Presentation on theme: "Nathan A Ledeboer Assistant Professor of Pathology"— Presentation transcript:
1Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Nathan A LedeboerAssistant Professor of PathologyMedical College of Wisconsin andMedical Director, Microbiology and Molecular PathologyDynacare Laboratories and Froedtert HospitalMilwaukee, WI
2Outline Overview of Technology Are MALDI-TOF instruments all they described to be?How can I justify the cost?Applications in DevelopmentWhat is the Impact for Patient CareThe Future of Mass SpectrometryConclusionsQuestions
3DisclosuresDr. Ledeboer will discuss products that are not FDA clearedFinancial DisclosuresConsultant:Nanosphere, IncThermoFisher Scientific, IncLabCorpCepheid (Scientific Advisory Board)iCubateHonorariaBruker DaltonicsMeridian
6Step 1: Target Preparation, continued Direct Smear Method:Touch colony with transfer device, such as toothpickTransfer a small amount onto spotLet air dryCover with 1 µL of MALDI matrix, let air dryAnalyzeResearch use only – not for use in diagnostic procedures
7Inactivation of pathogens Ethanol-Formic Acid Extraction(if required – low score value after direct smear)Inactivation of pathogensEthanolFormic acid,acetonitrileAnalyzesupernatantdH2OEthanol10 minResearch use only – not for use in diagnostic procedures
8Matrix Assisted Laser Desorption/Ionization Matrix: HCCA (a-Cyano-4-hydroxycinnamic acid)Solvent: Acetonitrile, TFA (trifluoroacetic acid)Lyses cell walls and extract proteinSeparates protein molecules (proteins are “sticky”)After sample preparationTo bring in some MALDI in the runsample overlain with matrix solution contains special small organic molecules critical for soft ionizationEvaporation OS > formation of co-crystal of sample and matrix molecules1 µL MatrixAnalyte (organism)Target plateResearch use only – not for use in diagnostic procedures
9Matrix Assisted Laser Desorption/Ionization Laser light pulsesMatrix molecules readily absorb laser light (photon energy), creating an excited energy stateThe matrix is acidic, and donates positive charge to the analytesLaser fired, absorbed by matrix moleculesResearch use only – not for use in diagnostic procedures
10Matrix Assisted Laser Desorption/Ionization Localized heating causes micro-explosion of materialCollisions with neutral sample facilitate charge transfer to/from excited matrix moleculesIons “desorb” from the target surfaceMatrixEnergy transfer > formation of a cloud ionsOf notice: most sample protein ions result from protonationSingly positively chargedResearch use only – not for use in diagnostic procedures
11TOF – Time of Flight Detector m/zIntensityFollowing acceleration, the charged ions are allowed to drift through a free field toward the detectorThe speed of travel (time of flight) is proportional to the ion’s mass (smaller ions reach the detector first)Drift regionAs heavier molecules travel at lower speed they need more time to reach detectorToF > mass to charge ratio calculatedSingly charged > m/z=MWResearch use only – not for use in diagnostic procedures1111
12MALDI: Results outputRaw profile spectrumRefined profile spectrumResults are analyzed by a computer, cleaned-up and the spectrum is searched against a database with known spectra.
13MALDI identification result Secure genus and species identificationProbable genus identificationUnreliable identification
15Are MALDI-TOF instruments all they are described to be?
16MALDI publications:More than 200 peer reviewed publications, mainly in high-ranking microbiology journals as of September Topics include:Particular groups of bacteria(e.g. anaerobes, Listeria, Neisseria, Yeast...)Routine applicationRare and difficult to analyse microorganisms (e.g. Prothotheca)Highly pathogenic bacteria (e.g. Francisella, Brucella)Blood culture direct analysisUrine direct analysisSub-typingResistance or Virulence FactorsMycobacteriaFilamentous Fungi
17In a study by Benagli et al In a study by Benagli et al., the authors compared performance of MALDI-TOF to biochemical ID and resolved discrepancies with sequencing. The results follow.Benagli C et al. PLoS One. 6(1).
18Clinical Application - Bacteroides species 277 Clinical Bacteroides Isolates from a European study:270 isolates (97,5%) identified with significant score, 7 isolates not in Reference Library (e.g. Bacteriodes distasonis)MALDI ID discrepant sequenced*Bacteroides fragilisBacteroides thetaiotaomicronBacteroides ovatusBacteroides vulgatusBacteroides uniformisBacteroides eggerthiiBacteroides nordiiBacteroides salyersiaeBacteroides massiliensis*only IDs with log(score)<2.516S rDNA Sequenceing Confirmed 10 of 11 Discrepant MALDI Results, 1 Case Only “Bacteroides spec.“Nagy et al., Clin Microbiol Infect 2009; 15: 796–802
19Clinical Application: Yeast No incorrect Yeast Identification by the Respective Molecular FingerprintMarklein et al., JCM, Vol
21System and application (no. of isolates tested) SaramisBiotyper% Correctly identifiedP value Routine (986)a83.8 vs 92.7<0.001NAdNA86.9 vs 95.512.8 vs 3.20.3 vs 1.2<0.01Vitek MS83.8 vs 93.292.7 vs 93.20.60886.9 vs 93.695.5 vs 93.6<0.0512.8 vs 5.83.2 vs 5.80.3 vs 0.411.2 vs 0.4Martiny, et al, JCM, 2012, 50
22Bruker Biotyper vs Vitek MS PropertyMicroflex LTVitek MS RUOVitek MS IVDRemarksUser friendliness Ready-to use Matrix solutionNoYes Facility of preparing smearVery easyEasyFor Vitek-MS systems, matrix solution must be deposed each two spots Disposable targets Reusable targets SoftwareEasy to useNot easy to useVery easy to useTime for 96 identifications Time to prepare work list (min)<55–10NDa Time to load target and make vacuum25 Time for analysis (min)4055 Time for 16 identifications (min)ND15No ID before success of QC at end of run (each 16 IDs)Quality IVD RUONeed for validation before clinical reporting Quality managementCostc Device+NAb++ Reactants+++NABased on catalog prices MaintenanceImplementation NoiseSilentNoisy SizeSmallerBulkierConnectivityVia LISVia MylaCapacity1 × 964 × 48Martiny, et al, JCM, 2012, 50
23*FTE cost/hour $41.35 Time/ test (hour) FTE Cost/test* Supply Time/ test(hour)FTECost/test*SupplyCost/testTotalCostRapid Biochemicals0.10$4.14$0.29$4.43Automated Biochemicals0.14$5.79$9.59$15.38Long Biochemicals0.33$13.65$5.32$18.97Sequencing0.73$30.19$20.02$50.21Mass Spectrometry0.05$2.07$0.24$2.31*FTE cost/hour $41.35Slide courtesy of Robin Patel, MD
24Cost Savings Comparison Instrument Included* Assumes a 3 year depreciation of instrument and an instrument cost of $200,000Tests per Year50001000020000300004000050000Instrument Add*$13.33$6.66$3.33$2.22$1.66$1.33MALDI-TOF ID Consumables and Labor$2.31Biochemical ID Consumables and Labor$15.38Difference per test-$0.26$6.41$9.74$10.85$11.41$11.74Cost Savings per year-$1,300.00$64,100.00$296,337.55$448,005.18$471,128.03$484,753.99Return on Investment (in years)6.063.120.670.450.420.41No Instrument$13.07$65,350.00$130,700.00$397,652.14$539,670.763.061.530.500.37
25Cost-effectivenesss of switching to MALDI-TOF MS for routine bacterial identification Galliot O, Blondiaux N, Lorez C, Wallet F, Lemaitre N, Herwegh S and Courcol RSeptember 2009Switched from conventional biochemicals (Vitek 2 and API) to MALDI-TOF MS (Bruker)Cost analysis performedOctober 2008-September 2009October 2009-September 2010Isolates Tested33,32038,624Biochemical Costs$193,754$5,374MALDI-TOF-$15,836TOTAL$21, 210Avg Cost/ID$5.81$.54Annual Savings = $177, 090“allowed decrease of 89.3% of the cost of bacterial identification in the first year.”In addition:Decreased waste from 1,424kg to 44kgDecreased subculture media of $1,102Decreased sequencing cost of $1,650JCM epub ahead of print
28MALDI – blood culture direct analysis Detailed results and effect of adopted processing< 1.63,9%11,7%9,1%< 1.710,4%5,2%27,3%20,8%> 1.885,7%83,1%63,6%67,5%> 2.04000300039784211666644952NEW - ThresholdsORIGINAL - Thresholds77 samples< 1.65,4%7,5%11,8%< 1.78,6%9,7%15,1%26,9%> 1.886,0%82,8%73,1%61,3%> 2.040003000571189142580776857NEW - ThresholdsORIGINAL - Thresholds93 samplesNo false positive result in the “yellow“ and “green“ log(score) range3000/4000 –detection boundary increased for protein to reduce background
29Proof Point: Blood Culture Innovative Extraction 99% of tested strains were correctly identified directly from Blood Culture
30ACN + silica beads, vortex 1 min. Sample PreparationPositive culture75% EtOHWater50ulWaterWater95°C30 min2 ml50ulACN + silica beads, vortex 1 min.FA50ul100%EtOH~60 min.
31ACN + silica beads, vortex 1 min. Sample PreparationPositive culture75% EtOHWater50ulWaterWater95°C30 min2 ml50ulACN + silica beads, vortex 1 min.FA50ul100%EtOH~60 min.
32Heat Kill Test Days to positivity Following heat inactivation step, TREK Myco bottles were inoculated and held 6 weeks.Days to positivityM. che/abs grM. fortuitum grMAIMTB
33ACN + silica beads, vortex 1 min. Sample PreparationPositive culture75% EtOHWater50ulWaterWater95°C30 min2 ml50ulACN + silica beads, vortex 1 min.FA50ul100%EtOH~30 min.
34Effect of Bead-Beat on MALDI Spectrum No Bead-beat stepBead-beat step
39Data Summary 90.3% (65/72) with acceptable ID score (>1.7) Middlebrook 7H1090.3% (65/72) with acceptable ID score (>1.7)98.3% (59/60) correct species IDTrek Myco91.7% (66/72) with acceptable ID score (>1.7) 98.4% (60/61) correct species ID
41ID Filamentous Fungi - workflow 1. Direct Transfer of “Front Mycelium“ (1 min)if successful: ID is FINISHEDe.g. A.niger2. Ethanol Extraction of “Front Mycelium“ (10 min)if successful: ID is FINISHED3. Broth Cultivation (approx. 1 additional day) & extractionID is possible for agar adhering filamentous fungiID is possible for slow or fast sporulating fungiID is possible for every kind of filamentous fungiALL matches against the SAME Filamentous Fungi DB
44Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD
45Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD Organism-groupnMean # of days isolate identified earlierProportion identified earlier by MALDI-protocol,by number of days of workup<0da0db1d2d3d4d5d6d>6d(days)(%)S. aureus1091.351.818.104.22.168.9Other Staphc261.197.765.426.9BHSd720.601.438.958.3VGSe70.5742.957.1S. anginosus171.1241.229.45.923.5S. pneumoniae60.3366.733.3Other GPCf3.3316.7Enterococcus sp.781.641.351.334.69.02.6Enterobacteriaceae2841.3469.422.214.171.124.4P. aeruginosa771.8241.649.4Other NF GNBg392.5930.835.915.45.1Haemophilus sp.101.4080.020.0Other GNCBh0.1485.714.3Corynebacterium sp.91.6722.2Other GPRi84.1312.537.5Anaerobic GNj2.543.819.2Anaerobic GPk142.6421.428.67.1C. albicans520.0492.31.9Other Candida sp.561.938.9126.96.36.199Other yeasts3.7525.0All organisms9111.4513.552.7188.8.131.52Tan KE, et al, JCM, In Press – Kindly provided by K. Carroll, MD
46Patient ImpactRetrospective chart review of patients with positive blood cultures obtained during validation of MALDI-TOF Biotyper and Sepsityper KitTime to ID (MALDI-TOF v traditional culture methods) and start/stop times of antibiotics reviewedTheoretical reduction in empiric antibiotic duration and cost difference (AWP) calculatedPaul J., et al. IDSA
47Time to Antimicrobial De-Escalation Traditional IDMALDI-TOF Direct IDDifference (in hours)PEmperic Antibiotic Duration ( IQR, hrs)66.6( )15.5( )51.6( )<0.01Antibiotic Cost (USD)$245.24$88.48$156.76Paul J., et al. IDSA
50Van Belkum, et al, JCM, 2012, 50 Characteristic MALDI-TOF LC-ESI-QqQ-MSIonizationSoft ionization with matrixSoft ionization with solvents and electronebulizationFragmentationNo (intact molecules)YesSampleSolid form (or liquid allowed to dry on target)Liquid form (downstream of a liquid chromatography step)MoleculesMainly proteins, large glycopeptides, oligonucleotides, carbohydratesDifferent molecules, especially peptidesTurnaround time20-30 s per sample at laser frequency of 50 Hz to generate a spectrumMinutes or hours depending on liquid chromatography adsorption/elution timesMinutes from sample preparation to resultThroughputDisposable target with multiple spots (48 to 96)No batch mode at the moment due to LC stepReagentChemical matrixChemical reagents for chromatographic separation and elutionExternal calibrantInternal calibrantMaterialDisposable targetChromatographic column and precolumnVials for LC injectionQuantificationNot well suitedFully adaptedSpecificityDepending on MS specificity and proteins testedUsually higher than MALDI when selected reaction monitoring (MS2) is usedSensitivityBacterial ID: 105 CFU using fingerprint approachTo be exploredUrine sample after purification without culture: 105 CFU/mlIntegration into microbiology lab workflowYes (IVD-compliant systems)No (research applications)Today's clinical microbiology applicationsMicrobiology: identification of bacteria, yeast, and moldsNoneQuantitative assays for small molecules, such as vitamin D (outside microbiology field)Van Belkum, et al, JCM, 2012, 50
52ConclusionsData demonstrates excellent performance of MALDI-TOF MS for identification of bacteria and yeast from plates and from positive blood cultures.High Capital Cost can be overcome by consumable savingsUse of technology can result in a significant reduction in laboratory turnaround and significant antimicrobial cost savingsMALDI-TOF and current technologies represent the beginning of protein revolution