MALDI-TOF
Matrix assisted laser desorption ionization –
time of flight
MALDI-TOF: Bringing
Bacteriology into the 21st
Century
Ross Davidson PhD, FCCM, D(ABMM)
Director, Bacteriology
Dept. Of Pathology & Laboratory Medicine
CDHA
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
Biochemical to MALDI-TOF 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
6
FASTER, BETTER, CHEAPER, BUT NOT PERFECT!
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 antiPseudomonal therapy 20-24 hours earlier
MALDI TOF Sample Preparation
Step 1
Spot target slide with
direct colony (can be
up to 5 days old).
Step 2
Add matrix
solution*
Step 3
Step 4
Load target slides
NOTE:
Other sample types:
- sediment from positive blood cultures
- sediment from certain specimen (e.g. urines)
Air dry for 1-2 min.
Bacteria, molds,
yeasts,
Mycobacteria
Target 8Slide
48 wells
Matrix Solution: (0.5 µl -cyano-4-hydroxycinnamic acid)
Create Spectra
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
1. Sample (A) is mixed
with excess matrix (M)
and dried on a MALDI
target
+
m
m
m
a
+
a
m a
m
+
a m
a
m
a
+
m m
m+
2. Laser ionises matrix
molecules
3. Sample molecules are
ionized by proton
transfer from matrix:
MH+ + A  M + AH+.
Principle of MALDI-TOF
Time of Flight
Molecular masses
The Workflow: Measurement
automated spectrum
acquisition ~ 60 sec
ionization of intact proteins and
molecular weight measurement
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5000
7000
11000
11906
11572
10226
10347
9000
Mass (m /z)
10608
10
9654
8450
9330
7863
7367
20
7092
3581
40
6837
50
5931
5015
60
10939
9994
7980
70
% Intensity
t
9453
8744
8074
7075
80
0
3000
a
1648.1
90
30
D
3775
<
13000
Low influence of culture
conditions
Psdm. oleovorans B396_Medium 360
1000
0
Psdm. oleovorans B396_Medium 464
1000
0
Psdm. oleovorans B396_Medium 53
1000
0
Psdm. oleovorans B396_Medium 65
1000
0
Psdm. oleovorans B396_Medium 98
1000
500
0
Psdm. oleovorans B396_MRS10
2000
1000
0
Psdm. oleovorans B396_YPD
2000
1000
0
4000
5000
6000
7000
8000
9000
10000
11000
m/z
Bacterial Identification by Mass Spectrometry
Concordance between Conventional Routine Identification
(Vitek) and Matrix-Assisted Laser Desorption Ionization Timeof-Flight (MALDI-TOF) Mass Spectrometry
84.1%
95.4%
Seng P et al. Clin Infect Dis. 2009;49:543-551
Bizzini et al. J Clin Microbiol 2010;48:1549
van Veen et al. J Clin Microbiol 2010:48:900
Retrospective Validation
Maldi Correlation Summary
Mismatch,
3%
Total Tested
838
Correlates
810 97%
Mismatch
28
Correlates
Mismatch
3%
Correlates,
97%
16s rDNA used as gold standard
Prospective Validation
Total Tested
Correlates
Mismatch
No Growth
No Data
356
342
7
--7
96%
1.9%
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%
380
*0.8%
Citrobacter
92.5% of
positive 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
#
Negatives
48,862
Positives
14,707
ChromAgar Chrom +
MALDI
$30,294
Yeast
Chrom
Blood
Agar
BL +
MALDI
$13,192
$5047
$17,102
E.coli
8140
KES
1716
$1956
$1356
Enterococcus
1680
$1915
$1325
Bp B Strep
668
$762
$528
Proteus
630
$718
$497
Candida
404
$461
$319
Misc Pos
1462
$1667
$1178
$251
+/- Cost
$6430
+$1382
-$600
-$590
-$235
-$221
-$489
Projected cost savings of moving back to Blood agar is approximately $18,000 / year
Cost calculations
Throat 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 $5000 / year Saving!!
What Does an Instrument Cost?
Instrument
Service Contract
Disposables
~ cost per test
~$ 220,000
~$ 20,000 / yr
~ $3-7,000 / yr
~ $ 0.51 / test
We currently spend ~$ 65 – 75,000 / yr
on Vitek ID panels
Impact of MALDI-TOF MS
Study 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.
Time to therapy adjustment
60
48
P = 0.04
Hours
40
20
17
26
0
Perez KK, et al. Arch Pathol Lab Med. 2012
Pre-intervention
Intervention
EFFECTS ON HEALTH CARE COST
 Hospitalization cost
reduction of $19,547/patient
27
Perez KK, et al. Arch Pathol Lab Med. 2012
MALDI – TOF Laboratory Integration
E
X
P
E
C
T
A
T
I
O
N
S
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)
Pre MALDI - Good Clinical Microbiology
Begins With Good specimens –
Garbage In = Garbage Out
• 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
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 MALDI
Purpose: Separate human and bacterial/yeast ribosomal proteins
Methods: Lysis/centrifugation or membrane filtration
Journal of Clinical Microbiology 51;805-809, 2013
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
Journal of Clinical Microbiology 48;1584-1591, 2010
Potential Options
for Direct detection
from clinical
specimens
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