MALDI-TOF Mass Spectrometry Hype or Revolution? Will Probert, Ph.D. Microbial Diseases Laboratory Branch California Department of Public Health

Transcription

1 MALDI-TOF Mass Spectrometry Hype or Revolution? Will Probert, Ph.D. Microbial Diseases Laboratory Branch California Department of Public Health

2 Matrix Assisted Laser Desorption Ionization (MALDI) Sample Analytes Most abundant bacterial proteins between 2 20 kda (e.g. Ribosomal Proteins) Matrix Mixed with sample to form crystalline structure Converts laser energy to heat energy Facilitates vaporization of the analyte Transfer protons to analytes (ionization) Laser Energy source for vaporization of the sample

3 Time of Flight (TOF) Electrostatic field focuses analytes (ions) into a vacuum tube Analytes (ions) travel through the vacuum tube according to mass/charge value (smaller molecules move faster)

4 Mass Spectra Most Abundant Proteins Bacterial Ribosome

5 MALDI-TOF Mass Spectrometry - Workflow

6 Instrumentation Bruker Daltonics Biotyper FDA-cleared for Gram negative bacteria Database size > 4000 spectra biomerieux Vitek MS FDA-cleared for 193 microorganisms (bacteria and yeast) Database size >25,000 spectra

7 MALDI-TOF MS Instrument Comparison Property Biotyper Vitek MS Reusable Target Plates Yes No Software Easy to use Very easy to use Time for 96 Identifications 45 min 65 min Quality Management Easy Very Easy Instrument Cost $$ $$$ Consumable Cost $$ $ Maintenance Cost $$ $$$ Noise Silent Noisy Size Smaller Bulkier Connectivity Via LIS Via Myla Adapted from: Martiny, D et. al. J Clin Microbiol (4): 1313

8 Sample Preparation Direct Smear Thin film of bacterial cells applied directly to target Rapid, but less reliable identification Direct, On-Plate Formic Acid Prep Formic Acid/Acetonitrile Extraction Releases soluble proteins and removes cell debris Adds 20 min to process, but higher scores and more reliable identification FA/A Extraction + Beads Mycobacteria ID Solid growth or MGIT culture 30 min heat inactivation step Blood Culture Bottles Cell lysis and capture microorganisms on a filter Sepsityper Urine monomicrobial >10 6 CFU/ml

9 Data Analysis Bruker Daltonics Biotyper Comparative spectral algorithm Score values from 0 to >2.0 = species-level identification 1.7 to = genus-level identification <1.7 = unreliable identification 10% Rule Guideline for excluding closely related species listed in the top 10 matches Exclusion of any species scoring >10% below the top scoring match biomerieux Vitek MS Weighted bin algorithm Displays identifications with confidence value (0-99.9) Confidence value 60% % Good ID = species-level identification 60% % Low Discrimination = genus-level identification < 60% No ID

10 Applications Bacterial ID Generally good performance Exceptions Shigella, V. cholerae not in Bruker Database S. pneumoniae/s. mitis group Yeast/Fungi ID generally good performance Biothreat Agents Inactivation step Custom library needed β-lactamase Confirmation Detection of β-lactam degradation products Salmonella serotyping Specific biomarkers?

11 MDL Verification Study Bruker Biotyper Study Design 262 Isolates representing 208 species Best score recorded as identification Score <1.4 recorded as no reliable identification Performance 92% Accuracy at species level 100% Reproducibility Limit of Detection = 10 6 organisms Implemented in 2012

12 MDL Workflow Integration

13 Hype or Revolution? Advantages: Rapid turnaround time Automated high throughput User-expandable database Reusable, environmentfriendly steel target plates Small instrument footprint Broad applicability to bacteria and fungi Cost effective (~$0.50/sample) Limitations: Requires isolated colony for analysis Lack of susceptibility information Limited FDA approval ID limited by reference spectra in database Inability to differentiate among certain closely related organisms Repeat analysis may be required Source -

14 Questions?