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ESCMID Online Lecture Library © by Author Current challenges with fungal identification using MALDI‐TOF – how to improve? Professor© by Renaud author Piarroux ESCMID OnlineAix‐Marseille Lecture University Library The challenge © by author ESCMID Online Lecture Library Diversity© by of clinical author conditions ESCMID Online Lecture Library © by author Species diversity ESCMIDin Online medical mycology Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Identifying mould species Aspergillus flavus Aspergillus ochraceus © by author ESCMIDConidies Online : 3 et 6µ Lecture Conidies : 3µ Library Conidiophore « slighlty » echinulated Conidiophore markedly echinulated A first attempt of MLDI‐TOF MS identification © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Mould routine identification : proof of concept © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Moulds identification LS threshold Results: Consecutive clinical isolates betwen 2010 July 1,9 and November © by author ESCMID Online Lecture Library Graphical representation of the best‐match LS values for each of the 4 spots issued from identification of the156 clinical isolates. (The dark line represents the best‐match LS values of the concordant spots whereas the gray line shows the best‐match LS values of the discordant ones). Mold identification protocol in Marseille’s Mycology laboratory From culture to the best identification conceivable © by author ESCMID Online Lecture Library … 31/12/2011 01/01/2012 … Culture Culture Mass Spectrometric identification Macroscopic and After 3 to 4 culture days microscopic characterisation After 4 to 7 culture days Macroscopic and microscopic characterisation After 3 to 7 culture days DNA sequencing for: © by author Scedosporium DNA sequencing for : Fusarium MS identification <1.9 uncharacteristic Aspergillus spp. ESCMID Online LectureFusarium Library Scedosporium uncharacteristic Aspergillus spp. Routine identification of dermatophytes © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Routine identification of yeasts © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Improving the reference database © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Improving the decision algorithm for identification © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Performance of 10 different decision algorithm in crrectly identifying moulds and dermatophytes © by author ESCMID Online Lecture Library Application to blood cultures © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Some «emergent » pathogens better detected by MALDI‐TOF MS © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Schizophyllum commune Detection of mixed infections © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Some pathogens difficult to identify © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Typing fungi using MALDI‐TOF MS © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Routine MALDI‐TOF MS identification in medical laboratories © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library Day " x "DMS results ay " x+1 " MS results Matches microscopic examination 3 matching ‐> results VALIDATION AND at least one logscore >1.9 Doubt regarding Sequencing microscopic characteristics Culture on Scraping of the colony, Sabouraud Matches (Chloramphenicol / MS microscopic Gentamicin) agar treatment, examination plate 4 deposits ‐> 3 matching VALIDATION results AND at least one logscore Scraping of the >1.9 Less than 3 Doubt colony, regarding matching Sequencing results OR © by authorMS treatment, microscopic characteristics no LS>1.9 4 deposits Less than 3 matching Sequencing results OR ESCMID Online Lectureno LS>1.9Library Filamentous fungi: 16 months Routine laboratory review Isolates: 1115 Non compliant : 8 Identification : 2 spots No sequencing Identification : 1 spot No Mass Spectrometry Validated assay: 1107 No identification: 2 (0.1%) Mass spectrometry identification© : 1090 by (98.5%) author Identification at the genus level: 15 (1.3%) ESCMIDMicroscopic error : 10% OnlineDNA confirmation Lecture : 61 Library January to April 2011 (302 samples subcultured) Positive Subcultures : 247 Initial identification (2011) Identification : 66.8% 16 distinct species Yes No 165 82 MALDI-TOF MS-based MALDI-TOF MS-based identification (2013 database) identification (2013 database) Identification : 97.6% MALDI-TOF MS-based Concordant Initial Identification identification misidentification*© by authoridentification failure 76 150 15 6 DNA sequence-based Identification ESCMIDTotal identification Online : 98,8% Lecture Library 42 distinct species Yes Penicillium madriti No Penicillium sp P.Aurantiogriseum 3 Phoma sp 3 Tilletiopsis albescens Scedosporium sp. Fungal diversity in the first four months of 2011, initial identification vs MS identification 2011, without MS 2011, with MS Alternaria alternata Exophiala dermatitidis Alternaria alternata Exophiala dermatitidis Aspergillus calidoustus Paecilomyces lilacinus Aspergillus flavus Mucor circinelloides Aspergillus flavus Mucor circinelloides Aspergillus hortai Penicillium allii Aspergillus fumigatus Penicillium chrysogenum Aspergillus fumigatus Penicillium chrysogenum Aspergillus lentulus Penicillium aurantiogriseum Aspergillus nidulans Penicillium glabrum Aspergillus nidulans Penicillium glabrum Aspergillus puniceus Penicillium citrinum Aspergillus niger Radulidium subulatum Aspergillus niger Radulidium subulatum Aspergillus sclerotiorum Penicillium crustosum Aspergillus niveus Scedosporium apiospermum Aspergillus niveus Scedosporium apiospermum Aspergillus tubingensis Penicillium funiculosum Aspergillus ochraceus Scopulariopsis brevicaulis Aspergillus© ochraceus by author Scopulariopsis brevicaulis Penicillium purpurogenum Penicillium griseofulvum Aspergillus sydowii Tilletiopsis albescens Aspergillus sydowii Alternaria malorum Fusarium. oxysporum Penicillium madriti Aspergillus terreus Aspergillus terreus Phaeoacremonium parasticum Fusarium proliferatum Penicillium nalgiovense Aspergillus versicolor Aspergillus ustusESCMID Online LectureUlocladium atrum LibraryFusarium solani Penicillium piceum Aspergillus versicolor Tilletiopsis albescens Trichoderma longibrachiatum Fusarium verticillioides Penicillium pinophilum Filamentous fungi Routine laboratory review in 2012 © by author ESCMID Online Lecture Library 109 species Aspergillus spp. © by author ESCMID Online Lecture Library 16 species / 17 taxa Penicillium & Paecilomyces spp. © by author ESCMID Online Lecture Library 15 species / 18 taxa Origin and distribution of the mold identified in the laboratory during a 16 months period © by author ESCMID Online Lecture Library Routine MALDI‐TOF MS identification to manage a collection of fungi © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library On the way to online application © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library The algorithms © by author ESCMID Online Lecture Library © by author ESCMID Online Lecture Library The databases © by author ESCMID Online Lecture Library Aphanoascus 2 Beauveria 1 Hamigera 1 Radulidium 1 Auxarthron 2 Bipolaris 1 Hanseniaspora 1 Rhizomucor 1 Chaetomium 2 Bjerkandera 1 Hexagonia 1 Rhodotorula 1 List of fungal genera in the community databse and number of species per Chrysonilia 2 Blastobotrys 1 Histoplasma 1 Saprochaete 1 Eurotium 2 Boeremia 1 Hypocrea 1 Sarocladium 1 genus Gallactomyces 2 Botrytis 1 Hypoxylon 1 Schizophyllum 1 Geosmithia 2 Brachycladium 1 Kazachstania 1 Scolecobasidium 1 Gymnascella 2 Byssochlamys 1 Kloeckera 1 Scytalidium 1 Aspergillus; 71 Metschnikowia 2 Chalastospora 1 Kluyveromyces 1 Sordaria 1 Neosartorya 2 Chrysosporium 1 Kodamaea 1 Sporopachydermia 1 Nigrospora 2 Colletotrichum 1 Lecythophora 1 Sporotrichum 1 Phaeoacremonium 2 Coniothyrium 1 Macrophomina 1 Sydowia 1 Phanerochaete 2 Coprinellus 1 Merimbla 1 Syncephalastrum 1 Phoma 2 Cunninghamella 1 Mortierella 1 Talatomyces 1 Sporothrix 2 Dicyma 1 Mycotypha 1 Thanatephorus 1 Stachybotrys 2 Doratomyces 1 Myriodontium 1 Tilletiopsis 1 Trametes 2 Dothideomyces 1 Myrmecridium 1 Torula 1 Trichurus 2 Engyodontium 1 Neoscytalidium 1 Torulaspora 1 Unidentified 2 Epicoccum 1 Onychocola 1 Trichothecium 1 Zygosaccharomyces 2 Epidermophyton 1 Periconia 1 Tritirachium 1 Absidia 1 Eupenicillium 1 Pestalotiopsis 1 Tyromyces 1 Acrodontium 1 Eutypella 1 Phaeosphaeria 1 Ustilago 1 Aphanocladium 1 Exserohilum 1 Phialophora 1 Verticillium 1 Penicillium; 62 Apophysomyces 1 Fomes 1 Pithomyces 1 Westerdykella 1 Arthrocladium 1 Ganoderma 1 Pleospora 1 Wickerhamomyces 1 Arthrographis 1 Geomyces 1 Pleurostomophora 1 Wolfiporia 1 Arxula 1 Gloeophyllum 1 Porostereum 1 Ybotromyces 1 Aureobasidium 1 Gloeostereum 1 Saccharomyces; 3 Fomitopsis; 3 Phlebia; 3 Exophiala; 3 Debaryomyces;
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