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

Aspergillus Life-cycle Germination Spores inhaled Hyphal elongation and branching Mass of hyphae (plateau phase) www.aspergillus.man.ac.uk

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.

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)

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.

Small vessel angioinvasion Unequivocal ‘Halo sign’ surrounding a nodule Halo Small vessel angioinvasion Herbrecht, Denning et al, NEJM 2002;347:408-15.

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

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

Pulmonary nodules a useful feature if invasive pulmonary aspergillosis CT features in 48 CTs of which 17 IPA IPA Other Halo 13/17 0/31 Nodules 14/17 11/31 Masses 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

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

Microscopy Fluorescent brighteners such as Calcufluor white, Blankophor increase sensitivity and speed Ruchel R, www.aspergillus.man.ac.uk/images

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.

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.

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) - 83.8% in at risk patients Negative 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

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.

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 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)

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

Aspergilloma Severo on www.aspergillus.man.ac.uk

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

Chronic Cavitary Pulmonary Aspergillosis Patient JA Feb 2002

Chronic Cavitary Pulmonary Aspergillosis Patient JA April 2003

Chronic Cavitary Pulmonary Aspergillosis Patient JA July 2003

Chronic cavitary pulmonary aspergillosis an example of radiographic failure Patient SS April 2004 Patient SS July 2004, despite receiving itraconazole for 3 months www.aspergillus.man.ac.uk

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 Caillot et al, J Clin Oncol 2001;19:253 (unpublished data)

Antibody diagnosis of invasive aspergillosis In house ELISA method Definite IA 20/31 (64.5) Probable IA 11/67 (16.4) Possible IA 14/55 (25.5) All episodes 45/153 (29.4) Herbrecht et al, J Clin Microbiol 2002;20:1898-906

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