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

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

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) -? -? -? +/- +

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) -? -? -? +/- +

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.

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) -? -? -? +/- +

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

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.

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)

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

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

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

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)

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

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

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

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

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.

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;116:803-811.

Comparison of BAL antigen and real-time PCR Culture Antigen PCR Proven/probable IA All haem malignancy 6/20 20/20 18/20 Sanguinetti, Clin Microbiol. 2003;41:3922-5.

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

% 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 [All 41 83 100 Tarrand, 2003] AlloBMT 17 0 17 Roychowdhury, 2006

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

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.

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

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

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