Galactomannan testing: lessons from the last decadeClaudio ViscoliProfessor of Infectious Disease, University of GenovaChief, Division of Infectious Disease, San Martino University Hospital, Genova, Italy

Galactomannan antigen detection Platelia Aspergillus ELISA (Bio-Rad)

Galactomannan antigenPlatelia Aspergillus (Bio-Rad)Sensitivity highly variable (29-100%)Specificity generally better (81-98%)FDA approvedImportant tool in the diagnosis of aspergillosis (EORTC-MSG definitions of IA (Ascioglu 2002)May be positive before the occurrence of clinical and radiological signs/symptomsTwo main strategies of use:Serial collection of samples (2 or 3 times/week) in high risk patientsIntensive testing in symptomatic patients (unexplained persistent fever unresponsive to broad spectrum antibiotics )

ControversiesDifferent cut-off used: 0.5, 0.7, 1, 1.5DrawbacksFalse positive and false negative results Too low sensitivity according to some authors (Pinel 2003, Allan 2005)Galactomannan antigenPlatelia Aspergillus (Bio-Rad)

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 5083%,specificity 10073,7% compared with BIO-RAD cut-off (Marr 2004)Galactomannan antigen CUT-OFF FOR POSITIVITYSingle test Index > 0.7Two consecutive test Index > 0.5(Maertens 2004)Static cut-offDynamic cut-off

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

Grafico1

332

1238

909

1308

1450

1892

2100

1897

2418

3209

4402

2938

year

Galactomannana determinations

Foglio1

annonum esami

1998332

19991238

2000909

20011308

20021450

20031892

20042100

20051897

20062418

20073209

20084402

July 20092938

media2007.75

max4402

min332

Foglio1

0

0

0

0

0

0

0

0

0

0

0

0

year

Galactomannana determinations

Foglio2

Foglio3

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. 1999 to May 2003Total number of patients420Total number of serum samples4702Median samples per patient 7 (1-64)Median samples per month85 (35-146)Median positivity rate per monthJan. 1999 - Jan. 2003 9% (0-18)Feb. 2003 - May 200324% (20-44)

36% of patients and 28% of specimens were positive

Patient receiving piperacillin-tazobactam

11%89% 26%74%Patient NOT receiving piperacillin-tazobactam

Platelia Aspergillus Test results by administration of Piperacillim-Tazobactamp < 0,001Pipera-tazo YES= since at least 24 hrsViscoli et al ICAAC 2003; CID 2004

Platelia Aspergillus teston piperacillin-tazobactam six batches of Tazocin taken from the hospital pharmacy were testedtwo 4.5 g. vials per batchdiluted with 100 ml NaCl 0.9%five of six batches tested positivemedian GM index 4.7 (1.5-5.7)

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)Galactomannan antigen FALSE POSITIVE IN PEDIATRIC PATIENTS

Clinical Microbiology and Infection, in press

Why we have false negative results?Low prevalence of the diseaseConcomitant use of antifungalsLittle angioinvasion (HSCT)Presence of anti-aspergillus antibodiesLow fungal burdenInappropriate cut-offInappropriate useTestingSamplingStorage

Pfeiffer et al., CID, 2006

Antifungal therapy1,510,50,511,5(Marr 2005)YesNo

Conventional methodFiltration and use of a larger volume of serumVerwej 2005

Galactomannan in other body fluids

GM in CSF (Klont RR, CID, 2004)Cerebral aspergillosis 10%-20% of all acses of invasive aspergillosis

Not validatedCut-off?

Aspergillus galactomannan antigen detection in cerebral aspergillosis

Cerebral aspergillosis Control patients (5 pts; 8 samples) (16 pts; 33 samples)

GM index in CSF

Box-plot analysis of CSF GM in patients with or without cerebral aspergillosis, demonstrating the specificity of the test when performed in the CSF

(Viscoli et al. 2001)

Galactomannan as a surrogate marker of efficacy

Galactomannan levels in serum and CSF samplesSample / cut-off OD indexDays from BMT(Machetti et al. 2000)

Chart3

50501

3.45621

2.27641

3.55691

3711

2.95761

4.45791

3.55831

2.27851

2.23901

1.8921

2.1971

1.9625991

616.18

712.586

931.161

Clinical sign of aspergillosis

Index = 29,45

Elisa serum result

Elisa CSF result

Elisa positivity threshold

Sheet1

Elisa CSF resultElisa serum resultElisa positivity threshold

501

623.451

642.271

693.551

7131

762.951

794.451

833.551

852.271

902.231

921.81

972.11

991.96251

616.18

712.586

931.161

Thank you for your attention

Pfeiffer et al., CID, 2006

PCR screening twice weekly during stay in hospital and once weekly after discharge until D100 Antifungal therapy initiationPCR group: in PCR+ patients with signs of infection and in patients with 2 consecutive PCR +Empirical treatment group: 5d of febrile neutropenia PCR based Empiricn = 196n = 207Antifungal therapy109 (56%)76 (37%)(p