Presentation on theme: "Diagnostic advances for distinct patient populations"— Presentation transcript:
1 Diagnostic advances for distinct patient populations Prof. Jean-Pierre GANGNEUXParasitology and Mycology, Rennes Teaching HospitalBrittany, FRANCEEA 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 interactionAspergillus 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 examinationCultureCytology- Time-consuming- Needs expertise- Variable sensitivityPositive culture means either infection or colonisationShows vascular invasionNo 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 backgroundFalse positivity3. PCR and mass spectrometry:- Still need a standardization- Less and less costly4. Markers of allergy: Eosinophils, PMN, total IgE, specific IgE- Specificity?
5 Diagnostic tools available and their limits 5. Imaging:Radiography CT scanSensitivity?Specificity?Improved performancesMore delayed and costlyFlow rupture
6 Variable contribution of diagnostic tools according to the disease Mycology, PCR, MSAnti-Aspergillus antibodiesAspergillus antigensAllergic markersImagingChronic pulmonary aspergillosis+++-RadiographyInvasive aspergillosisCT scanAllergic 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 disseminationHematological malignancies represent the most important risk factorsHematological malignanciesSensitivity of mycology30-67%Specificityof mycology72%GM antigenemiaMeta-analysis58% all patients65% BMT(25%-100%)b1-3-D-glucan55%-68%PCR54%-88%Imaging : CT scanHalo sign/ air- crescentAntifungals decreased the sensitivity of culture Prophylaxis and empirical antifungal strategies must be known to interpret the results of mycologyImpact of neutropenia< 100 PMN/L(n=18)> 100 PMN/L(n=81)PSensitivity GM61%19%0.001Reichenberger BMT 1999; Maertens JCM 1999; Pfeiffer CID 2006; Cordonnier CMI 2009; Koo CID 2009; Mengoli Lancet ID 2009 ; White JCM 2010
9 Solid organ recipients Invasive aspergillosisInvasive aspergillosis must be recognised in non hematological patientsSolid organ recipientsCOPDSensitivity of mycology40%-50%83%Specificityof mycology5-8% (Lung transplant)22%GM antigenemia22%-60%42%-48%b1-3-D-glucanInsufficient evaluationPCRAntibodies (precipitins)?+ImagingMainly consolidation and nodulesDecreased specificity« but must not be trivialised »*TransplantGM antigenemiaLung22%-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
10 Heterogeneous population Risk factors for IPA in non-neutropenic critically ill patients in the ICUSolid 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 interpretRadiological diagnosis clouded by underlying lung pathologiesAspergillus isolation infection /colonisation?Antibody detection often weak in patients on long-term steroid therapyFalse positivity of galactomannan detection (serum and BAL): Beta-lactam antibiotics, other fungi, dietary antigens, pediatricsSpecific ICU false positivity of galactomannan detection (serum and BAL): hemodialysis, cirrhosis, bacteriemia, IV Ig, cellulose, antitumor polysaccharides, abdominal surgery
11 Invasive aspergillosis: Summary Hematological patientsMycologyCytologyGM Agb-glucanPCR(blood)BAL (culture-Ag-PCRImagingAntibodiesCriteria for Dg+-Markers to exclude infectionNon hematological patientsMycologyCytologyGM Agb-glucanPCR(blood)BAL (culture-Ag-PCRImagingAntibodiesCriteria for Dg++/-(less sensitive)(less specific)Markers to exclude infection?
12 precipitin antibodies Chronic Pulmonary AspergillosisUnderlying condition + colonisation chronic destruction of lung tissue Cavitary or fibrosing lesions associated to an overexpressed immune host responsemycology/cytologyorprecipitin antibodies+Permission DW DenningAspergillomaChronic 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
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)- EosinophiliaPrecipiting antibodies (IgG) in serum- IgE in serum > IU/mLCentral bronchiectasis- Serums A. fumigatus-specific IgG and IgE- Aspergillus in sputum- Expectoration of brownish black mucus plugs- Skin reaction type III to Aspergillus antigenComplex diagnosisBecause colonisation and sensitisation may precede ABPA for many years, treatment has a hard (impossible??) task to act against long-term immunological disorders and tissue damageRosenberg Ann Int Med 1977 ; Patterson Arch Int Med 1986
15 Which markers for early patient screening? - ABPA during asthmaAspergillus skin test in patients with bronchial asthma (Agarwal Chest 2009)- ABPA during cystic fibrosisIgE (total and anti-Aspergillus)Precipiting IgGAspergillus 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-colonised38 colonised with Aspergillus- 27 ABPA comparative performances of Aspergillus detection in sputum and of classical biological markers in the diagnosis of ABPASensitivitySpecificityPositivepredictive valueNegativePositive sputum for Aspergillus- By mycological examination- By real time PCR41.7%50%63.3%31,3%28,6%73.1%71.4%Positive anti-A. fumigatus antibodies62.5%71.7%57,7%82.7%Total IgE (>500 UI/microL)91.7%75.9%62,9%95.3%Positive anti-A. fumigatus IgE95.8%94.4%88,5%98.1%Eosinophil polymorphonuclear counts (>500/L)25%89.5%73,9%1. Specific anti-Aspergillus IgE2. 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 patients3319- 16 %Patients colonised with Aspergillus2738+ 13 %ABPA patients24+ 3 %Screening for colonisation: An early step for the management of ABPAInterest of real time PCR in sputum?Positive sputum for AspergillusN = 208 (84 patients)SensitivitySpecificityPositivepredictive valueNegativeBy mycological examination- By real time PCR41.7%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 CFPark et al. : 54 sputum samples from ABPA, CPA and volunteersNCulture +PCR +Culture –PCR –Baxter et al.1043342 (40%)29Park et al.741431 (41%)Clinical value of culture – PCR + patients?Baxter et al.: 40% of PCR positive patients had serological sensitisation46% had serological infection without sensitisation
19 Detection of antifungal resistance in Aspergillus Detection of antifungal resistance in Aspergillus ? => MIC determinationA. fumigatusA. terreusAmB : SAmB : R
20 Two difficulties exist The validation of breakpointsThe 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 negativesS. Park et al., 2010Amplification 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