Diagnostik invasiver Pilzinfektionen
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1 Diagnostik invasiver Pilzinfektionen Cornelia Lass-Flörl Medizinische Universität Innsbruck Bonn, April 2005
2 The medically most important opportunistic mycoses in Europe are caused by Aspergillus spp. and Candida spp. Aspergillus spp. A. fumigatus is the most common etiologic agent (80-90%), other species are A.flavus (10-15%), A.terreus (2-5%), A.niger (1-2%). Candida spp. C. albicans, with an increasing incidence of C. glabrata, C. tropicalis and C. krusei. Kullberg et al. Epidemiology of opportunistic invasive mycosis. Europ. J Med. Research, 2002
3 Approaches to diagnosis Non-culture methods Serology Nucleic acid amplification Radiology/MR imaging Culture methods Microscopic examination
4 Non-culture approaches to fungal diagnosis Candida Aspergillus Detection Cell wall components Mannans 1,3-β-D-glucans chitin Galactomannan 1,3-β-D-glucans chitin LA ELISA RIA Amebocyte lysate assay Spectrophotometry Cytoplasmic antigens Enolase HSP-90 ELISA Immunoblot Metabolites arabinitol D-mannitol GLC Massspectrosco. Genomic DNA sequences C-14 lanosterol demethylase Chitin synthase Actin Aspartate proteinase Ribosomal RNA genes C-14-lanosterol demethylase Alkaline protease Mitochondrial DNA HSP-90 Ribosomal RNA genes PCR
5 Why the lack of progress? Clinical manifestations are non-specific Conventional diagnostic tests insensitive, positive late Inability to perform invasive diagnostic procedures
6 Comparison of diagnostic tests in proven invasive aspergillosis 1 Finding Chest radiograph Sensitivity (N=18) 94% Specificity (N=82) 60% CT of lung 2 Any abnormality Halo sign BAL growing Aspergillus 3 Aspergillus Galactomannan EIA 1 result > 2 results 78% 28% 50% 94% 94% 7% 93% 92% 85% 99% 1 data from maertens; 2 CT done in 15 cases and 15 controls; 3 BAL performed in 16 cases and 26 controls
7 Sensitivity and specificity of Platelia aspergillus galactomannan immunoassay Reference Sensitivity Positive/total (%) Specificity Negative/total (%) Comment Maertens 02 Prov 17/18 (94) Prob 0/0 Pos 0/6 (0) 72/73 (99) Prospetive monitoring allo-hsct, 2x/week, cut-off 1.5, cons. Positive Herbrecht 02 Prov 20/31 (64) Prob (16) Pos 14/55 (25) 607/640 (95) Prospective monitoring and diagnostic evaluation of suspected IA, cut off 1.5, single positive Pinel 03 Prov 0/3 (0) Prob 14/31 (45) 748/751 (99) Prospective monitoring during neutropenia, cut off 1.5 Becker 03 Serum 8/17 (47) BAL 17/17 (100) BAL 19/22 ( /143 (93) 143/143 (100) 176 /176 (100) Prospective diagnostic evaluation during neutropenia, CT guided BAl, cut off 1.0 McLaughlin 04 Prov 12/13 (92) Prob 8/11 (73) 589/607 (97) Retrospective, cut off 0.5, single positive
8 Causes for false-positive Ag-EIA Condition Reproducible low-positive results by negative results GVHD Mechanism Concomitant antifungal treatment, low fungal burden Auto-Antibodies Reference Bentsen 05 Hamaki 01 Allogeneic HSCT Contamination with cotton Intestinal break down-dietary galactomannan Shared glucopyranose Herbrecht 02 Dalle 02 Penicillium contamination Fungemia or bacteremia Medications Cyclophosphamide treatment Shared galactomannan Cross reactive antigen Contaminating galactomannan Cross-reactive metabolite Kappe 93, Stynen 92 Swanink 97, Herbrecht 02 Pinel 03, Ansorg 97, Viscoli 05 Hashiguchi 94
9 Detection of Fungal DNA by PCR Einsele, 1997; Buchheidt 2004; Bialek 2001; Lass-Flörl 2004;... Clinical specimens! blood! serum! tissue biopsies! BAL! CSF Different protocols DNA extraction PCR design Amplicon Primer 2004: UK (Williams) Ringversuch PCR: 7 Labors, PCR Übereinstimmung: 56%
10 Problems with PCR Contamination -extraction -setup, probes -reagents -environmental Lack of standardisation -in-house assays -comparisons difficult Lack of interpretation of the data
11 The performance of (1, 3)-beta-D-glucan and Aspergillus galactomannan measurement for early diagnosis of invasive aspergillosis in patients with hematological diseases!.based on the analysis of 69 results with confirmed disease status, theoverallperformanceof the Fungitec, the Wako, and the ELISA test were as follows: sensitivity was 0.78, 0.63, and 0.50, respectively, whereas the specificity was 0.85, 0.98, and 1.0, respectively. (Horiguchi I 2004)
12 Proven invasive moulds infections 12 Asp.spp A.terreus zygomycosis Open window hospital Laminar flow unit construction construction CT guided biopsies 1. No single method for diagnostic 2. CT guided biopsies: sensitivity 89%, specificity 90% 3. Shift: Aspergillus to Mucor 4. Shift: Heamatology to ICUs 5. A. terreus strains endemic, different strains 6. 10% better outcome for VOR, CAS in comp. to AMB First line: CAS, VOR Lass-Flörl, 05
13 Kosten der antifungalen und antibakteriellen Therapie am LKI Datum Euro
14 Puzzle diagnostic Clinical features Microscopic Examination COMBINATION Culture Antigen tests
15 ...lots of work...! Thank you!
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