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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 and Amé Maters, MS, MT ASCP) The Johns Hopkins Hospital, Baltimore MD USA.

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Applications of mass spectrometry in clinical microbiology

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


Applications of mass spectrometry in clinical microbiology1
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


Applications of mass spectrometry in clinical microbiology2
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


Applications of mass spectrometry in clinical microbiology3
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


Applications of mass spectrometry in clinical microbiology4
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.


Applications of mass spectrometry in clinical microbiology5
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


Applications of mass spectrometry in clinical microbiology6
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.


Principles of maldi tof ms
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


Detector

Vacuum Tube

Laser Desorption Ionization

Figure & animation adapted from Bruker Daltonics, Inc.

Electrodes

Matrix-Assisted


Intensity

m/z

Mass Spectrometry

Detector

Time-of- Flight

Vacuum Tube

Laser Desorption Ionization

Electrodes

Matrix-Assisted

Figure & animation adapted from BrukerDaltonics


Applications of mass spectrometry in clinical microbiology7
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


Maldi tof mass spectroscopy
MALDI-TOF Mass Spectroscopy

Smear colonies on target;

apply matrix

Pick 1-2 colonies

Insert target into instrument



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



Johns hopkins hospital microbiology laboratory
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


Study data collection
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


Standard algorithm
Standard Algorithm

Compared to MALDI-TOF Algorithm

vs

Software v 3.0

Database v 3.1.2


Maldi tof protocol
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.


Accuracy
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%






Time to identification finalized
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%



Time to identification tti summary
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


Costs
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


Maldi application rules
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


Example of cost calculations cost per staphylococcus aureus isolate
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


Cost analysis summary
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


Summary
Summary

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


Practical rules of maldi
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


Applications of mass spectrometry in clinical microbiology8
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


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