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Update on glucan detection

Update on glucan detection. Malcolm Richardson PhD, FIBiol, FRCPath Department of Bacteriology & Immunology University of Helsinki malcolm.richardson@helsinki.fi. Pathogenesis of aspergillosis. Fungal cell wall. b -(1,6)-glucan. Fungal cell wall. Phospholipid bilayer of the fungal cell

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Update on glucan detection

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

  2. Pathogenesis of aspergillosis

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

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

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

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

  7. Factor C Factor C {activated} Proclotting enzyme Clotting enzyme Boc-Leu-Gly-Arg-OH p--nitroaniline The ‘G’ test 1->3-ß-D-glucan Boc-Leu-Gly-Arg--p-nitroanilide Diazo coupling 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 • limulus factor G reacts with  1-3-D-glucan • 5 pg/ml glucan lower limit of detectability • 11 candidaemia: 150 pg/ml • 4 suspected candidaemia: 1006.8 pg/ml • 1 invasive aspergillosis: 312 pg/ml • 12 aspergilloma: 55.6 pg/ml • range: 20.1-138.0 pg/ml

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

  12. Glucan and IPA Patient # Diag Day Glucan (pg/ml) Pastorex ELISA 1 AML 0 895.0 1X ND 2 516.0 8X 19.0 4 939.0 32X 17.7 2 HL 0 27.0 1X 7.4 9 246.0 2X 15.0 3 AA 0 49.4 ND 2.5 15 113.4 2X 11.2 21 521.0 2X 12.2 27 150.0 1X ND

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

  14. Glucatell - Fungitell

  15. Glucatell® (1.3)-Beta-DGEstablishing Cut-off • 30 healthy adults • 30 patients nonneutropenic with symptomatic Candidemia Odabasi et al. CID’04  Manuel Cuenca-Estrella

  16. Only 2 serum samples > 60 pg/ml Only 1 serum sample < 60 pg/ml > 60 pg/ml Glucatell® (1.3)-Beta-DGEstablishing Cut-off • 30 healthy adults • 30 patients nonneutropenic with symptomatic Candidemia  Manuel Cuenca-Estrella Odabasi et al. CID’04

  17. 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 • 283 patients on prophylaxis • 1 or more serum samples positive 10 days before clinical diagnosis: 100% of definite IFI cases • Absence of glucan 100% negative predictive value • Specificity: • 90% single test • 96% for 2 sequential positive results CONCLUSION: “highly sensitive and specific” Odabasi et al. CID 2004; 39: 199-205.

  18. Probable fungal pneumonia Organism Max BG level No +BG  tests AF 2105 4 +18 AF 81 1 -4 AT and CA 108 1 +23 Fusarium sp. 207 1 +4 =no. days pt was BG pos before clin diag. Odabasi et al 2005 CID

  19. The performance of glucan and Aspergillus GM measurement for early diagnosis of IA in patients with hematological diseases • Comparison of Fungitec G test-MK and WAKO test • 8 cases of IA Test Sensitivity Specificity Cut-off Fungitec 0.88 0.85 24.9 pg/ml WAKO 0.63 0.98 7.3 pg/ml Horiguchi. Kansenshogaku Zasshi 2004. (in Japanese)

  20. 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

  21. Sensitivity Specificity PPV NPV 87.5% 89.6% 70% 96.3% False positivity: 10.3% (Previous reported sensitivities: 50-63%)

  22. Kinetics of BG

  23. Temporal onset of BG in patients with proven and probable IA Time point n No (%) Days between BG + BG + and time point median First day PUO 7 2 (28.2) 5 (4-6) First day clin signs 7 4 (57.1) 10.7 (4-21) Pulmonary HRCT 7 7 (100) 9.3 (1-21) Start antifungal Tx 7 5 (71.4) 14 (4-25) Pazos et al. JCM 2005.

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

  25. False positive BG kinetics

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

  27. Clearance of glucan

  28. (13)-ß-D-glucans: kinetics • Receptor recognition • clearance from bloodstream • triggering of immune responses • masking or underexposure • Only soluble glucans are released Brown and Gordon, Cellular Microbiology, 2005.

  29. (13)-ß-D-glucans Receptors identified on: • monocytes • macrophages • neutrophils • Langerhans cells • eosinophils • NK cells • endothelial cells • alveolar epithelial cells

  30. (13)-ß-D-glucans Cellular receptors • CR3 • lactosylceramide • scavenger receptors • Dectin-1 Brown and Gordon, Cellular Microbiology 2005.

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

  32. Fungal glucans are very readily internalized by host effector cells

  33. Panfungal Detection  Manuel Cuenca-Estrella

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