SEROLOGY OF FUNGAL INFECTIONS. Biological infection Pathological changes Empirical/targeted therapy...

SEROLOGY OF FUNGAL SEROLOGY OF FUNGAL INFECTIONSINFECTIONS

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

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

• 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

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)

• 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

• Aspergillosis

• Candidiasis

• Cryptococcosis

Opportunisticpathogens

Truepathogens

Antigen &antibody (?)

detection

Antigen &antibody (?)

detection

• Histoplasmosis

• Blastomycosis

• Coccidioidomycosis

• Paracoccidioidomycosis

Mycotic diseasesMycotic diseases

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.

Treatment strategies based on the pathophysiology of aspergillosis

McCormick et al. 2010

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

DIAGNOSTIC TOOLS 1976DIAGNOSTIC TOOLS 1976


Sandwich-ELISA•galactomannan•mannanHigh-resolution CT-scanUltrasoundBronchoalveolar lavagesBiopsy techniques(BLOOD)CULTURESFungitell (-1-3-D-glucan)PCR


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 antigenhighly 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

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


ELISA, LA; Cut off 0.5, except neutopenic groupELISA, LA; Cut off 0.5, except neutopenic group

(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)



- 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

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

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

Aspergillus precipitin test

Strong reactions: indicative of aspergilloma

ImmunoCAP

• IgE

• IgG

• ABPA:– asthma– cystic fibrosis– COPD– cavitary disease

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:

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



- 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)


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


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


Sensitivity may not be relevant in Sensitivity may not be relevant in immunocompromisedimmunocompromised individualsindividuals

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)


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 (%)

The future?

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

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