1. Invasive aspergillosis : Update on conventional diagnosis
Jacques Bille, MD
Institut of Microbiology
University Hospital
Lausanne, Switzerland

2. Invasive aspergillosis : update on conventional diagnosis
The problems
The disease is evolving
Clinical signs/ symptoms are non specific
Conventional diagnosis is insensitive or too late
Non conventional diagnosis is "promising"

3. Invasive aspergillosis : update on conventional diagnosis
The disease is evolving
A continuum of clinical presentations
New (?) hosts
Underdiagnosed ante-mortem

4. Invasive aspergillosis : update on conventional diagnosis
Hope, WW, Walsh TJ, Denning DW. The invasive and saprophytic syndromes due to Aspergillus species. Medical Mycology 2005, 43: S

5. Invasive aspergillosis : update on conventional diagnosis
Clinical signs/symptoms are non specific
fever
dyspnea
non productive cough
hemoptysis
chest pain
pleural rib
In a proper host
rapidly progressive

6. Invasive aspergillosis : update on conventional diagnosis
• direct examination
of tissue
of indirect clinical specimens (sputum, BAL)
sputum/BAL tissue
unstained wet prep ± KOH
routine stains Gram HE fungal stains GMS, PAS GMS, PAS
fluorescent Calcofluor white
dyes Uvitex 2B
Blankophor

7. In: Hope et al., Lancet Infectious Diseases 5: 609, 2005
Invasive aspergillosis : update on conventional diagnosis
In: Hope et al., Lancet Infectious Diseases 5: 609, 2005

8. Invasive aspergillosis : update on conventional diagnosis
Histopathologic diagnosis of Aspergillosis
Improvement by immunohistochemical detection
Monoclonal antibodies
• WF-AF-1 (Dako) 1)
• EB-A1 2)
Sensitivity in culture-proven cases : 89-94%
Genus or species specific results
1) Choi JK et al., Am J Clin Pathol 121: 18, 2004
2) Pierard GE et al., Am J Clin Pathol 96: 373, 1991
Verweij PE et al., Am J Clin Pathol 49: 798, 1996

9. Invasive aspergillosis : update on conventional diagnosis
In: Choi JK, Am J Clin Pathol 121: 18, 2004

10. Invasive aspergillosis : update on conventional diagnosis
Culture
Isolation: Sabouraud (+ antibiotics)
Media blood agar, chocolate agar
Identification : malt-extract, corn-meal agar
Czapek agar
Incubation
temperature 25-30°C  improvement ?
athmosphere aerobic
duration 2-6 weeks

11. In: Andreoni et al., Medical Mycology Atlas
Invasive aspergillosis : update on conventional diagnosis
In: Andreoni et al., Medical Mycology Atlas

12. Invasive aspergillosis : update on conventional diagnosis
Culture
• Can we improve the sensitivity ? (in tissue)
• What is the best indirect specimen ?
Sputum or BAL
• What are the PPV of a positive culture for IA, and the sensitivity of culture to diagnose IA ?

13. Invasive aspergillosis : update on conventional diagnosis
Performance of a diagnostic test
Caveat
timing of sampling - evolution of the disease
per test vs per episode analysis
patient population
Prior antifungal prophylaxis or therapy

14. Invasive aspergillosis : update on conventional diagnosis
Yield of culture for molds in histopathologic positive samples
n= culture positive
Autopsy samples %
Surgical or biopsy tissue %
Tarrand JJ et al., Am J Clin Pathol 2003; 119: 854

15. Invasive aspergillosis : update on conventional diagnosis
Culture
Can we do better ?
Mimic physiologic termperature and decreased oxygen environment : 35°C, 6% O2 -10% CO2
 significant increase of Aspergillus spp
from autopsy tissue and various clinical samples (+ 31%)
Tarrand JJ et al., J Clin Microbiol 2005; 43: 382

16. Diagnostic yield of bronchoscopy specimen in histologically proven IPA
Invasive aspergillosis : update on conventional diagnosis
Culture : BAL is better than sputum (?)
Overall sensitivity 50%
Adapted from Reichenberger et al., Bone Marrow Transplantation 1999; 24: 1195
Diagnostic yield of bronchoscopy specimen in histologically proven IPA
No. of cases Bronchoscopy diagnostic
Albeda %
Treger %
Kahn %
Saito %
Levy %
Mc Whinney %
Saugier-Weber %
Von Eiff %
Horvath %
Caillot %
Baron %
Reichenberger %
Overall %

17. Invasive aspergillosis : update on conventional diagnosis
The paradox of a positive sputum culture for Aspergillus
low sensitivity for IA
low specificity for IA
Can we improve ?

18. Invasive aspergillosis : update on conventional diagnosis
How to increase the PPV for IA of a positive sputum culture
• at (high) risk patient
• multiple positive samples 1)
• quantitative culture 1) 2)
• use of a score 2) 3)
1) Nalesnik et al., J Clin Microbiol 1980; 11: 370
2) Greub and Bille, Clin Microbiol Infect 1998; 4: 710
3) Bouza and Muñoz, J Clin Microbiol 2005; 43: 2075

19. Invasive aspergillosis : update on conventional diagnosis
Positive predictive value of a positive sputum culture for IA
Highly variable (15-77%)
Depends on host factors
allo BMT 60%
leukemia + neutropenia 70-80%
SOT 20-60%
HIV/AIDS 14-20%
Corticosteroids 20%
Perfect et al. (MSG), Clin Infect Dis 2001; 33: 1824
Yu et al., Am J Med 1986; 81: 249
Horvath and Dummer, Am J Med 1996; 100: 171

20. Invasive aspergillosis : update on conventional diagnosis
Prospective assessment of the clinical signi- ficance of isolating A.fumigatus by culture
404 A.fumigatus positive cultures (260 patients)
90 (22.3%) from 31 (12%) patients with IA
6% if 1+ cult.
18% 2+
38% 3+
10% if 1-2 score
40% 3-4
70%  5
Bouza et al., J Clin Microbiol 2005; 43: 2075
Score
Criteria points
Invasive procedure 1
 2 + cultures 1
Leukemia 2
Corticosteroids 2
Neutropenia 5

21. Invasive aspergillosis : update on conventional diagnosis
Radiology
2 interesting recent findings :
Systematic CT • is more sensitive than galactomannan for early detection of IA1)
• reduces the amount of antifungal therapy when combined to GM screening2)
Angio CT has a higher specificity than CT for IA3)
1) Weisser et al., Clinical Infectious Diseases 2005; 41: 1143
2) Maertens et al., Clinical Infectious Diseases 2005; 41: 1242
3) Sonnet et al., Am J Roentgenol 2005; 184: 746

22. Invasive aspergillosis : update on conventional diagnosis
Conclusions
Conventional diagnosis of IA is :
suboptimal
indispensable
 genus, species
 AFST
perfectible