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

4. Why use serology
Antigens and antibodies are easier to detect than finding the organism directly
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. Antibodies and antigens
Available tests
Antibodies
Immunodiffusion
Radioallergosorbent Test (RAST)
Antigens
Latex Agglutination
Radioimmunoassay (RIA)
Antibodies and antigens
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. Mycotic diseases Aspergillosis Candidiasis Cryptococcosis
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.

10. Treatment strategies based on the pathophysiology of aspergillosis

11. McCormick et al. 2010

12. Serological Diagnosis
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.

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 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

17. Aspergillosis Antigen Tests: Galactomannan Patient group
Sensitivity (%)
Specificity (%)
Allo HSCT, neutropenia, all on steroids 96 99
Neutropenia, suspected IA, GVHD, steroids
100 93
Allo HSCT 81 89
Haematologic malignancy 80 82
Neutropenia, Cut Off 1.5 88 90
ELISA, LA; Cut off 0.5, except neutopenic group
Wheat L.J, Transplant Infect Dis (2006), 8:128

18. 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

19. Aspergillosis Antigen Tests: (1→3)-β-D-glucan Patient group
Sensitivity (%)
Specificity (%)
Haematologic disease 88 85
Neutropenia, suspected IA, GVHD, steroids 55 95
Neutropenia, Cut off 120 pg/ml 90
Cut off 60 pg/ml, except neutorpenic group
Wheat L.J, Transplant Infect Dis (2006), 8:128

20. 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

21. Aspergillus precipitin test
Strong reactions: indicative of aspergilloma

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

24. 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)

25. 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

26. 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

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

28. 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)

29. 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

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 clinicians to follow the progress of the disease – however may be difficult to obtain appropriate specimens
Kits are expensive making continuous monitoring difficult