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

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 1900 1970 2000 5 5

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!

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

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)

General schematic for MS analysis of ionized microbiological isolates Clark A E et al. Clin. Microbiol. Rev. 2013;26:547-603

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

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 12

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

Bacterial Identification by Mass Spectrometry

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;49:543-551

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

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

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 +$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

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? ~$ 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 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

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

MALDI – TOF Laboratory Integration X P C T A 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)

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

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

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;805-809, 2013 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