ESCMID Online Lecture Library. by author

Transcription

1 Current challenges with fungal identification using MALDI TOF how to improve? Professor Renaud Piarroux ESCMID Online Lecture Library Aix Marseille University

2 The challenge

3 Diversity of clinical conditions

4 Species diversity in medical mycology

5

6

7

8 Identifying mould species Aspergillus flavus Conidies : 3 et 6µ Conidies : 3µ Aspergillus ochraceus ESCMID Online Lecture Library Conidiophore «slighlty» echinulated Conidiophore markedly echinulated

9 A first attempt of MLDI TOF MS identification

10

11 Mould routine identification : proof of concept

12

13

14 1,9 Moulds identification LS threshold Results: Consecutive clinical isolates betwen 2010 July and November 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).

15 Mold identification protocol in Marseille s Mycology laboratory From culture to the best identification conceivable ESCMID Online Lecture Library

16 Culture Macroscopic and microscopic characterisation After 4 to 7 culture days DNA sequencing for: Scedosporium Fusarium 31/12/2011 uncharacteristic Aspergillus spp. Culture 01/01/2012 Mass Spectrometric identification After 3 to 4 culture days Macroscopic and microscopic characterisation After 3 to 7 culture days DNA sequencing for : MS identification <1.9 Fusarium Scedosporium uncharacteristic Aspergillus spp. ESCMID Online Lecture Library

17 Routine identification of dermatophytes

18

19 Routine identification of yeasts

20

21 Improving the reference database

22

23

24

25 Improving the decision algorithm for identification

26

27 Performance of 10 different decision algorithm in crrectly identifying moulds and dermatophytes ESCMID Online Lecture Library

28 Application to blood cultures

29

30 Some «emergent» pathogens better detected by MALDI TOF MS

31

32

33

34 Schizophyllum commune ESCMID Online Lecture Library

35 Detection of mixed infections

36

37 Some pathogens difficult to identify

38

39

40 Typing fungi using MALDI TOF MS

41

42

43 Routine MALDI TOF MS identification in medical laboratories

44

45

46 Culture on Sabouraud (Chloramphenicol / Gentamicin) agar plate Day " x " Day " x+1 " Scraping of the colony, MS treatment, 4 deposits MS results 3 matching results AND at least one logscore >1.9 Less than 3 matching results OR no LS>1.9 Matches microscopic examination > VALIDATION Doubt regarding microscopic characteristics Sequencing Scraping of the colony, MS treatment, 4 deposits MS results 3 matching results AND at least one logscore >1.9 Less than 3 matching results OR no LS>1.9 Matches microscopic examination > VALIDATION Doubt regarding microscopic characteristics ESCMID Online Lecture Library Sequencing Sequencing

47 Filamentous fungi: 16 months Routine laboratory review Isolates: 1115 Validated assay: 1107 Non compliant : 8 Mass spectrometry identification : 1090 (98.5%) Microscopic error : 10% Identification : 2 spots No sequencing Identification : 1 spot No Mass Spectrometry No identification: 2 (0.1%) Identification at the genus level: 15 (1.3%) DNA confirmation : 61 ESCMID Online Lecture Library

48 January to April 2011 (302 samples subcultured) Yes 165 MALDI-TOF MS-based identification (2013 database) Concordant identification 150 Positive Subcultures : 247 Initial identification (2011) Identification : 97.6% Initial misidentification* 15 No 82 MALDI-TOF MS-based identification (2013 database) Identification 76 Yes 3 MALDI-TOF MS-based identification failure 6 DNA sequence-based Identification Penicillium madriti P.Aurantiogriseum Tilletiopsis albescens No 3 Identification : 66.8% 16 distinct species ESCMID Online Lecture Library Total identification : 98,8% 42 distinct species Penicillium sp Phoma sp Scedosporium sp.

49 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 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 ustus ESCMID Online Lecture Library Aspergillus versicolor Ulocladium atrum Fusarium solani Penicillium piceum Aspergillus versicolor Tilletiopsis albescens Trichoderma longibrachiatum Fusarium verticillioides Penicillium pinophilum

50 Filamentous fungi Routine laboratory review in species

51 Aspergillus spp. 16 species / 17 taxa ESCMID Online Lecture Library

52 Penicillium & Paecilomyces spp. 15 species / 18 taxa ESCMID Online Lecture Library

53 Origin and distribution of the mold identified in the laboratory during a 16 months period ESCMID Online Lecture Library

54 Routine MALDI TOF MS identification to manage a collection of fungi ESCMID Online Lecture Library

55

56 On the way to online application

57

58 The algorithms

59

60 The databases

61 Aphanoascus 2 Beauveria 1 Hamigera 1 Radulidium 1 Auxarthron 2 Bipolaris 1 Hanseniaspora 1 Rhizomucor 1 Chaetomium 2 Bjerkandera 1 Hexagonia 1 Rhodotorula 1 Chrysonilia 2 Blastobotrys 1 Histoplasma 1 Saprochaete 1 Eurotium 2 Boeremia 1 Hypocrea 1 Sarocladium 1 Gallactomyces 2 Botrytis 1 Hypoxylon 1 Schizophyllum 1 Geosmithia 2 Brachycladium 1 Kazachstania 1 Scolecobasidium 1 Gymnascella 2 Byssochlamys 1 Kloeckera 1 Scytalidium 1 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 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; 3 Curvularia; 3 Arthroderma; 3 Alternaria; 3 Scedosporium; 4 Rhizopus; 4 Microsporum; 4 Malassezia; 4 Arthrinium; 4 Trichoderma; 5 Paecilomyces; 5 Mucor; 5 Cladosporium; 5 Pseudallescheria; 6 List of fungal genera in the community databse and number of species per genus Pichia; 6 Scopulariopsis; 7 Cryptococcus; 7 Aspergillus; 71 Fusarium; 22 Talaromyces; 13 Acremonium; 7 Trichosporon; 11 Trichophyton; 10 Candida; 42 Penicillium; 62 ESCMID Online Lecture Library

62

63

64

65

66 The website

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82 Reliability of results (community database)

83 Evaluation panel 1274 isolates collected in 5 hospitals in France and Belgium. Bordeaux, Marseille, Nice AZ Sint Jan (Brugge), Universitair Ziekenhuis Brussel 108 taxa (moulds and dermatophytes) 38,77% des isolates were sequenced for species confirmation ESCMID Online Lecture Library 768 isolats identified only by their morphology

84 juste complexe erreur sous seuil Assessment of the application reliability nombre d'isolats % des isolats totaux nombre d'isolats % des isolats totaux nombre d'isolats % des isolats totaux nombre d'isolats % des isolats totaux BRUKER % % % % BRUKER IHEM/MRS % % % % ESCMID Online Lecture Library MSI COMMUNAUTAIRE % % % % Seuil 1,7 Seuil 25

85 The current users in France

86

87 CHU MARSEILLE; date de début : 01/08/2016 Nb spectres total Nb spectres >25 Nb spectres lev>25 Nb spectres fil> OK OK Levure Analyse phénotypique SM BRUKER PAS OK PAS OK Pousse Filamenteux OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK Dermatophyte OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK ESCMID Online Lecture Library MSI MSI MSI MSI MSI MSI MARSEILLE MARSEILLE

88 CHU BORDEAUX; date de début : 01/08/2016 Nb spectres total Nb spectres >25 Nb spectres lev>25 Nb spectres fil> OK OK Levure Analyse phénotypique SM BRUKER PAS OK PAS OK Pousse Filamenteux OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK Dermatophyte OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK ESCMID Online Lecture Library MSI MSI MSI MSI MSI BORDEAUX BORDEAUX

89 CHU ROUEN; date de début : 01/08/2016 Nb spectres total Nb spectres >25 Nb spectres lev>25 Nb spectres fil> OK OK Levure Analyse phénotypique SM BRUKER PAS OK PAS OK Pousse Filamenteux OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK Dermatophyte OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK ESCMID Online Lecture Library MSI MSI MSI MSI MSI MSI ROUEN ROUEN

90 CHU-LHUPM, CAYENNE; date de début : 01/08/2016 Nb spectres total Nb spectres >25 Nb spectres lev>25 Nb spectres fil> OK OK Levure Analyse phénotypique SM BRUKER PAS OK PAS OK Pousse Filamenteux * OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK Dermatophyte OK OK Analyse phénotypique SM BRUKER PAS OK PAS OK ESCMID Online Lecture Library MSI MSI MSI MSI MSI MSI CAYENNE CAYENNE * Pb de livraison en milieux de culture pendant la période de l étude

91 The next steps Securise the application (website, algorithms, databases, fast identification) Publish the results Develop new algorithms Better/faster identification Detect mixed cultures Detect spread of fungal spread Application to other needs Parasitology (Leishmania) Entomology (mosquitoes, sandflies) ESCMID Online Lecture Library Develop epidemiological tools based on historical databases analyses

92 Thank you to Laboratoire de Mycologie de La Timone à Marseille Carole Cassagne Magali Gautier Anne Cécile Normand Coralie L Ollivier Stéphane Ranque Renaud Piarroux BCCM/IHEM Brussels, Belgium Marijke Hendricks Ann Packeu Sam Roesems David Triest Dirk Stubbe Pierre Becker Partners using the application Bichat : Christine Bonnal, Sandrine Houze Bordeaux : Frédéric Gabriel, Isabelle Acroceberry, Laurence Delhaes Cayenne : Cécile Nabet, Magalie Demar Montpellier : Laurence Lachaud Nice : Martine Gari Toussaint, Lilia Hasseine, Pierre Marty Rouen : Loic Favennec Saint Etienne : Hélène Raberin Toulouse : Sophie Cassaing Winnipeg : Markus Stein ESCMID Online Lecture Library

93 Thank you for your attention! hm.fr