Jacques Bille, MD
Institut of Microbiology
University Hospital
Lausanne, Switzerland

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"

Invasive aspergillosis : update on conventional diagnosis

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 207-238

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

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

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

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)
2)
Choi JK et al., Am J Clin Pathol 121: 18, 2004
Pierard GE et al., Am J Clin Pathol 96: 373, 1991
Verweij PE et al., Am J Clin Pathol 49: 798, 1996

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

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

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

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 ?

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

Invasive aspergillosis : update on conventional diagnosis
Yield of culture for molds in histopathologic positive samples
Autopsy samples n= culture positive
23
Surgical or biopsy tissue 30
12
9
52%
30%
Tarrand JJ et al., Am J Clin Pathol 2003; 119: 854

Invasive aspergillosis : update on conventional diagnosis
Culture
Can we do better ?
Mimic physiologic termperature and decreased oxygen environment : 35 °C, 6% O
2
-10% CO
2
 significant increase of Aspergillus spp from autopsy tissue and various clinical samples (+ 31%)
Tarrand JJ et al., J Clin Microbiol 2005; 43: 382

Invasive aspergillosis : update on conventional diagnosis
Culture :
BAL is better than sputum (?)
Overall sensitivity 50%
Diagnostic yield of bronchoscopy specimen in histologically proven IPA
No. of cases Bronchoscopy diagnostic
Albeda 1984 15
Treger 1985 16
Kahn 1986 17
Saito 1988 18
Levy 1992 11
Mc Whinney 1993 19
Saugier-Weber 1993 2
Von Eiff 1995 12
Horvath 1996 8
Caillot 1997 20
Baron 1998 4
Reichenberger 1999 9
12
29
18
13
23
14
12
27
9
16
12
10
6
11
8
8
7
9
6
3
5
8
13
0
36 %
67 %
48 %
0 %
56 %
50 %
30 %
50 %
38 %
45 %
61 %
30 %
Overall 195 84 43 %
Adapted from Reichenberger et al., Bone Marrow Transplantation 1999; 24: 1195

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

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)
2)
3)
Nalesnik et al., J Clin Microbiol 1980; 11: 370
Greub and Bille, Clin Microbiol Infect 1998; 4: 710
Bouza and Muñoz, J Clin Microbiol 2005; 43: 2075

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
HIV/AIDS
Corticosteroids
20-60%
14-20%
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

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

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

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