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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 [email protected]

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Page 1: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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]

Page 2: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

- Aspergillus is a fungus responsible for a wide range of diseases

- Aspergillosis results from a complex host-pathogen interaction

Page 3: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 4: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 5: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

5. Imaging:

Radiography CT scan

- Sensitivity?- Specificity?

- Improved performances- More delayed and costly- Flow rupture

Diagnostic tools available and their limits

Page 6: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 7: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 8: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

57% : hematological malignancies

But !! 43% nonneutropenic nonhematological patients

59%

89%

Mortality

Page 9: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 10: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 11: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

+ ? +

Page 12: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 13: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 14: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 15: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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?

Page 16: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 17: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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%

Page 18: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 19: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

A. fumigatus A. terreus

AmB : S AmB : R

Detection of antifungal resistance in Aspergillus ? => MIC determination

Page 20: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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

Page 21: Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUX Parasitology and Mycology, Rennes Teaching Hospital Brittany, FRANCE EA

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