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

Galactomannan antigen detection Platelia Aspergillus – ELISA (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 )

Galactomannan antigen Platelia Aspergillus (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)

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) Static cut-off Single test Index > 0.7 (Maertens 2004) Dynamic cut-off Two consecutive test Index > 0.5

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.

Why we have false positive results?

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)

Fungal organism likely testing positive with the Platelia test

Routine use of the GM test at the BMT Unit in Genova from Jan 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)

36% of patients and 28% of specimens were positive

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

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)

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)

Clinical Microbiology and Infection, in press

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 Testing Sampling Storage

Pfeiffer et al., CID, 2006

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

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

Galactomannan in other body fluids

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

Aspergillus galactomannan antigen detection in cerebral aspergillosis

Galactomannan as a surrogate marker of efficacy

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

Thank you for your attention

Background.  A double-sandwich enzyme-linked immunosorbent galactomannan assay has been approved for surveillance for invasive aspergillosis in immunocompromised patients. We undertook a meta-analysis to assess the accuracy of a galactomannan assay for diagnosing invasive aspergillosis. Methods.  Studies of the galactomannan assay that used the European Organization for Research and Treatment of Cancer or similar criteria as a reference standard and provided data to calculate sensitivity and specificity were included. Pooled sensitivity and specificity and summary measures of accuracy, Q* (the upper left-most point on the summary receiver-operating characteristic curve), mean D (a log odds ratio), and Youden index were calculated. Subgroup analyses were performed to explore heterogeneity. Results.  Twenty-seven studies from 1966 to 28 February 2005 were included. Overall, the galactomannan assay had a sensitivity of 0.71 (95% confidence interval [CI], 0.68–0.74) and specificity of 0.89 (95% CI, 0.88–0.90) for proven cases of invasive aspergillosis. The Youden index, mean D, and Q* were 0.54 (95% CI, 0.41–0.65), 2.74 (95% CI, 21.12–3.36), and 0.80 (95% CI, 0.74–0.86), respectively, indicating moderate accuracy. Subgroup analyses showed that the performance of the test differed by patient population and type of reference standard used. Significant heterogeneity was present. Conclusions.  The galactomannan assay has moderate accuracy for diagnosis of invasive aspergillosis in immunocompromised patients. The test is more useful in patients who have hematological malignancy or who have undergone hematopoietic cell transplantation than in solid-organ transplant recipients. Further studies with attention to the impact of antifungal therapy, rigorous assessment of false-positive test results, and assessment of the utility of the test under nonsurveillance conditions are needed. Pfeiffer et al., CID, 2006

There has been minimal clinical experience with the use of the Aspergillus galactomannan enzyme-linked immunosorbent assay (ELISA) for patients receiving echinocandin therapy. We reviewed the experience with the galactomannan ELISA for 17 patients in a study of caspofungin treatment for invasive aspergillosis. The rate of successful outcomes for these patients was similar to that overall for participants in the study. Trends in antigenemia levels correlated with clinical and radiographic findings.

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 = 196 n = 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)