Circulating fungal glycans in sera of ICU patients. Clinical relevance and development of physico-chemical methods of detection. J.POISSY 1-2, B.SENDID 2-3, S.DAMIENS 2, R.FAVORY 1, N.FRANCOIS 3, N.GHALEM 3, S.JAWHARA 2, T.JOUAULT 1, D.MATHIEU 1, Y.GUERARDEL 4, D.POULAIN 2-3 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 Adapted from Kumar A et col. ICAAC K 2174 All patientsPatients 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 circulating in 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: 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=8 N=34 N=32 N=18 N=16 N=13 N=9 N=6 p= 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 fungal biomarkers? U995 Journées André Verbert 11 Sept 2012

Blood culture Incubation Mass spectrometry Spectral profile Culture Susceptibility to ATFs Isolation/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°C Centrifugation Supernatent with less proteins, Hb, lipids C18 column preconditionned by H2O+acétonitrile After washing : Oligosaccharides, glucose and salts Solid phase extraction Activated 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 patients with Candidemia. With reference to peak 361 (Matrix) H 2 Trehalose (Positive internal spiked control) U995 Journées André Verbert 11 Sept 2012

365 MS fungal signature in clinical samples DiseasePositive samples365 vs 361 Healthy subjects0/15 (0%)None Candidemia12/12 (100%)+++ Aspergillosis5/5 (100%)+++ Pneumocystis pneumonia3/5 (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