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SEROLOGY OF FUNGAL INFECTIONS. Biological infection Pathological changes Empirical/targeted therapy Fungitell Aspergillus PCR Aspergillus GM Current diagnostic.

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Presentation on theme: "SEROLOGY OF FUNGAL INFECTIONS. Biological infection Pathological changes Empirical/targeted therapy Fungitell Aspergillus PCR Aspergillus GM Current diagnostic."— Presentation transcript:

1 SEROLOGY OF FUNGAL INFECTIONS

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

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

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 Immunodiffusion Latex Agglutination Antibodies Antigens Radioimmunoassay (RIA) Complement fixation Enzyme-linked immunosorbent assay (ELISA) Antibodies and antigens Radioallergosorbent Test (RAST) Enzyme Immunoassay (EIA)

6 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 Measures of Accuracy in Serology Assays

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

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

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10 Treatment strategies based on the pathophysiology of aspergillosis

11 McCormick et al. 2010

12 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.Aspergillosis

13 DIAGNOSTIC TOOLS 1976

14 DIAGNOSTIC TOOLS 1979

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

16 Aspergillosis Galactomannan (GM) - polysaccharide component of the cell wall However... - GM presence in patient’s blood is determined by multiple factors Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755 - highly immunogenic antigen - present in most Aspergilli - exo-antigen that can be detected in serum, BAL or CSF - 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) - monitoring of GM during antifungal therapy allows progression of treatment to be measured - several commercially available ELISA tests (Platelia, Pastorex) Diagnosis of invasive disease

17 Antigen Tests: Patient groupSensitivity (%)Specificity (%) Allo HSCT, neutropenia, all on steroids 9699 Neutropenia, suspected IA, GVHD, steroids Allo HSCT8189 Haematologic malignancy8082 Neutropenia, Cut Off Galactomannan Wheat L.J, Transplant Infect Dis (2006), 8:128 Aspergillosis ELISA, LA; Cut off 0.5, except neutopenic group

18 (1→3)-β-D-glucan - widely distributed in nature (fungi, yeast, algae, bacteria, plants) However... - false-positive results may occur (60% of bacteraemic patients) Aspergillosis Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755 - 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

19 Aspergillosis Antigen Tests: Patient groupSensitivity (%)Specificity (%) Haematologic disease8885 Neutropenia, suspected IA, GVHD, steroids 5595 Neutropenia, Cut off 120 pg/ml 8890 Wheat L.J, Transplant Infect Dis (2006), 8:128 (1→3)-β-D-glucan Cut off 60 pg/ml, except neutorpenic group

20 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 Aspergillosis Diagnosis of allergic Aspergillosis Antibody Test

21 Aspergillus precipitin test Strong reactions: indicative of aspergilloma

22 ImmunoCAP IgE IgG ABPA: –asthma –cystic fibrosis –COPD –cavitary disease

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

25 Mannan - highly immunogenic antigen However... - negative results of the tests do not exclude infection Candidiasis Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755 - 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 Antigen Test - commercially available tests: Pastorex and Platelia (ELISA)

26 Antigen Test LA mannan (Pastorex) MAb Yeo & Wong, Clin Micro Rev (2002) 15:465 ELISA mannan (Platelia) MAb β-glucan (enzymatic – Fungitec-G) Sensitivity (%)Specificity (%) Test MAb – monoclonal antibody ELISA mannan (Platelia) PAb PAb – polyclonal antibody Candidiasis

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

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

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

30 The future?

31 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 clinician s 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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