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Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Nathan A Ledeboer Assistant Professor of Pathology Medical College of Wisconsin.

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Presentation on theme: "Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Nathan A Ledeboer Assistant Professor of Pathology Medical College of Wisconsin."— Presentation transcript:

1 Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Nathan A Ledeboer Assistant Professor of Pathology Medical College of Wisconsin and Medical Director, Microbiology and Molecular Pathology Dynacare Laboratories and Froedtert Hospital Milwaukee, WI 1

2 Outline Overview of Technology Are MALDI-TOF instruments all they described to be? How can I justify the cost? Applications in Development What is the Impact for Patient Care The Future of Mass Spectrometry Conclusions Questions 2

3 Disclosures Dr. Ledeboer will discuss products that are not FDA cleared Financial Disclosures – Consultant: Nanosphere, Inc ThermoFisher Scientific, Inc LabCorp Cepheid (Scientific Advisory Board) iCubate – Honoraria Bruker Daltonics Meridian 3

4 OVERVIEW OF TECHNOLOGY 4

5 Bruker Biotyper vs Vitek MS 5

6 Direct Smear Method: Touch colony with transfer device, such as toothpick Transfer a small amount onto spot Let air dry Cover with 1 µL of MALDI matrix, let air dry Analyze Step 1: Target Preparation, continued Research use only – not for use in diagnostic procedures

7 dH2O Ethanol-Formic Acid Extraction (if required – low score value after direct smear) Research use only – not for use in diagnostic procedures Inactivation of pathogens Ethanol Formic acid, acetonitrile Analyze supernatant 10 min

8 Target plate Analyte (organism) 1 µL Matrix Matrix Assisted Laser Desorption/Ionization Matrix: HCCA (  -Cyano-4-hydroxycinnamic acid) Solvent: Acetonitrile, TFA (trifluoroacetic acid) Lyses cell walls and extract protein Separates protein molecules (proteins are “sticky”) Research use only – not for use in diagnostic procedures

9 Matrix Assisted Laser Desorption/Ionization Laser light pulses Matrix molecules readily absorb laser light (photon energy), creating an excited energy state The matrix is acidic, and donates positive charge to the analytes Research use only – not for use in diagnostic procedures

10 Matrix Assisted Laser Desorption/Ionization Matrix Localized heating causes micro-explosion of material Collisions with neutral sample facilitate charge transfer to/from excited matrix molecules Ions “desorb” from the target surface Research use only – not for use in diagnostic procedures

11 Drift region Detector TOF – Time of Flight m/z Intensity Following acceleration, the charged ions are allowed to drift through a free field toward the detector The speed of travel (time of flight) is proportional to the ion’s mass (smaller ions reach the detector first) Research use only – not for use in diagnostic procedures

12 MALDI: Results output Raw profile spectrumRefined profile spectrum 12 Results are analyzed by a computer, cleaned-up and the spectrum is searched against a database with known spectra.

13 Secure genus and species identification Probable genus identification Unreliable identification MALDI identification result

14

15 ARE MALDI-TOF INSTRUMENTS ALL THEY ARE DESCRIBED TO BE? 15

16 MALDI publications: More than 200 peer reviewed publications, mainly in high-ranking microbiology journals as of September 2012 Topics include: - Particular groups of bacteria -(e.g. anaerobes, Listeria, Neisseria, Yeast...) - Routine application - Rare and difficult to analyse microorganisms (e.g. Prothotheca) - Highly pathogenic bacteria (e.g. Francisella, Brucella) - Blood culture direct analysis - Urine direct analysis - Sub-typing - Resistance or Virulence Factors -Mycobacteria -Filamentous Fungi

17 17 Benagli C et al. PLoS One. 6(1). In a study by Benagli et al., the authors compared performance of MALDI-TOF to biochemical ID and resolved discrepancies with sequencing. The results follow.

18 MALDI ID discrepant sequenced* Bacteroides fragilis Bacteroides thetaiotaomicron Bacteroides ovatus Bacteroides vulgatus Bacteroides uniformis 5 32 Bacteroides eggerthii 1 0 Bacteroides nordii 4 31 Bacteroides salyersiae 1 0 Bacteroides massiliensis *only IDs with log(score)< Clinical Bacteroides Isolates from a European study: 270 isolates (97,5%) identified with significant score, 7 isolates not in Reference Library (e.g. Bacteriodes distasonis) 16S rDNA Sequenceing Confirmed 10 of 11 Discrepant MALDI Results, 1 Case Only “Bacteroides spec.“ Nagy et al., Clin Microbiol Infect 2009; 15: 796–802 Clinical Application - Bacteroides species

19 Marklein et al., JCM, Vol Clinical Application: Yeast No incorrect Yeast Identification by the Respective Molecular Fingerprint

20 Bruker Biotyper vs Vitek MS 20

21 21 Martiny, et al, JCM, 2012, 50

22 Bruker Biotyper vs Vitek MS 22 Martiny, et al, JCM, 2012, 50

23 23 Time/ test (hour) FTE Cost/test* Supply Cost/test Total Cost Rapid Biochemicals0.10$4.14$0.29$4.43 Automated Biochemicals 0.14$5.79$9.59$15.38 Long Biochemicals0.33$13.65$5.32$18.97 Sequencing0.73$30.19$20.02$50.21 Mass SpectrometryMass Spectrometry0.05$2.07$0.24$2.31 *FTE cost/hour$41.35 Slide courtesy of Robin Patel, MD

24 24 Cost Savings Comparison Instrument Included * Assumes a 3 year depreciation of instrument and an instrument cost of $200,000 Tests per Year Instrument Add* $13.33$6.66$3.33$2.22$1.66$1.33 MALDI-TOF ID Consumables and Labor $2.31 Biochemical ID Consumables and Labor $15.38 Difference per test -$0.26$6.41$9.74$10.85$11.41$11.74 Cost Savings per year-$1,300.00$64,100.00$296,337.55$448,005.18$471,128.03$484, Return on Investment (in years) No Instrument Tests per Year MALDI-TOF ID Consumables and Labor $2.31 Biochemical ID Consumables and Labor $15.38 Difference per test $13.07 Cost Savings per year$65,350.00$130,700.00$397,652.14$539, Return on Investment (in years)

25 Cost-effectivenesss of switching to MALDI-TOF MS for routine bacterial identification Galliot O, Blondiaux N, Lorez C, Wallet F, Lemaitre N, Herwegh S and Courcol R  September 2009  Switched from conventional biochemicals (Vitek 2 and API) to MALDI-TOF MS (Bruker)  Cost analysis performed October September 2009 October September 2010 Isolates Tested33,32038,624 Biochemical Costs $193,754$5,374 MALDI-TOF-$15,836 TOTAL$193,754$21, 210 Avg Cost/ID$5.81$.54 JCM epub ahead of print Annual Savings = $177, 090 “allowed decrease of 89.3% of the cost of bacterial identification in the first year.” In addition: Decreased waste from 1,424kg to 44kg Decreased subculture media of $1,102 Decreased sequencing cost of $1,650

26 COMING APPLICATIONS 26

27 MALDI– blood culture direct analysis 27

28 MALDI – blood culture direct analysis < 1.63,9%11,7%9,1%11,7%< ,4%5,2%27,3%20,8% > 1.885,7%83,1%63,6%67,5%> < < > > NEW - ThresholdsORIGINAL - Thresholds 77 samples < 1.65,4%7,5%11,8% < ,6%9,7%15,1%26,9% > 1.886,0%82,8%73,1%61,3%> < < > > NEW - ThresholdsORIGINAL - Thresholds 93 samples Detailed results and effect of adopted processing No false positive result in the “yellow“ and “green“ log(score) range 3000/4000 –detection boundary increased for protein to reduce background

29 Proof Point: Blood Culture Innovative Extraction 99% of tested strains were correctly identified directly from Blood Culture

30 2 ml Positive culture ~60 min. Sample Preparation Water 75% EtOH Water 50ul 95°C 30 min 100% EtOH 50ul ACN + silica beads, vortex 1 min. FA 50ul

31 2 ml Positive culture ~60 min. Sample Preparation Water 75% EtOH Water 50ul 95°C 30 min 100% EtOH 50ul ACN + silica beads, vortex 1 min. FA 50ul

32 Days to positivity M. che/abs gr M. fortuitum gr MAI MTB Heat Kill Test Following heat inactivation step, TREK Myco bottles were inoculated and held 6 weeks.

33 2 ml Positive culture ~30 min. Sample Preparation Water 75% EtOH Water 50ul 95°C 30 min 100% EtOH 50ul ACN + silica beads, vortex 1 min. FA 50ul

34 No Bead-beat step Bead-beat step Effect of Bead-Beat on MALDI Spectrum

35 Mycobacteria v1.0 Database

36 HPLCSequencing ID# Middlebrook 7H10Trek Myco < – 2.0> 2.0< – 2.0> 2.0 MAI (14)M. avium (1)10 M. intracellularae MTBC (6)M. tuberculosis* M. chelonae/ abcessus (9) M. abscessus M. chelonae M. immunogenum M. fortuitum (16) M. fortuitum M. peregrinum M. porcinum M. peregrinumM. conceptionense M. xenopi (6)M. xenopi M. szulgai (1)M. szulgai

37 HPLCSequencing ID# Middlebrook 7H10Trek Myco < – 2.0> 2.0< – 2.0> 2.0 M. gordonae M. mucogenicum (14) M. mucogenicum M. phocaicum M. llatzerense M. peregrinum M.muco/phocaicum M. phocaicum M. malmoense M. phlei (1) M. phlei M. smegmatis M. porcinum Total727 (9.7%) 31 (43.1%) 34 (47.2%) 6 (8.3%) 14 (19.4%) 52 (72.2%) Agreement (species)80.0%96.6%100%83.3%92.9%100%

38 HPLCSequencing ID# Middlebrook 7H10Trek Myco < – 2.0> 2.0< – 2.0> 2.0 M. gordonae M. mucogenicum (14) M. mucogenicum M. phocaicum M. llatzerense M. peregrinum M.muco/phocaicum M. phocaicum M. malmoense M. phlei (1) M. phlei M. smegmatis M. porcinum Total727 (9.7%) 31 (43.1%) 34 (47.2%) 6 (8.3%) 14 (19.4%) 52 (72.2%) Agreement (species)85.7%96.4%100%83.3%92.3%100%

39 Middlebrook 7H % (65/72) with acceptable ID score (>1.7)  98.3% (59/60) correct species ID Trek Myco 91.7% (66/72) with acceptable ID score (>1.7)  98.4% (60/61) correct species ID Data Summary

40 Time: Clinical Impact t = 0 t = 3-5 days t = 1 day t = 1-2 days t = 1 hr

41 ID Filamentous Fungi - workflow 1. Direct Transfer of “Front Mycelium“ (1 min)  if successful: ID is FINISHED e.g. A.niger 2. Ethanol Extraction of “Front Mycelium“ (10 min)  if successful: ID is FINISHED 3. Broth Cultivation (approx. 1 additional day) & extraction  ID is possible for agar adhering filamentous fungi  ID is possible for slow or fast sporulating fungi  ID is possible for every kind of filamentous fungi  ALL matches against the SAME Filamentous Fungi DB

42 IMPACT ON PATIENT CARE 42

43 Work flow and delay for matrix-assisted laser desorption ionization time-of-flight (MALDI- TOF) mass spectrometry identification of bacteria in this study. Seng P et al. Clin Infect Dis. 2009;49: © 2009 by the Infectious Diseases Society of America

44 44 Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD

45 45 Organism-groupn Mean # of days isolate identified earlier Proportion identified earlier by MALDI-protocol, by number of days of workup <0d a 0d b 1d2d3d4d5d6d>6d (days) (%) S. aureus Other Staph c BHS d VGS e S. anginosus S. pneumoniae Other GPC f Enterococcus sp Enterobacteriaceae P. aeruginosa Other NF GNB g Haemophilus sp Other GNCB h Corynebacterium sp Other GPR i Anaerobic GN j Anaerobic GP k C. albicans Other Candida sp Other yeasts All organisms Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD

46 Patient Impact Retrospective chart review of patients with positive blood cultures obtained during validation of MALDI-TOF Biotyper and Sepsityper Kit Time to ID (MALDI-TOF v traditional culture methods) and start/stop times of antibiotics reviewed Theoretical reduction in empiric antibiotic duration and cost difference (AWP) calculated 46 Paul J., et al. IDSA. 2011

47 Time to Antimicrobial De- Escalation 47 Traditional ID MALDI-TOF Direct ID Difference (in hours) P Emperic Antibiotic Duration ( IQR, hrs) 66.6 ( ) 15.5 ( ) 51.6 ( ) <0.01 Antibiotic Cost (USD) $245.24$88.48$156.76<0.01 Paul J., et al. IDSA. 2011

48 THE FUTURE OF MASS SPECTROMETRY 48

49 Electrospray Mass Spectrometry

50 50 Van Belkum, et al, JCM, 2012, 50

51 Microcantilevers

52 Conclusions Data demonstrates excellent performance of MALDI-TOF MS for identification of bacteria and yeast from plates and from positive blood cultures. High Capital Cost can be overcome by consumable savings Use of technology can result in a significant reduction in laboratory turnaround and significant antimicrobial cost savings MALDI-TOF and current technologies represent the beginning of protein revolution 52

53 Questions ?


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