MALDI-TOFMatrix assisted laser desorption ionization –

time of flight

MALDI-TOF: Bringing Bacteriology into the 21st

Century

Ross Davidson PhD, FCCM, D(ABMM)Director, BacteriologyDept. Of Pathology & Laboratory MedicineCDHA

Disclosure

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

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

Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

Advances in 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

Biochemical to MALDI-TOF Bacterial Identification

FASTER, BETTER, CHEAPER, BUT NOT PERFECT!FASTER, BETTER, CHEAPER, BUT NOT PERFECT!

6

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

Add matrix solution*

Air dry for 1-2 min.

MALDI TOF Sample Preparation

Create Spectra

Target Slide48 wells

Step 1 Step 2 Step 3 Step 4

Bacteria, molds, yeasts,Mycobacteria

Spot target slide with direct colony

(can be up to 5 days old).

Load target slides

• Matrix Solution: (0.5 µl -cyano-4-hydroxycinnamic acid)

NOTE:Other sample types:- sediment from positive blood cultures- sediment from certain specimen (e.g. urines)

8

General schematic for MS analysis of ionized microbiological isolates

Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

MALDI Mechanism

Laser

matrix & analyte

Sample support

aa

mm

mm

mm

mm

mm

mmmm

mm aa

aa

aa

aa

++

++

++

++mm

aamm++

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+.

Principle of MALDI-TOF

Molecular massesTime of Flight

3000 5000 7000 9000 11000 13000

Mass (m/z)

1648.1

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

< < C :\D o k u m e n te u n d E in s te llu n g e n \m .e rh a rd \E ig e n e D a te ie n \A n a g n o s T e c -d c \Z w is c h e n a b la g e \D o k u m e n t B a n n e r.tx t> > S p e c [B P = 3

3775

7075

8074

8744

1093

9

9453

9994

7980

1034

7

5015

7863

1157

2

7092

5931

9654

3581

6837

9330

7367

1022

6

8450

1060

8

1190

6

automated spectrum acquisition ~ 60 secionization of intact proteins and molecular weight measurement

The Workflow: Measurement

Psdm. oleovorans B396_Medium 360

0

1000

Psdm. oleovorans B396_Medium 464

0

1000

Psdm. oleovorans B396_Medium 53

0

1000

Psdm. oleovorans B396_Medium 65

0

1000

Psdm. oleovorans B396_Medium 98

0500

1000

Psdm. oleovorans B396_MRS10

010002000

Psdm. oleovorans B396_YPD

010002000

4000 5000 6000 7000 8000 9000 10000 11000m/z

Low influence of culture conditions

Bacterial Identification by Mass Spectrometry

Concordance between Conventional Routine Identification (Vitek) and Matrix-Assisted Laser Desorption Ionization Time-

of-Flight (MALDI-TOF) Mass Spectrometry

Seng P et al. Clin Infect Dis. 2009;49:543-551

84.1%95.4%

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

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

Retrospective Validation

Total Tested 838

Correlates 810 97%

Mismatch 28 3%

16s rDNA used as gold standard

Prospective Validation

Total Tested 356Correlates 342 96%Mismatch 7 1.9%No Growth ---No Data 7

Urinary Tract Isolates (2010 – 2012)

Organisms Number Percentage

TOTAL Specimens 190,757 --

TOTAL Negatives 146,586 77%

TOTAL Positives 44,121 23%

E.coli 24,415 *55%

KES 5,148 *12%

Enterococcus 5,036 *11%

Gp B Streptococcus 2,005 *4.5%

Proteus 1,890 *4.0%

Candida albicans 1,211 *2.7%

S.saprophyticus 1,006 *2.3%

Citrobacter 380 *0.8%

92.5% ofpositive cultures

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

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)

Cost Calculations Urine cultures

# ChromAgar Chrom + MALDI

Yeast Chrom

Blood Agar

BL + MALDI

+/- Cost

Negatives 48,862 $30,294 $13,192 -$17,102

Positives 14,707

E.coli 8140 $5047 $6430 +$1382KES 1716 $1956 $1356 -$600Enterococcus 1680 $1915 $1325 -$590Bp B Strep 668 $762 $528 -$235Proteus 630 $718 $497 -$221Candida 404 $251 $461 $319

Misc Pos 1462 $1667 $1178 -$489

Projected cost savings of moving back to Blood agar is approximately $18,000 / year

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 = $1629 vs $231 (MALDI) = $1398 SAV

• Gp G Strep: Costs $377 for a neg PYR $314 extraction $458 for a neg Gp C latex $458 Gp G latex = $1607 vs $163 (MALDI) = $1444 SAV

• Approx $5000 / year Saving!!

What Does an Instrument Cost?

Instrument ~$ 220,000

Service Contract ~$ 20,000 / yr

Disposables ~ $3-7,000 / yr

~ cost per test ~ $ 0.51 / test

We currently spend ~$ 65 – 75,000 / yr on Vitek ID panels

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 = 0.04P = 0.04

Perez KK, et al. Arch Pathol Lab Med. 2012

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EFFECTS ON HEALTH CARE COST

Hospitalization cost reduction of $19,547/patient

Perez KK, et al. Arch Pathol Lab Med. 201227

MALDI – TOF Laboratory Integration

EXPECTATIONS

Current Level

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

No Test is perfect!

• E.coli vs Shigella

• Acinetobacter baumanii-calcoaceticus complex (A. baumanii, A. calcoaceticus, A. genospecies 3, A. genospecies 13)

• 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

Pre MALDI - Good Clinical Microbiology Begins With Good specimens – Garbage In = Garbage Out

31

Future Direction

• Direct specimen applications

(already blood / urine data)

• Ability to resolve poly-microbial

specimens

• Antimicrobial resistance determination

(already MRSA, carbapenemase)

• Strain typing

Direct Detection for Positive Blood Culture Bottles By MALDIDirect Detection for Positive Blood Culture Bottles By MALDI

Purpose: Separate human and bacterial/yeast ribosomal proteinsMethods: Lysis/centrifugation or membrane filtration

Journal of Clinical Microbiology 51;805-809, 2013

Journal of Clinical Microbiology 48;1584-1591, 2010

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?

33

Potential Optionsfor Direct detectionfrom clinicalspecimens

Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

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

MALDI-TOF Limitations

• Databases : still in their infancy

• High initial capital expenditure

• New approaches (business models)

• Potential instrument downtime

- single instrument

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 bacteria

including anaerobes, yeasts, fungi)

• COST SAVINGS

Questions

General schematic for MS analysis of ionized microbiological isolates

Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603