Update on glucan detectionUpdate on glucan detection

Malcolm Richardson PhD, FIBiol, FRCPathMalcolm Richardson PhD, FIBiol, FRCPath

Department of Bacteriology & ImmunologyDepartment of Bacteriology & ImmunologyUniversity of HelsinkiUniversity of Helsinki

malcolm.richardson@helsinki.fimalcolm.richardson@helsinki.fi

Malcolm Richardson PhD, FIBiol, FRCPathMalcolm Richardson PhD, FIBiol, FRCPath

Department of Bacteriology & ImmunologyDepartment of Bacteriology & ImmunologyUniversity of HelsinkiUniversity of Helsinki

malcolm.richardson@helsinki.fimalcolm.richardson@helsinki.fi

Pathogenesis of aspergillosisPathogenesis of aspergillosis

Phospholipid bilayerof the fungal cell

membrane

Fungalcell wall

-(1,3)-glucan

-(1,6)-glucan

-(1,3)-glucan synthase Ergosterol

Fungal cell wallFungal cell wall

(13)-ß-D-glucans(13)-ß-D-glucans

non-allergeneicnon-allergeneic water-insolublewater-insoluble most fungi, some bacteria, most higher most fungi, some bacteria, most higher

plants, many lower plantsplants, many lower plants Up to 60% dry weight of fungal cells wallUp to 60% dry weight of fungal cells wall Glucose polymersGlucose polymers linked to proteins, lipids, mannan, chitinlinked to proteins, lipids, mannan, chitin

Douwes, Indoor Air 2005Douwes, Indoor Air 2005

non-allergeneicnon-allergeneic water-insolublewater-insoluble most fungi, some bacteria, most higher most fungi, some bacteria, most higher

plants, many lower plantsplants, many lower plants Up to 60% dry weight of fungal cells wallUp to 60% dry weight of fungal cells wall Glucose polymersGlucose polymers linked to proteins, lipids, mannan, chitinlinked to proteins, lipids, mannan, chitin

Douwes, Indoor Air 2005Douwes, Indoor Air 2005

(13)-ß-D-glucans(13)-ß-D-glucans

independent of growth conditionsindependent of growth conditionswide range of biological responses:wide range of biological responses:

stimulation of RESstimulation of RES activation of neutophils, macrophages, activation of neutophils, macrophages,

complementcomplementenhancement of host-mediated enhancement of host-mediated

resistance to infectionresistance to infection

Douwes, Indoor Air 2005.Douwes, Indoor Air 2005.

independent of growth conditionsindependent of growth conditionswide range of biological responses:wide range of biological responses:

stimulation of RESstimulation of RES activation of neutophils, macrophages, activation of neutophils, macrophages,

complementcomplementenhancement of host-mediated enhancement of host-mediated

resistance to infectionresistance to infection

Douwes, Indoor Air 2005.Douwes, Indoor Air 2005.

(13)-ß-D-glucans(13)-ß-D-glucans

AssaysAssays Glucan-specific limulus amebocyte lysate Glucan-specific limulus amebocyte lysate

(LAL) assay(LAL) assay ImmunoassaysImmunoassays

inhibition ELISAinhibition ELISAsandwich ELISAsandwich ELISA

Due to water-insolubility NaOH or heat Due to water-insolubility NaOH or heat extractionextraction

AssaysAssays Glucan-specific limulus amebocyte lysate Glucan-specific limulus amebocyte lysate

(LAL) assay(LAL) assay ImmunoassaysImmunoassays

inhibition ELISAinhibition ELISAsandwich ELISAsandwich ELISA

Due to water-insolubility NaOH or heat Due to water-insolubility NaOH or heat extractionextraction

The ‘G’ test1->3-ß-D-glucan1->3-ß-D-glucan

Boc-Leu-Gly-Arg--Boc-Leu-Gly-Arg--p-p-nitroanilidenitroanilide

Boc-Leu-Gly-Arg-OHBoc-Leu-Gly-Arg-OHpp--nitroaniline--nitroaniline

Diazo couplingDiazo coupling

Factor CFactor C Factor CFactor C{activated}{activated}

ProclottingProclottingenzymeenzyme

ClottingClottingenzymeenzyme

Obayashi et al. Lancet 1995; 345:17

Panfungal DetectionPanfungal Detection

Manuel Cuenca-Estrella

Development historyDevelopment history

Wako Pure Chemical Industries (Tokyo)

Seikagaku Kogyo Corporation (Tokyo)

Associates of Cape Cod (Falmouth, USA)

Fungitec G testFungitec G test

detection of detection of 1-3-D-glucan 1-3-D-glucan limulus factor G reacts with limulus factor G reacts with 1-3-D-glucan 1-3-D-glucan 5 pg/ml glucan lower limit of detectability5 pg/ml glucan lower limit of detectability 11 candidaemia: 150 pg/ml11 candidaemia: 150 pg/ml 4 suspected candidaemia: 1006.8 pg/ml4 suspected candidaemia: 1006.8 pg/ml 1 invasive aspergillosis: 312 pg/ml1 invasive aspergillosis: 312 pg/ml 12 aspergilloma: 55.6 pg/ml12 aspergilloma: 55.6 pg/ml range: 20.1-138.0 pg/mlrange: 20.1-138.0 pg/ml

detection of detection of 1-3-D-glucan 1-3-D-glucan limulus factor G reacts with limulus factor G reacts with 1-3-D-glucan 1-3-D-glucan 5 pg/ml glucan lower limit of detectability5 pg/ml glucan lower limit of detectability 11 candidaemia: 150 pg/ml11 candidaemia: 150 pg/ml 4 suspected candidaemia: 1006.8 pg/ml4 suspected candidaemia: 1006.8 pg/ml 1 invasive aspergillosis: 312 pg/ml1 invasive aspergillosis: 312 pg/ml 12 aspergilloma: 55.6 pg/ml12 aspergilloma: 55.6 pg/ml range: 20.1-138.0 pg/mlrange: 20.1-138.0 pg/ml

Detection of D-glucan in a rat model of aspergillosisDetection of D-glucan in a rat model of aspergillosis

ImmunosImmunosuupressed ratspressed ratsTranstracheal challengeTranstracheal challengeDay 6:Day 6:

370370178 pg/ml untreated controls178 pg/ml untreated controls154 154 43 pg/ml 0.5 mg/kg amphotericin B43 pg/ml 0.5 mg/kg amphotericin B

Day 11:Day 11:2, 5902, 590± 2, 940 pg/ml untreated± 2, 940 pg/ml untreated448 ± 442 pg/ml treated448 ± 442 pg/ml treated

ImmunosImmunosuupressed ratspressed ratsTranstracheal challengeTranstracheal challengeDay 6:Day 6:

370370178 pg/ml untreated controls178 pg/ml untreated controls154 154 43 pg/ml 0.5 mg/kg amphotericin B43 pg/ml 0.5 mg/kg amphotericin B

Day 11:Day 11:2, 5902, 590± 2, 940 pg/ml untreated± 2, 940 pg/ml untreated448 ± 442 pg/ml treated448 ± 442 pg/ml treated

Glucan and IPAGlucan and IPAPatient Patient ## DiagDiag DayDay Glucan (pg/ml)Glucan (pg/ml) PastorexPastorex ELISAELISA

11 AMLAML 00 895.0895.0 1X1X NDND22 516.0516.0 8X8X 19.019.044 939.0939.0 32X32X 17.717.7

22 HLHL 00 27.027.0 1X1X 7.47.499 246.0246.0 2X2X 15.015.0

33 AAAA 00 49.449.4 NDND 2.52.51515 113.4113.4 2X2X 11.211.22121 521.0521.0 2X2X 12.212.2

2727 150.0150.0 1X1X NDND

Patient Patient ## DiagDiag DayDay Glucan (pg/ml)Glucan (pg/ml) PastorexPastorex ELISAELISA

11 AMLAML 00 895.0895.0 1X1X NDND22 516.0516.0 8X8X 19.019.044 939.0939.0 32X32X 17.717.7

22 HLHL 00 27.027.0 1X1X 7.47.499 246.0246.0 2X2X 15.015.0

33 AAAA 00 49.449.4 NDND 2.52.51515 113.4113.4 2X2X 11.211.22121 521.0521.0 2X2X 12.212.2

2727 150.0150.0 1X1X NDND

Fungitell: technical considerationsFungitell: technical considerations

Analytical senstivity: 1 pg/mlAnalytical senstivity: 1 pg/ml inhibitors of serine proteases in serum inhibitors of serine proteases in serum

have to be removed: alkali pre-have to be removed: alkali pre-treatmenttreatment

endotoxin removal: factor C used to endotoxin removal: factor C used to deplete limulus lysate deplete limulus lysate

Analytical senstivity: 1 pg/mlAnalytical senstivity: 1 pg/ml inhibitors of serine proteases in serum inhibitors of serine proteases in serum

have to be removed: alkali pre-have to be removed: alkali pre-treatmenttreatment

endotoxin removal: factor C used to endotoxin removal: factor C used to deplete limulus lysate deplete limulus lysate

Glucatell - Fungitell

Glucatell® (1.3)-Beta-DGEstablishing Cut-off

Glucatell® (1.3)-Beta-DGEstablishing Cut-off

Odabasi et al. CID’04

HealthyHealthy CandidemiaCandidemia

Mean Mean ++ SD SD RangeRange Mean Mean ++ SD SD RangeRange

17 17 ++ 34 pg/ml 34 pg/ml 0-86 0-86 pg/mlpg/ml

2999 2999 ++ 51905190

36-22,26336-22,263

30 healthy adults30 healthy adults30 patients nonneutropenic with 30 patients nonneutropenic with symptomatic Candidemia symptomatic Candidemia

30 healthy adults30 healthy adults30 patients nonneutropenic with 30 patients nonneutropenic with symptomatic Candidemia symptomatic Candidemia

Manuel Cuenca-Estrella

30 healthy adults30 healthy adults 30 patients nonneutropenic with 30 patients nonneutropenic with symptomatic Candidemia symptomatic Candidemia

30 healthy adults30 healthy adults 30 patients nonneutropenic with 30 patients nonneutropenic with symptomatic Candidemia symptomatic Candidemia

Glucatell® (1.3)-Beta-DGEstablishing Cut-off

Glucatell® (1.3)-Beta-DGEstablishing Cut-off

Odabasi et al. CID’04

HealthyHealthy CandidemiaCandidemia

Mean Mean ++ SD SD RangeRange Mean Mean ++ SD SD RangeRange

17 17 ++ 34 pg/ml 34 pg/ml 0-86 0-86 pg/mlpg/ml

2,999 2,999 ++ 5,1905,190

36-22,26336-22,263

Only 2 serum

samples > 60 pg/ml

Only 1 serum

sample < 60 pg/ml

> 60 pg/ml

Manuel Cuenca-Estrella

ß-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic sundrome

ß-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic sundrome

Cutoff: Cutoff: 60 pg/ml60 pg/ml 283 patients on prophylaxis283 patients on prophylaxis 1 or more serum samples positive 10 days before 1 or more serum samples positive 10 days before

clinical diagnosis: 100% of definite IFI casesclinical diagnosis: 100% of definite IFI cases Absence of glucan 100% negative predictive valueAbsence of glucan 100% negative predictive value Specificity: Specificity:

90% single test90% single test 96% for 96% for 2 sequential positive results2 sequential positive results

CONCLUSION: “highly sensitive and specific”CONCLUSION: “highly sensitive and specific”

Odabasi et al. CID 2004; 39: 199-205.Odabasi et al. CID 2004; 39: 199-205.

Cutoff: Cutoff: 60 pg/ml60 pg/ml 283 patients on prophylaxis283 patients on prophylaxis 1 or more serum samples positive 10 days before 1 or more serum samples positive 10 days before

clinical diagnosis: 100% of definite IFI casesclinical diagnosis: 100% of definite IFI cases Absence of glucan 100% negative predictive valueAbsence of glucan 100% negative predictive value Specificity: Specificity:

90% single test90% single test 96% for 96% for 2 sequential positive results2 sequential positive results

CONCLUSION: “highly sensitive and specific”CONCLUSION: “highly sensitive and specific”

Odabasi et al. CID 2004; 39: 199-205.Odabasi et al. CID 2004; 39: 199-205.

Probable fungal pneumoniaProbable fungal pneumoniaOrganismOrganism Max BG levelMax BG level No +BGNo +BG

teststests

AFAF 21052105 44 +18+18AFAF 8181 11 -4-4AT and CAAT and CA 108108 11 +23+23FusariumFusarium sp. sp. 207207 11 +4+4

=no. days pt was BG pos before clin diag.=no. days pt was BG pos before clin diag.

Odabasi et al 2005 CIDOdabasi et al 2005 CID

OrganismOrganism Max BG levelMax BG level No +BGNo +BG teststests

AFAF 21052105 44 +18+18AFAF 8181 11 -4-4AT and CAAT and CA 108108 11 +23+23FusariumFusarium sp. sp. 207207 11 +4+4

=no. days pt was BG pos before clin diag.=no. days pt was BG pos before clin diag.

Odabasi et al 2005 CIDOdabasi et al 2005 CID

Comparison of Fungitec G test-MK and WAKO testComparison of Fungitec G test-MK and WAKO test 8 cases of IA8 cases of IA

TestTest SensitivitySensitivity SpecificitySpecificity Cut-offCut-off

FungitecFungitec 0.880.88 0.850.85 24.9 pg/ml24.9 pg/mlWAKOWAKO 0.630.63 0.980.98 7.3 pg/ml7.3 pg/ml

Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese)Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese)

Comparison of Fungitec G test-MK and WAKO testComparison of Fungitec G test-MK and WAKO test 8 cases of IA8 cases of IA

TestTest SensitivitySensitivity SpecificitySpecificity Cut-offCut-off

FungitecFungitec 0.880.88 0.850.85 24.9 pg/ml24.9 pg/mlWAKOWAKO 0.630.63 0.980.98 7.3 pg/ml7.3 pg/ml

Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese)Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese)

The performance of glucan and Aspergillus GM measurement for early diagnosis of IA in patients with hematological diseases

The performance of glucan and Aspergillus GM measurement for early diagnosis of IA in patients with hematological diseases

•5 proven IA

•3 probable IA

•3 possible IA

•glucan calibration curve: 6.25 to 100 pg/ml

•Positive score: ≥120 pg/ml in 1 or more samples

SensitivitySensitivity SpecificitySpecificity PPVPPV NPVNPV

87.5%87.5% 89.6%89.6% 70%70% 96.3%96.3%

False positivity: 10.3%False positivity: 10.3%

(Previous reported sensitivities: 50-63%)(Previous reported sensitivities: 50-63%)

SensitivitySensitivity SpecificitySpecificity PPVPPV NPVNPV

87.5%87.5% 89.6%89.6% 70%70% 96.3%96.3%

False positivity: 10.3%False positivity: 10.3%

(Previous reported sensitivities: 50-63%)(Previous reported sensitivities: 50-63%)

Kinetics of BGKinetics of BG

Temporal onset of BG in patients with proven and probable IATemporal onset of BG in patients with proven and probable IA

Time pointTime point nn No (%)No (%) Days betweenDays betweenBG +BG + BG + and timeBG + and time

point medianpoint median

First day PUOFirst day PUO 77 2 (28.2)2 (28.2) 5 (4-6)5 (4-6)First day clin signsFirst day clin signs 77 4 (57.1)4 (57.1) 10.7 (4-21)10.7 (4-21)Pulmonary HRCTPulmonary HRCT 77 7 (100)7 (100) 9.3 (1-21)9.3 (1-21)Start antifungal TxStart antifungal Tx 77 5 (71.4)5 (71.4) 14 (4-25)14 (4-25)

Pazos et al. JCM 2005.Pazos et al. JCM 2005.

Time pointTime point nn No (%)No (%) Days betweenDays betweenBG +BG + BG + and timeBG + and time

point medianpoint median

First day PUOFirst day PUO 77 2 (28.2)2 (28.2) 5 (4-6)5 (4-6)First day clin signsFirst day clin signs 77 4 (57.1)4 (57.1) 10.7 (4-21)10.7 (4-21)Pulmonary HRCTPulmonary HRCT 77 7 (100)7 (100) 9.3 (1-21)9.3 (1-21)Start antifungal TxStart antifungal Tx 77 5 (71.4)5 (71.4) 14 (4-25)14 (4-25)

Pazos et al. JCM 2005.Pazos et al. JCM 2005.

Source of false positives

Surgical gauseSurgical gause Immunoglobulin productsImmunoglobulin productsHaemodialysis with cellulose filtersHaemodialysis with cellulose filtersEnvironmental fungal glucan: dustEnvironmental fungal glucan: dust

Surgical gauseSurgical gause Immunoglobulin productsImmunoglobulin productsHaemodialysis with cellulose filtersHaemodialysis with cellulose filtersEnvironmental fungal glucan: dustEnvironmental fungal glucan: dust

False positive BG kineticsFalse positive BG kinetics

Environmental sources of glucanEnvironmental sources of glucan

airairsettled dustsettled dustfabricsfabricsorganic waste - indoor organic waste - indoor

composterscomposters

Douwes Indoor Air 2005Douwes Indoor Air 2005

airairsettled dustsettled dustfabricsfabricsorganic waste - indoor organic waste - indoor

composterscomposters

Douwes Indoor Air 2005Douwes Indoor Air 2005

Clearance of glucanClearance of glucan

(13)-ß-D-glucans: kinetics(13)-ß-D-glucans: kinetics

Receptor recognitionReceptor recognition clearance from bloodstreamclearance from bloodstream triggering of immune responsestriggering of immune responses

masking or underexposuremasking or underexposureOnly soluble glucans are releasedOnly soluble glucans are released

Brown and Gordon, Cellular Microbiology, 2005.Brown and Gordon, Cellular Microbiology, 2005.

Receptor recognitionReceptor recognition clearance from bloodstreamclearance from bloodstream triggering of immune responsestriggering of immune responses

masking or underexposuremasking or underexposureOnly soluble glucans are releasedOnly soluble glucans are released

Brown and Gordon, Cellular Microbiology, 2005.Brown and Gordon, Cellular Microbiology, 2005.

(13)-ß-D-glucans(13)-ß-D-glucans

Receptors identified on:Receptors identified on: monocytesmonocytes macrophagesmacrophages neutrophilsneutrophils Langerhans cellsLangerhans cells eosinophilseosinophils NK cellsNK cells endothelial cellsendothelial cells alveolar epithelial cellsalveolar epithelial cells

Receptors identified on:Receptors identified on: monocytesmonocytes macrophagesmacrophages neutrophilsneutrophils Langerhans cellsLangerhans cells eosinophilseosinophils NK cellsNK cells endothelial cellsendothelial cells alveolar epithelial cellsalveolar epithelial cells

(13)-ß-D-glucans(13)-ß-D-glucans

Cellular receptorsCellular receptors

CR3CR3 lactosylceramidelactosylceramidescavenger receptorsscavenger receptorsDectin-1Dectin-1

Brown and Gordon, Cellular Microbiology 2005.Brown and Gordon, Cellular Microbiology 2005.

Cellular receptorsCellular receptors

CR3CR3 lactosylceramidelactosylceramidescavenger receptorsscavenger receptorsDectin-1Dectin-1

Brown and Gordon, Cellular Microbiology 2005.Brown and Gordon, Cellular Microbiology 2005.

Dectin-1Dectin-1

type II transmembrane glycoproteintype II transmembrane glycoprotein Expressed on:Expressed on:

monocytesmonocytes macrophagesmacrophages neutrophilsneutrophils expression profoundly influenced by cytokines and expression profoundly influenced by cytokines and

microbial productsmicrobial products major role: pathogen surveillancemajor role: pathogen surveillance

glucansglucans intact fungal cellsintact fungal cells

mediates phagocytosismediates phagocytosis

type II transmembrane glycoproteintype II transmembrane glycoprotein Expressed on:Expressed on:

monocytesmonocytes macrophagesmacrophages neutrophilsneutrophils expression profoundly influenced by cytokines and expression profoundly influenced by cytokines and

microbial productsmicrobial products major role: pathogen surveillancemajor role: pathogen surveillance

glucansglucans intact fungal cellsintact fungal cells

mediates phagocytosismediates phagocytosis

Fungal glucans are very readily internalized by host effector cells

Panfungal DetectionPanfungal Detection

Manuel Cuenca-Estrella