Applications of mass spectrometry in clinical microbiology
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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


Applications of mass spectrometry in clinical microbiology

Detector

Vacuum Tube

Laser Desorption Ionization

Figure & animation adapted from Bruker Daltonics, Inc.

Electrodes

Matrix-Assisted


Applications of mass spectrometry in clinical microbiology

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


Applications of mass spectrometry in clinical microbiology

Spectral Pattern


Applications of mass spectrometry in clinical microbiology

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


Maldi algorithm

MALDI Algorithm


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

    • Urine̶ Stool

    • Aerobic wound̶ 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%


Performance gpc

Performance - GPC


Performance gnb

Performance - GNB


Performance other bacteria

Performance – Other Bacteria


Applications of mass spectrometry in clinical microbiology

Discrepant Analysis


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

Time-to-Identification


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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