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
[email protected]@helsinki.fi
Malcolm Richardson PhD, FIBiol, FRCPathMalcolm Richardson PhD, FIBiol, FRCPath
Department of Bacteriology & ImmunologyDepartment of Bacteriology & ImmunologyUniversity of HelsinkiUniversity of Helsinki
[email protected]@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