Advances in the diagnosis of invasive aspergillosis Paul E. Verweij, MD Department of Medical Microbiology Nijmegen University Center of Infectious Diseases, UMC St Radboud

Advances……… Understanding release and kinetics of surrogate markers Comparison of surrogate markers Comparison of strategies

Design Sensi (%) Spec (%) Ref Pan-fungal 100 98 JCM 1997;35:1353-60 75 96 BJH 2001;113:180-4 Asp. sp. 65 JID 2000;181:1713-9 91.7 81.3 CID 2001;33:428-35 79 92 CID 2001;33:1504-12

Performance of GM detection in published studies specificity sensitivity

BG in invasive fungal infection AML, MDS Receiving antifungal prophylaxis Clin Infect Dis 2004;39:199-205

Prospective monitoring of blood by PCR Study design : prospective screening Study population : 165 hematology patients + HSCT Samples : 1522 whole blood (3-5 ml) Strategy : 6.4 – 17 samples per neutropenic episode Method : nested PCR Aspergillus sp. specific (18 S rRNA) positive samples analyzed by Light Cycler PCR galactomannan ELISA Br J Haematol 2004;125:196-202

Prospective monitoring of blood by PCR Performance sensitivity specificity Single + 7 of 11 (63.6%) 66/104 (63.5%)  2 + 4 of 11 (36.4%) 96/104 (92.3%) GM 33.3% 98.9% Br J Haematol 2004;125:196-202

EORTC/MSG Proven Prob. Poss. No IA Samples 67 46 781 681 Patients 8 3 90 104 PCR+ 8 5 56 50 PCR- 59 41 725 576 “false” positives “false” negatives Br J Haematol 2004;125:196-202

colonisation EORTC/MSG definitions colonisation infection disease

-/+ -/+ - Invasive opportunistic mycoses (IM) Host: high risk colonisation infection disease CT-scan positive negative negative Class.: No IM No IM/possible Possible/probable/ proven IM -/+ -/+ - PCR

117 nested PCR + -> 25 (21.4%) LightCycler + Clinical studies 117 nested PCR + -> 25 (21.4%) LightCycler + LightCycler Copies/ml Proven IA 0 - Probable IA 2 5,270 – 1,902,099 Possible IA 16 52 – 410, 667 No IA 7 37 – 90,909 Br J Haematol 2004;125:196-202

Galactomannan variability: Published factors: False positive reactivity……….. Piperacillin-tazobactam LTA of bifidobacteria False negative reactivity………… N Engl J Med 2003;349:24-5 Lancet 2004;363:325-7

Factors that influence performance Biological factors Site of infection Aspergillus species Microenvironment at site of infection (nutrients, pH, etc) Molecule structure of released GM Underlying condition / immune suppression Exposure to antifungals Renal clearance, hepatic metabolism Presence of GM antibodies Lancet Infect Dis 2004;4:349-57

Factors that influence performance-continued…. Biological factors Storage of sample Pre-treatment procedure Epidemiological factors Patient population Prevalence of infection Sampling strategy Definitions (positive result (cut-off), positive patient) Lancet Infect Dis 2004;4:349-57

GM cut-off 986 serum samples from 67 patients with hematological malignancy J Infect Dis 2004;190:641-9

Effect of exposure to mould-active antifungals J Infect Dis 2004;190:641-9

Release of surrogate markers is a dynamic process

I II III Growth phases of A. fumigatus in vitro Release of markers by the fungus I II III Carbon source (glucose) Excretion of organic acids Decrease of pH Logaritmic growth No glucose Re-use of organic acids Increase of pH Decreased growth No glucose No acids Stable pH Lysis and cannibalism Arch Aller Appl Immun 1980;62:252-64

Concentration Release of surrogate markers I II III hours glucose 7 6 5 4 3 2 1 I II III Concentration 24 48 72 96 120 144 168 192 hours glucose Dry weight GM

- - - - - - - - + Concentration Release of surrogate markers hours PCR 7 6 5 4 3 2 1 24 48 72 96 120 144 168 192 hours Release of surrogate markers Concentration - - - - - - - - + PCR (supernatant) Need damage of fungus by host defences?

GM versus PCR versus BG ROC analysis: GM > PCR and GM > BG Comparative studies GM versus PCR versus BG Adult hematology patients on mould active antifungal prophylaxis Prospective study, 149 treatment episodes, 96 patients Weekly sampling ROC analysis: GM > PCR and GM > BG J Clin Microbiol 2004;42:2733-41

Need for strategic studies: which test to use PCR Both? Antigen

PCR,GM and BG may be useful for the early detection of invasive mycoses when intensive sampling is achieved. Some progress has been made in understanding the kinetics of surrogate markers Correlation with other diagnostics and optimal sequence of testing needs to be determined Conclusions