Transcript 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
Empirical/targeted therapy

SEROLOGICAL TARGETS
Serology methods utilise the reactions and properties of serum
• ANTIBODIES
(Use of commercially available
antigens)
Exo-antigen
Antigen
• ANTIGENS
(Use of specific antibodies)
Antibody
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
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)
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
Mycotic diseases
• Aspergillosis
• Candidiasis
Opportunistic
pathogens
Antigen &
antibody (?)
detection
True
pathogens
Antigen &
antibody (?)
detection
• Cryptococcosis
• Histoplasmosis
• Blastomycosis
• Coccidioidomycosis
• Paracoccidioidomycosis
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.
Treatment strategies based on the
pathophysiology of aspergillosis
McCormick et al. 2010
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.
DIAGNOSTIC TOOLS 1976
DIAGNOSTIC TOOLS 1979
DIAGNOSTIC TOOLS 2011
Sandwich-ELISA
•galactomannan
•mannan
High-resolution CT-scan
Ultrasound
Bronchoalveolar lavages
Biopsy techniques
(BLOOD)CULTURES
Fungitell (-1-3-D-glucan)
PCR
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
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
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
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
88
90
Cut off 60 pg/ml, except neutorpenic group
Wheat L.J, Transplant Infect Dis (2006), 8:128
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
Aspergillus precipitin test
Strong reactions: indicative of aspergilloma
ImmunoCAP
• IgE
• IgG
• ABPA:
– asthma
– cystic fibrosis
– COPD
– cavitary disease
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)
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
Candidiasis
Antigen Test
Test
LA mannan (Pastorex) MAb
Sensitivity (%)
Specificity (%)
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
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
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 LatexCryptococcus antigen assay)
Cryptococcosis
Antigen Test
- detection in serum, BAL or CSF
- false-positive results may be caused by rheumatoid factor or crossreactive 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
The future?
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