maldi-tof: bringing bacteriology into the 21st century
TRANSCRIPT
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-TOFAt 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!
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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)
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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 13000Mass (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 11000 m/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 PercentageTOTAL 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 14,707E.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 $319Misc 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 $5000 / year Saving!!
What Does an Instrument Cost?
Instrument ~$ 220,000Service Contract ~$ 20,000 / yrDisposables ~ $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 = 0.04
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
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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?
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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