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Galactomannan testing: lessons from the last decade Claudio Viscoli Professor of Infectious Disease, University of Genova Chief, Division of Infectious Disease, San Martino University Hospital, Genova, Italy Slide 2 Galactomannan antigen detection Platelia Aspergillus ELISA (Bio-Rad) Slide 3 Galactomannan antigen PlateliaAspergillus (Bio-Rad) Galactomannan antigen Platelia Aspergillus (Bio-Rad) Sensitivity highly variable (29-100%) Specificity generally better (81-98%) FDA approved Important tool in the diagnosis of aspergillosis (EORTC-MSG definitions of IA (Ascioglu 2002) May be positive before the occurrence of clinical and radiological signs/symptoms Two main strategies of use: Serial collection of samples (2 or 3 times/week) in high risk patients Intensive testing in symptomatic patients (unexplained persistent fever unresponsive to broad spectrum antibiotics ) Slide 4 Controversies Different cut-off used: 0.5, 0.7, 1, 1.5 Drawbacks False positive and false negative results Too low sensitivity according to some authors (Pinel 2003, Allan 2005) Galactomannan antigen PlateliaAspergillus (Bio-Rad) Galactomannan antigen Platelia Aspergillus (Bio-Rad) Slide 5 Test result as GM index = sample OD/cut-off OD (1 ng/ml ) Index > 1.5 in 2 consecutive samples (BIO-RAD) Index > 1 (Verweij 1998; Maertens 2001; Sulahian 2001; Ascioglu 2002) Index > 0.7 (sensitivity+24%;specificity-5.5%compared with BIO-RAD cut-off) (Herbrecht 2002) Index > 0,5 (sensitivity 50 83%,specificity 100 73,7% compared with BIO-RAD cut-off (Marr 2004) Galactomannan antigen CUT-OFF FOR POSITIVITY Single test Index > 0.7 Two consecutive test Index > 0.5 (Maertens 2004) Static cut-off Dynamic cut-off Slide 6 From 1998 to July 2009: 24.093 Galactomannan determinations with Platelia Aspergillus (ELISA) (mean: 2007 determinations/year; min 332, max 4402) Galactomannan antigen We perform GM test in serum, BAL, sputum, CSF, pleural fluid, tracheal aspirate fluid and synovial fluid. Slide 7 Slide 8 Why we have false positive results? Slide 9 Aspergillus galactomannan Aspergillus galactomannan False positive results Transient antigenemia (non invasive infections?) Cross reactivity with exoantigens (bacteria-fungi) Induction by cyclophosphamide (Hashiguchi et al. 1994) Premature infants (83%) (Siemann et al. 1998) Cotton swabs (Dalle et al. 2002) Absorption of galactomannan through a damaged intestinal mucosa (Letscher-Bru et al. 1998) During caspofungin therapy (Petraitiene et al. 2002) Galactomannan in antibiotics (Ansorg et al. 1997; Viscoli et al 2003) Slide 10 Fungal organism likely testing positive with the Platelia test Slide 11 Routine use of the GM test at the BMT Unit in Genova from Jan. 1999 to May 2003 Total number of patients420 Total number of serum samples4702 Median samples per patient 7 (1-64) Median samples per month85 (35-146) Median positivity rate per month Jan. 1999 - Jan. 2003 9% (0-18) Feb. 2003 - May 200324% (20-44) Slide 12 36% of patients and 28% of specimens were positive Slide 13 Patient receiving piperacillin-tazobactam 11% 89% 26% 74% Patient NOT receiving piperacillin-tazobactam Platelia Aspergillus Test results by administration of Piperacillim-Tazobactam p < 0,001 Pipera-tazo YES= since at least 24 hrs Viscoli et al ICAAC 2003; CID 2004 Slide 14 Platelia Aspergillus test on piperacillin-tazobactam six batches of Tazocin taken from the hospital pharmacy were tested two 4.5 g. vials per batch diluted with 100 ml NaCl 0.9% five of six batches tested positive median GM index 4.7 (1.5-5.7) Slide 15 False positive GM test in 83% of premature infants (prolonged ICU and birth weight of 400-1320 g) (Siemann 1998) Passage of food-GM through damaged intestinal mucosa of BMT children (Letscher-Bru 1998) Neonates milk formula, false positive GM test (Gangneux 2002) Bifidobacterium sp. lipoteichoic acid (bacteria that heavily colonize neonatal gut) produces false positive GM test(Mennink-Kersten 2004) 0 Galactomannan antigen FALSE POSITIVE IN PEDIATRIC PATIENTS Slide 16 Clinical Microbiology and Infection, in press Slide 17 Slide 18 Why we have false negative results? Low prevalence of the disease Concomitant use of antifungals Little angioinvasion (HSCT) Presence of anti-aspergillus antibodies Low fungal burden Inappropriate cut-off Inappropriate use lTesting lSampling lStorage Slide 19 Pfeiffer et al., CID, 2006 Slide 20 Antifungal therapy 0 1,5 0101 0 0,5 0101 0 1,5 (Marr 2005) Yes No Slide 21 Conventional method Filtration and use of a larger volume of serum Verwej 2005 Slide 22 Galactomannan in other body fluids Slide 23 Slide 24 Slide 25 Slide 26 GM in CSF (Klont RR, CID, 2004) (Klont RR, CID, 2004) Cerebral aspergillosis 10%-20% of all acses of invasive aspergillosis Not validated Cut-off? Slide 27 Aspergillus galactomannan antigen detection in cerebral aspergillosis Slide 28 Galactomannan as a surrogate marker of efficacy Slide 29 Galactomannan levels in serum and CSF samples Sample / cut-off OD index Days from BMT (Machetti et al. 2000) Slide 30 Slide 31 Slide 32 Thank you for your attention Slide 33 Pfeiffer et al., CID, 2006 Slide 34 Slide 35 Slide 36 PCR screening twice weekly during stay in hospital and once weekly after discharge until D100 Antifungal therapy initiation lPCR group: in PCR+ patients with signs of infection and in patients with 2 consecutive PCR + lEmpirical treatment group: 5d of febrile neutropenia PCR based Empiric n = 196n = 207 Antifungal therapy109 (56%)76 (37%)(p