Circulating fungal glycans in sera of ICU patients. Clinical relevance and development of physico-chemical methods of detection. J.POISSY 1-2, B.SENDID

Circulating fungal glycans in sera of ICU patients. Clinical relevance and

development of physico-chemical methods of detection.

J.POISSY1-2, B.SENDID2-3, S.DAMIENS2, R.FAVORY1, N.FRANCOIS3, N.GHALEM3, S.JAWHARA2, T.JOUAULT1,

D.MATHIEU1, Y.GUERARDEL4, D.POULAIN2-3

1: Pôle de Réanimation, CHRU Lille; 2: UMR Inserm U995 Eq 2, Lille 2; 3: Laboratoire de Parasitologie-Mycologie CHRU Lille;

4: UGSF, UMR 8576 CNRS, Lille 1.

U995 Journées André Verbert 11 Sept 2012

Morrell M et col. AAC. 2005 Adapted from Kumar A et col. ICAAC 2007. K 2174

All patients Patients with septic shock


Candidemia in ICU patients: delay in initiation of antifungal treatment has a high impact on mortality.

An early diagnosis is needed

Prevalence : 7/1000. Mortality : 42.6%

Schematic representation of yeast cell wall (Bennett JE. NEJM. 2006)

Detection of cell wall molecules circulatingin patients’ sera as biomarkers of

invasion

U995

Immunological detection of Mannans Platelia® ELISA tests.

Based on immunocapture with a monoclonal antibody.

Introduced in 2003 (Sendid et al).

Biochemical detection of glucans Fungitell®test.

Based on derivation of Limulus test enzymic cascade rendered specific for glucans.

Recently introduced in France.

Journées André Verbert 11 Sept 2012

Aims of the study

• Evaluation of the clinical relevance of β-D-1,3-glucans detection in ICU and comparison with mannan detection (Diagnostic, theranostic)

• Development of methods for detection of circulating fungal glycans by Mass Spectrometry


Patients and methods

• Selection of Patients from Clinical Mycology Laboratory registry– Patients with candidemia from ICU department, CHRU de

Lille, from 2005 to 2010 (117 patients)– Sera available at least one week before and one week after

blood culture : 43 selected patients, 363 sera.

• Controls: – Patients hospitalized in the same ward with no evidence of

Candida infection (According to EORTC) : 135 patients and 324 sera


Fungitell®: Sensitive but non specific test (cut-off=80 pg/mL)


Receiver Operating Characteristics curve (ROC)


Area under the curve: 0.750

Sp=90% for BDG>1150 pg/mL

Se/Sp=77/68% for a cut-off at 205

Median rate patients vs controls:

425 vs 119

Higher than cut-off recommanded by manufacturer

Early presence but slow decrease of glucanemia (little value for treatment follow-

up)

N=8N=34

N=32

N=18

N=16

N=13

N=9

N=6

p=0.0019


Median delay of positivity

10 days before blood culture

(Cut-off 80 pg/mL)

Rapid evolution of clinical biology.Mass spectrometry: a new standard

equipment in Medical Mycology Laboratories.

Adaptation for detection of fungalbiomarkers?


Blood culture Incubation

Mass spectrometry

Spectral profile

Culture Susceptibility to ATFsIsolation/identification

Previous basic studies as a reference for seeking a Candida glucan signal by using

MALDI TOF Jawhara S et al; Plos One, 2012


Development of an original extraction procedure* in order to reduce the signal/noise ratio

EDTA+120°CCentrifugation

Supernatent with less proteins, Hb, lipids

C18 column preconditionned

by H2O+acétonitrile

After washing : Oligosaccharides, glucose

and salts

Solid phase extractionActivated coal

After washing and elution :oligosaccharides

* Patent CHRU/PRES Nord de France/CNRS


Spiking of purified F2 in serum generates a 365 signal even for very low quantities


Nb: no correlation between MALDI-TOF and Fungitel test (different molecules detected)

The 365 signal is present in sera from patientswith Candidemia. With reference to peak 361

(Matrix) H 2 Trehalose (Positive internal spiked control)

365

361

365

361

375


365 MS fungal signature in clinical samples

Disease Positive samples 365 vs 361

Healthy subjects 0/15 (0%) None

Candidemia 12/12 (100%) +++

Aspergillosis 5/5 (100%) +++

Pneumocystis pneumonia 3/5 (60%) +++


Conclusion/Perspectives

• There is a « 365 signature » of candidemia in patients sera

• Explore the range and the nature of «glycans circulating molecular species »(Mannans, glucans, chitin) their diagnostic and pathophysiological significance

• Development of animal model (rabbit) to speed-up and complement these researches

• Connection between this R&D project and the ongoing constitution of a cohort to analyse genetic risks factors of candidemia (Promotor code: 2011_63)


Documents

Circulating fungal glycans in sera of ICU patients. Clinical relevance and development of physico-chemical methods of detection. J.POISSY 1-2, B.SENDID