circulating fungal glycans in sera of icu patients. clinical relevance and development of...
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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
U995 Journées André Verbert 11 Sept 2012
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
U995 Journées André Verbert 11 Sept 2012
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
U995 Journées André Verbert 11 Sept 2012
Fungitell®: Sensitive but non specific test (cut-off=80 pg/mL)
U995 Journées André Verbert 11 Sept 2012
Receiver Operating Characteristics curve (ROC)
U995 Journées André Verbert 11 Sept 2012
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
U995 Journées André Verbert 11 Sept 2012
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?
U995 Journées André Verbert 11 Sept 2012
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
U995 Journées André Verbert 11 Sept 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
U995 Journées André Verbert 11 Sept 2012
Spiking of purified F2 in serum generates a 365 signal even for very low quantities
U995 Journées André Verbert 11 Sept 2012
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
U995 Journées André Verbert 11 Sept 2012
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%) +++
U995 Journées André Verbert 11 Sept 2012
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)
U995 Journées André Verbert 11 Sept 2012