SEROLOGY OF FUNGAL INFECTIONS

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SEROLOGY OF FUNGAL INFECTIONS
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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
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SEROLOGICAL TARGETS
Serology methods utilise the reactions and
properties of serum

• ANTIBODIES
• (Use of commercially available
• antigens)
• ANTIGENS
• (Use of specific antibodies)

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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

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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)

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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

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Mycotic diseases

• Aspergillosis
• Candidiasis
• Cryptococcosis

Antigen antibody (?) detection
Opportunistic pathogens

• Histoplasmosis
• Blastomycosis
• Coccidioidomycosis
• Paracoccidioidomycosis

Antigen antibody (?) detection
True pathogens
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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.

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Treatment strategies based on the pathophysiology
of aspergillosis
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McCormick et al. 2010
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Serological Diagnosis
Aspergillosis

• 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.

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DIAGNOSTIC TOOLS 1976
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DIAGNOSTIC TOOLS 1979
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DIAGNOSTIC TOOLS 2011

• Sandwich-ELISA
• galactomannan
• mannan
• High-resolution CT-scan
• Ultrasound
• Bronchoalveolar lavages
• Biopsy techniques
• (BLOOD)CULTURES
• Fungitell (?-1-3-D-glucan)
• PCR

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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 patients 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) 26755
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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), 8128
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Aspergillosis
(1?3)-ß-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) 26755
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Aspergillosis
Antigen Tests
(1?3)-ß-D-glucan
Patient group Sensitivity () Specificity ()
Haematologic disease 88 85
Neutropenia, suspected IA, GVHD, steroids 55 95
Neutropenia, Cut off 120 pg/ml 88 90
Cut off 60 pg/ml, except neutorpenic group
Wheat L.J, Transplant Infect Dis (2006), 8128
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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
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Aspergillus precipitin test
Strong reactions indicative of aspergilloma
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ImmunoCAP

• IgE
• IgG
• ABPA
• asthma
• cystic fibrosis
• COPD
• cavitary disease

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Candidiasis
Primary aetiological agents

• C. albicans,

• C. parapsilosis,
• C. glabrata,

• C. tropicalis.

Diagnosis

• Based on detection of
• antigen gt ß-glucan (Fungitec-G enzymatic
  assay) gt Mannan (Pastorex, Platelia - ELISA)
• - antibody (?) (IgA, IgG ELISA, Immunodiffusion)

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Candidiasis
Antigen Test
Mannan

• highly immunogenic antigen

- immunologically more active then ß-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) 26755
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Candidiasis
Antigen Test
Sensitivity ()
Specificity ()
Test
LA mannan (Pastorex) MAb
25-28
100
ELISA mannan (Platelia) MAb
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93-98
ELISA mannan (Platelia) PAb
21-84
98-100
ß-glucan (enzymatic Fungitec-G)
71-97
54-96
MAb monoclonal antibody
PAb polyclonal antibody
Yeo Wong, Clin Micro Rev (2002) 15465
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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
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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)
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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) 15465
Santangelo, Med Mycol (2005) 43335
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The future?
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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