SEROLOGY OF FUNGAL INFECTIONS. Biological infection Pathological changes Empirical/targeted therapy...
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SEROLOGY OF FUNGAL SEROLOGY OF FUNGAL INFECTIONSINFECTIONS
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Biological infection
Pathological changes
Empirical/targeted therapy
FungitellAspergillus PCRAspergillus GM Current diagnostic methods
INFECTION
Clinicalinfection
Targeted prophylaxis/Pre-emptive therapy
Diagnosis in the setting of increasing fungal burden
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Exo-antigen
Antibody
Antigen
•ANTIBODIES(Use of commercially availableantigens)
•ANTIGENS(Use of specific antibodies)
SEROLOGICAL TARGETSSEROLOGICAL TARGETSSerology methods utilise the reactions and properties of
serum
Exo-antigen
Antigen
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• Antigens and antibodies are easier to detect than finding the organism directly
Why use serologyWhy 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
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Available testsAvailable tests
• Immunodiffusion
• Latex Agglutination
AntibodiesAntibodies
AntigensAntigens
• Radioimmunoassay (RIA)
• Complement fixation
• Enzyme-linked immunosorbent assay (ELISA)
AntibodiesAntibodies andand antigensantigens
• Radioallergosorbent Test (RAST)
• Enzyme Immunoassay (EIA)
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• 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 AccuracyMeasures of Accuracyin Serology Assaysin Serology Assays
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• Aspergillosis
• Candidiasis
• Cryptococcosis
Opportunisticpathogens
Truepathogens
Antigen &antibody (?)
detection
Antigen &antibody (?)
detection
• Histoplasmosis
• Blastomycosis
• Coccidioidomycosis
• Paracoccidioidomycosis
Mycotic diseasesMycotic diseases
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AspergillosisAspergillosisPrimary Primary aaetiological agents:etiological agents:
A. flavus,
A. fumigatus,
A. niger, A. terreus.
Aspergillus spp. have a global distribution: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
• 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.
AspergillosisAspergillosis
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DIAGNOSTIC TOOLS 1976DIAGNOSTIC TOOLS 1976
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DIAGNOSTIC TOOLS 1979DIAGNOSTIC TOOLS 1979
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DIAGNOSTIC TOOLS 2011DIAGNOSTIC TOOLS 2011
Sandwich-ELISA•galactomannan•mannanHigh-resolution CT-scanUltrasoundBronchoalveolar lavagesBiopsy techniques(BLOOD)CULTURESFungitell (-1-3-D-glucan)PCR
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AspergillosisAspergillosis
Galactomannan (GM)Galactomannan (GM)
- polysaccharide component of the cell wallpolysaccharide component of the cell wall
However...However...- GM presence in patient’s blood is determined by multiple factorsGM presence in patient’s blood is determined by multiple factors
Kedzierska et al, Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755Eur J Clin Microbiol Infect Dis (2007) 26:755
- highly immunogenic antigen- highly immunogenic antigen- present in most Aspergilli- present in most Aspergilli- exo-antigen that can be detected in serum, BAL or CSF - exo-antigen that can be detected in serum, BAL or CSF
- sensitivity of GM detection depends on the site of infection- sensitivity of GM detection depends on the site of infection- certain antibiotics (e.g. ampicillin, amoxicillin, amoxicillin-clavulanate)- certain antibiotics (e.g. ampicillin, amoxicillin, amoxicillin-clavulanate)may give false-positive resultsmay give false-positive results
- Aspergillosis should also be confirmed by other diagnostic tools (CT)Aspergillosis should also be confirmed by other diagnostic tools (CT)
- monitoring of GM during antifungal therapy allows progression of monitoring of GM during antifungal therapy allows progression of treatment to be measuredtreatment to be measured- several commercially available ELISA tests (Platelia, Pastorex)several commercially available ELISA tests (Platelia, Pastorex)
Diagnosis of invasive diseaseDiagnosis of invasive disease
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Antigen Tests:Antigen Tests:
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
GalactomannanGalactomannan
Wheat L.J, Wheat L.J, Transplant Infect Dis (2006), 8:128Transplant Infect Dis (2006), 8:128
AspergillosisAspergillosis
ELISA, LA; Cut off 0.5, except neutopenic groupELISA, LA; Cut off 0.5, except neutopenic group
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(1→3)-(1→3)-ββ-D-glucan-D-glucan
- widely distributed in nature (fungi, yeast, algwidely distributed in nature (fungi, yeast, algaae, bacteria, plants)e, bacteria, plants)
However...However...
- false-positive results may occur (60% of bacterfalse-positive results may occur (60% of bacteraaemic patients)emic patients)
AspergillosisAspergillosis
Kedzierska et al, Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755Eur J Clin Microbiol Infect Dis (2007) 26:755
- not presentnot present (or low) (or low) in in CryptococcusCryptococcus species, zygomycetes and species, zygomycetes and
humanshumans
- exo-antigen- exo-antigen
- may also be used in diagnosis of candidiasis or fusariosis- may also be used in diagnosis of candidiasis or fusariosis
- commercially available kits: Fungitec-G, Fungitell- commercially available kits: Fungitec-G, Fungitell
- may be used as a complementary test to GM- may be used as a complementary test to GM
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AspergillosisAspergillosisAntigen Tests:Antigen Tests:
Patient group Sensitivity (%) Specificity (%)
Haematologic disease 88 85
Neutropenia, suspected IA, GVHD, steroids
55 95
Neutropenia, Cut off 120 pg/ml 88 90
Wheat L.J, Wheat L.J, Transplant Infect Dis (2006), 8:128Transplant Infect Dis (2006), 8:128
(1→3)-(1→3)-ββ-D-glucan-D-glucan
Cut off 60 pg/ml, except neutorpenic groupCut off 60 pg/ml, except neutorpenic group
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AspergillusAspergillus antibodies can only be detected in ABPA, Aspergilloma antibodies can only be detected in ABPA, Aspergilloma and CCPA patients.and CCPA patients.
Less reliable than antigen tests due to the presence of anti-Less reliable than antigen tests due to the presence of anti-AspergillusAspergillus antibodies in healthy individuals.antibodies in healthy individuals.
High level of precipitating antibodies does not prove the presence of High level of precipitating antibodies does not prove the presence of ongoing diseaseongoing disease
AspergillosisAspergillosisDiagnosis of allergic AspergillosisDiagnosis of allergic Aspergillosis
Antibody TestAntibody Test
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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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Primary Primary aaetiological agents:etiological agents: C. albicans,C. albicans, C. C. parapsilosis,parapsilosis, C. C. glabrata,glabrata, C. tropicalis.C. tropicalis.
Based on detection of:Based on detection of:-antigen:antigen: > > ββ-glucan (F-glucan (Fuungitec-G – enzymatic assay)ngitec-G – enzymatic assay) > Mannan (Pastorex, Platelia - ELISA) > Mannan (Pastorex, Platelia - ELISA)
- antibody (?) (IgA, IgG – ELISA, Immunodiffusion)- antibody (?) (IgA, IgG – ELISA, Immunodiffusion)
CandidiasisCandidiasis
Diagnosis:Diagnosis:
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MannanMannan
- highly immunogenic antigenhighly immunogenic antigen
However...However...
- negative results of the tests do not exnegative results of the tests do not excclude infectionlude infection
CandidiasisCandidiasis
Kedzierska et al, Kedzierska et al, Eur J Clin Microbiol Infect Dis (2007) 26:755Eur J Clin Microbiol Infect Dis (2007) 26:755
- immunologically more active then - immunologically more active then ββ-glucan -glucan
- polysaccharide component of the cell wall of - polysaccharide component of the cell wall of CandidaCandida spp. spp.
- positive results may be obtained 2-15 days before positive bloodpositive results may be obtained 2-15 days before positive blood
culturescultures
Antigen TestAntigen Test
- commercially available tests: Pastorex and Platelia (ELISA)commercially available tests: Pastorex and Platelia (ELISA)
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Antigen TestAntigen Test
LA mannan (Pastorex) MAb
Yeo & WongYeo & Wong, Clin Micro Rev (2002) 15:465, Clin Micro Rev (2002) 15:465
ELISA mannan (Platelia) MAb
β-glucan (enzymatic – Fungitec-G)
Sensitivity (%) Specificity (%)
25-28 100
42 93-98
Test
71-97
MAb – monoclonal antMAb – monoclonal antiibodybody
ELISA mannan (Platelia) PAb 21-84
54-96
98-100
PAb – polyclonal antPAb – polyclonal antiibodybody
CandidiasisCandidiasis
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Antibody TestAntibody Test
Anti-Anti-CandidaCandida antibodies may also be present in healthy individuals antibodies may also be present in healthy individuals and cause false possitive resultsand cause false possitive results
IMMYIMMY laboratory manuals laboratory manuals
Sensitivity ~80% in Sensitivity ~80% in immunocompetentimmunocompetent individualsindividuals
CandidiasisCandidiasis
Sensitivity may not be relevant in Sensitivity may not be relevant in immunocompromisedimmunocompromised individualsindividuals
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CryptococcosisCryptococcosisPrimary Primary aaetiological agent:etiological agent: C. neoformansC. neoformans
Only based on detection of Only based on detection of capsular polysaccharidecapsular polysaccharide((glucuronoxylomannanglucuronoxylomannan) antigen) antigen
No antNo antiibody tests performedbody tests performed
Diagnosis:Diagnosis:
Latex agglutination (PREMIER Cryptococcal antigen assay)Latex agglutination (PREMIER Cryptococcal antigen assay)
Several tests:Several tests:
Enzyme Immunoassay (Pastorex Crypto Plus, IMMY Latex-Enzyme Immunoassay (Pastorex Crypto Plus, IMMY Latex-Cryptococcus antigen assay)Cryptococcus antigen assay)
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Antigen TestAntigen Test
Latex AgglutinationLatex Agglutination
SantangeloSantangelo, Med Mycol (2005) 43:335, Med Mycol (2005) 43:335
ELISA ELISA MAbMAb
9090 9595
9090 70-8070-80
TestTest
Yeo & WongYeo & Wong, Clin Micro Rev (2002) 15:465, Clin Micro Rev (2002) 15:465
- detection in serum, BAL or CSF- detection in serum, BAL or CSF
- false-positive results may be caused by rheumatoid factor or cross-- false-positive results may be caused by rheumatoid factor or cross-reactive organisms (reactive organisms (Trichosporon asahiiTrichosporon asahii) )
CryptococcosisCryptococcosis
Sensitivity (%) Specificity (%)
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The future?
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ConclusionConclusion• 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