1. David W. Denning Wythenshawe Hospital University of Manchester
Getting to the diagnosis of aspergillosis: Tests and their interpretation
David W. Denning
Wythenshawe Hospital
University of Manchester

2. Aspergillus Life-cycle
Germination
Spores inhaled
Hyphal elongation and branching
Mass of hyphae (plateau phase)

3. CLASSIFICATION OF ASPERGILLOSIS
Invasive aspergillosis
Acute (<1 month course)
Subacute/chronic necrotising (1-3 months)
Airways/nasal exposure to airborne Aspergillus
Chronic aspergillosis (>3 months)
Chronic cavitary pulmonary
Aspergilloma of lung
Chronic fibrosing pulmonary
Chronic invasive sinusitis
Maxillary (sinus) aspergilloma
Allergic
Allergic bronchopulmonary (ABPA)
Extrinsic allergic (broncho)alveolitis (EAA)
Asthma with fungal sensitisation (SAFS)
Allergic Aspergillus sinusitis (eosinophilic fungal rhinosinusitis)
Persistence
without disease
colonisation of
the airways or nose/sinuses
Exposure to individual Aspergillus spores or conidia is almost constant. If eradicated immediately, as is usual in normal people, no disease results. If colonisation occurs, it may be short or long term. The pattern of disease is mostly determined by the host group (see next slide), with probably a component of the inoculum size contributing to invasive disease.

4. CLASSIFICATION OF ASPERGILLOSIS
Invasive aspergillosis
Acute (<1 month course)
Subacute/chronic necrotising (1-3 months)
Airways/nasal exposure to airborne Aspergillus
Chronic aspergillosis (>3 months)
Chronic cavitary pulmonary
Aspergilloma of lung
Chronic fibrosing pulmonary
Chronic invasive sinusitis
Maxillary (sinus) aspergilloma
Persistence without disease - colonisation of the airways or nose/sinuses
Exposure to individual Aspergillus spores or conidia is almost constant. If eradicated immediately, as is usual in normal people, no disease results. If colonisation occurs, it may be short or long term. The pattern of disease is mostly determined by the host group (see slide 8), with probably a component of the inoculum size contributing to invasive disease.
Acute and subacute disease is usually associated with immunocompromised patients eg AIDS, chemotherapy, transplant etc. and these will be discussed first.
Allergic
Allergic bronchopulmonary (ABPA)
Extrinsic allergic (broncho)alveolitis (EAA)
Asthma with fungal sensitisation
Allergic Aspergillus sinusitis (eosinophilic fungal rhinosinusitis)

5. Early diagnosis of invasive aspergillosis is important
Treatment started <10d >11d
Mortality % 90%
Von Eiff et al, Respiration 1995;62:241-7.

6. Modalities for early diagnosis of invasive aspergillosis
CT scanning
Microscopy
Antigen (blood or respiratory fluid)
PCR (blood or respiratory fluid)

7. Investigations for diagnosis of IPA
Abnormal/All %
Chest X-ray 89/98 (91)
Focal disease /98 (59)
Cavitation /98 ( 5)
Diffuse/multiple 26/98 (27)
Chest CT scan 23/23 (100)
Focal disease /23 (13)
Cavitation /23 (17)
Diffuse/multiple /23 (70)
Bronchoalveolar lavage 36/61 (59)
Transbronchial biopsy 4/6 (67)
Open lung biopsy 4/8 (50)
Denning et al, J Infection 1998;37:

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

9. Criteria for Halo Sign n gg
“Perimeter of ground-glass opacity surrounding a nodular lesion”
Identified early in angio-invasive aspergillosis
Differentiate from nodular lesions with unsharp margination that lack a perimeter of ground-glass n gg
The Halo Sign is a perimeter of ground-glass opacity on computed tomography that surrounds a nodular lesion.
It is seen in angioinvasive aspergillosis.
Ground-glass opacity on CT refers to an intermediate density between completely aerated lung and solid lung opacification such that bacground pulmonary vessels can be seen through it.
gg = ground-glass halo
n = nodular lesion
Greene et al, ECCMID 2003

10. Criteria for Air Crescent Sign
“Crescent of gas surmounting
soft tissue sequestrum within
a nodular or cavitary lesion”
Differentiate from non-specific thick- or thin-walled cavities lacking sequestra
Usually appear late in angio-invasive aspergillosis after recovery from neutropenia ac s
ac = air crescent
S = sequestrum
Greene et al, ECCMID 2003

11. Pulmonary nodules a useful feature if invasive pulmonary aspergillosis
CT features in 48 CTs of which 17 IPA
IPA Other
Halo 13/ /31
Nodules 14/17 11/31
Masses 6/17 2/31
Kami, Mycoses 2002;45:

12. Pulmonary nodules a useful feature if invasive pulmonary aspergillosis
CT features in 235 CTs in patients with IPA
Macronodule (>1cm) (94%)
Halo (60%)
Consolidation (30%)
Macro-nodule, infarct shaped (27%)
Cavitary lesion (20%)
Air bronchograms (16%)
Clusters of small nodules (<1cm) (11%)
Pleural effusion (11%)
Air crescent sign (10%)
Non-specific ground glass (9%)
Greene submitted, from Herbrecht N Engl J Med 2002:347:408.

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

14. % 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

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

16. Sputum Cultures for Fungus
Bacteriological media inferior to fungal media – 32% higher yield on fungal media
At least 3 sputum specimens should be submitted for fungal culture whenever fungal infection is suspected. On a survey of routine bacterial culture versus fungal culture, many positive cultures for Aspergillus were found on the routine cultures, but 32% of instances of Aspergillus were only seen in the fungal culture medium. Horvath & Dummer, Am J Med 1996;100:171-8.
Horvath & Dummer, Am J Med 1996;100:171-8.

17. Aspergillus workload and significance
3 year survey in Spanish teaching hospital
404 isolates from 260 patients
1/1000 micro samples positive
31/260 (12%) had invasive disease
Point score system for IA developed:
Invasive sample positive 1
> 2 positive samples 2
leukaemia 2
neutropenia 5
corticosteroid Rx 2
Score of 1 or 2 = 10.3%, of 3 or 4 = 40%, of >5 = 70%
Bouza J Clin Microbiol 2005;43:2075.

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. BSMM proposed standards of care
All bronchoscopy fluids from patients suspected of infection should be examined microscopically for hyphae and cultured on specialised media.
All clinical isolates of Aspergillus should be identified to species level
Denning, Barnes and Kibbler. Lancet Infect Dis 2003;3:230.

20. Aspergillus Antigen Test
Diagnosis or surveillance?
Only blood, or BAL, CSF etc
Best OD cut-off - 0.7
False positives in kids / antibiotics
False negative with antifungal
prophylaxis
Not as useful for non-hematology
Not useful if pre-existing antibody
Herbrecht et al, J Clin Microbiol 2002;20: ; and others

21. Aspergillus Antigen in BAL
13/17 (76%) in acute leukaemia with CT abnormality
5/20 (25%) in suspected IFIs
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
Becker, Br J Haem 2003;121:448; Sanguinetti, JCM 2003;41:3922; Musher, JCM 2004;42:5517.

22. 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 IA patients without haem malignancy:
Culture +ve in 56/67 (84%)
Aspergillus antigen +ve 27/51 (53%)
Autopsy +ve for hyphae in 27/41 (66%)
Predicted mortality = 48%, actual 91%
Meersemann et al, Am J Resp Med Crit Care 2004;170:621.

23. CLASSIFICATION OF ASPERGILLOSIS
Invasive aspergillosis
Acute (<1 month course)
Subacute/chronic necrotising (1-3 months)
Airways/nasal exposure to airborne Aspergillus
Chronic aspergillosis (>3 months)
Chronic cavitary pulmonary
Aspergilloma of lung
Chronic fibrosing pulmonary
Chronic invasive sinusitis
Maxillary (sinus) aspergilloma
Persistence without disease - colonisation of the airways or nose/sinuses
Exposure to individual Aspergillus spores or conidia is almost constant. If eradicated immediately, as is usual in normal people, no disease results. If colonisation occurs, it may be short or long term. The pattern of disease is mostly determined by the host group (see next slide), with probably a component of the inoculum size contributing to invasive disease.
However chronic disease if usually seen in patients with apparently normal immune systems.
Allergic
Allergic bronchopulmonary (ABPA)
Extrinsic allergic (broncho)alveolitis (EAA)
Asthma with fungal sensitisation
Allergic Aspergillus sinusitis (eosinophilic fungal rhinosinusitis)

24. Simple aspergilloma Patient RT December 2002 Cough (mild) & tired
Wythenshawe Hospital

25. Aspergilloma
Severo on

26. Chronic Cavitary Pulmonary Aspergillosis Normal smoking 30 year woman
Patient JA
Jan 2001

27. Chronic Cavitary Pulmonary Aspergillosis
Patient JA
Feb 2002

28. Chronic Cavitary Pulmonary Aspergillosis
Patient JA
April 2003

29. Chronic Cavitary Pulmonary Aspergillosis
Patient JA
July 2003

30. Chronic cavitary pulmonary aspergillosis an example of radiographic failure
Patient SS
April 2004
Patient SS
July 2004, despite receiving itraconazole for 3 months

31. Chronic pulmonary aspergillosis - serology
All 18 patients had positive Aspergillus precipitins (1+-4+)
All 18 patients had elevated inflammatory markers, CRP, PV and / or ESR
14 of 18 (78%) had elevated total IgE (>20), 13 >200 and 7 >400
9 of 14 (67%) had Aspergillus specific IgE (RAST)
Denning DW et al, Clin Infect Dis 2003; 37:S265

32. 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)

33. Antibody diagnosis of invasive aspergillosis
In house ELISA method
Definite IA /31 (64.5)
Probable IA /67 (16.4)
Possible IA /55 (25.5)
All episodes /153 (29.4)
Herbrecht et al, J Clin Microbiol 2002;20: