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1 The goal of teaching: 1. Principles of LDG of SFI: Hair Nail Skin Mucosal Eye Ear 3. Methods for fungal identification Test methods for fungal susceptibility testing Diffusion Dilution Etest 2. Principles of LDg of IFI: agglutination, precipitation, ELISA, PCR, PH invasive aspergillosis (IA) invasive candidiasis (IC) zygomycosis Pneumocystis pneumonia 4. Epidemiology of FI Risk groups of patients: Endemic FI (dimorphic and subcutaneous mycoses) Mycoallergens and mycoallergosis Mycotoxins and mycotoxicosis
2 BASIC PRINCIPLES FOR DIAGNOSIS OF FI Standard mycological methods (Conventional methods) 1. Microscopic examination of patients samples (direct microscopy) - preparation (DMP) - histopathology (PH) 2. Isolation of fungi from patient samples - mycological cultures (MC) IMMUNOLOGICAL METHODS: - Detection of antibodies (Ab) - Detection of antigen (Ag) - DETECTION OF BIOMARKERS MOLECULAR METHODS - Evidence of DNA (PCR) golden standard PROVEN FI PROBABLY/POSSIBLE FI
3 BASIC PRINCIPLES FOR DIAGNOSIS OF IFI non culture metods methods of choice for LDg of IFI Standard mycological methods (Conventional methods) - Blood cultures for fungi - biopsy IMMUNOLOGICAL METHODS: - Detection of antibodies (Ab) - Detection of antigen (Ag) - Detection of biomarkers MOLECULAR METHODS - Evidence of DNA (PCR) Problem: rarely positive, invasive methods of sampling, long lasting Problem: Ab interpretation in immunodeficient individuals Problem: possible contamination of samples Not standardized Advantage: PROVEN IFI Advantage : Early Dg of IFI Sensitive Fast Advantage: Early Dg of IFI High sensitive Fast
4 New strategy for Dg of IFI Host Clinical factors + features + Micology tissue = Proven IFI Host Clinical Micology factors + features + = Probable IFI Host factors + Clinical features Negative Not done = Host factors Negative + Micology = Possible IFI *EORTC/MSG criteria
5 Ideal diagnostic test Sensitive and specific High positive predictive value (PPV) High negative predictive value (NPV) Simple Cheap Fast
6 Why non cultural methods? (detection of biomarkers)? Principles of tests
7 Why biomarkers? Antigeni and antitela se lakše otkrivaju nego same gljive Antibodies are produced in large quantities and they are circulating Soluble fungal Ag often pass from tissue into the circulation Soluble fungal Ag can be found in the urine Soluble fungal Ag can be detected in a very small quantity Mycological cultures have low sensitivity: The small number of fungi in tissue or blood many samples are required
8 Fungal biomarkers and non culture methods Fungi structure Candida Aspergillus Methods Cell wall Cytoplasmic membrane Mannans 1,3-β-D-glucan chitin Enolase HSP-90 Galactomannan 1,3-β-D-glucan chitin LA ELISA RIA Amebocid lisat spectrophotometry ELISA Immunoblot Metabolites arabinitol D-mannitol GLC Mas. spectroscop. DNA/RNA C-14 lanosterol demethylase Chitin synthase Actin Aspartat proteinase Ribosomal RNA C-14-lanosterol demethylase Alcaline protease Mitochondrial DNA HSP-90 Ribosomal RNA genes PCR
9 Non cultural methods (agglutination, immunodiffusion, ELISA) Principles of Ag-Ab tests
10 Antigen Test Latex agglutination (LA) principles of test + Latex coated Ab Sample (+) fungal Ag Agglutinat Antibody Test + Sample Latex coat (+) fungal Ab Agglutinat
11 Latex agglutination (LA) use Application of test detection of Ag: Galactomannan Aspergillus Ag (IA*) Mannan Candida Ag (IC**) Glucuroxylomannan Cryptococcus Ag Application of test detection of Ab: Anti-galactomannan-Ab Anti-mannan-Ab Anti-glucuroxylomannan-Ab LA interpretation of finding *Invasive aspergillosis ** Invasive candidiasis
12 Immunodiffusion (ID) principles of test Immunobiology, 4 th Edition
13 Immunodiffusion (ID) principles of test Concentration
14 Immunodiffusion (ID) - principles of test Concentration
15 Immunodiffusion (ID) principles of test Kuby Immunology, 4 th Edition
16 Immunodiffusion (ID) principles of test Kuby Immunology, 4 th Edition
17 Immunodiffusion (ID) principles of test Kuby Immunology, 4 th Edition
18 Immunodiffusion (ID) use Using of ID: Aspergillosis (Ag, IgG) Subcutaneous mycosis (Ag, IgG) Endemic mycosis (Ag, IgG) Positive finding - precipitation
19 Enzyme-Linked Immunosorbent Assay (ELISA) Sandwich ELISA Principle of test detection of fungal Ag Adapted from Kuby Immunology, 4 th Edition
20 Enzyme-Linked Immunosorbent Assay (ELISA) Sandwich ELISA Principle of test detection of fungi Ag Add Specimen Adapted from Kuby Immunology, 4 th Edition
21 Enzyme-Linked Immunosorbent Assay (ELISA) Sandwich ELISA Principle of test detection of fungi Ag Add Specimen Adapted from Kuby Immunology, 4 th Edition
22 Enzyme-Linked Immunosorbent Assay (ELISA) Sandwich ELISA principle of test detection of fungi Ag Add Specimen Containing Ag Application of test: Galactomannan Aspergillus Ag (IA) Mannan Candida Ag (IK) Glucuroxylomannan Cryptococcus Ag Adapted from Kuby Immunology, 4 th Edition
23 Enzyme-Linked Immunosorbent Assay (ELISA) Principle of test detection of Ab Adapted from Kuby Immunology, 4 th Edition
24 Enzyme-Linked Immunosorbent Assay (ELISA) Principle of test detection of Ab (Specimen) Adapted from Kuby Immunology, 4 th Edition
25 Enzyme-Linked Immunosorbent Assay (ELISA) Principle of test detection of Ab (Specimen) Adapted from Kuby Immunology, 4 th Edition
26 Enzyme-Linked Immunosorbent Assay (ELISA) Principle of test detection of Ab Application of test: anti-aspergillus IgA, IgM, IgG, IgE (IA, aspergilloma, ABPA, asthma) anti-candida (IC) IgA, IgM, IgG anti-cryptococcus IgM, IgG Specific Ab in suspected endemic mycosis
27 Non cultural methods (PCR) Principles of PCR test
28 PCR FOR DIAGNOSIS OF IFI IS NOT STANDARDIZED!! Samples: blood serum tissue BAL CSF Protocols DNK extraction PCR design Amplification Species/genus specific sequence HSP 90 Lanosterol demethylase chitin synthase aspartic proteinase actin Application of PCR: Aspergillosis Candidiasis Pneumocystis pneumonia
29 Automated method of DNA extraction of fungi from blood DNA extraction - Automated - Test duration 16 h - 4 h - In vitro sensitivity (1cfu/ml) Contamination (a problem) - during extraction - during the test - during the preparation of probes - during the preparation of reagents - Environment!
30 Methods of pathohistology in the diagnosis of IFI Special fungal staining
31 FUNGI special staining (HP) H&E: Advantage: well detect inflammatory respons Disadvantage: poor visualization of fungi Gomori methenamin silver (GMS): Advantage: good visualization of fungi Fungi: dark brown ground green Periodic acid-shiff (PAS): Advantage: good visualization of fungi Fungi: red or pink ground green Mucin (mucicarmin): Advantage: good visualization of capsula of fungi Capsula: rose ground gray-rose Fontana-Masson: Advantage: good visualization of cell wall of fungi and melanin Fungi/melanin: darkbrown ground light
32 Fungi histopathology (HP) GMS H&E Hope et al., Lancet Infectious Diseases 5: 609, 2005
33 Fungi histopathology (HP) Lung (mucicarmin) Lung (PAS)
34 Fungi immunohistopathology (IHP) Immunohistohemia (mab) WF-AF-1 (Dako) EB-A1 (Dako) Choi JK, Am J Clin Pathol 121: 18, 2004
35 Pneumocystis jiroveci - Giemsa Microscopic findings of octet cystic formations (400X) BAL, biopsy of lung tissue
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