1. Diagnostic advances for distinct patient populations
Prof. Jean-Pierre GANGNEUX
Parasitology and Mycology, Rennes Teaching Hospital
Brittany, FRANCE
EA 4427 Signalisation et réponse aux agents infectieux et chimiques,
IRSET – Institut de Recherche Santé Environnement Travail – IFR 140, Université Rennes 1

2. Which diagnostic marker for which disease?
- Aspergillus is a fungus responsible for a wide range of diseases
- Aspergillosis results from a complex host-pathogen interaction
Aspergillus is a fungus responsible for a wide range of diseases as exposed by DD. They are the consequences of a complex host-pathogen interaction

3. Diagnostic tools available and their limits
1. Mycology and cytology:
Direct examination
Culture
Cytology
- Time-consuming
- Needs expertise
- Variable sensitivity
Positive culture means either infection or colonisation
Shows vascular invasion
No identification (Aspergillus sp., Fusarium sp., Scedosporium sp.)
008, IJP

4. Diagnostic tools available and their limits
2. Serology:
Antibody and antigen detection (Galactomannan and b1-3-D-glucan)
Variable sensitivity according to the patient / immune background
False positivity
3. PCR and mass spectrometry:
- Still need a standardization
- Less and less costly
4. Markers of allergy: Eosinophils, PMN, total IgE, specific IgE
- Specificity?

5. Diagnostic tools available and their limits
5. Imaging:
Radiography CT scan
Sensitivity?
Specificity?
Improved performances
More delayed and costly
Flow rupture

6. Variable contribution of diagnostic tools according to the disease
Mycology, PCR, MS
Anti-Aspergillus antibodies
Aspergillus antigens
Allergic markers
Imaging
Chronic pulmonary aspergillosis + ++ -
Radiography
Invasive aspergillosis
CT scan
Allergic aspergillosis
+/-
Strategies with combined tests adapted to the disease and the patient
 warrant an early diagnosis and appropriate treatment

7. Invasive aspergillosis
Tissue invasion  rapid damage  angioinvasion  dissemination
Hematological malignancies represent the most important risk factors
Hematological malignancies
Sensitivity of mycology
30-67%
Specificity
of mycology
72%
GM antigenemia
Meta-analysis
58% all patients
65% BMT
(25%-100%)
b1-3-D-glucan
55%-68%
PCR
54%-88%
Imaging : CT scan
Halo sign/ air- crescent
Antifungals decreased the sensitivity of culture
 Prophylaxis and empirical antifungal strategies must be known to interpret the results of mycology
Impact of neutropenia
< 100 PMN/L
(n=18)
> 100 PMN/L
(n=81) P
Sensitivity GM
61%
19%
0.001
Reichenberger BMT 1999; Maertens JCM 1999; Pfeiffer CID 2006; Cordonnier CMI 2009; Koo CID 2009; Mengoli Lancet ID 2009 ; White JCM 2010

8. 57% : hematological malignancies
But !! 43% nonneutropenic nonhematological patients
59%
89%
Mortality

9. Solid organ recipients
Invasive aspergillosis
Invasive aspergillosis must be recognised in non hematological patients
Solid organ recipients
COPD
Sensitivity of mycology
40%-50%
83%
Specificity
of mycology
5-8% (Lung transplant)
22%
GM antigenemia
22%-60%
42%-48%
b1-3-D-glucan
Insufficient evaluation
PCR
Antibodies (precipitins) ? +
Imaging
Mainly consolidation and nodules
Decreased specificity
« but must not be trivialised »*
Transplant
GM antigenemia
Lung
22%-60%
Liver
56%
Bulpa ERJ 2007*; Singh CMR 2005; Cornelius JCM 2007; Pfeiffer CID 2006; Guinea CMI 2009; Meersseman CCM 2004; Cornillet CID 2006; Husain Transplantation 2007

10. Heterogeneous population
Risk factors for IPA in non-neutropenic critically ill patients in the ICU
Solid Organ Transplantation
COPD
High-dose systemic corticosteroids (Prednisone equivalent >20 mg/day) > 3 weeks
Chronic renal failure
Liver cirrhosis/acute hepatic failure
Diabetes mellitus
Systemic disease requiring immunosuppressive therapy
Near-drowning, severe burns, etc…
Beware of confusing factors for the diagnosis :
Mechanical ventilation  clinical signs difficult to interpret
Radiological diagnosis  clouded by underlying lung pathologies
Aspergillus isolation  infection /colonisation?
Antibody detection  often weak in patients on long-term steroid therapy
False positivity of galactomannan detection (serum and BAL):
 Beta-lactam antibiotics, other fungi, dietary antigens, pediatrics
Specific ICU false positivity of galactomannan detection (serum and BAL):
 hemodialysis, cirrhosis, bacteriemia, IV Ig, cellulose, antitumor polysaccharides, abdominal surgery

11. Invasive aspergillosis: Summary
Hematological patients
Mycology
Cytology
GM Ag
b-glucan
PCR
(blood)
BAL (culture-Ag-PCR
Imaging
Antibodies
Criteria for Dg + -
Markers to exclude infection
Non hematological patients
Mycology
Cytology
GM Ag
b-glucan
PCR
(blood)
BAL (culture-Ag-PCR
Imaging
Antibodies
Criteria for Dg +
+/-
(less sensitive)
(less specific)
Markers to exclude infection ?

12. precipitin antibodies
Chronic Pulmonary Aspergillosis
Underlying condition + colonisation  chronic destruction of lung tissue  Cavitary or fibrosing lesions associated to an overexpressed immune host response
mycology/cytology or
precipitin antibodies +
Permission DW Denning
Aspergilloma
Chronic cavitary pulmonary aspergillosis (CCPA)
Chronic fibrosing pulmonary aspergillosis (CFPA)
IgE more informative on the underlying condition than for the diagnosis?
Immunocompetent patients with a chronic clinical and radiological evolution (>3 months)
Denning CID 2003; Smith & Denning ERJ 2010

13. ABPA
Genetic predisposition (asthma, cystic fibrosis) + sensitisation to Aspergillus
 Pulmonary eosinophilic inflammation and airway remodeling
Histopathologic findings in a patient with allergic bronchopulmonary aspergillosis
Agarwal R Chest 2009;135:
©2009 by American College of Chest Physicians

14. Rosenberg and Patterson criteria for the diagnosis of ABPA
Major Criteria
« ARTEPICS »
Minor criteria
- Asthma
- Roentgenographic fleeting pulmonary opacities
- Skin test positive for Aspergillus (HS type I)
- Eosinophilia
Precipiting antibodies (IgG) in serum
- IgE in serum > IU/mL
Central bronchiectasis
- Serums A. fumigatus-specific IgG and IgE
- Aspergillus in sputum
- Expectoration of brownish black mucus plugs
- Skin reaction type III to Aspergillus antigen
Complex diagnosis
Because colonisation and sensitisation may precede ABPA for many years, treatment has a hard (impossible??) task to act against long-term immunological disorders and tissue damage
Rosenberg Ann Int Med 1977 ; Patterson Arch Int Med 1986

15. Which markers for early patient screening?
- ABPA during asthma
Aspergillus skin test in patients with bronchial asthma (Agarwal Chest 2009)
- ABPA during cystic fibrosis
IgE (total and anti-Aspergillus)
Precipiting IgG
Aspergillus detection in sputum
. Clinical value during ABPA?
. Clinical value before ABPA?

16. Rennes Teaching Hospital CF centers: Long-term follow up of 84 CF patients since 2005
19 non-colonised
38 colonised with Aspergillus
- 27 ABPA
 comparative performances of Aspergillus detection in sputum and of classical biological markers in the diagnosis of ABPA
Sensitivity
Specificity
Positive
predictive value
Negative
Positive sputum for Aspergillus
- By mycological examination
- By real time PCR
41.7%
50%
63.3%
31,3%
28,6%
73.1%
71.4%
Positive anti-A. fumigatus antibodies
62.5%
71.7%
57,7%
82.7%
Total IgE (>500 UI/microL)
91.7%
75.9%
62,9%
95.3%
Positive anti-A. fumigatus IgE
95.8%
94.4%
88,5%
98.1%
Eosinophil polymorphonuclear counts (>500/L)
25%
89.5%
73,9%
1. Specific anti-Aspergillus IgE
2. 50% of the patients benefited from an antifungal treatment (+/-corticosteroids)
=> Aspergillus detection : marker of infection + efficacy of antifungals

17. Evolution of the clinical status of our cohort of 84 patients between 2005 and 2007
Non-colonised patients 33 19
- 16 %
Patients colonised with Aspergillus 27 38
+ 13 %
ABPA patients 24
+ 3 %
Screening for colonisation: An early step for the management of ABPA
Interest of real time PCR in sputum?
Positive sputum for Aspergillus
N = 208 (84 patients)
Sensitivity
Specificity
Positive
predictive value
Negative
By mycological examination
- By real time PCR
41.7%
50%
63.3%
31.3%
28.6%
73.1%
71.4%

18. Identification of patients with Aspergillus colonisation using real time PCR
Baxter et al. : 104 patients with CF
Park et al. : 54 sputum samples from ABPA, CPA and volunteers N
Culture +
PCR +
Culture –
PCR –
Baxter et al.
104 33
42 (40%) 29
Park et al. 74 14
31 (41%)
Clinical value of culture – PCR + patients?
Baxter et al.: 40% of PCR positive patients had serological sensitisation
46% had serological infection without sensitisation

19. Detection of antifungal resistance in Aspergillus
Detection of antifungal resistance in Aspergillus ? => MIC determination
A. fumigatus
A. terreus
AmB : S
AmB : R

20. Two difficulties exist
The validation of breakpoints
The low culture positive rates observed during invasive aspergillosis, CPA and ABPA : 30%-60%
 What is the level of resistance in non-culturable Aspergillus ?
30 positive sputum for Aspergillus amplification (MycAssayTM) but were culture negatives
S. Park et al., 2010
Amplification of the CYP51A gene using a nested PCR + analysis of azole resistance SNPs (single nucleotid polymorphisms)
However, low culture positive rates are observed during IA, CPA and ABPA as we have seen previously, and this raises the lack of knowledge on antifungal resistance in patients with culture negative. Here again, molecular amplificationallows the study of non cukturable Aspergillosis as demonstrated in the group of D. Denning. Among 54 samples assyed, 30 were PCR positive. DNA amplified was the used for a nested PCR followed by the analysis of SNP on the CYP51A gene .
18/30 (60%) with an azole resistant mutation  Clinical value??
- some of the patients had documented treatment failure after single azole/panazole therapy
- some of the patients had never received triazole therapy
- need to be evaluated in large cohorts

21. Predictive markers for Aspergillus infection?
The future of biology:
Predictive markers for Aspergillus infection?
Bochud PY et al, NEJM 2008
- TLR4 haplotypes in unrelated donors are associated with an increased risk of IA among recipients of allogeneic hematopoietic-cell transplants
- Polymorphisms in genes encoding IL-1, IL-10, TNF r2, TLR1, TLR6…
Seo, BMT 2005; Kesh Ann N Y Acad Sci 2005; Sainz Immunol lett 2007; Sainz Human Immunol 2007; Vaid Clin Chem Lab Med 2007; Sainz J Clin Immunol 2008