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Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA.

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Presentation on theme: "Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA."— Presentation transcript:

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 - Aspergillus is a fungus responsible for a wide range of diseases - Aspergillosis results from 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 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 5. Imaging: Radiography CT scan - Sensitivity? - Specificity? - Improved performances - More delayed and costly - Flow rupture Diagnostic tools available and their limits

6 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

7 Invasive aspergillosis Tissue invasion rapid damage angioinvasion dissemination Hematological malignancies Sensitivity of mycology 30-67% Specificity of mycology 72% GM antigenemia Meta-analysis 58% all patients 65% BMT (25%-100%) 1-3-D-glucan 55%-68% PCR Meta-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

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

9 Solid organ recipients COPD Sensitivity of mycology 40%-50%83% Specificity of 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 Lung22%-60% Liver56% 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

10 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? 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

11 Invasive aspergillosis: Summary Hematological patients Mycology Cytology GM Ag -glucanPCR (blood) BAL (culture- Ag-PCR ImagingAntibodies Criteria for g ++++- Markers to exclude infection ++- Non hematological patients Mycology Cytology GM Ag -glucanPCR (blood) BAL (culture- Ag-PCR ImagingAntibodies Criteria for g ++/- (less sensitive) +/- (less specific) ++/- Markers to exclude infection +?+

12 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/cytology or precipitin antibodies + Permission DW Denning

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;135:805-826 ©2009 by American College of Chest Physicians

14 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 criteria for 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

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 SensitivitySpecificityPositive predictive value Negative predictive 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 of 84 CF patients since 2005 - 19 non-colonised - 38 colonised with Aspergillus 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 Clinical status20052007 2005-2007 Non-colonised patients3319- 16 % Patients colonised with Aspergillus2738+ 13 % ABPA patients2427+ 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) SensitivitySpecificityPositive predictive value Negative predictive value -By mycological examination - By real time PCR 41.7% 50% 63.3% 50% 31.3% 28.6% 73.1% 71.4%

18 Baxter et al. : 104 patients with CF Park et al. : 54 sputum samples from ABPA, CPA and volunteers NCulture + PCR + Culture – PCR + Culture + PCR – Culture – PCR – Baxter et al.1043342 (40%)029 Park et al.741431 (41%)029 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 colonisation using real time PCR

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

20 1.The validation of breakpoints 2.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 (MycAssay TM ) but were culture negatives S. Park et al., 2010

21 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


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