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Diapositive 1 ESCMID Postgraduate Education Course Cluj-Napoca, Romania. State-of-the-art in Emerging Fungal Infections 8th-9th September 2011 Diagnosis of zygomycosis in the clinical microbiology laboratory Eric DANNAOUI • Université Paris Descartes © by author • Unité de Parasitologie-Mycologie, Service de Microbiologie Hôpital Européen Georges Pompidou (HEGP) ESCMID Online Lecture Library Introduction Why is it important to have a diagnosis of zygomycosis and to identify the species ? Specific treatment of zygo vs other fungi (e.g. Aspergillus)1 Some species are contamination (e.g. R. stolonifer) 2 Sometimes nosocomial infection Species differ for antifungal susceptibility Current problems Identification of the species© by inauthor culture Identification of the species directly from tissues when cultures are negative Multiresistance to antifungals ESCMID Online Lecture1. Spellberg Library et al. CID 2009 48:1743 2. Rammaert et al. ICAAC 2009 Introduction Zygomycetes Ubiquitous in environment From Antarctica1 to hot geothermal soil2 Some species seem to be more restricted geographically Tropical areas (Saksenaea / Apophysomyces) Europe3 (Lichtheimia spp.) China4 (Rh. variabilis) © by author 1. Lawley B., et al.ESCMID 2004. Appl. Environ. Online Microbiol. 70: 5963. Lecture 3. Nicco Library E., et al. 2011. ICAAC. M-1513. 2. Redman RS., et al. 1999. Appl. Environ. Microbiol. 65: 5193. 4. Lu XL., et al. 2009. CID. 49: e39. Zygomycetes : ecology Ecology, pathogenic species Group of filamentous fungi characterized by non-septate mycelium Saprobes soil decaying organic matter fruits various grains © by author Sporulation ++, fungal spores in the air ESCMID Frequently isolatedOnline as Lecturea culture contaminant Library Pathogenic species Pathogenic species : ca. 20 species / 10 genera Family Genus Species Mucoraceae Rhizopus R. oryzae R. microsporus Mucor M. circinelloides M. indicus Rhizomucor R. pusillus Apophysomyces A. elegans Lichtheimiaceae Lichtheimia L. corymbifera © by author L. ramosa Cunninghamellaceae Cunninghamella C. bertholletiae Saksenaeaceae Saksenaea S. vasiformis ESCMID Online Lecture Library Pathogenic species Correlation species / clinical form Distribution of zygomycetes species Rhinocebral Rhizopus oryzae responsible for human infections in France, 2003-2008 Pulmonary Rhizopus microsporus Rhizomucor pusillus Cunninghamella spp. Lichtheimia spp. Cutaneous © by author Mucor circinelloides Lichtheimia spp. Saksenaea vasiformis ESCMID Online Lecture Library Abstract P810. ECCMID April 2010 Diagnostic strategy Samples Microbiology Histology Lab Lab Fixative / Inclusion Direct Culture Slice cuts Exam MB/ FFPE Staining Staining MB/ fresh molecular morpho ID biopsies tissues ID myco / IHC myco© by author No serology, no antigen detection (beta-glucan neg), no PCR kitESCMID Online Lecture Library Processing of samples and interpretation of results Garcia-Hermoso D., et al. Manual of Clinical Microbiology 2011, ASM Press sample Histopathology lab Mycology lab Collection of sampleCollection Tissue section (PE) Direct stained with HE, PAS, - - - Culture + examination + + Techique GMS Typical hyphae Typical hyphae Sporulating Zygomycete yes no yes no yes no •Morphology •Special •Immunohisto •MB •Max growth temp media •MB •MB •MB Confront with direct Species Results andResults additional tests Confront with culture © by authorexam / histology results identification results Histo / direct exam Culture Interpretation Positive Positive Definite diagnosis Positive Negative Definite diagnosis (for biopsies) NegativeESCMID OnlinePositive LectureConfront Library with clinical context Negative Negative Obtain new samples Interpretation of resultsInterpretation Mycological diagnosis Samples: BA, BAL, biopsies, others, (depending on localization) Direct examination: important ++ Chlorazol black, Calcofluor, Gomori-Methenamine-Silver (GMS)… Cultures: risk of false positive (contaminants) Many media suitable, but cultures often negative Media without cycloheximide++ Incubation 37°C, 30°C Biopsies : no homogeneization+++, breakdown of hyphae Histopathology: Definite diagnosis © by author No species identification ESCMID Online Lecture Library Current issues cultures often negative, or not done © by author ESCMID OnlineDiagnostic Lecture in tissues Library mandatory Roden M. et al. 2005. CID 41:634. Identification: the classical way Identification of Zygomycetes from culture The classical way Phenotypic identification Mainly based on morphological characteristics Remains difficult and sometimes needs expertise of a reference laboratory R. oryzae R. microsporus Absidia Mucor Rhizomucor © by author Some species (Apophysomyces, Saksenaea) fail to sporulate on routine media ESCMID Online Lecture Library Identification: the classical way Culture on standard media macroscopy Observation stereomicroscope Architecture of the colony © by author Wet mount / lactophenol blue ESCMID microscopy Online Lecture Library Identification: the classical way Mucoraceae - Lichtheimiaceae - In culture (general characteristics) Sporangium Sporangiospores Columella Apophysis Collerette Sporangiophore © by author Rhizoïds ESCMID Online Lecture Library Identification: the classical way Identification of Zygomycetes from culture The classical way • Evaluation of maximum temperature of growth • Specialized tests : SEM mating physiology © by author Morphological-based identification erroneous in > 20% of the cases1 ESCMIDNew Online identification Lecture methods Library are needed 1. Kontoyiannis, D. P., et al. 2005. JID 191:1350. Identification: new tools Zygomycetes : physiological tests as an adjunct to identification Carbon source assimilation: ID32C® A 0 1 2 3 4 5 6 7 8 9 A B C D E F A. corymbifera GAL ACT SAC NAG LAT ARA CEL RAF MAL TRE 2KG MDG MAN LAC INO 0 SOR XYL RIB GLY RHA PLE ERY MEL GRT MLZ GNT LVT GLU SBE GLN ESC B 0 1 2 3 4 5 6 7 8 9 A B C D E F R. microsporus GAL ACT SAC NAG LAT ARA CEL RAF MAL TRE 2KG MDG MAN LAC INO 0 SOR XYL RIB GLY RHA PLE ERY MEL ©GRT MLZby GNT author LVT GLU SBE GLN ESC C 0 1 2 3 4 5 6 7 8 9 A B C D E F S. racemosum GAL ACT ESCMIDSAC NAG LAT ARA CEL Online RAF MAL TRE Lecture2KG MDG MAN LAC LibraryINO 0 SOR XYL RIB GLY RHA PLE ERY MEL GRT MLZ GNT LVT GLU SBE GLN ESC Identification: new tools ID32C Zygomycetes : Assimilation + physiological Assimilation - identification Cunninghamella bertholletiae Syncephalastrum racemosum Rhizomucor pusillus Absidia corymbifera Mucor © by authorRhizopus microsporus Rhizopus oryzae ESCMID Online Lecture Library Schwarz P. et al. JCM 2007, 45, 1433. Molecular techniques © by author ESCMID Online Lecture Library Molecular identification in culture Zygomycetes : molecular identification of species1 Reliable identification (DNA barcoding) needs2 An informative DNA target Working for all species in the group Low intra-specific and high inter-specific variability A validated sequence database for comparison (public,© commercial)by author MicroSeq D2 large-subunit DNA sequencing kit: misidentification of Zygo in ~50%3 1. Dannaoui, E.ESCMID 2009. Clin Microbiol InfectOnline 15 Suppl 5: 66Lecture-70. Library 2. Balajee A. et al. 2007. Med. Mycol 45:475. 3. Hall L. et al, 2004. JCM 42:622. Identification: new tools Zygomycetes : molecular identification of species Sequencing ITS region : intra and inter-species variability V9D LS266 SSU (18S) 5.8S LSU (28S) ITS1 ITS2 Species n Rhizopus oryzae 54 strains, known agents of (T) 17 zygomycosis, 16 species Rhizopus microsporus (T) 7 Mostly clinical origin © byAbsidia author corymbifera 6 Diverse geographic areas Rhizomucor pusillus (T) 7 Including type strains Mucor circinelloides (T) 5 Other 13 ESCMID Online (T)Lecture Including type strain Library Identification: new tools ITS sequencing - Intra-specific variability Species % similarity R. oryzae 99 – 100 R. microsporus 99-100 Rh. pusillus 100 A. corymbifera 99-100 M. circinelloides © by author99 Low variability within a given species ESCMID Online Lecture Library Schwarz P. et al. 2006. JCM 44:340. Molecular identification Intra species sequence variability for strains of R. oryzae (Schwarz P. et al. 2006. JCM 44:340) R. oryzae CCT - - TAG GAGTACC GGTGATG TTTTTTT CAGTAAC CCTCTTAG GAGCACC GGTAATG TTTCTTT CAGCAAC ITS 1 ITS 4 ITS 1 ITS 2 5.8S 0 50 169 224 382 409 512 554 629 New proposed species: R. delemar© by author Abe A. et al. 2007. Mycologia 99:714-22 ESCMID Online Lecture Library Identification: new tools ITS sequencing Species R. o A. c M. c R. p A. corymbifera (A. c) 36 Percentage of similarities M. circinelloides (M. c) 48 37 of the ITS region between species of zygomycetes R. pusillus (R. p) 37 37 42 R. microsporus (R. m) 61 34 47 33 Heterogeneous between genera : similarity ≤ 68% Heterogeneous between species : similarity ≤ 95% few exceptions © by author Homogenous within a given species : similarity 99% Sequencing of ITS region is a suitable tool for DNA ESCMIDbarcoding Online the zygomycetes Lecture Libraryspecies Molecular identification in culture Current guidelines The Clinical Laboratory Standards Institute (CLSI) proposed guidelines for fungal identification by DNA Target Sequencing Microorganism or Group: Zygomycetes ITS Target Comments for ITS Target Alternative DNA Target Resolution to Good discrimination of genera; D1/D2 region may genus and interspecies variation 0- 40%; provide resolution usually to intraspecies differences 0-2%. to species species Five species of Rhizopus can be distinguished.© by author CLSI. InterpretiveESCMID
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