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Nathan A Ledeboer Assistant Professor of Pathology

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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

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

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

4 Overview of Technology

5 Bruker Biotyper vs Vitek MS

6 Step 1: Target Preparation, continued
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 Research use only – not for use in diagnostic procedures

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

8 Matrix Assisted Laser Desorption/Ionization
Matrix: HCCA (a-Cyano-4-hydroxycinnamic acid) Solvent: Acetonitrile, TFA (trifluoroacetic acid) Lyses cell walls and extract protein Separates protein molecules (proteins are “sticky”) After sample preparation To bring in some MALDI in the run sample overlain with matrix solution contains special small organic molecules critical for soft ionization Evaporation OS > formation of co-crystal of sample and matrix molecules 1 µL Matrix Analyte (organism) Target plate 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 Laser fired, absorbed by matrix molecules Research use only – not for use in diagnostic procedures

10 Matrix Assisted Laser Desorption/Ionization
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 Matrix Energy transfer > formation of a cloud ions Of notice: most sample protein ions result from protonation Singly positively charged Research use only – not for use in diagnostic procedures

11 TOF – Time of Flight Detector
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) Drift region As heavier molecules travel at lower speed they need more time to reach detector ToF > mass to charge ratio calculated Singly charged > m/z=MW Research use only – not for use in diagnostic procedures 11 11

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

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

14

15 Are MALDI-TOF instruments all they are described to be?

16 MALDI publications: More than 200 peer reviewed publications, mainly in high-ranking microbiology journals as of September 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 In a study by Benagli et al
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. Benagli C et al. PLoS One. 6(1).

18 Clinical Application - Bacteroides species
277 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) MALDI ID discrepant sequenced* Bacteroides fragilis Bacteroides thetaiotaomicron Bacteroides ovatus Bacteroides vulgatus Bacteroides uniformis Bacteroides eggerthii Bacteroides nordii Bacteroides salyersiae Bacteroides massiliensis *only IDs with log(score)<2.5 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

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

20 Bruker Biotyper vs Vitek MS

21 System and application (no. of isolates tested)
Saramis Biotyper % Correctly identified P value     Routine (986)a 83.8 vs 92.7 <0.001 NAd NA 86.9 vs 95.5 12.8 vs 3.2 0.3 vs 1.2 <0.01 Vitek MS 83.8 vs 93.2 92.7 vs 93.2 0.608 86.9 vs 93.6 95.5 vs 93.6 <0.05 12.8 vs 5.8 3.2 vs 5.8 0.3 vs 0.4 1 1.2 vs 0.4 Martiny, et al, JCM, 2012, 50

22 Bruker Biotyper vs Vitek MS
Property Microflex LT Vitek MS RUO Vitek MS IVD Remarks User friendliness     Ready-to use Matrix solution No Yes     Facility of preparing smear Very easy Easy For Vitek-MS systems, matrix solution must be deposed each two spots     Disposable targets     Reusable targets     Software Easy to use Not easy to use Very easy to use Time for 96 identifications     Time to prepare work list (min) <5 5–10 NDa     Time to load target and make vacuum 2 5     Time for analysis (min) 40 55     Time for 16 identifications (min) ND 15 No ID before success of QC at end of run (each 16 IDs) Quality     IVD     RUO Need for validation before clinical reporting     Quality management Costc     Device + NAb ++     Reactants +++ NA Based on catalog prices     Maintenance Implementation     Noise Silent Noisy     Size Smaller Bulkier Connectivity Via LIS Via Myla Capacity 1 × 96 4 × 48 Martiny, et al, JCM, 2012, 50

23 *FTE cost/hour $41.35 Time/ test (hour) FTE Cost/test* Supply
Time/ test (hour) FTE Cost/test* Supply Cost/test Total Cost Rapid Biochemicals 0.10 $4.14 $0.29 $4.43 Automated Biochemicals 0.14 $5.79 $9.59 $15.38 Long Biochemicals 0.33 $13.65 $5.32 $18.97 Sequencing 0.73 $30.19 $20.02 $50.21 Mass Spectrometry 0.05 $2.07 $0.24 $2.31 *FTE cost/hour $41.35 Slide courtesy of Robin Patel, MD

24 Cost Savings Comparison
Instrument Included * Assumes a 3 year depreciation of instrument and an instrument cost of $200,000 Tests per Year 5000 10000 20000 30000 40000 50000 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 $484,753.99 Return on Investment (in years) 6.06 3.12 0.67 0.45 0.42 0.41 No Instrument $13.07 $65,350.00 $130,700.00 $397,652.14 $539,670.76 3.06 1.53 0.50 0.37

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 2008-September 2009 October 2009-September 2010 Isolates Tested 33,320 38,624 Biochemical Costs $193,754 $5,374 MALDI-TOF - $15,836 TOTAL $21, 210 Avg Cost/ID $5.81 $.54 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 JCM epub ahead of print

26 Coming applications

27 MALDI– blood culture direct analysis

28 MALDI – blood culture direct analysis
Detailed results and effect of adopted processing < 1.6 3,9% 11,7% 9,1% < 1.7 10,4% 5,2% 27,3% 20,8% > 1.8 85,7% 83,1% 63,6% 67,5% > 2.0 4000 3000 3 9 7 8 4 21 16 66 64 49 52 NEW - Thresholds ORIGINAL - Thresholds 77 samples < 1.6 5,4% 7,5% 11,8% < 1.7 8,6% 9,7% 15,1% 26,9% > 1.8 86,0% 82,8% 73,1% 61,3% > 2.0 4000 3000 5 7 11 8 9 14 25 80 77 68 57 NEW - Thresholds ORIGINAL - Thresholds 93 samples 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 ACN + silica beads, vortex 1 min.
Sample Preparation Positive culture 75% EtOH Water 50ul Water Water 95°C 30 min 2 ml 50ul ACN + silica beads, vortex 1 min. FA 50ul 100% EtOH ~60 min.

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

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

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

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

35 Mycobacteria v1.0 Database

36 HPLC Sequencing ID # Middlebrook 7H10 Trek Myco < 1.7 1.7 – 2.0 > 2.0 MAI (14) M. avium 14 4 10 4(1) M. intracellularae MTBC (6) M. tuberculosis* 6 3 M. chelonae/ abcessus (9) M. abscessus 5 1 M. chelonae 2 M. immunogenum M. fortuitum (16) M. fortuitum 13 9 M. peregrinum M. porcinum M. conceptionense M. xenopi (6) M. xenopi M. szulgai (1) M. szulgai

37 HPLC Sequencing ID # Middlebrook 7H10 Trek Myco < 1.7 1.7 – 2.0 > 2.0 M. gordonae 3 M. mucogenicum (14) 1 M. phocaicum 2 M. llatzerense 5 4 M. peregrinum M.muco/phocaicum M. malmoense M. phlei (1) M. phlei M. smegmatis M. porcinum Total 72 7 (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%

38 HPLC Sequencing ID # Middlebrook 7H10 Trek Myco < 1.7 1.7 – 2.0 > 2.0 M. gordonae 3 M. mucogenicum (14) 1 M. phocaicum 2 M. llatzerense 5 4 M. peregrinum M.muco/phocaicum M. malmoense M. phlei (1) M. phlei M. smegmatis M. porcinum Total 72 7 (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%

39 Data Summary 90.3% (65/72) with acceptable ID score (>1.7)
Middlebrook 7H10 90.3% (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

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

43 Work flow and delay for matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry identification of bacteria in this study. 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 Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD

45 Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD
Organism-group n Mean # of days isolate identified earlier Proportion identified earlier by MALDI-protocol, by number of days of workup <0da 0db 1d 2d 3d 4d 5d 6d >6d (days) (%) S. aureus 109 1.35 1.8 66.1 28.4 2.8 0.9 Other Staphc 26 1.19 7.7 65.4 26.9 BHSd 72 0.60 1.4 38.9 58.3 VGSe 7 0.57 42.9 57.1 S. anginosus 17 1.12 41.2 29.4 5.9 23.5 S. pneumoniae 6 0.33 66.7 33.3 Other GPCf 3.33 16.7 Enterococcus sp. 78 1.64 1.3 51.3 34.6 9.0 2.6 Enterobacteriaceae 284 1.34 69.4 23.2 2.1 1.1 0.4 P. aeruginosa 77 1.82 41.6 49.4 Other NF GNBg 39 2.59 30.8 35.9 15.4 5.1 Haemophilus sp. 10 1.40 80.0 20.0 Other GNCBh 0.14 85.7 14.3 Corynebacterium sp. 9 1.67 22.2 Other GPRi 8 4.13 12.5 37.5 Anaerobic GNj 2.54 3.8 19.2 Anaerobic GPk 14 2.64 21.4 28.6 7.1 C. albicans 52 0.04 92.3 1.9 Other Candida sp. 56 1.93 8.9 67.9 3.6 5.4 Other yeasts 3.75 25.0 All organisms 911 1.45 13.5 52.7 23.6 3.7 2.7 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 Paul J., et al. IDSA

47 Time to Antimicrobial De-Escalation
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 Paul J., et al. IDSA

48 The Future of Mass Spectrometry

49 Electrospray Mass Spectrometry

50 Van Belkum, et al, JCM, 2012, 50 Characteristic MALDI-TOF
LC-ESI-QqQ-MS Ionization Soft ionization with matrix Soft ionization with solvents and electronebulization Fragmentation No (intact molecules) Yes Sample Solid form (or liquid allowed to dry on target) Liquid form (downstream of a liquid chromatography step) Molecules Mainly proteins, large glycopeptides, oligonucleotides, carbohydrates Different molecules, especially peptides Turnaround time 20-30 s per sample at laser frequency of 50 Hz to generate a spectrum Minutes or hours depending on liquid chromatography adsorption/elution times Minutes from sample preparation to result Throughput Disposable target with multiple spots (48 to 96) No batch mode at the moment due to LC step Reagent Chemical matrix Chemical reagents for chromatographic separation and elution External calibrant Internal calibrant Material Disposable target Chromatographic column and precolumn Vials for LC injection Quantification Not well suited Fully adapted Specificity Depending on MS specificity and proteins tested Usually higher than MALDI when selected reaction monitoring (MS2) is used Sensitivity Bacterial ID: 105 CFU using fingerprint approach To be explored Urine sample after purification without culture: 105 CFU/ml Integration into microbiology lab workflow Yes (IVD-compliant systems) No (research applications) Today's clinical microbiology applications Microbiology: identification of bacteria, yeast, and molds None Quantitative assays for small molecules, such as vitamin D (outside microbiology field) 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

53 Questions ?


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