1. Matrix assisted laser desorption ionization –
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

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

3. 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

4. Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

5. Advances in Bacterial Identification
1900
1970
2000 5 5

6. 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
FASTER, BETTER, CHEAPER, BUT NOT PERFECT!

7. 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 hours earlier

8. MALDI TOF Sample Preparation
Step 1
Step 2
Step 3
Step 4
Add matrix solution*
Air dry for 1-2 min.
Load target slides
Bacteria, molds, yeasts,
Mycobacteria
Spot target slide with direct colony (can be up to 5 days old).
Create Spectra
NOTE:
Other sample types:
sediment from positive blood cultures
sediment from certain specimen (e.g. urines)
Target Slide
48 wells
Matrix Solution: (0.5 µl -cyano-4-hydroxycinnamic acid)

9. General schematic for MS analysis of ionized microbiological isolates
Clark A E et al. Clin. Microbiol. Rev. 2013;26:

10. MALDI Mechanism
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+. + m m +
Laser a m a + m a m m + a m a m a + m m
matrix & analyte
Sample support

11. Principle of MALDI-TOF
Time of Flight
Molecular masses

12. The Workflow: Measurement
automated spectrum acquisition ~ 60 sec
ionization of intact proteins and molecular weight measurement 12

13. Low influence of culture conditions
Psdm. oleovorans B396_Medium 360
1000
Psdm. oleovorans B396_Medium 464
1000
Psdm. oleovorans B396_Medium 53
1000
Psdm. oleovorans B396_Medium 65
1000
Psdm. oleovorans B396_Medium 98
1000
500
Psdm. oleovorans B396_MRS10
2000
1000
2000
Psdm. oleovorans B396_YPD
1000
4000
5000
6000
7000
8000
9000
10000
11000
m/z

14. Bacterial Identification by Mass Spectrometry

15. Concordance between Conventional Routine Identification (Vitek) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry
84.1%
95.4%
Concordance between Conventional Routine Identification (Vitek; bioMérieux) and Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry Identification (Brucker Mass Spectrometer and Database Complemented with Local Database)‏
Seng P et al. Clin Infect Dis. 2009;49:

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

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

18. Retrospective Validation
Total Tested
838
Correlates
810
97%
Mismatch 28 3%
16s rDNA used as gold standard

19. Prospective Validation
Total Tested
356
Correlates
342
96%
Mismatch 7
1.9%
No Growth
---
No Data

20. 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% of
positive cultures

21. Cost Calculations Urine cultures
Cost of Chrom-agar vs Blood agar + MALDI
Crom-agar $ / Blood agar $0.27
MALDI $0.51 (plate, matrix, toothpicks, pipette tips, etc.)
LAP $ / PYR $1.95 / Ox $ / PH B $ / Strep A,C,G $1.46

22. 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 ,862 x 0.27 ($30,294 - $13,192 = $17,102 SAV)

23. 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
+$1382
KES
1716
$1956
$1356
-$600
Enterococcus
1680
$1915
$1325
-$590
Bp B Strep
668
$762
$528
-$235
Proteus
630
$718
$497
-$221
Candida
404
$251
$461
$319
Misc Pos
1462
$1667
$1178
-$489
Projected cost savings of moving back to Blood agar is approximately $18,000 / year

24. 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 = $ 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 = $ vs $163 (MALDI) = $1444 SAV
Approx $5000 / year Saving!!

25. What Does an Instrument Cost?
~$ 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

26. 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.
The recent study by the Dr. Musser’s group at the Methodist hospital in Houston Texas integrated rapid identification of gram negative bacter from blood culture by MALDI-TOF MS with active antimicrobial stewardship using an infectious disease pharmacist as direct liaison to the clinicians. The intervention period was compared to the pre-intervention period. All blood culture results were called to the ID pharmacist 24/7 and the pharmacist would work with the clinicians to recommend de-escalation or adjustment of therapy based on the rapid ID and faster susceptibility results.
When looking at the time to inactive/no antibiotics, the difference between the pre-intervention and intervention period was significant at 24 and 48 h.
In addition, the time to adjusted therapy was significantly reduced by 31 h.
P = 0.04
Perez KK, et al. Arch Pathol Lab Med. 2012

27. EFFECTS ON HEALTH CARE COST
Hospitalization cost reduction of $19,547/patient
The study also looked at the differences in length of stay, and estimated annual cost savings.
They found that the impact of combining MALDI-TOF with antimicrobial stewardship on hospital length of stay after bloodstream infection onset was 1.8 days with cost savings of $19,547/patient. Significantly, they estimated a annual cost savings of 18 million dollars.
Other study – 11% increase at 24 hours for MALDI IDs pts receiving appropriate therapy.
35%
Perez KK, et al. Arch Pathol Lab Med. 2012

28. MALDI – TOF Laboratory IntegrationX P C T A I O N S
Current Level

29. 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

30. No Test is perfect!
E.coli vs Shigella
Acinetobacter baumanii-calcoaceticus complex (A. baumanii, A. calcoaceticus, A. genospecies 3, A. genospecies 13)

31. Control of sample acceptability
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

32. Direct specimen applications (already blood / urine data)
Future Direction
Direct specimen applications (already blood / urine data)
Ability to resolve poly-microbial specimens
Antimicrobial resistance determination (already MRSA, carbapenemase)
Strain typing

33. Direct Detection for Positive Blood Culture Bottles By MALDI
Purpose: Separate human and bacterial/yeast ribosomal proteins
Methods: Lysis/centrifugation or membrane filtration
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?
Journal of Clinical Microbiology 51; , 2013
Journal of Clinical Microbiology 48; , 2010

34. Potential Options for Direct detection from clinical specimens
Clark A E et al. Clin. Microbiol. Rev. 2013;26:

35. 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

36. MALDI-TOF Limitations
Databases : still in their infancy
High initial capital expenditure
New approaches (business models)
Potential instrument downtime - single instrument

37. 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

38. Questions

39. General schematic for MS analysis of ionized microbiological isolates
Clark A E et al. Clin. Microbiol. Rev. 2013;26: