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

Diagnosis of pulmonary aspergillosis(ignoring allergy)

David W. DenningWythenshawe Hospital

University of Manchester

Conceptual framework

Aspergilloma - CCPA - CNPA/subacute IPA - acute IPA

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Hyphal load in tissue

Normal

Massive

Vascular invasion, necrosis,

disseminationGranulomas, acute

inflammation, central necrosis

Chronic inflammation and fibrosis

Testing performance?Im

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Culture + +/- +/- +/- +/-Antigen - - - + ++Glucan +/- +? +? ++ +/-Antibody +++ +++ ++ +? -PCR (resp) ++ +? ++? ++? ++PCR (blood) -? -? -? +/- +

Aspergilloma - CCPA - CNPA/subacute IPA - acute IPA

Testing performance?

Pulmonary defect + innate immune defect

corticosteroidsneutrophil defect

neutropeniamultiple defects

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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?

Pulmonary defect + innate immune defect

corticosteroidsneutrophil defect

neutropeniamultiple defects

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Culture (+) +/- +/- +/- +/-Antigen (-) - - + ++Glucan (+/-) +? +? ++ +/-Antibody (+++) +++ ++ +? -PCR (resp) (++) +? ++? ++? ++PCR (blood) -? -? -? +/- +

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

• 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.

Aspergillus Antigen in BAL

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

Unequivocal ‘Halo sign’ surrounding a nodule

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

Small vessel angioinvasion

Halo

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

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

Test sensitivity important:

Microscopy methodology

Culture versus PCR

Histopathology versus culture

Test sensitivity important:

Microscopy methodology

Culture versus PCR

Histopathology versus culture

Microscopy

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

Fluorescent brighteners such as Calcufluor white,

Blankophor increase sensitivity and speed

Test sensitivity important:

Microscopy methodology

Culture versus PCR

Histopathology versus culture

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.

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

• 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

Sanguinetti, Clin Microbiol. 2003;41:3922-5.

Culture Antigen PCRProven/probable IAAll haem malignancy 6/20 20/20 18/20

Perlin , unpublished

Real time PCR to distinguish Aspergillus

speciesA. terreus resistant to

amphotericin B

Additional sensitivity will allow species detection and possibly resistance detection on culture negative clinical specimens

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[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

www.aspergillus.man.ac.uk