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MALDI-TOF Matrix assisted laser desorption ionization – time of flight

MALDI-TOF Matrix assisted laser desorption ionization – time of flight. MALDI-TOF: Bringing Bacteriology into the 21 st Century. Ross Davidson PhD, FCCM, D(ABMM) Director, Bacteriology Dept. Of Pathology & Laboratory Medicine CDHA. Disclosure.

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MALDI-TOF Matrix assisted laser desorption ionization – time of flight

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  1. MALDI-TOF Matrix assisted laser desorption ionization – time of flight MALDI-TOF: Bringing Bacteriology into the 21st Century Ross Davidson PhD, FCCM, D(ABMM) Director, Bacteriology Dept. Of Pathology & Laboratory Medicine CDHA

  2. Disclosure • I have NO affiliation, financial or otherwise, with any company whose products or devices are discussed within this presentation.

  3. MALDI-TOF At the end of this session, participants will be able to: • Understand the principles of MALDI-TOF • Understand the application and integration of MALDI-TOF into the clinical laboratory • Describe the benefits of MALDI-TOF for patient care and potential cost savings for the laboratory • Describe potential future applications of MALDI-TOF

  4. Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

  5. Advances in Bacterial Identification

  6. Biochemical to MALDI-TOF Bacterial Identification • Most significant advance in Clinical Microbiology (Bacteriology) in 30 years! • Rapid and cost effective identification of bacteria directly from isolated colonies and positive culture bottles based on protein biomarkers • Protein biomarkers measured are highly expressed proteins responsible for housekeeping functions, such as ribosomal (16S) and transcription/translation factor proteins FASTER, BETTER, CHEAPER, BUT NOT PERFECT!

  7. Conventional ID vs MALDI • Monday, 12pm, Mr. J’s blood culture flags positive • Bottle removed, gram stain /culture prepared • Gram negative rods seen, floor called at 1:10pm • 3pm – Mr. J started on Ceftriaxone • Tuesday, 10:30am P. aeruginosa identified • Floor called 10:45am • Mr J started on Pip/tazo • MALDI ID would have seen Mr J on appropriate anti-Pseudomonal therapy 20-24 hours earlier

  8. MALDI TOF Sample Preparation Step 4 Step 1 Step 2 Step 3 Load target slides Add matrix solution* Air dry for 1-2 min. Create Spectra • NOTE: • Other sample types: • sediment from positive blood cultures • sediment from certain specimen (e.g. urines) Spot target slide with direct colony (can be up to 5 days old). Bacteria, molds, yeasts, Mycobacteria Target Slide 48 wells • Matrix Solution: (0.5 µl -cyano-4-hydroxycinnamic acid)

  9. General schematic for MS analysis of ionized microbiological isolates Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

  10. MALDI Mechanism m m m a a a m m m m m a a a m m • 1. Sample (A) is mixed with excess matrix (M) and dried on a MALDI target • 2. Laser ionises matrix molecules • 3. Sample molecules are ionized by proton transfer from matrix: • MH+ + A  M + AH+. + + Laser + + + matrix & analyte Sample support

  11. Principle of MALDI-TOF Time of Flight Molecular masses

  12. The Workflow: Measurement automated spectrum acquisition ~ 60 sec ionization of intact proteins and molecular weight measurement

  13. Low influence of culture conditions Psdm. oleovorans B396_Medium 360 1000 0 Psdm. oleovorans B396_Medium 464 1000 0 Psdm. oleovorans B396_Medium 53 1000 0 Psdm. oleovorans B396_Medium 65 1000 0 Psdm. oleovorans B396_Medium 98 1000 500 0 Psdm. oleovorans B396_MRS10 2000 1000 0 Psdm. oleovorans B396_YPD 2000 1000 0 4000 5000 6000 7000 8000 9000 10000 11000 m/z

  14. Bacterial Identification by Mass Spectrometry

  15. Concordance between Conventional Routine Identification (Vitek) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry 84.1% 95.4% Seng P et al. Clin Infect Dis. 2009;49:543-551

  16. Bizzini et al. J Clin Microbiol 2010;48:1549

  17. van Veen et al. J Clin Microbiol 2010:48:900

  18. Retrospective Validation 16s rDNA used as gold standard

  19. Prospective Validation

  20. Urinary Tract Isolates (2010 – 2012) 92.5% of positive cultures

  21. Cost Calculations Urine cultures • Cost of Chrom-agar vs Blood agar + MALDI • Crom-agar $0.62 / Blood agar $0.27 • MALDI $0.51 (plate, matrix, toothpicks, pipette tips, etc.) • LAP $1.19 / PYR $1.95 / Ox $0.94 / PH B $2.69 / Strep A,C,G $1.46

  22. Cost Calculations Urine cultures • Total # of specimens / year: 63,585 • Total # of negatives / year: 48,862 • Total # of positives / year: 14,707 • Cost of a negative culture: 48,862 x 0.62 - 48,862 x 0.27($30,294 - $13,192 = $17,102 SAV)

  23. Cost Calculations Urine cultures Projected cost savings of moving back to Blood agar is approximately $18,000 / year

  24. Cost calculationsThroat cultures • Gp A Strep $2996 (PYR) vs $1298 (MALDI) = $1698 SAV • Gp C Strep: Costs $534 for a neg PYR $445 extraction $650 Gp C latex = $1629vs$231 (MALDI) = $1398 SAV • Gp G Strep: Costs $377 for a neg PYR $314 extraction $458 for a negGp C latex $458 Gp G latex = $1607vs $163 (MALDI) = $1444 SAV • Approx $5000 / year Saving!!

  25. What Does an Instrument Cost? We currently spend ~$ 65 – 75,000 / yr on Vitek ID panels

  26. Impact of MALDI-TOF MSStudy from Methodist Hospital, Huston TX • Intervention arm (Gram Negative Bacilli): • Integrated rapid ID with active antimicrobial stewardship • Results called to infectious diseases pharmacist 24/7 • Pharmacist recommends de-escalation or adjustment of therapy based on the rapid ID • Time to adjusted therapy was significantly reduced by 31 hrs. P = 0.04 Perez KK, et al. Arch Pathol Lab Med. 2012

  27. EFFECTS ON HEALTH CARE COST • Hospitalization cost reduction of $19,547/patient Perez KK, et al. Arch Pathol Lab Med. 2012

  28. MALDI – TOF Laboratory Integration E X P E C T A T I O N S Current Level

  29. MALDI – TOF Laboratory Integration Challenges • Technologist buy-in • Spotting plates an “art”, not a science • Updated nomenclature (New names)-Wohlfahrtiimonas chitinclastica (Wool farti WHAT??)-isolated from 3rd stage larvae of Wohlfahrtia magnifica • Workflow

  30. No Test is perfect! • E.coli vs Shigella • Acinetobacter baumanii-calcoaceticus complex (A. baumanii, A. calcoaceticus, A. genospecies 3, A. genospecies 13)

  31. Pre MALDI - Good Clinical Microbiology Begins With Good specimens – Garbage In = Garbage Out • Control of sample acceptability • Verification that appropriate sample(s) collected • Correct volume submitted • Sample placed promptly in correct transport media • Optimal and timely transport conditions • Sample handled properly in laboratory • Shared samples • Reflexed samples

  32. Future Direction • Direct specimen applications(already blood / urine data) • Ability to resolve poly-microbial specimens • Antimicrobial resistance determination(already MRSA, carbapenemase) • Strain typing

  33. Direct Detection for Positive Blood Culture Bottles By MALDI Purpose: Separate human and bacterial/yeast ribosomal proteins Methods: Lysis/centrifugation or membrane filtration • Issues: • Removal of human proteins • Extraction protocol required • Bacterial concentration • need~107/mL • Polymicrobial specimens • Seen on Gram stain? • Charcoal • Antibiotic resistance genes • Yeasts? • Unique database, different cutoffs? Journal of Clinical Microbiology 51;805-809, 2013 Journal of Clinical Microbiology 48;1584-1591, 2010

  34. Potential Optionsfor Direct detectionfrom clinicalspecimens Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

  35. MALDI-TOF Bacterial ID • Minimal sample preparation • Cost effective - low consumable cost • Powerful bioinformatic approaches • Species to strain resolution • Non-expert identification possible • Dedicated databases continue to expand

  36. MALDI-TOF Limitations • Databases : still in their infancy • High initial capital expenditure • New approaches (business models) • Potential instrument downtime - single instrument

  37. MALDI-TOF in the Clinical Laboratory • Rapid turn around time, high throughput - impact on appropriate emperic therapy • Single colony requirement- direct from blood culture • Low exposure risk –sample inactivation • Broad applicability (all types bacteriaincluding anaerobes, yeasts, fungi) • COST SAVINGS

  38. Questions

  39. General schematic for MS analysis of ionized microbiological isolates Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

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