Malcolm Richardson PhD, FIBiol, FRCPath Department of

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Update on glucan detection

• Malcolm Richardson PhD, FIBiol, FRCPath
• Department of Bacteriology Immunology
• University of Helsinki
• malcolm.richardson_at_helsinki.fi

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Pathogenesis of aspergillosis
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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
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(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

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(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.

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(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

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The G test
1-gt3-ß-D-glucan
Boc-Leu-Gly-Arg--p-nitroanilide
Diazo coupling
Obayashi et al. Lancet 1995 34517
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Panfungal Detection
? Manuel Cuenca-Estrella
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Development history
Wako Pure Chemical Industries (Tokyo) Seikagaku
Kogyo Corporation (Tokyo) Associates of Cape Cod
(Falmouth, USA)
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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

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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

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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

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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

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

• 30 healthy adults
• 30 patients nonneutropenic with symptomatic
  Candidemia

Odabasi et al. CID04
? Manuel Cuenca-Estrella
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Glucatell (1.3)-Beta-DGEstablishing Cut-off
Only 2 serum samples gt 60 pg/ml
Only 1 serum sample lt 60 pg/ml

• 30 healthy adults
• 30 patients nonneutropenic with symptomatic
  Candidemia

? Manuel Cuenca-Estrella
Odabasi et al. CID04
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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.

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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

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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)

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• 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

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• Sensitivity Specificity PPV NPV
• 87.5 89.6 70 96.3
• False positivity 10.3
• (Previous reported sensitivities 50-63)

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Kinetics of BG
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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.

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Source of false positives

• Surgical gause
• Immunoglobulin products
• Haemodialysis with cellulose filters
• Environmental fungal glucan dust

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False positive BG kinetics
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Environmental sources of glucan

• air
• settled dust
• fabrics
• organic waste - indoor composters
• Douwes Indoor Air 2005

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Clearance of glucan
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(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.

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(1?3)-ß-D-glucans

• Receptors identified on
• monocytes
• macrophages
• neutrophils
• Langerhans cells
• eosinophils
• NK cells
• endothelial cells
• alveolar epithelial cells

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(1?3)-ß-D-glucans

• Cellular receptors
• CR3
• lactosylceramide
• scavenger receptors
• Dectin-1
• Brown and Gordon, Cellular Microbiology 2005.

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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

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Fungal glucans are very readily internalized by
host effector cells
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Panfungal Detection
? Manuel Cuenca-Estrella