Getting to the diagnosis of aspergillosis: Tests and their

interpretation

David W. DenningWythenshawe Hospital

University of Manchester

Spores inhaled

Aspergillus Life-cycle

www.aspergillus.man.ac.uk

Hyphal elongation and branching

Germination

Mass of hyphae (plateau phase)

CLASSIFICATION OF ASPERGILLOSIS

Airways/nasal exposure to airborne Aspergillus

Invasive aspergillosis• Acute (<1 month course)• Subacute/chronic necrotising (1-3 months)

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)

Persistencewithout disease- colonisation of the airways or nose/sinuses

CLASSIFICATION OF ASPERGILLOSIS

Persistence without disease - colonisation of the airways or nose/sinuses

Airways/nasal exposure to airborne Aspergillus

Invasive aspergillosis• Acute (<1 month course)• Subacute/chronic necrotising (1-3 months)

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• Allergic Aspergillus sinusitis (eosinophilic

fungal rhinosinusitis)

Early diagnosis of invasive aspergillosis is important

Treatment started <10d>11d

Mortality 40% 90%

Von Eiff et al, Respiration 1995;62:241-7.

Modalities for early diagnosis of invasive aspergillosis

• CT scanning• Microscopy• Antigen (blood or respiratory fluid)• PCR (blood or respiratory fluid)

Investigations for diagnosis of IPA

Abnormal/All %Chest X-ray 89/98 (91) Focal disease 58/98 (59) Cavitation 5/98 ( 5) Diffuse/multiple 26/98 (27)

Chest CT scan 23/23 (100) Focal disease 3/23 (13) Cavitation 4/23 (17) Diffuse/multiple 16/23 (70)

Bronchoalveolar lavage 36/61 (59)Transbronchial biopsy 4/6 (67)Open lung biopsy 4/8 (50)

Denning et al, J Infection 1998;37:173-80.

Unequivocal ‘Halo sign’ surrounding a noduleUnequivocal ‘Halo sign’ surrounding a nodule

Herbrecht, Denning et al, NEJM 2002;347:408-15.

Small vessel angioinvasion

Halo

Criteria for Halo Sign

gggg

ggggnn

n = nodular lesiongg = ground-glass halo

Identified early in angio-invasive aspergillosis

Differentiate from nodular lesions with unsharp

margination that lack a perimeter of ground-glass

“Perimeter of ground-glass opacity

surrounding a nodular lesion”

Greene et al, ECCMID 2003

Criteria for Air Crescent Sign

ss

Usually appear late in angio-invasive aspergillosis after

recovery from neutropenia

S = sequestrumac = air crescent

acac

acac

Differentiate from non-specificthick- or thin-walled cavities

lacking sequestra

“Crescent of gas surmounting soft tissue sequestrum within a nodular or cavitary lesion”

Greene et al, ECCMID 2003

Pulmonary nodules a useful feature if invasive pulmonary aspergillosis

CT features in 48 CTs of which 17 IPA

IPA OtherHalo 13/17 0/31Nodules 14/17 11/31Masses 6/17 2/31

Kami, Mycoses 2002;45:287-94.

Pulmonary nodules a useful feature if invasive pulmonary aspergillosis

CT features in 235 CTs in patients with IPA

Macronodule (>1cm) 221 (94%)Halo 143 (60%)Consolidation 71 (30%)Macro-nodule, infarct shaped 63 (27%)Cavitary lesion 48 (20%)Air bronchograms 37 (16%)Clusters of small nodules (<1cm) 25 (11%)Pleural effusion 25 (11%)Air crescent sign 24 (10%)Non-specific ground glass 21 (9%)

Greene submitted, from Herbrecht N Engl J Med 2002:347:408.

Contribution of CT scans and antigen testing to rapid diagnosis of IA

Caillot et al, J Clin Oncol 2001;19:253

Bronchoalveolar lavage for diagnosis

of invasive pulmonary aspergillosis% positive result in all those with definite or probable aspergillosis

Patients BAL BAL Either Referenceculture cytology or both

Acute leukaemia - - 50 Albeda, 1984Leukaemia 23 53 59 Kahn, 1986Leukaema 0 0 0 Saito, 1988 Leukaemia, BMT, 40 64 67 Levy, 1992 OncologyBMT focal 0 0 0 McWhinney, diffuse 100 0 100 1993

Microscopy

Ruchel R, www.aspergillus.man.ac.uk/images

Fluorescent brighteners such as Calcufluor white,

Blankophor increase sensitivity and speed

Sputum Cultures for Fungus

Horvath & Dummer, Am J Med 1996;100:171-8.

Bacteriological media inferior to fungal media –

32% higher yield on fungal media

Aspergillus workload and significance

3 year survey in Spanish teaching hospital404 isolates from 260 patients1/1000 micro samples positive

31/260 (12%) had invasive disease

Point score system for IA developed:Invasive sample positive 1> 2 positive samples 2leukaemia 2neutropenia 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.

Prospective study of 197 bronchial washes in 176 patients (most leukaemia, most lung infiltrates on X-ray)

Results

PCR detection of Aspergillus (rRNA target)

31 6 0 5

2 102 0 30

+ve PCR

-ve PCR

Immunocom-promised pts IA not IA

‘normal’ pts IA not IA

Positive predictive value (PPV) - 83.8% in at risk patientsNegative predictive value (NPV) - 98.1% in at risk patients

Buchheidt Br J Haematol 2002;116:803-811.

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.

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;20:1898-906; and others

• 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.

Aspergillus Antigen in BAL

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.

CLASSIFICATION OF ASPERGILLOSIS

Persistence without disease - colonisation of the airways or nose/sinuses

Airways/nasal exposure to airborne Aspergillus

Invasive aspergillosis• Acute (<1 month course)• Subacute/chronic necrotising (1-3 months)

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• Allergic Aspergillus sinusitis (eosinophilic

fungal rhinosinusitis)

Simple aspergilloma

Patient RTDecember 2002

Cough (mild) &tired

Wythenshawe Hospital

AspergillomaAspergilloma

Severo on www.aspergillus.man.ac.uk

Chronic Cavitary Pulmonary Aspergillosis

Normal smoking 30 year woman

Patient JAJan 2001

Chronic Cavitary Pulmonary Aspergillosis

Patient JAFeb 2002

Chronic Cavitary Pulmonary Aspergillosis

Patient JAApril 2003

Chronic Cavitary Pulmonary Aspergillosis

Patient JAJuly 2003

Chronic cavitary pulmonary aspergillosis an example of radiographic failure

Patient SSApril 2004

www.aspergillus.man.ac.uk

Patient SSJuly 2004, despite receiving itraconazole for 3 months

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

Contribution of CT scans and antibody testing to rapid diagnosis

of IA

Caillot et al, J Clin Oncol 2001;19:253 (unpublished data)

Pre Oct ‘91 Post Oct ‘91 P value

Patients 22 19

Mean time from IPA sign to diagnosis

6.8 + 5 days

2.2 + 2.3 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 NS

Antibody diagnosis of invasive aspergillosis

Herbrecht et al, J Clin Microbiol 2002;20:1898-906

In house ELISA method

Definite IA20/31 (64.5)

Probable IA11/67 (16.4)

Possible IA14/55 (25.5)

All episodes 45/153 (29.4)

www.aspergillus.man.ac.uk