Diagnostic advances for distinct patient populations
Prof. Jean-Pierre GANGNEUXParasitology and Mycology, Rennes Teaching Hospital
Brittany, FRANCEEA 4427 Signalisation et réponse aux agents infectieux et chimiques,
IRSET – Institut de Recherche Santé Environnement Travail – IFR 140, Université Rennes [email protected]
- Aspergillus is a fungus responsible for a wide range of diseases
- Aspergillosis results from a complex host-pathogen interaction
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
3. PCR and mass spectrometry:- Still need a standardization- Less and less costly
2. Serology: Antibody and antigen detection (Galactomannan and 1-3-D-glucan)- Variable sensitivity according to the patient / immune background - False positivity
4. Markers of allergy: Eosinophils, PMN, total IgE, specific IgE- Specificity?
Diagnostic tools available and their limits
5. Imaging:
Radiography CT scan
- Sensitivity?- Specificity?
- Improved performances- More delayed and costly- Flow rupture
Diagnostic tools available and their limits
Mycology, PCR, MS
Anti-Aspergillus antibodies
Aspergillus antigens
Allergic markers
Imaging
Chronic pulmonary aspergillosis
+ ++ - - Radiography
Invasive aspergillosis
++ - ++ - CT scan
Allergic aspergillosis
+/- + - ++ Radiography
Strategies with combined tests adapted to the disease and the patient
warrant an early diagnosis and appropriate treatment
Variable contribution of diagnostic tools according to the disease
Invasive aspergillosis
Tissue invasion rapid damage angioinvasion dissemination
Hematological malignancies
Sensitivity of mycology
30-67%
Specificityof mycology
72%
GM antigenemiaMeta-analysis
58% all patients65% BMT(25%-100%)
1-3-D-glucan 55%-68%
PCRMeta-analysis
54%-88%
Imaging : CT scan
Halo sign/air- crescent
Reichenberger BMT 1999; Maertens JCM 1999; Pfeiffer CID 2006;Cordonnier CMI 2009; Koo CID 2009; Mengoli Lancet ID 2009 ; White JCM 2010
Impact of neutropenia
< 100 PMN/L(n=18)
> 100 PMN/L(n=81)
P
Sensitivity GM 61% 19% 0.001
Antifungals decreased the sensitivity of culture Prophylaxis and empirical antifungal strategies
must be known to interpret the results of mycology
Hematological malignancies represent the most important risk factors
57% : hematological malignancies
But !! 43% nonneutropenic nonhematological patients
59%
89%
Mortality
Solid organ recipients
COPD
Sensitivity of mycology
40%-50% 83%
Specificityof mycology
5-8%(Lung transplant)
22%
GM antigenemia
22%-60% 42%-48%
1-3-D-glucan Insufficient evaluation
PCR Insufficient evaluation
Antibodies (precipitins)
? +
Imaging Mainly consolidation and nodules
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
Decreased specificity« but must not be trivialised »*
Invasive aspergillosis
Invasive aspergillosis must be recognised in non hematological patients
Risk factors for IPA in non-neutropenic critically ill patients in the ICU
Solid Organ TransplantationCOPDHigh-dose systemic corticosteroids (Prednisone equivalent >20 mg/day) > 3 weeksChronic renal failureLiver cirrhosis/acute hepatic failureDiabetes mellitusSystemic disease requiring immunosuppressive therapyNear-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?
Heterogeneous population
-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
Invasive aspergillosis: Summary
Hematological patients
Mycology
Cytology
GM Ag -glucan
PCR(blood)
BAL (culture-Ag-PCR
Imaging
Antibodies
Criteria for g
+ + + + -
Markers to exclude infection
+ + -
Non hematological patients
Mycology
Cytology
GM Ag -glucan
PCR(blood)
BAL (culture-Ag-PCR
Imaging
Antibodies
Criteria for g
+ +/-(less
sensitive)
+/-(less
specific)
+ +/-
Markers to exclude infection
+ ? +
Chronic Pulmonary Aspergillosis
Underlying condition + colonisation chronic destruction of lung tissue Cavitary or fibrosing lesions associated to an overexpressed immune host response
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
mycology/cytologyor
precipitin antibodies+
Permission DW Denning
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;135:805-826
©2009 by American College of Chest Physicians
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 > 1.000 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
Rosenberg Ann Int Med 1977 ; Patterson Arch Int Med 1986
Rosenberg and Patterson criteriafor the diagnosis of ABPA
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
Which markers for early patient screening?
- ABPA during asthmaAspergillus 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?
Sensitivity Specificity Positivepredictive value
Negativepredictive value
Positive sputum for Aspergillus- By mycological examination- By real time PCR
41.7%
50%
63.3%
50%
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% 50% 73,9%
- 27 ABPA comparative performances of Aspergillus detection in sputum and of classical biological markers in the diagnosis of ABPA
Rennes Teaching Hospital CF centers: Long-term follow up of84 CF patients since 2005
- 19 non-colonised
- 38 colonised with Aspergillus
1. Specific anti-Aspergillus IgE2. 50% of the patients benefited from an antifungal treatment (+/-corticosteroids)=> Aspergillus detection : marker of infection + efficacy of antifungals
Evolution of the clinical status of our cohort of 84 patients between 2005 and 2007
Clinical status 2005 2007 2005-2007
Non-colonised patients 33 19 - 16 %
Patients colonised with Aspergillus 27 38 + 13 %
ABPA patients 24 27 + 3 %
Screening for colonisation: An early step for the management of ABPA Interest of real time PCR in sputum?
Positive sputum for AspergillusN = 208 (84 patients)
Sensitivity Specificity Positivepredictive value
Negativepredictive value
-By mycological examination
- By real time PCR
41.7%
50%
63.3%
50%
31.3%
28.6%
73.1%
71.4%
Baxter et al. : 104 patients with CFPark et al. : 54 sputum samples from ABPA, CPA and volunteers
N Culture +PCR +
Culture –PCR +
Culture +PCR –
Culture –PCR –
Baxter et al. 104 33 42 (40%) 0 29
Park et al. 74 14 31 (41%) 0 29
Clinical value of culture – PCR + patients?Baxter et al.: 40% of PCR positive patients had serological sensitisation
46% had serological infection without sensitisation
Identification of patients with Aspergillus colonisationusing real time PCR
A. fumigatus A. terreus
AmB : S AmB : R
Detection of antifungal resistance in Aspergillus ? => MIC determination
1. The validation of breakpoints2. The low culture positive rates observed during invasive
aspergillosis, CPA and ABPA : 30%-60% What is the level of resistance in non-culturable
Aspergillus ?
Two difficulties exist
Amplification of the CYP51A gene using a nested PCR + analysis of azole resistance SNPs (single nucleotid polymorphisms)
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
30 positive sputum for Aspergillus amplification (MycAssayTM) but were culture negatives
S. Park et al., 2010
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
