Paul E. Verweij, MDDepartment of Medical MicrobiologyNijmegen University Center of Infectious Diseases, UMC St Radboud

Advances in the diagnosis of invasive aspergillosis

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

Pan-fungal 75 96 BJH 2001;113:180-4

Asp. sp. 100 65 JID 2000;181:1713-9

Asp. sp. 91.7 81.3 CID 2001;33:428-35

Asp. sp. 79 92 CID 2001;33:1504-12

40

50

60

70

80

90

100

1994 1999 2004

sensitivity

specificity

Performance of GM detection in published studies

specificity

sensitivity

Clin Infect Dis 2004;39:199-205

BG in invasive fungal infection

AML, MDS

Receiving antifungal prophylaxis

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

Prospective monitoring of blood by PCR

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

Br J Haematol 2004;125:196-202

“false” positives“false” negatives

colonisation

infection

disease

colonisation

EORTC/MSG

definitions

Invasive opportunistic mycoses (IM)

colonisation infection disease

Class.: No IM No IM/possible Possible/probable/

proven IM

Host: high risk

CT-scan negative negative positive

PCR -/+- -/+

Clinical studies

LightCycler

Proven IA 0 -

Probable IA 2 5,270 – 1,902,099

Possible IA 16 52 – 410, 667

No IA 7 37 – 90,909

117 nested PCR + -> 25 (21.4%) LightCycler +

Copies/ml

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) Lance

t In

fect

Dis

20

04

;4:3

49

-57

J Infect Dis 2004;190:641-9

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

Release of surrogate markers is a dynamic process

Growth phases of A. fumigatus in vitro

I

Carbon source (glucose)

Excretion of organic acids

Decrease of pH

Logaritmic growth

II

No glucose

Re-use of organic acids

Increase of pH

Decreased growth

III

No glucose

No acids

Stable pH

Lysis and cannibalism

Arch Aller Appl Immun 1980;62:252-64

Release of markers by the fungus

Con

cen

trati

on

7

6

5

4

3

2

1

0 24 48 72 96 120 144 168 192hours

Release of surrogate markers

glucose Dry weight GM

I II III

Con

cen

trati

on

7

6

5

4

3

2

10 24 48 72 96 120 144 168 192hours

Release of surrogate markers

PCR

(supernatant

)

- - - - - - - - +Need damage of fungus by host defences?

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

PCR Antigen

Need for strategic studies: which test to use

Both?

Conclusio

ns

•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