9/5/2017. MALDI-ToF Mass Spectrometry and the Future of Clinical Microbiology Laboratories. Objectives

Transcription

1 Objectives At the end of this presentation, the learner will be able to: Mass Spectrometry and the Future of Clinical Microbiology Laboratories Gerald A. Capraro, Ph.D., D(ABMM) Director, Clinical Microbiology Laboratory Carolinas Pathology Group Carolinas HealthCare System Charlotte, North Carolina Describe the basic considerations laboratories encounter when implementing MS for identification of microorganisms Create a plan for utilization of MS in his/her own laboratory Discuss the benefits and risks of potential future applications of SWACM Annual Meeting September 6, 2017 St. Louis, MO Carolinas HealthCare System 2017 Carolinas HealthCare System Laboratory Scope of Services CHS laboratory provides testing services to acute care facilities (metro), Physician Practices (outreach), and free standing Emergency Departments. Testing locations 12 Acute Care Hospitals 6 Free Standing ED Laboratories Phlebotomy Services 19 Patient Services Centers (outpatient blood draws) 16 physician practices 21 skilled nursing facilities Reference Laboratory Testing 2,978 providers located in 938 medical practices Laboratory Departments 1) Hematology 2) Chemistry/Toxicology 3) Microbiology 4) Histology/Cytology 5) Blood Bank 6) Cytogenetics 7) Molecular Diagnostics 8) HLA Transplant 9) Coagulation Microbiology Testing Volumes Full Service Lab Bacteriology Mycobacteriology Mycology Virology Molecular Micro: C. difficile, positive blood cultures Minimal parasitology: EIA, WORM, INSECT Core Lab Levine Cancer Institute Levine Children s Hospital Reference Lab for non-system hospitals in the region Teaching: Med Tech students, Peds ID residents/msiv Frequent site for method comparison and workflow studies High sample volume allows utilization of continuous flow TEST Annual Volume Urine Cultures 300,000 GAS Cultures 82,000 Exudate/Wound Cultures 31,000 Respiratory Cultures 29,000 GBS Screens 21,000 Positive Blood Cultures 17,000 Sterile Fluid Cultures 16,000 Stool Cultures 14,000 C. difficile Testing 14,000 Fungal Cultures 13,000 Anaerobic Cultures 10,000 Virology Cultures 9,000 AFB Cultures 7,000 Parasitology Testing 6,000 MRSA Screens 4,000 Microbiology Staffing We staff 24/7 and modify based on volume demand Technologists per shift per day of the week: Shift M T W F Sat Sun 1 st nd rd

2 Microbiology Production TEST 1 st SHIFT 2 nd SHIFT 3 rd SHIFT Hood Processing Processing of Positive Blood Cultures Gram Stains Cdiff EIA Respiratory Bench Anaerobe Bench Blood Bench (x) Virology AFB Mycology Urine Bench Exudate Bench Fungal Smears Stool Bench Cdiff PCR AFB Processing AFB Smears Stool Antigen Testing Parasite Antigen Testing Specimen receipt Culture setup Current Identification Methods Culture reading Setup ID/AST testing Time Organism ID AST results Microbiology Takes Time There are very few STAT tests in Microbiology Rapid antigen tests STAT Gram stains Malaria smears Organism Generation Time (h) Escherichia coli 0.33 Staphylococcus aureus 0.5 Pseudomonas aeruginosa 0.67 Mycobacterium tuberculosis 12 Need for Rapid Assays (we are at the mercy of the growth rate of these organisms) Global Approach Microorganism Current State Goal Bacteria 3 days 1 day # Yeast 3 days 1 day # Mycobacteria 4-6 weeks 1 day # Filamentous Mould 4-6 weeks 1 day # # From culture growth M A L D I Matrix-Assisted Laser Desorption Ionization Time of Flight T o F Identification of microbes based on detection of protein biomarkers by mass spectrometry, rather than by biochemical reactions Identification results on the first day of culture growth 2

3 3 Functional Units 1. Ion Source ionizes sample molecules and transfers into gas phase soft ionization proteins remain intact 2. Mass Analyzer separates the ions by mass-to-charge ratio (m/z) 3. Detection Device records the separated ions Spot isolate onto target. Let air dry. Overlay with matrix. Let air dry. Target Plate Laser light pulses Matrix molecules readily absorb laser light (photon energy), creating an excited energy state The matrix is acidic, and donates positive charge to the analytes Localized heating causes micro-explosion of analyte Collisions with neutral sample facilitate charge transfer to/from excited matrix molecules Ions desorb from the target surface Matrix Proteins Detector The speed of travel (time of flight) is proportional to the ion s mass (smaller ions reach the detector first) Drift region Detected Proteins (Biomarkers) Abundant, basic, and hydrophobic Primarily cytoplasmic proteins, like ribosomes hsp, DNA binding, chaperonins Highly evolutionarily conserved, allowing for discrete characterization of individual species Intensity m/z 3

4 Intens. [a.u.] x Intens. [a.u.] Intens. [a.u.] m/z 9/5/2017 Creation of spectra (protein fingerprint) for the unknown organism Gram-negative and Gram-positive bacteria, mycobacteria, yeast, filamentous fungi Escherichia coli Bacillus subtilis Candida albicans m/z Aspergillus fumigatus The last step is to compare the collected spectra from the unknown to a database that contains pre-identified spectra for comparison and identification Two Commercially Available Systems Bruker BioTyper (Size: 0.5 x 1.1 x 0.68 m) BioMerieux Vitek MS (Size: 0.7 x 1.92 x 0.85 m) Target Plates Target Plates Bruker BioTyper BioMerieux Vitek MS Bruker BioTyper BioMerieux Vitek MS 4

5 Interfaces Bruker vs. Vitek MS Performance Bruker can be interfaced through: LabPro Connect (MicroScan) BD EpiCenter (Phoenix) Vitek MS can be interfaced through biomerieux s middleware, Myla % Correct (Biotyper) % Correct (Vitek MS) Bacteria (#) Genus Species Genus Species Routine Isolates (317) Gram positive Gram negative Total Challenge Isolates (296) Gram positive Gram negative Total Carbonelle et al J Microbiol Methods 89: Workflow Workflow Pre-Analytical Processing Individual colonies spotted directly to target or extracted with ethanol Broth cultures can be concentrated and processed Strong organic acids can be used to disrupt the cell wall Acetonitrile (matrix solvent) is used for protein extraction Tube extraction products spotted to target and allowed to dry Workflow Target Plates Reusable or disposable slides 48 spots per target Reusable wash with distilled water and tri-fluoroacetic acid Room temperature storage Workflow Direct Spot Touch the colony with a toothpick Transfer onto target spot duplicate (?) Air dry Cover with 1 ul of matrix and let dry 5

6 Workflow On-Target Extraction Touch the colony with a toothpick Transfer onto target spot and air dry Add 1 ul of 70% formic acid to spot and air dry Cover with 1 ul of matrix and let dry Workflow Tube Extraction Transfer colonies to tube of 70% EtOH to make a slurry Pellet cells and remove EtOH Resuspend pellet in formic acid/acetonitrile and centrifuge Transfer 1 ul of supernatant to target spot and air dry Cover with 1 ul of matrix and let dry Accurate, Specific Identifications Workflow ID accuracy published for routine bacteria, yeasts, mycobacteria, and molds Score depends largely on isolate processing, number of cells analyzed, and database Non-IDs typically result from: the organism is not (widely) represented in the database too few cells present in the spot Positive Blood Culture Bottles Key: removal of RBCs, serum proteins, hemoglobin Centrifuge to pellet bacterial cells Wash pellet with sterile H2O to remove serum proteins Continue processing as with the Tube Extraction method Spectral profiles of the same strain of Escherichia coli obtained from blood culture (bottom) and from one isolated colony (top). Impact Agnès Ferroni et al JCM 48:

7 Compared to Conventional Tests Compared to Conventional Tests 12 week testing period 952 isolates from 2214 specimens Time to ID: 1.45 d sooner 12 week testing period 952 isolates from 2214 specimens Time to ID: 1.45 d sooner Significant decrease in reagent cost No significant change in labor time Tan et al. JCM. 2012: Compared to Conventional Tests Impact on Clinical Outcomes 28.8 h 12 week testing period 952 isolates from 2214 specimens Time to ID: 1.45 d sooner No significant change in labor time 11.3% Annual cost per ID decreased by 57% (>$102K) Vlek et al PLoS One 7: e32589 Clinical Outcomes Clinical Outcomes Huang et al CID 57: Perez et al Arch Path Lab Med 137: Decreased hospital LOS by 2.6 d Decreased ICU LOS by 1 d Decreased overall hospital costs by > $19K Decreased time to ID 1.25 d Decreased 30-day all-cause mortality 8% Decreased LOS (ICU) 7 d Decreased time to effective/optimal therapy 10 h/2 d 7

8 Clinical Outcomes (Pediatrics) Retrospective chart review of pediatric patients with positive BLDCU 3-month period in 3 consecutive years Aim: Assess impact of MALDI and short-incubation MALDI on patient care, compared to conventional culture ID Conventional: BAP, MAC, CHOC at 8, 18 h, with Vitek2 MALDI: BAP, MAC, CHOC at 8, 18 h, with BioTyper SIMI: BAP surface swabbed at 3 h, with BioTyper LIS Data: time to ID, time to appropriate AB initiation, hospital LOS Delport et al PLoS ONE 11(8): e Clinical Outcomes (Pediatrics) 92 patients (conventional) (MALDI) (SIMI) MALDI decreased time to ID by h decreased LOS 3.07 d SIMI - further reduction decreased time to ID by h decreased time to optimized AB by 20.2 h decreased LOS further by 8.92 d RR of sepsis-associated mortality was 4x higher with conventional ID compared to SIMI, and if AB needed to be optimized, this RR was 7x higher Delport et al PLoS ONE 11(8): e Rapid Diagnostic Methods Limitations Mixed Cultures can only identify isolates from pure cultures Specimen receipt Culture reading Organism ID Culture setup Setup ID/AST testing AST results Time Spectral peaks from multiple organisms will likely result in No ID If multiple organisms mixed, may only get result for the one in highest abundance Culture isolates AST results Limitations E. coli vs. Shigella These organisms are nearly identical MALDI will return a result of E. coli/shigella Have a plan to deal with these Backup with biochemical testing or sequencing (?) Result as presumptive Shigella if from stool (?) Regulatory Issues CAP checklist items regarding CAP Micro Checklist

9 Regulatory Issues Regulatory Issues CAP Micro Checklist 2017 CAP Micro Checklist 2017 Summary allows for routine ID of clinically relevant bacteria and yeast Time to ID and cost per ID are significantly decreased Laboratories must decide and validate a routine approach for utilization of for ID Utilization of has been shown to improve patient outcomes MALDI-specific requirements are now included on CAP inspection checklists Applications Mycobacteria Mycobacteria processing ID by represents an improvement in both TAT and specificity Water 75% EtOH Water Water 50ul 95 C 30 min 100% EtOH 50ul ACN + silica beads, vortex 1 min. FA 50ul Cells difficult to lyse and extract protein biomarkers Processing requires both heating and sonication of cells 2 ml Growth medium impacts the overall score result Buchan et al AJCP 141: Separate library for Mycobacterium species 9

10 Effect of Bead-Beat on MALDI Spectrum No Bead-beat step Bead-beat step Buchan et al AJCP 141: Buchan et al AJCP 141: Comparison of spectral quality generated using the UW and BM extraction protocols. Mycobacteria processing Water 75% EtOH Water Water 50ul 95 C 30 min 100% EtOH 50ul ACN + silica beads, vortex 1 min. FA 50ul 2 ml Buchan et al AJCP 141: % EtOH 95 C 30 min 10 min 2 min FA 50ul + ACN 300ul water + silica beads Mather et al JCM 52: Mather et al JCM 52: Filamentous Molds Filamentous Molds processing ID by represents an improvement in both TAT and specificity Cells difficult to lyse and extract protein biomarkers FA 50ul 75% 50ul EtOH ACN Processing requires both heating and sonication of cells Growth medium impacts the overall score result 500 ul EtOH + silica beads Separate library for molds Lau SK et al BMC Microbiol 16: 36 Lau AF et al JCM 51:

11 Filamentous Molds performance Filamentous Molds performance Custom NIH Mold Database 180 Hyaline 70 Dematiaceous 27 Mucorales 7 Dermatophytes 10 Dimorphic Fungi Clinical isolates recovered from NIH patients 76 genera, 152 species Grown on SAB at 25C for 5 days Library (ies) # (%) of isolates with the indicated score >= 2.0 <=1.99 and >=1.7 <1.7 Combined NIH and Bruker 144 (92.3) 4 (2.6) 8 (5.1) NIH alone 144 (92.3) 4 (2.6) 8 (5.1) Bruker alone 3 (1.9) 26 (16.7) 127 (81.4) Overall performance of the NIH mold database and the Bruker library when adjusted to evaluate only isolates (n = 156) for which there were representative spectra in the manufacturer's library Lau AF et al JCM 51: Lau AF et al JCM 51: Direct Specimen Testing Direct Specimen Testing DeMarco & Burnham AJCP 141: Urine (1 ml) Centrifuge (30 s, 2000g) Transfer supernatant in new cup Centrifuge (5 min., 15500g) Dry pellet Wash once with de-ionized water Standard extraction protocol for bacterial profiling Spot 1 µl onto MALDI target, overlay with HCCA matrix MALDI Biotyper analysis ID Decreased time to ID of uropathogens to 2 3 hours Established a limit of detection of cfu/ml Performance: Sensitivity 67% Specificity 100% PPV 100% NPV 94% Promising, but not quite ready for prime-time Detection of Antimicrobial Resistance Detection of Antimicrobial Resistance Detection of MRSA-specific peaks Detection of beta-lactam hydrolysis by carbapenemases, ESBLs, and AmpC Not quite ready for prime-time Szabados F. et al J Infection 65:

12 Detection of Antimicrobial Resistance Functional assay for detection of cleavage of the b-lactam ring by b-lactamases? + 18 Da - 44 Da Detection of Antimicrobial Resistance Detection of Antimicrobial Resistance (A and B) MALDI-TOF MS spectra of piperacillin after incubation with the β- lactamase-negative E. coli strain DH5α (A) and a β- lactamase-producing strain (B). Sparbier K et al JCM 50: Zimmermann, ECCMID 2012, London ß-lactamase positive Summary allows for ID of clinically relevant mycobacteria and filamentous molds Libraries need to be enhanced Standardization of mold processing is needed Time to ID and cost per ID are significantly decreased Utilization of for detection of pathogens directly from specimens and detection of antimicrobial resistance is in the development stage Need to be operationalized for routine workflow Not all antimicrobial resistance determinants are detectable 12