Galactomannan testing: lessons from the last decade

1. Galactomannan testing: lessons from the last decade Claudio Viscoli Professor ofInfectiousDisease, Universityof Genova Chief, DivisionofInfectiousDisease, San Martino University Hospital, Genova, Italy

2. Galactomannan antigen detectionPlatelia Aspergillus – ELISA (Bio-Rad)

3. Galactomannan antigenPlateliaAspergillus (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 )

4. Galactomannan antigenPlateliaAspergillus (Bio-Rad) 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)

5. Galactomannan antigen CUT-OFF FOR POSITIVITY 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) Single test Index > 0.7 Static cut-off (Maertens 2004) Two consecutive test Index > 0.5 Dynamic cut-off

6. Galactomannan antigen From 1998 to July 2009: 24.093 Galactomannan determinations with Platelia Aspergillus (ELISA) (mean: 2007 determinations/year; min 332, max 4402) We perform GM test in serum, BAL, sputum, CSF, pleural fluid, tracheal aspirate fluid and synovial fluid.

8. Whywehave false positive results?

9. Aspergillus galactomannanFalse 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)

10. Fungal organism likely testing positive with the Platelia test

11. Routine use of the GM test at the BMT Unit in Genova from Jan. 1999 to May 2003 • Total number of patients 420 • Total number of serum samples 4702 • Median samples per patient 7 (1-64) • Median samples per month 85 (35-146) • Median positivity rate per month • Jan. 1999 - Jan. 2003 9% (0-18) • Feb. 2003 - May 2003 24% (20-44)

12. 36% of patients and 28% of specimens were positive

13. Platelia Aspergillus Test results by administration of Piperacillim-Tazobactam Patient receiving piperacillin-tazobactam Patient NOT receiving piperacillin-tazobactam 26% 89% 11% 74% Pipera-tazo YES= since at least 24 hrs p < 0,001 Viscoli et al ICAAC 2003; CID 2004

14. Platelia Aspergillus teston 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)

15. Galactomannan antigen FALSE POSITIVE IN PEDIATRIC PATIENTS • 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)

16. Clinical Microbiology and Infection, in press

18. Whywehave false negative results? • Low prevalenceof the disease • Concomitantuseofantifungals • Little angioinvasion (HSCT) • Presenceofanti-aspergillusantibodies • Low fungalburden • Inappropriate cut-off • Inappropriate use • Testing • Sampling • Storage

19. Pfeiffer et al., CID, 2006

20. Antifungal therapy Yes No • 0,5 • 1 • 1,5 • 0,5 • 1 • 1,5 (Marr 2005)

21. Filtration and use of a larger volume of serum Conventional method Verwej 2005

22. Galactomannan in other body fluids

26. GM in CSF (Klont RR, CID, 2004) Cerebral aspergillosis 10%-20% of all acses of invasive aspergillosis • Not validated • Cut-off?

27. Aspergillus galactomannan antigen detection in cerebral aspergillosis

28. Galactomannan as a surrogate marker of efficacy

29. Galactomannan levels in serum and CSF samples Sample / cut-off OD index Days from BMT (Machetti et al. 2000)

32. Thankyouforyourattention

33. Pfeiffer et al., CID, 2006

36. Comparison of empirical and PCR-based preemptive antifungal therapy in 408 allogeneic stem cell transplant recipients • PCR screening twice weekly during stay in hospital and once weekly after discharge until D100 • Antifungal therapy initiation • PCR group: in PCR+ patients with signs of infection and in patients with 2 consecutive PCR + • Empirical treatment group: 5d of febrile neutropenia PCR based Empiric n = 196n = 207 Antifungal therapy 109 (56%) 76 (37%) (p<0.05) Proven invasive aspergilosis 11 16 • Reduction in early mortality (D30) in patients receiving PCR-based therapy but no difference in mortality at D100 and D180 • (Hebart et al. ASH 2004)