matrix-assisted laser desorption ionization time of …...corynebacterium kroppenstedtii b...

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Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry in Clinical Microbiology

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Presenter:

Robin Patel, M.D.Professor of Medicine and MicrobiologyChair, Division of Clinical Microbiology and the Elizabeth P. and Robert E. Allen Professor of Individualized Medicine

Department of Laboratory Medicine and Pathology

at Mayo Clinic, Rochester, Minnesota

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Disclosures

• Dr. Patel reports grants from CD Diagnostics, BioFire, Curetis, Merck, Contrafect, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, EnBiotix, Contrafect and The Medicines Company. Dr. Patel is or has been a consultant to Curetis, Specific Technologies, Selux Dx, GenMark Diagnostics, PathoQuest, Heraeus Medical, and Qvella; monies are paid to Mayo Clinic. In addition, Dr. Patel has a patent on Bordetellapertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. Dr. Patel receives travel reimbursement from ASM and IDSA and an editor’s stipend from ASM and IDSA, and honoraria from the NBME, Up-to-Date and the Infectious Diseases Board Review Course.

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Objectives

• Explain how matrix-assisted laser desorption ionization time of flight (MALDI ToF) mass spectrometry works

• Explain the role of MALDI ToF mass spectrometry in the clinical laboratory

• Highlight our experience with MALDI ToF mass spectrometry in clinical microbiology

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How Does Mass Spectrometry Work?

• Principle – Separation of m/z ratio• Usually z (charge) constant• Ionization, separation of ions, detection

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Types of Mass Spectrometry

Matrix-assisted laser desorption/ionizationElectron impactSpark sourceThermal ionizationPhoto-ionizationChemical ionizationField ionizationField desorptionMultiphoton ionizationFast atom bombardmentPlasma desorption Infrared laser desorptionNanosprayElectrospray ionization

Time of flightMagneticDouble-focusingReversed geometryQuadrupoleQuadrupole ion trapTriple quadrupoleFour sectorHybridRadio frequencyFourier transformMagnetic sectorElectric sectorLinear ion trapIon cyclotron resonance

Faraday cupSecondary electronmultiplierScintillatorMicro channel plate

IonSource

MassAnalyzer Detector

Ion formation → Ion separation → Ion detection

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1) Sample• Colony

2) Add matrix (1–2 µL)

• Dissolved in acetonitrile (50%)• & 2.5% trifluoroacetic acid

3) Dry – room air 5 min

Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

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Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry - Bruker Biotyper

Put plate into instrument and analyze

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Matrix-Assisted Laser Desorption Ionization

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Accelerating potential

`

Drift region

Detector

Time of Flight

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Mass Spectrum Generated Compared with Library (Database)

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Laboratory Workflow of the Past

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Laboratory Workflow Since 2011

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Aerobic Gram-Positive BacteriaAbiotrophia defectiva Corynebacterium xerosisB Micrococcus lylaeB Staphylococcus warneri

Aerococcus sanguinicolaB Dermabacter hominisB Pediococcus acidilactici Staphylococcus xylosusB

Aerococcus urinaeB Dermacoccus nishinomiyaensisB Pediococcus pentosaceusB Streptococcus agalactiae

Aerococcus viridans Enterococcus avium Rothia dentocariosaB Streptococcus anginosus

Alloiococcus otitisB Enterococcus casseliflavus Rothia mucilaginosa Streptococcus canisB

Arthrobacter cumminsiiB Enterococcus durans Rothia aeriaB Streptococcus equiB

Brevibacterium caseiB Enterococcus faecalis Staphylococcus auricularisB Streptococcus constellatus

Corynebacterium accolensB Enterococcus faecium Staphylococcus aureus Streptococcus dysgalactiaeB

Corynebacterium afermentans groupB Enterococcus gallinarum Staphylococcus capitis Streptococcus dysgalactiae ssp

dysgalactiaeV

Corynebacterium amycolatumB Enterococcus hiraeB Staphylococcus capraeB Streptococcus dysgalactiae spp

equisimilisV

Corynebacterium aurimucosum groupB Enterococcus mundtiiB Staphylococcus carnosusB Streptococcus gallolyticus ssp gallolyticusV

Corynebacterium bovisB Enterococcus raffinosusB Staphylococcus cohniiB Streptococcus gallolyticusB

Corynebacterium coyleaeB Facklamia hominisB Staphylococcus cohnii ssp cohniiV Streptococcus gordoniiB

Corynebacterium diphtheriaeB Gardnerella vaginalis Staphylococcus cohnii ssp urealyticusV Streptococcus infantarius ssp coli

(Str.lutetiensis)V

Corynebacterium freneyiB Gemella haemolysans Staphylococcus delphiniB Streptococcus infantarius ssp infantariusV

B = FDA-approved/cleared for the MALDI Biotyper CA system onlyV = FDA-approved/cleared for the Vitek MS system only


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Aerobic Gram-Positive Bacteria, ContinuedCorynebacterium glucuronolyticumB Gemella morbillorum Staphylococcus epidermidis Streptococcus intermediusB

Corynebacterium glutamicumB Gemella sanguinisB Staphylococcus equorumB Streptococcus lutetiensisB

Corynebacterium jeikeium Granulicatella adiacens Staphylococcus felisB Streptococcus mitis/Streptococcus oralisV

Corynebacterium kroppenstedtiiB Helcococcus kunziiB Staphylococcus haemolyticus Streptococcus mitis/oralis groupB

Corynebacterium macginleyiB Kocuria kristinaeB Staphylococcus hominis Streptococcus mutans

Corynebacterium minutissimumB Kytococcus sedentariusB Staphylococcus intermediusB Streptococcus parasanguinisB

Corynebacterium mucifaciens / ureicelerivorans

groupB

Lactobacillus jenseniiB Staphylococcus lentusB Streptococcus pneumoniae

Corynebacterium propinquumB Lactobacillus rhamnosusB Staphylococcus lugdunensis Streptococcus pyogenes

Corynebacterium pseudodiphtheriticumB Lactococcus garvieae Staphylococcus pasteuriB Streptococcus salivarius / vestibularis groupB

Corynebacterium pseudotuberculosisB Lactobacillus gasseriB Staphylococcus pettenkoferiB Streptococcus salivarius ssp salivariusi

Corynebacterium resistensB Lactococcus lactis Staphylococcus pseudintermediusB Streptococcus sanguinis

Corynebacterium riegeliiB Leuconostoc citreumB Staphylococcus saccharolyticusB Streptococcus sobrinusB

Corynebacterium striatum groupB Leuconostoc mesenteroides Staphylococcus saprophyticus Streptococcus thermophilusB

Corynebacterium tuberculostearicumB Leuconostoc pseudomenteroides Staphylococcus schleiferi Trueperella bernardiaeB

Corynebacterium ulceransB Listeria monocytogenes Staphylococcus sciuri Turicella otitidisB

Corynebacterium urealyticumB Macrococcus caseolyticusB Staphylococcus simulans Vagococcus fluvialisB

Micrococcus luteus Staphylococcus vitulinusB



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Gram-Negative Bacteria, EnterobacteriaceaeCitrobacter amalonaticusV Enterobacter cancerogenusV Leclercia adecarboxylota Serratia fonticola

Citrobacter amalonaticus complexB Enterobacter cloacaeV Morganella morganii Serratia liquefaciens

Citrobacter braakiiV Enterobacter cloacae complexB Pantoea agglomerans Serratia marcescens

Citrobacter freundiiV Pluralibacter gergoviae Plesiomonas shigelloidesB Serratia odorifera

Citrobacter freundii complexB Escherichia coli Proteus mirabilis Serratia plymuthicaB

Citrobacter koseri Escherichia fergusoniiV Proteus penneriV Serratia rubidaeaB

Citrobacter youngaeV Escherichia hermannii Proteus vulgarisV Yersinia enterocolitica

Cronobacter sakazakiiV Escherichia vulnerisB Proteus vulgaris groupB Yersinia frederiksenii

Cronobacter sakazakii groupB Ewingella americana Providencia rettgeri Yersinia intermedia

Edwardsiella hoshinaeV Hafnia alvei Providencia stuartii Yersinia kristensenii

Edwardsiella tarda Klebsiella oxytocaV Raoultella ornitholyticaV Yersinia pseudotuberculosis

Enterobacter aerogenes Klebsiella oxytoca / Raoultella

ornithinolyticaB

Raoultella planticolaV

Enterobacter amingenusB Klebsiella pneumoniae Salmonella groupV

Enterobacter asburiaeV Klebsiella variicolaB Salmonella spB



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Gram-Negative Bacteria, Non-EnterobacteriaceaeAchromobacter denitrificansV Aeromonas sp.B Chryseobacterium indologenes Pseudomonas putidaV

Achromobacter xylosoxidans Alcaligenes faecalisB Cupriavidus pauculus groupB Pseudomonas putida groupB

Acinetobacter baumannii complexV Alcaligenes faecalis ssp faecalisV Delftia acidovorans groupB Pseudomonas stuzeri

Acinetobacter baumannii/nosocomialis groupB Bordetella groupB Elizabethkingia meningosepticaV Ralstonia pickettii

Acinetobacter calcoaceticusB Bordetella hinziiB Elizabethkingia meningoseptica groupB Rhizobium radiobacter

Acinetobacter haemolyticus Bordetella parapertussisV Fluoribacter bozemaniiB Sphingobacterium multivorum

Acinetobacter johnsoniiB Bordetella pertussisV Mannheimia haemolytica groupB Sphingobacterium spiritivorum

Acinetobacter junii Brevundimonas dimunutaV Myroides odoratimimusB Sphingomonas paucimobilisV

Acinetobacter lwoffii Brevundimonas dimunuta groupB Myroides odoratusB Sphingomonas paucimobilis groupB

Acinetobacter pittiiB Burkholderia cepacia complexB Ochrobactrum anthropi Stenotrophomonas maltophilia

Acinetobacter radioresistensB Burkholderia gladioliB Pasteurella multocida Sutterella wadsworthensisB

Acinetobacter ursingiiB Burkholderia multivorans Pseudomonas aeruginosa Vibrio choleraeV

Aeromonas hydrophila/caviaeV Capnocytophaga ochraceaB Pseudomonas fluorescensV Vibrio parahaemolyticus

Aeromonas salmonicidaB Capnocytophaga sputigenaB Pseudomonas fluorescens groupB Vibrio vulnificus

Aeromonas sobriaV Chryseobacterium gleumB Pseudomonas oryzihabitansB Weeksella virosaB


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Fastidious Gram-Negative BacteriaAggregatibacter actinomycetemcomitans Haemophilus influenzae Moraxella catarrhalisB Neisseria gonorrhoeae

Aggregatibacter aphrophilus Haemophilus parahaemolyticusV Moraxella (Neisseria) ovisV Neisseria lactamicaB

Aggregatibacter segnis Haemophilus parahaemolyticus

groupB

Moraxella nonliquefaciensB Neisseria meningitidis

Campylobacter coli Haemophilus parainfluenzae Moraxella osloensisB Neisseria mucosa/siccaV

Campylobacter jejuni Kingella denitrificans Neisseria bacilliformisB Neisseria sicca groupB

Campylobacter ureolyticusB Kingella kingae Neisseria cinerea Neisseria weaveriB

Eikenella corrodens Legionella longbeachaeB Neisseria elongataB Oligella ureolytica

Haemophilus haemolyticusB Legionella pneumophila Neisseria flavescens/subflava groupB Oligella urethralis


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Anaerobic BacteriaActinomyces europaeusB Bacteroides fragilis Clostridium clostridioforme groupB Parabacteroides distasonisB

Actinomyces funkeiB Bacteroides nordiiB Clostridium clostridioformeV Parabacteroides johnsonii / merdae groupB

Actinomyces graevenitziiB Bacteroides ovatus/xylanisolvensV Clostridium difficile Parabacteroides goldsteiniiB

Actinomyces hyovaginalisB Bacteroides ovatus groupB Clostridium innocuumB Parvimonas micra

Actinomyces meyeri Bacteroides pyogenesB Clostridium paraputrificumB Peptoniphilus asaccharolyticusV

Actinomyces neuii Bacteroides salyersiaeB Clostridium perfringens Peptoniphilus harei groupB

Actinomyces odontolyticus Bacteroides stercoris groupB Clostridium ramosum Peptostreptococcus anaerobius

Actinomyces radingaeB Bacteroides thetaiotaomicronVClostridium septicumB Porphyromonas gingivalisB

Actinomyces turicensisB Bacteroides thetaiotaomicron groupB Clostridium sordelliiB Porphyromonas someraeB

Actinomyces urogenitalisB Bacteroides uniformis Clostridium sporogenes / Clostridium

botulinum (group I)B

Prevotella bivia

Actinomyces orisB Bacteroides vulgatusV Clostridium tertiumB Prevotella buccae

Actinotignum schaalii groupB Bacteroides vulgatus groupB Finegoldia magna Prevotella denticola

Alloscardovia omnicolensB Bifidobacterium breveB Fusobacterium canifelinumB Prevotella intermedia

Anaerococcus murdochiiB Clostridium beijerinckiiB Fusobacterium necrophorum Prevotella melaninogenica

Anaerococcus vaginalisB Clostridium bifermentansB Fusobacterium nucleatum Propionibacterium acnes

Bacteroides caccae Clostridium butyricumB Mobiluncus curtsii Veillonella parvula groupB


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Mycobacteria and Nocardia speciesMycobacterium abscessusV Mycobacterium kansasiiV Mycobacterium szulgaiV Nocardia farcinicaV

Mycobacterium aviumV Mycobacterium lentiflavumV Mycobacterium tuberculosis

complexV

Nocardia farcinica groupB

Mycobacterium chelonaeV Mycobacterium malmoenseV Mycobacterium xenopiV Nocardia otitidiscaviarum

Mycobacterium fortuitum groupV Mycobacterium marinumV Nocardia abscessusV Nocardia paucivoransV

Mycobacterium gordonaeV Mycobacterium mucogenicumV Nocardia nova Nocardia pseudobrasiliensisV

Mycobacterium haemophilumV Mycobacterium scrofulaceumV Nocardia asteroidesV Nocardia transvalensisV

Mycobacterium immunogenumV Mycobacterium simiaeV Nocardia brasiliensis Nocardia veteranaV

Mycobacterium intracellulareV Mycobacterium smegmatisV Nocardia cyriacigeorgica Nocardia wallaceiV


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Yeasts

Candida albicans Candida krusei Candida tropicalis Kodamaea/Pichia ohmeri***

Candida boidiniiB Candida lambica Candida utilis/ Cyberlindnera jadinii* Malassezia furfur

Candida dubliniensis Candida lipolytica Candida validaB Malassezia pachydermatis

Candida duobushaemuloniiB Candida lusitaniae Candida zeylanoides Rhodotorula mucilaginosa

Candida famata Candida metapsilosisB Cryptococcus gattiiB Saccharomyces cerevisiae

Candida glabrata Candida norvegensis Cryptococcus neoformansV Trichosporon asahii

Candida guilliermondii Candida orthopsilosisB Cryptococcus neoformans var grubiiB Trichosporon inkin

Candida haemulonii Candida parapsilosis Cryptococcus neoformans var

neoformansB

Trichosporon mucoidesV

Candida inconspicua Candida pararugosaB Geotrichum candidumB Trichosporon mucoides groupB

Candida intermedia Candida pelliculosa Geotrichum capitatum/ Saprochaete

capitate**

Candida kefyr Candida rugosaV Kloeckera apiculata

*Cyberlindnera jadinii (teleomorph) is approved/cleared on the MALDI Biotyper CA system, whereas Candida utilis (anamorph) is approved/cleared on the Vitek MS system.**Geotrichum capitatum is approved/cleared on the MALDI Biotyper CA system, whereas Saprochaete capitate is approved/cleared on the Vitek MS system*****Kodamaea ohmeri is approved/cleared on the Vitek MS system whereas Pichia ohmeri is approved/cleared on the MALDI Biotyper CA system.


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Filamentous Fungi FDA-approved/cleared Vitek MS systemAcremonium sclerotigenum Blastomyces dermatitidis Histoplasma capsulatum Rhizopus arrhizus complex

Alternaria alternata Cladophialophora bantiana Lecythophora hoffmannii Rhizopus microsporus complex

Aspergillus brasiliensis Coccidioides immitis/posadasii Lichtheimia corymbifera Sarocladium kiliense

Aspergillus calidoustus Curvularia hawaiiensis Microsporum audouinii Scedosporium apiospermum

Aspergillus flavus/oryzae Curvularia spicifera Microsporum canis Scedosporium prolificans

Aspergillus fumigatus Epidermophyton floccosum Microsporum gypseum Sporothrix schenckii complex

Aspergillus lentulus Exophiala dermatitidis Mucor racemosus complex Trichophyton interdigitale

Aspergillus nidulans Exophiala xenobiotica Paecilomyces variotii complex Trichophyton rubrum

Aspergillus niger complex Exserohilum rostratum Penicillium chrysogenum Trichophyton tonsurans

Aspergillus sydowii Fusarium oxysporum complex Pseudallescheria boydii Trichophyton verrucosum

Aspergillus terreus complex Fusarium proliferatum Purpureocillium lilacinum Trichophyton violaceum

Aspergillus versicolor Fusarium solani complex Rasamsonia argillacea complex

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Beyond FDA-Approved/Cleared Systems

• Bruker• Research use only (RUO) database • Filamentous Fungi Library• Mycobacteria Library • “Security Relevant” database

• bioMérieux• VITEK MS RUO (SARAMIS) databases

• User-constructed databases

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MALDI TOF Mass Spectrometry: Strengths

• Cost effective – low consumable costs

• Green

• Rapid turnaround time, high throughput

• Automated, robust, interlaboratory reproducibility

• Single colony requirement

• Small footprint

• Low exposure risk – sample inactivation

• Broad applicability (all types bacteria including anaerobes, fungi)

• Adaptable - open system, expandable by user

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MALDI TOF Mass Spectrometry: Limitations

• No susceptibility information

• Not useful for direct testing of clinical specimens (except urine)

• Repeat analyses, additional processing (extraction)

• Cutoffs vary – does “one size fit all”?

• Challenging organisms, relatives, media, colony age

• Databases vary – taxonomy, inclusiveness, security (Bruker)

• Comparison of data from different companies’ instruments?

• Financial loss on existing equipment

• Instrument maintenance downtime (single instrument)

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References1. Alatoom AA, Cazanave CJ, Cunningham SA, Ihde SM, and Patel R. Identification of non-diphtheriae Corynebacterium by use of matrix-assisted laser

desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012 Jan;50(1):160-163.

2. Alatoom AA, Cunningham SA, Ihde SM, et al. Comparison of direct colony method versus extraction method for identification of Gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2011 Aug;49(8):2868-2873.

3. Carroll K and Patel R. Systems for identification of bacteria and fungi. Manual of Clinical Microbiology. 12th ed. ASM Press.

4. Cunningham SA, Mainella JM, and Patel R. Misidentification of Neisseria polysaccharea as Neisseria meningitidis with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2014 Jun;52(6):2270-2271 .

5. Marko DC, Saffert RT, Cunningham SA, et al. Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of nonfermenting gram-negative bacilli isolated from cultures from cystic fibrosis patients. J Clin Microbiol. 2012 Jun;50(6):2034-2039.

6. Mushtaq A, Chen DJ, Strand GJ, et al. Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles—a retrospective chart review. Diagn Microbiol Infect Dis. 2016 Jul;85(3):372-376.

7. Patel R. MALDI-TOF mass spectrometry: Transformative proteomics for clinical microbiology. Clin Chem. 2013 Feb;59(2):340-342.

8. Patel R. Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology. Clin Infect Dis. 2013 Aug;57(4):564-572.

9. Saffert RT, Cunningham SA, Ihde SM, et al. Comparison of Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometer to BD Phoenix automated microbiology system for identification of Gram-negative bacilii. J Clin Microbiol. 2011 Mar;(49)3:887-892.

10.Schmitt BH, Cunningham SA, Dailey AL, Gustafson DR, and Patel R. Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation. J Clin Microbiol. 2013 Mar;51(3):782-786.

11.Theel ES, Schmitt BH, Hall L, et al. Formic acid-based direct, on-plate testing of yeast and Corynebacterium species by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2012 Sep;50(9):3093-3095.

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Acknowledgements• Ryan T. Saffert, Ph.D.• Scott A. Cunningham, M.S.• Nancy L. Wengenack, Ph.D.• Adnan A. Alatoom, M.D., Ph.D.• Elitza S. Theel, Ph.D.• Brian H. Schmitt, M.D.• Jill M. Mainella• Brenda L. Dylla• Derrick J. Chen, M.D.• Ammara Mushtaq, M.D.• Clinical Bacteriology & Mycology Laboratory

Staff• Bruker Daltonics• bioMérieux

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