1. Diagnosis of pulmonary aspergillosis (ignoring allergy)
David W. Denning
Wythenshawe Hospital
University of Manchester

2. Conceptual framework Normal Massive Hyphal load in tissue
Immune function
Hyphal load in tissue
Normal
Massive
Chronic inflammation and fibrosis
Vascular invasion, necrosis, dissemination
Granulomas, acute inflammation, central necrosis
Aspergilloma - CCPA - CNPA/subacute IPA - acute IPA

3. Aspergilloma - CCPA - CNPA/subacute IPA - acute IPA
Testing performance?
Immune function
Hyphal load in tissue
Normal
Massive
Aspergilloma - CCPA - CNPA/subacute IPA - acute IPA
Culture + +/- +/- +/ /-
Antigen
Glucan +/- +? ? /-
Antibody ?
PCR (resp) ++ +? ? ++?
PCR (blood) -? -? ? +/

4. Testing performance? Culture + +/- +/- +/- +/- Antigen - - - + ++
Immune function
Hyphal load in tissue
Normal
Massive
Pulmonary defect
+ innate immune defect
corticosteroids
neutrophil defect
neutropenia
multiple defects
Culture + +/- +/- +/ /-
Antigen
Glucan +/- +? ? /-
Antibody ?
PCR (resp) ++ +? ? ++?
PCR (blood) -? -? ? +/

5. Invasive aspergillosis in ICU
127 of 1850 (6.9%) consecutive medical ICU admissions with IA or colonisation (micro/histol).
89/127 (70%) did not have haematological malignancy
67/89 proven/probable IA, 33 of 67 (50%) COPD
In 67
Culture +ve in 56/67 (84%)
Aspergillus antigen +ve 27/51 (53%)
Meersemann et al, Am J Resp Med Crit Care 2004;170:621.

6. Testing performance? Culture (+) +/- +/- +/- +/- Antigen (-) - - + ++
Immune function
Hyphal load in tissue
Normal
Massive
Pulmonary defect
+ innate immune defect
corticosteroids
neutrophil defect
neutropenia
multiple defects
Culture (+) +/- +/- +/ /-
Antigen (-)
Glucan (+/-) +? ? /-
Antibody (+++) ?
PCR (resp) (++) +? ? ++?
PCR (blood) -? -? ? +/

7. Organism/antigen/marker. performance will vary by fungal
Organism/antigen/marker performance will vary by fungal load (in lung, but not necessarily blood) and possibly treatment

8. Aspergillus Antigen in BAL
13/17 (76%) in acute leukaemia with CT abnormality
17/17 (100%) in neutropenic patients before antifungal Rx, 0% after 3d antifungal therapy
20/20 (100%) in haem-onc pts with IPA
37/49 (76%) in HSCT & haem-onc with IPA
6 of 11 (55%) immunocompromised (8 of 11 +ve by PCR)
5/20 (25%) in suspected IFIs
Becker, Br J Haem 2003;121:448; Sanguinetti, JCM 2003;41:3922; Musher, JCM 2004;42:5517.

9. Organism/antigen/marker. performance will vary by fungal
Organism/antigen/marker performance will vary by fungal load (in lung, but not necessarily blood) and possibly treatment Antibody and imaging performance will be more independent of organism load to the same extent Antibody takes time to form (and tests are not standardised)

10. Contribution of CT scans and antigen testing to rapid diagnosis of IA
Caillot et al, J Clin Oncol 2001;19:253

11. Small vessel angioinvasion
Unequivocal ‘Halo sign’ surrounding a nodule
Halo
Small vessel angioinvasion
Herbrecht, Denning et al, NEJM 2002;347:

12. CT scan enlargement of IA on treatment despite good outcomes
Caillot et al, J Clin Oncol 2001;19:253

13. Contribution of CT scans and antibody testing to rapid diagnosis of IA
Pre Oct ‘91
Post Oct ‘91
P value
Patients 22 19
Mean time from IPA sign to diagnosis
days
days
0.002
Pre-IPA Dx antibody tests positive 16 6
0.008
Post-IPA Dx antibody tests positive
16/19
14/19 NS
Antigen tests positive
8/14
7/19
Caillot et al, J Clin Oncol 2001;19:253 (unpublished data)

14. Test sensitivity important: Microscopy methodology Culture versus PCR Histopathology versus culture

15. Test sensitivity important: Microscopy methodology Culture versus PCR Histopathology versus culture

16. Microscopy
Fluorescent brighteners such as Calcufluor white, Blankophor increase sensitivity and speed
Ruchel R,

17. Test sensitivity important: Microscopy methodology Culture versus PCR Histopathology versus culture

18. PCR detection of Aspergillus (rRNA target)
Prospective study of 197 bronchial washes in 176 patients (most leukaemia, most lung infiltrates on X-ray)
Results
Immunocom-promised pts
IA not IA
‘normal’ pts
IA not IA 31 6 5 2
102 30
+ve PCR
-ve PCR
Positive predictive value (PPV) % in at risk patients
Negative predictive value (NPV) % in at risk patients
Buchheidt Br J Haematol 2002;116:

19. PCR detection of Aspergillus (rRNA target)
Immunocom-promised pts
IA not IA
‘normal’ pts
IA not IA 31 6 5 2
102 30
+ve PCR
-ve PCR
Proven, probable and possible was 12, 13 and 5, of whom all proven and probable cases had abnormal chest CT scans,
11 had positive cultures from BAL (9) or sputum (2), 14 had positive cytology from BAL or sputum but were culture negative,
3 had positive galactomannan antigen tests and 3 had histological confirmation.
20 of the 31 patients died.
Buchheidt Br J Haematol 2002;116:

20. Comparison of BAL antigen and real-time PCR
Culture Antigen PCR Proven/probable IA All haem malignancy 6/ /20 18/20
Sanguinetti, Clin Microbiol. 2003;41:

21. Real time PCR to distinguish Aspergillus species
Additional sensitivity will allow species detection and possibly resistance detection on culture negative clinical specimens
Real time PCR to distinguish Aspergillus species
A. terreus resistant to amphotericin B
Perlin , unpublished

22. % positive result in all those with definite or probable aspergillosis
Bronchoalveolar lavage for diagnosis of invasive pulmonary aspergillosis
% positive result in all those with
definite or probable aspergillosis
Patients BAL BAL Either Reference
culture cytology or both
Acute leukaemia Albeda, 1984
Leukaemia Kahn, 1986
Leukaema Saito, 1988 Leukaemia, BMT, Levy, 1992
Oncology
BMT focal McWhinney,
diffuse
[All Tarrand, 2003]
AlloBMT Roychowdhury, 2006

23. Test sensitivity important: Microscopy methodology Culture versus PCR Histopathology versus culture/antigen

24. Invasive aspergillosis in ICU
127 of 1850 (6.9%) consecutive medical ICU admissions with IA or colonisation (micro/histol).
89/127 (70%) did not have haematological malignancy
67/89 proven/probable IA, 33 of 67 (50%) COPD
In 67
Culture +ve in 56/67 (84%)
Aspergillus antigen +ve 27/51 (53%)
Autopsy +ve for hyphae in 27/41 (66%)
Meersemann et al, Am J Resp Med Crit Care 2004;170:621.

25. Respiratory samples +ve for Aspergillus in ICU
Vandewoude KH. Critical Care 2006;10:R31

26. Respiratory samples +ve for Aspergillus in ICU
Vandewoude KH. Critical Care 2006;10:R31