Applications of mass spectrometry in clinical microbiology

1. Applications of mass spectrometry in clinical microbiology J. Stephen Dumler, MD Department of Pathology University of Maryland School of Medicine Baltimore, MD USA (special thanks to Karen Carroll, MD andAmé Maters, MS, MT ASCP) The Johns Hopkins Hospital, Baltimore MD USA

2. Applications of mass spectrometry in clinical microbiology Outcome of infectious disease often linked to: • Time to treatment of infectious agent • Time to recovery and identification of infectious agent Good antibiotic stewardship mandates • Using effective antibiotics only • Avoid using broad spectrum antibiotics when the infectious agent is unknown

3. Applications of mass spectrometry in clinical microbiology The impact of early appropriate therapy is dramatic with severe infections • Mortality when treated within 1h <0.3 times that of longer intervals

4. Applications of mass spectrometry in clinical microbiology • Empiric antibiotic prescribing can drive antimicrobial resistance • Even without increased mortality, prolonged morbidity is expensive, impacting health care costs for all Methods to reduce time to appropriate antibiotic treatment are critically needed

5. Applications of mass spectrometry in clinical microbiology Routine identification of bacterial and fungal pathogens on average requires 2 days or longer • Day 1 – culture and identification • Day 2 – antibiotic susceptibility testing Automated systems (e.g. blood culture) has reduced time recovery and pathogen identification • 12-16h recovery times reduced to as short as 4-8h.

6. Applications of mass spectrometry in clinical microbiology Therefore, methods to “speed up” microbiology are desperately needed. • Antigen detection methods • FISH and PNA-FISH • Directed molecular diagnostics • PCR and broad range PCR • NASBA, other nucleic acid amplification tests

7. Applications of mass spectrometry in clinical microbiology Therefore, methods to “speed up” microbiology are desperately needed. • The only technology to rapidly improve microbiology identification in recent years is matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectroscopy.

8. Principles of MALDI-TOF MS Ions travel in vacuum electrical field based on mass to charge ratio (m/z) Bacterial ribosomal proteins ionize Photons activate matrix Bacteria + Matrix

9. Detector Vacuum Tube Laser Desorption Ionization Figure & animation adapted from Bruker Daltonics, Inc. Electrodes Matrix-Assisted

10. Intensity m/z Mass Spectrometry Detector Time-of- Flight Vacuum Tube Laser Desorption Ionization Electrodes Matrix-Assisted Figure & animation adapted from BrukerDaltonics

11. Applications of mass spectrometry in clinical microbiology Advantages of MALDI-TOF MS for microbiology • Very little sample preparation • Rapid • 10-20 seconds for acquisition • 15-30 seconds for database comparisons • Total time after culture 1-2 minutes Disadvantages of MALDI-TOF MS for microbiology • Limited to pure cultures • Hard to detect new organisms • Difficult to quantitate

12. MALDI-TOF Mass Spectroscopy Smear colonies on target; apply matrix Pick 1-2 colonies Insert target into instrument

13. Spectral Pattern

15. Spot Isolates in Duplicates from Primary Plates (>2 spots if mucoid, sticky, uncertain) MALDI How about those with poor scores, but have a single organism ID? Top 2 scores: ≥ 2.0, ≥ 1.7? NO Subculture or re-inc, then repeat MALDI YES NO Same species/ group? YES Scores acceptable? NO Full Extraction & repeat MALDI YES Accept ID & Scores

16. MALDI Algorithm

18. Johns Hopkins HospitalMicrobiology Laboratory • Full service CAP accredited laboratory • Provides testing for 3 hospitals, 21 clinics • Processes 175,000 specimens annually for bacterial and fungal cultures • Bacterial, yeast cultures read once daily, on day shift, 7 d/week • 8 workstations (benches) • Blood ̶ Sterile body fluids • Urine ̶ Stool • Aerobic wound ̶ Respiratory • Anaerobic wound/tissue ̶ Cystic fibrosis

19. Study Data Collection • 12 weeks of prospective data: • MALDI vs. standard identification • Reagents consumed (costs) • Timing required for testing • Recorded data on: • Reagent costs • Hands-on time required per assay • Estimated annual costs based on prevalence of organisms from 2011

20. Standard Algorithm Compared to MALDI-TOF Algorithm vs Software v 3.0 Database v 3.1.2

21. MALDI-TOF Protocol • Incorporated manufacturer’s recommendations and observations from recent studies • Allowed multiple attempts • Allowed incorporation of supplementary tests Justesen US, et al J ClinMicrobiol2011:49:4314-18; Neville SA, et al J ClinMicrobiol2011; 49:2980-84; Saffert RT, et al J ClinMicrobiol2011; 49:887-92.

22. Accuracy 952 Isolates Tested by MALDI Protocol 912 (95.8%) concordant 33 discordant & with discrepancy analysis 7 discordant & but no discrepancy analysis 17 MALDI correct 16 MALDI incorrect 4 where standard protocol correct MALDI-TOF method accuracy = 929/945 = 98.3% Current standard method accuracy = 932/945 = 98.6%

23. Performance - GPC

24. Performance - GNB

25. Performance – Other Bacteria

26. Discrepant Analysis

27. Time-to-Identification Finalized 911 isolates included for analysis 87.2%ID on 1st day 10.6% ID on 2nd day 2.2% ID after 2nd day Corresponding values for Standard Protocol 9.4% 52.1% 26.5%

28. Time-to-Identification

29. Time to Identification (TTI) Summary • Overall TTI was 1.43 days earlier on average by MALDI • Likely underestimated as 2nd attempt was done next working day • Potentially may be done on 1st day • MALDI protocol will not affect time-to-susceptibility

30. Costs • Determined cost-per-isolate by standard methods and by MALDI • Based on reagent consumption data • Applied to the actual reagent-cost and hands-on-time • Determined the annual cost-per-species using the above and annual laboratory prevalence data

31. MALDI Application Rules Applied to “common” bacteria & yeasts • Encountered >50 times per year • 55 species in this study Not applied to: • Contaminants – CoNS, Corynebacteria, Lactobacilli, Enterococcus in mixed urine specimens • Uncommon organisms (>200 species) • At JHH represents 3.0% of annual organisms • Will require both MALDI and other methods

32. Example of Cost CalculationsCost-per-Staphylococcus aureus Isolate Standard Methods MALDI Method Each isolate required: 1.01 Direct MALDI ($0.36, 45s) per isolate 0.01 Extracted MALDI ($0.62, 4min)  $0.36 and 48s per isolate Each isolate required: • 0.28 catalase ($0.0005, 10s) • 1.03 latex-agglutination tests ($0.81, 30s) • 1.02 Phoenix-ID panels ($1.27, 0s)  $2.23 and 39s per isolate 7059 isolates identified annually: Reagent cost: $15,748$2,524 Labor-time: 76hrs 38mins 93hrs 22mins Labor-cost: $2,005$2,443

34. Cost AnalysisSummary • Savings of $102,413 (53.9%) • Does not include costs of susceptibility testing • Does not include indirect cost savings • Additional savings as more organisms are validated • P. aeruginosa, E. coli, S. aureus, GBS & E. faecalisrepresented 65.3% of the total savings

35. Summary Cherkaoui 2010 JCM 48:1169, Martiny 2012 JCM, Neville 2011 JCM 49:2980

36. Practical Rules of MALDI • Don’t MALDI everything • Use MALDI to confirm suspicion • Re-incubate plate if colonies are too small • Be careful of taxonomic complexes: • A. baumanii • C. freundii • E. cloacae • S. anginosus • If identification is unusual/ unexpected, repeat the MALDI

37. Applications of mass spectrometry in clinical microbiology • The future of MALDI-TOF MS in Clinical Microbiology Labs is the present • Can MALDI-TOF MS be used on direct samples? • Can MALDI-TOF MS be used as a discovery tool? • Will derivations of mass spectrometry replace MALDI-TOF MS and be the new “present” for Clinical Microbiology laboratories? • THE END! THANK YOU