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

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

3. Design Sensi (%) Spec (%) Ref
Pan-fungal
100 98
JCM 1997;35: 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:

4. Performance of GM detection in published studies
specificity
sensitivity

5. BG in invasive fungal infection
AML, MDS
Receiving antifungal prophylaxis
Clin Infect Dis 2004;39:

6. 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:

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

8. EORTC/MSG Proven Prob. Poss. No IA
Samples
Patients
PCR
PCR
“false” positives
“false” negatives
Br J Haematol 2004;125:

9. colonisation
EORTC/MSG
definitions
colonisation
infection
disease

10. -/+ -/+ - 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

11. 117 nested PCR + -> 25 (21.4%) LightCycler +
Clinical studies
117 nested PCR + -> 25 (21.4%) LightCycler +
LightCycler
Copies/ml
Proven IA
Probable IA ,270 – 1,902,099
Possible IA – 410, 667
No IA – 90,909
Br J Haematol 2004;125:

12. 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

13. 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

14. 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

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

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

17. Release of surrogate markers is a dynamic process

18. 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

19. Concentration Release of surrogate markers I II III hours glucose7 6 5 4 3 2 1 I II
III
Concentration 24 48 72 96
120
144
168
192
hours
glucose
Dry weight GM

20. - - - - - - - - + Concentration Release of surrogate markers hours PCR7 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?

21. 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:

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

23. 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