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Update on glucan detection Malcolm Richardson PhD, FIBiol, FRCPath Department of Bacteriology & Immunology University of Helsinki

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Presentation on theme: "Update on glucan detection Malcolm Richardson PhD, FIBiol, FRCPath Department of Bacteriology & Immunology University of Helsinki"— Presentation transcript:

1 Update on glucan detection Malcolm Richardson PhD, FIBiol, FRCPath Department of Bacteriology & Immunology University of Helsinki malcolm.richardson@helsinki.fi Malcolm Richardson PhD, FIBiol, FRCPath Department of Bacteriology & Immunology University of Helsinki malcolm.richardson@helsinki.fi

2 Pathogenesis of aspergillosis

3 Phospholipid bilayer of the fungal cell membrane Fungal cell wall -(1,3)-glucan -(1,6)- glucan -(1,3)-glucan synthase Ergosterol Fungal cell wall

4 (1 3)-ß-D-glucans non-allergeneic non-allergeneic water-insoluble water-insoluble most fungi, some bacteria, most higher plants, many lower plants most fungi, some bacteria, most higher plants, many lower plants Up to 60% dry weight of fungal cells wall Up to 60% dry weight of fungal cells wall Glucose polymers Glucose polymers linked to proteins, lipids, mannan, chitin linked to proteins, lipids, mannan, chitin Douwes, Indoor Air 2005 non-allergeneic non-allergeneic water-insoluble water-insoluble most fungi, some bacteria, most higher plants, many lower plants most fungi, some bacteria, most higher plants, many lower plants Up to 60% dry weight of fungal cells wall Up to 60% dry weight of fungal cells wall Glucose polymers Glucose polymers linked to proteins, lipids, mannan, chitin linked to proteins, lipids, mannan, chitin Douwes, Indoor Air 2005

5 (1 3)-ß-D-glucans independent of growth conditions independent of growth conditions wide range of biological responses: wide range of biological responses: stimulation of RES stimulation of RES activation of neutophils, macrophages, complement activation of neutophils, macrophages, complement enhancement of host-mediated resistance to infection enhancement of host-mediated resistance to infection Douwes, Indoor Air 2005. independent of growth conditions independent of growth conditions wide range of biological responses: wide range of biological responses: stimulation of RES stimulation of RES activation of neutophils, macrophages, complement activation of neutophils, macrophages, complement enhancement of host-mediated resistance to infection enhancement of host-mediated resistance to infection Douwes, Indoor Air 2005.

6 (1 3)-ß-D-glucans Assays Assays Glucan-specific limulus amebocyte lysate (LAL) assay Glucan-specific limulus amebocyte lysate (LAL) assay Immunoassays Immunoassays inhibition ELISA inhibition ELISA sandwich ELISA sandwich ELISA Due to water-insolubility NaOH or heat extraction Due to water-insolubility NaOH or heat extraction Assays Assays Glucan-specific limulus amebocyte lysate (LAL) assay Glucan-specific limulus amebocyte lysate (LAL) assay Immunoassays Immunoassays inhibition ELISA inhibition ELISA sandwich ELISA sandwich ELISA Due to water-insolubility NaOH or heat extraction Due to water-insolubility NaOH or heat extraction

7 The G test 1->3-ß-D-glucan Boc-Leu-Gly-Arg--p-nitroanilide Boc-Leu-Gly-Arg-OH p--nitroaniline Diazo coupling Factor C {activated} ProclottingenzymeClottingenzyme Obayashi et al. Lancet 1995; 345:17

8 Panfungal Detection Manuel Cuenca-Estrella

9 Development history Wako Pure Chemical Industries (Tokyo) Seikagaku Kogyo Corporation (Tokyo) Associates of Cape Cod (Falmouth, USA)

10 Fungitec G test detection of 1-3-D-glucan detection of 1-3-D-glucan limulus factor G reacts with 1-3-D-glucan limulus factor G reacts with 1-3-D-glucan 5 pg/ml glucan lower limit of detectability 5 pg/ml glucan lower limit of detectability 11 candidaemia: 150 pg/ml 11 candidaemia: 150 pg/ml 4 suspected candidaemia: 1006.8 pg/ml 4 suspected candidaemia: 1006.8 pg/ml 1 invasive aspergillosis: 312 pg/ml 1 invasive aspergillosis: 312 pg/ml 12 aspergilloma: 55.6 pg/ml 12 aspergilloma: 55.6 pg/ml range: 20.1-138.0 pg/ml range: 20.1-138.0 pg/ml detection of 1-3-D-glucan detection of 1-3-D-glucan limulus factor G reacts with 1-3-D-glucan limulus factor G reacts with 1-3-D-glucan 5 pg/ml glucan lower limit of detectability 5 pg/ml glucan lower limit of detectability 11 candidaemia: 150 pg/ml 11 candidaemia: 150 pg/ml 4 suspected candidaemia: 1006.8 pg/ml 4 suspected candidaemia: 1006.8 pg/ml 1 invasive aspergillosis: 312 pg/ml 1 invasive aspergillosis: 312 pg/ml 12 aspergilloma: 55.6 pg/ml 12 aspergilloma: 55.6 pg/ml range: 20.1-138.0 pg/ml range: 20.1-138.0 pg/ml

11 Detection of D-glucan in a rat model of aspergillosis Immunosupressed rats Immunosupressed rats Transtracheal challenge Transtracheal challenge Day 6: Day 6: 370 178 pg/ml untreated controls 370 178 pg/ml untreated controls 154 43 pg/ml 0.5 mg/kg amphotericin B 154 43 pg/ml 0.5 mg/kg amphotericin B Day 11: Day 11: 2, 590 ± 2, 940 pg/ml untreated 2, 590 ± 2, 940 pg/ml untreated 448 ± 442 pg/ml treated 448 ± 442 pg/ml treated Immunosupressed rats Immunosupressed rats Transtracheal challenge Transtracheal challenge Day 6: Day 6: 370 178 pg/ml untreated controls 370 178 pg/ml untreated controls 154 43 pg/ml 0.5 mg/kg amphotericin B 154 43 pg/ml 0.5 mg/kg amphotericin B Day 11: Day 11: 2, 590 ± 2, 940 pg/ml untreated 2, 590 ± 2, 940 pg/ml untreated 448 ± 442 pg/ml treated 448 ± 442 pg/ml treated

12 Glucan and IPA Patient #DiagDayGlucan (pg/ml)Pastorex ELISA 1AML0895.01XND 2516.08X19.0 4939.032X17.7 2HL027.01X7.4 9246.02X15.0 3AA049.4ND2.5 15113.42X11.2 21521.02X12.2 27150.01XND Patient #DiagDayGlucan (pg/ml)Pastorex ELISA 1AML0895.01XND 2516.08X19.0 4939.032X17.7 2HL027.01X7.4 9246.02X15.0 3AA049.4ND2.5 15113.42X11.2 21521.02X12.2 27150.01XND

13 Fungitell: technical considerations Analytical senstivity: 1 pg/ml Analytical senstivity: 1 pg/ml inhibitors of serine proteases in serum have to be removed: alkali pre- treatment inhibitors of serine proteases in serum have to be removed: alkali pre- treatment endotoxin removal: factor C used to deplete limulus lysate endotoxin removal: factor C used to deplete limulus lysate Analytical senstivity: 1 pg/ml Analytical senstivity: 1 pg/ml inhibitors of serine proteases in serum have to be removed: alkali pre- treatment inhibitors of serine proteases in serum have to be removed: alkali pre- treatment endotoxin removal: factor C used to deplete limulus lysate endotoxin removal: factor C used to deplete limulus lysate

14 Glucatell - Fungitell

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16 Glucatell® (1.3)-Beta-DG Establishing Cut-off Odabasi et al. CID04 HealthyCandidemia Mean + SD Range Range 17 + 34 pg/ml 0-86 pg/ml 2999 + 5190 36-22,263 30 healthy adults 30 healthy adults 30 patients nonneutropenic with symptomatic Candidemia 30 patients nonneutropenic with symptomatic Candidemia 30 healthy adults 30 healthy adults 30 patients nonneutropenic with symptomatic Candidemia 30 patients nonneutropenic with symptomatic Candidemia Manuel Cuenca-Estrella

17 30 healthy adults 30 healthy adults 30 patients nonneutropenic with symptomatic Candidemia 30 patients nonneutropenic with symptomatic Candidemia 30 healthy adults 30 healthy adults 30 patients nonneutropenic with symptomatic Candidemia 30 patients nonneutropenic with symptomatic Candidemia Glucatell® (1.3)-Beta-DG Establishing Cut-off Odabasi et al. CID04 HealthyCandidemia Mean + SD Range Range 17 + 34 pg/ml 0-86 pg/ml 2,999 + 5,190 36-22,263 Only 2 serum samples > 60 pg/ml Only 1 serum sample < 60 pg/ml > 60 pg/ml Manuel Cuenca-Estrella

18 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: 60 pg/ml Cutoff: 60 pg/ml 283 patients on prophylaxis 283 patients on prophylaxis 1 or more serum samples positive 10 days before clinical diagnosis: 100% of definite IFI cases 1 or more serum samples positive 10 days before clinical diagnosis: 100% of definite IFI cases Absence of glucan 100% negative predictive value Absence of glucan 100% negative predictive value Specificity: Specificity: 90% single test 90% single test 96% for 2 sequential positive results 96% for 2 sequential positive results CONCLUSION: highly sensitive and specific Odabasi et al. CID 2004; 39: 199-205. Cutoff: 60 pg/ml Cutoff: 60 pg/ml 283 patients on prophylaxis 283 patients on prophylaxis 1 or more serum samples positive 10 days before clinical diagnosis: 100% of definite IFI cases 1 or more serum samples positive 10 days before clinical diagnosis: 100% of definite IFI cases Absence of glucan 100% negative predictive value Absence of glucan 100% negative predictive value Specificity: Specificity: 90% single test 90% single test 96% for 2 sequential positive results 96% for 2 sequential positive results CONCLUSION: highly sensitive and specific Odabasi et al. CID 2004; 39: 199-205.

19 Probable fungal pneumonia OrganismMax BG levelNo +BG OrganismMax BG levelNo +BG tests AF21054+18 AF811-4 AT and CA1081+23 Fusarium sp.2071+4 =no. days pt was BG pos before clin diag. =no. days pt was BG pos before clin diag. Odabasi et al 2005 CID OrganismMax BG levelNo +BG OrganismMax BG levelNo +BG tests AF21054+18 AF811-4 AT and CA1081+23 Fusarium sp.2071+4 =no. days pt was BG pos before clin diag. =no. days pt was BG pos before clin diag. Odabasi et al 2005 CID

20 Comparison of Fungitec G test-MK and WAKO test Comparison of Fungitec G test-MK and WAKO test 8 cases of IA 8 cases of IA TestSensitivitySpecificityCut-off Fungitec0.880.8524.9 pg/ml WAKO0.630.987.3 pg/ml Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese) Comparison of Fungitec G test-MK and WAKO test Comparison of Fungitec G test-MK and WAKO test 8 cases of IA 8 cases of IA TestSensitivitySpecificityCut-off Fungitec0.880.8524.9 pg/ml WAKO0.630.987.3 pg/ml Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese) The performance of glucan and Aspergillus GM measurement for early diagnosis of IA in patients with hematological diseases

21 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

22 SensitivitySpecificityPPVNPV 87.5%89.6%70%96.3% False positivity: 10.3% (Previous reported sensitivities: 50-63%) SensitivitySpecificityPPVNPV 87.5%89.6%70%96.3% False positivity: 10.3% (Previous reported sensitivities: 50-63%)

23 Kinetics of BG

24 Temporal onset of BG in patients with proven and probable IA Time pointnNo (%)Days between BG +BG + and time point median First day PUO72 (28.2)5 (4-6) First day clin signs74 (57.1)10.7 (4-21) Pulmonary HRCT77 (100)9.3 (1-21) Start antifungal Tx75 (71.4)14 (4-25) Pazos et al. JCM 2005. Time pointnNo (%)Days between BG +BG + and time point median First day PUO72 (28.2)5 (4-6) First day clin signs74 (57.1)10.7 (4-21) Pulmonary HRCT77 (100)9.3 (1-21) Start antifungal Tx75 (71.4)14 (4-25) Pazos et al. JCM 2005.

25 Source of false positives Surgical gause Surgical gause Immunoglobulin products Immunoglobulin products Haemodialysis with cellulose filters Haemodialysis with cellulose filters Environmental fungal glucan: dust Environmental fungal glucan: dust Surgical gause Surgical gause Immunoglobulin products Immunoglobulin products Haemodialysis with cellulose filters Haemodialysis with cellulose filters Environmental fungal glucan: dust Environmental fungal glucan: dust

26 False positive BG kinetics

27 Environmental sources of glucan air air settled dust settled dust fabrics fabrics organic waste - indoor composters organic waste - indoor composters Douwes Indoor Air 2005 Douwes Indoor Air 2005 air air settled dust settled dust fabrics fabrics organic waste - indoor composters organic waste - indoor composters Douwes Indoor Air 2005 Douwes Indoor Air 2005

28 Clearance of glucan

29 (1 3)-ß-D-glucans: kinetics Receptor recognition Receptor recognition clearance from bloodstream clearance from bloodstream triggering of immune responses triggering of immune responses masking or underexposure masking or underexposure Only soluble glucans are released Only soluble glucans are released Brown and Gordon, Cellular Microbiology, 2005. Receptor recognition Receptor recognition clearance from bloodstream clearance from bloodstream triggering of immune responses triggering of immune responses masking or underexposure masking or underexposure Only soluble glucans are released Only soluble glucans are released Brown and Gordon, Cellular Microbiology, 2005.

30 (1 3)-ß-D-glucans Receptors identified on: monocytes monocytes macrophages macrophages neutrophils neutrophils Langerhans cells Langerhans cells eosinophils eosinophils NK cells NK cells endothelial cells endothelial cells alveolar epithelial cells alveolar epithelial cells Receptors identified on: monocytes monocytes macrophages macrophages neutrophils neutrophils Langerhans cells Langerhans cells eosinophils eosinophils NK cells NK cells endothelial cells endothelial cells alveolar epithelial cells alveolar epithelial cells

31 (1 3)-ß-D-glucans Cellular receptors CR3 CR3 lactosylceramide lactosylceramide scavenger receptors scavenger receptors Dectin-1 Dectin-1 Brown and Gordon, Cellular Microbiology 2005. Cellular receptors CR3 CR3 lactosylceramide lactosylceramide scavenger receptors scavenger receptors Dectin-1 Dectin-1 Brown and Gordon, Cellular Microbiology 2005.

32 Dectin-1 type II transmembrane glycoprotein type II transmembrane glycoprotein Expressed on: Expressed on: monocytes monocytes macrophages macrophages neutrophils neutrophils expression profoundly influenced by cytokines and microbial products expression profoundly influenced by cytokines and microbial products major role: pathogen surveillance major role: pathogen surveillance glucans glucans intact fungal cells intact fungal cells mediates phagocytosis mediates phagocytosis type II transmembrane glycoprotein type II transmembrane glycoprotein Expressed on: Expressed on: monocytes monocytes macrophages macrophages neutrophils neutrophils expression profoundly influenced by cytokines and microbial products expression profoundly influenced by cytokines and microbial products major role: pathogen surveillance major role: pathogen surveillance glucans glucans intact fungal cells intact fungal cells mediates phagocytosis mediates phagocytosis

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34 Fungal glucans are very readily internalized by host effector cells

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36 Panfungal Detection Manuel Cuenca-Estrella


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