, plants) • not present (or low) in Cryptococcus species, zygomycetes and humans - exo-antigen - may also be used in diagnosis of candidiasis or fusariosis - commercially available kits: Fungitec-G, Fungitell - may be used as a complementary test to GM However... • false-positive results may occur (60% of bacteraemic patients) Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755

  18. Aspergillosis Antigen Tests: (1→3)-β-D-glucan Cut off 60 pg/ml, except neutorpenic group Wheat L.J, Transplant Infect Dis (2006), 8:128

  19. Aspergillosis Diagnosis of allergic Aspergillosis Antibody Test Aspergillus antibodies can only be detected in ABPA, Aspergilloma and CCPA patients. Less reliable than antigen tests due to the presence of anti-Aspergillus antibodies in healthy individuals. High level of precipitating antibodies does not prove the presence of ongoing disease

  20. Aspergillus precipitin test Strong reactions: indicative of aspergilloma

  21. ImmunoCAP • IgE • IgG • ABPA: • asthma • cystic fibrosis • COPD • cavitary disease

  22. Candidiasis Primary aetiological agents: • C. albicans, • C. parapsilosis, • C. glabrata, • C. tropicalis. Diagnosis: Based on detection of: • antigen: > β-glucan (Fungitec-G – enzymatic assay) > Mannan (Pastorex, Platelia - ELISA) - antibody (?) (IgA, IgG – ELISA, Immunodiffusion)

  23. Candidiasis Antigen Test Mannan • highly immunogenic antigen - immunologically more active then β-glucan - polysaccharide component of the cell wall of Candida spp. • positive results may be obtained 2-15 days before positive blood cultures • commercially available tests: Pastorex and Platelia (ELISA) However... • negative results of the tests do not exclude infection Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755

  24. Candidiasis Antigen Test Test Sensitivity (%) Specificity (%) LA mannan (Pastorex) MAb 25-28 100 ELISA mannan (Platelia) MAb 42 93-98 ELISA mannan (Platelia) PAb 21-84 98-100 β-glucan (enzymatic – Fungitec-G) 71-97 54-96 MAb – monoclonal antibody PAb – polyclonal antibody Yeo & Wong, Clin Micro Rev (2002) 15:465

  25. Candidiasis Antibody Test Sensitivity ~80% in immunocompetentindividuals Anti-Candida antibodies may also be present in healthy individuals and cause false possitive results Sensitivity may not be relevant in immunocompromisedindividuals IMMY laboratory manuals

  26. Cryptococcosis Primary aetiological agent: • C. neoformans Diagnosis: Only based on detection of capsular polysaccharide(glucuronoxylomannan) antigen No antibody tests performed Several tests: Latex agglutination (PREMIER Cryptococcal antigen assay) Enzyme Immunoassay (Pastorex Crypto Plus, IMMY Latex-Cryptococcus antigen assay)

  27. Cryptococcosis Antigen Test - detection in serum, BAL or CSF - false-positive results may be caused by rheumatoid factor or cross-reactive organisms (Trichosporon asahii) Test Sensitivity (%) Specificity (%) Latex Agglutination 90 95 ELISA MAb 90 70-80 Yeo & Wong, Clin Micro Rev (2002) 15:465 Santangelo, Med Mycol (2005) 43:335

  28. The future?

  29. Conclusion • Serology is a useful tool for rapid diagnosis of fungal disease • Results may be obtained within a few hours without the need of culture • Results may also be obtained several days before clinical symptoms develop • More work needs to be done on candidosis serological testing • Continued screening allows clinicians to follow the progress of the disease – however may be difficult to obtain appropriate specimens • Kits are expensive making continuous monitoring difficult

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