IDENTIFICATION OF FUNGAL PATHOGENS BY MALDI-TOF MASS SPECTROMETRY. Alex van Belkum Delhi - March 19, 2015

Transcription

1 IDENTIFICATION OF FUNGAL PATHOGENS BY MALDI-TOF MASS SPECTROMETRY Alex van Belkum Delhi - March 19,

2 ASPERGILLUS SPECIES AND GENERA: THE HISTORICAL PERSPECTIVE NUMBER OF SPECIES DESCRIBED PER DECADE 1890: Koch s postulate 1942: Biological Species Concept 1953: DNA model 1983: PCR 1995: Saccharomyces genome 1995: Neurospora genome 41% of species many mould species have been described without modern, molecular techniques 69% of species 82% of species 2

3 MICROBIAL ID BY MALDI-TOF - HISTORY 1998: Anagnostec is founded as spin-off of TU Berlin develpment of SARAMIS starts 2004: first installation of SARAMIS in clinical lab 2005: Bruker Daltonics starts develpment MALDI Biotyper 2007: Acceptance for MALDI-ID increases rapidly 2009: biomérieux starts develpment own MS ID database 2010: biomérieux acquires assets from Anagnostec & partners with Shimadzu 2011: Launch of VITEK MS IVD in Europe, ASPAC, Canada 2012: Submission clinical data for FDA clearance 3 3

4 MALDI-TOF MS: BASIC PRINCIPLES AT A GLANCE Detection To Mass fingerprint Separation Acceleration Ionization Electrode Laser Desorption From sample Matrix (CHCA) 4 4

5 VITEK-MS DATABASE: DEVELOPMENT STRATEGY Strain collection Strain selection (10 isolates/species) LBM collection Vitek/API Sequencing St-Louis collection External culture collections Quality control Experimental plan Subculture in different conditions One obvious outlier Spectra acquisition 5 5

6 VITEK MS RUO VITEK MS PLUS: WORKFLOW o Research Use Only o o SARAMIS database + algorithms 1202 species Open SuperSpectra & ReferenceSpectra Cluster analysis LaunchPad Computer SARAMIS Server VITEK MS ( IVD ) o o IVD compliant / CE marked Submission for FDA cleared VITEK MS IVD" database + algorithm 751 species Closed Advanced Spectra Classifiers Acquisition Station Myla TM Server o Optimized Workflow & Integration w/ AST Prep Station 6 6

7 THREE SIMPLE STEPS Step Sample Preparation MALDI TOF MS Measurement Data analysis VITEK MS Sample Prep Station Disposable slide & ready to use reagents Growth on AGAR media No media requirements Direct smear method Automated measurement In a specific mass range Da With CHCA matrix Measurement of ribosomal proteins Peak detection Pattern matching against the database ASC (Advanced Spectra Classifiers) 7 7

8 MS MEASUREMENT: SPECTRUM PATTERN mass signals Cellular compounds detected: mostly proteins, but lipids and polysaccharides also possible Proteins detected : Those that are extractable, soluble, moderately hydrophilic, stable, and abundant In the mass range : mostly ribosomal proteins Proteins mass signal intensity depending on: Concentration of the protein Capacity for Ionisation (Proton affinity) Stability at the Ionisation/Acceleration/Flight Amino acid composition (Arg and Lys are efficiently protonated) %Int m/z ribosomal proteins, other peaks : mostly unknown proteins 8 8

9 ACQUISITION OF SPECIFIC SPECTRA: CONSERVED PEAK PATTERNS WITHIN SPECIES Mucor velutinosus Blastomyces dermatitidis Strains within a species show highly similar peak patterns, but 9 9

10 ACQUISITION OF SPECIFIC SPECTRA: DIFFERENT SPECIES DIFFERENT PATTERNS C. albicans C. tropicalis C. neoformans Examples of spectra obtained for Yeasts 10 10

11 ACQUISITION OF SPECIFIC SPECTRA: PEAK PATTERNS AND AGE OF CULTURE Spectra obtained after 2 and 8 days for Aspergillus niger 8 days 2 days 11 11

12 SAMPLE PREPARATION: EXTRACTION METHOD FOR MOULDS Ethanol Formic Acid 70% - Acetonitril Use a safety cabinet, wear laboratory coat/gloves/oversleeves Use the appropriate procedure/product to clean material Take some of the moulds using the wet swab Suspend moulds in a 2ml Eppendorf tube containing 300µl Medium Suspension This step could be done on the bench outside the safety cabinet Deposit 1 µl supernatant Dry completely Add 1 µl CHCA matrix Dry completely Analyze in VITEK MS Suspension Medium (sterile deionized water) for swab moistening 2&3 Add 0.9mL Ethanol and mix (vortex) Centrifugation 2& rpm (a) Discard the supernatant using a pipette 2&3 Centrifugation rpm (a) In 40 µl FA [70%] + Add 40µL Formic acid 70% and mix (vortex) 3 3 Add 40µL Acetonitril and mix (vortex) (a) Please note that depending on the instrument, the centrifugation time may need to be adapted (for example 2 30 min in order to have 2 min at 10,000 rpm). Moreover, if you need to put the tubes outside of the safety cabinet for the centrifugation steps, take care to clean the tubes with sporicidal product in order to avoid contamination

13 MOULDS: SIMPLE, SECURE PROTOCOL Extraction from agar plate, rapid in 8 simple steps, No specific material needed, use of standard and non teratogenic solvents, ID possible whatever the age of colony, on both mycelium and spores, no instrument contamination, The deposit can be performed outside of the safety cabinet in the preparation station

14 FDA CLINICAL TRIALS PERFORMANCE Ongoing Multicenter Clinical Study VITEK MS V2.0 Yeast # isolates Total OC LDCG OC+LDCG Site % 1.4% 91.8% Site % 0.7% 98.6% Site % 0.0% 98.2% Site % 3.2% 92.6% Site % 2.3% 95.3% Combined % 1.8% 94.7% All testing single spot / no prior extraction except for yeasts All organisms # isolates Total OC LDCG OC+LDCG Site % 7.7% 90.1% Site % 4.9% 94.5% Site % 6.2% 96.3% Site % 7.3% 91.7% Site % 8.3% 92.7% Combined % 7.3% 92.4% Species level Species + Genus level 14 14

15 VITEK MS IVD FDA CLINICAL TRIALS Rychert et al., 2013 JCM 51:2225 (GP), Richter et al EJCMID (GNE), Manji et al EJCMID (GNNE), Westblade et al JCM 51: 2267 (YST), Garner et al CMI (AN), Branda et al DMID (FST) Group No. Isolates % Correct to Species % Correct to Genus % Total Correct % Total MisID % Total No ID GP GNE GNNE YST AN FST

16 Pseudallescheria and Scedosporium species differentiation from agar cultures by MALDI-TOF MS 16

17 Master Class testing: study design Pseudallescheria angusta CBS Pseudallescheria boydii CBS /CBS /CBS Pseudallescheria ellipsoidea CBS Scedosporium aurantiacum CBS /CBS /CBS / Sub-cultured on 3 agar media & 2 ages of culture SGC2 (Sabouraud Gentamicin Chloramphenicol - biomérieux Ref ) SDA (Sabouraud Dextrose Agar biomérieux Ref ) PDA (Potatoes Dextrose Agar Homemade) 5 and 12 days incubation period at 30 C Moulds inactivation / extraction sample preparation Testing on VITEK MS RUO System and cluster analysis Extract spotted in duplicate 17 17

18 SCEDOSPORIUM AURANTIACUM COMPARISON WITH S.APIOSPERMUM & S.PROLIFICANS Scedosporium apiospermum Spectra included in the next VITEK MS IVD KB Scedosporium aurantiacum CBS /CBS /CBS / CBS Scedosporium prolificans Spectra included in the next VITEK MS IVD KB 18 18

19 Global view Pseudallescheria ellipsoidea CBS Pseudallescheria boydii CBS /CBS & Spectra included in the next VITEK MS IVD KB Pseudallescheria angusta CBS Pseudallescheria boydii CBS Scedosporium apiospermum Spectra included in the next VITEK MS IVD KB Scedosporium aurantiacum CBS /CBS /CBS / CBS Scedosporium prolificans Spectra included in the next VITEK MS IVD KB 21 19

20 Conclusion MALDI TOF Mass Spectrometry allows to work directly from agar plates independently of the culture media and the age of colony to obtain A clear differentation between Scedosporium and Pseudallescheria species Scedosporium species within Scedosporium genus Differenciation within Pseudallescheria complex 20 20

21 SOME ADDITIONAL FEATURES Diversity of spectra depending on strain origin: Trichophyton interdigitale Anthropophilic Zoophilic Need to take into account this diversity + diversity linked to culture conditions/operators/instruments 23 21

22 FOCUS ON VITEK MS FUNGI DATABASE SPECIES DISCRIMINATION Candida palmioleophila vs guilliermondii: antifungal therapy impact Highly Susceptible to Echinocandins C. palmioleophila Reduced Susceptibility to Echinocandins C. guilliermondii 24 22

23 FOCUS ON VITEK MS FUNGI DATABASE SPECIES DISCRIMINATION Aspergillus fumigatus vs lentulus: antifungal therapy impact Resistant profile Ampho. B Itraconazole Voriconazole Ravuconazole A. lentulus Susceptible profile Low MIC s to Ampho. B, Azole drugs Echinocandins A. fumigatus 25 23

24 TO TYPE OR NOT TO TYPE?? 24

25 ANTIBIOGRAMS OR NOT?? 25

26 SUMMARY AND CONCLUSIONS intra-specific variability of mass spectra is high in moulds mass spectra of mould strains vary in response to cultivation conditions databases need to include mass spectra variability databases need to be extended to rare species taxonomic issues need to be considered a standardized protocol is desirable mould identification by MALDI is possible but more complex than for bacteria 26

27 ACKNOWLEDGEMENTS Sophie De Respinis, Cincia Nobile, Mauro Tonolla Istituto Cantonale di Microbiologia, Bellinzona, CH Richard de Winter, Frank Janssen BaseClear, Leiden, NL Deanne Sutton, Nathan Wiederhold University of Texas, San Antonio, USA Bert Gerrits van den Ende, Sybren de Hoog Centraalbureau voor Schimmelcultures, Utrecht, NL Valérie Monnin, Victoria Girard, Sophie Polsinelli, Dave Pincus biomérieux, R&D Microbiology Regine Cuziat biomérieux strain collection 27 27