MALDI-TOF in Clinical Microbiologyour vision to future technology

1. MALDI-TOF IN CLINICAL MICROBIOLOGY OUR VISION TO FUTURE TECHNOLOGY Dr.T.V.Rao MD 4/1/2016 Dr.T.V.Rao MD 1

2. Why we need Newer Technologies in Diagnostic Microbiology■ Life is changing, Technology is changing and perception to diseases are changing ■ So we need better approaches to diagnose infectious diseases ■ Thoughts on how this technology will change the practice of Clinical Microbiology ■ Benefits of decreased time to detection of pathogens ■ Directed patient care ■ Antibiotic stewardship ■ Right drug, right dose, for the right duration 4/1/2016 Dr.T.V.Rao MD 2

3. Need for better Technologies in life threating Infections ■ Blood cultures are the best approach to establish the etiology of bloodstream infections and infectious endocarditis. Moreover, rapid identification of etiological agent of such severe infections are pivotal to guide antimicrobial therapy. Thus, the impact of timely microbiology laboratory reporting is maximal at the notification of positive blood cultures . The matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows the identification at the species level in few minutes of both Gram positive 4/1/2016 Dr.T.V.Rao MD 3

4. Changing understanding on Gram Negative Bacteria ■ Gram negative bacteria by measuring molecular masses of proteins and other bacterial components obtained from whole bacterial extracts 4/1/2016 Dr.T.V.Rao MD 4

5. Diagnostics changed with mass spectrometry ■ Since the early 1980s, mass spectrometry has emerged as a particularly powerful tool for analysis and characterization of proteins in research. Recently, bacteriologists have focused their attention on the use of mass spectrometry (MS) for bacterial identification, especially Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF). Moreover, recent publications have evaluated MALDI-TOF in microbiology laboratory for routine use. 4/1/2016 Dr.T.V.Rao MD 5

6. Trends of change with MALDI-TOF-MS ■ MALDI-TOF-MS is a rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Furthermore, it allows identification of bacteria directly from clinical samples (blood cultures for example).4/1/2016 Dr.T.V.Rao MD 6

7. Principles of MALDI- TOF■ Maldi is soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules such as DNA, proteins, peptides & sugar or polymers such as dendrimers and macromolecules • It is three steps method. I. The sample is mixed with a suitable matrix & applied to a metal plate. II. A pulsed laser irradiate a sample triggering desorption of matrix material. III. Ionization of analyte molecules 4/1/2016 Dr.T.V.Rao MD 7

8. What is happening in past with Phenotypic Methods ■ Traditional phenotypic based diagnostic methods for BSIs require the detection of bacterial growth in blood culture broths, followed by species identification and antimicrobial susceptibility testing (turnaround time 24–48 hours after initial growth). Pathogens with fastidious growth requirements and those difficult to identify by phenotypic methods require more time for identification 4/1/2016 Dr.T.V.Rao MD 8

9. What are the Recent advances in Blood culturing and Identification of pathogens ■ Rapid nucleic acid amplification methods such as real-time PCR using melting curve analysis, multiplex PCR, fluorescence in situ hybridization (FISH) and peptide nucleic acid- FISH (PNA-FISH) have been used to detect pathogens in blood cultures including Staphylococcus aureus, Enterococcus faecalis and Candida albicans . 4/1/2016 Dr.T.V.Rao MD 9

10. Target oriented Assays CHANGING FROM PHENOTYPIC TO NEWER METHODS ■ These assays, however, only target specific organisms; require technical expertise; and specimens are usually processed in batches. Turnaround times are up to 6 hours. 4/1/2016 Dr.T.V.Rao MD 10

11. A Rapid method to Investigate Bacteremia and Septicaemias■ Bruker‘s MALDI Sepsityper enables identification of gram- negative bacteria, gram-positive bacteria and yeast from positive blood cultures within 30 minutes. 4/1/2016 Dr.T.V.Rao MD 11

12. MALDI-TOF MS: History ■ Developed in 1980s by Karas and Hillenkamp ■ Detection of large molecules using TOF by Tanaka and Yoshida ■ Introduction of matrix compounds to analyze large molecules ■ First commercial instrument developed by Shimadzu ■ First commercial database developed by Anagnostec (1998) ■ Shimadzu scientist receives Nobel Prize in Chemistry – Kiochi Tanaka (2002) ■ Technology in use in Europe for >10 years. 124/1/2016 Dr.T.V.Rao MD

13. HISTORY OF MS • MALDI ■ Term was coined in 1985 by Franz Hillenkamp, Michael Karas • They found that amino acid alanine could be ionized easily if it was mixed with amino acid tryptophan & irradiated with pulsed 266nm laser. • Here, tryptophan absorbed the laser energy & helped to ionize the non absorbing alanine. 4/1/2016 Dr.T.V.Rao MD 13

14. A Rapid method to Investigate Bacteremia and Septicaemias■ MALDI Sepsityper enables faster results, which physicians can act upon to manage blood stream infections, engage in the fight against resistance, and improve patient outcomes. 4/1/2016 Dr.T.V.Rao MD 14

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16. Every Minute Counts to Life■ Bruker’s MALDI Sepsityper solution provides a rapid, highly accurate microbial identification directly from a positive blood culture 4/1/2016 Dr.T.V.Rao MD 16

17. Features of MALDI-TOF MS • Soft ionization - analyze intact biomolecules and synthetic polymers • Broad mass range - analyze a wide variety of biomolecules • Simple mixtures are okay • Relatively tolerant of buffers and salts • Fast data acquisition • Easy to use and maintain, no water or gas hook ups required • High sensitivity, superior mass resolution and accuracy 4/1/2016 Dr.T.V.Rao MD 17

18. What are the advantages of (MALDI- TOF MS)■ The matrix assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of pathogens in blood culture broths, with results available within 2 hours. Although it does not provide antimicrobial susceptibility data (with the exception of methicillin resistant Staphylococcus aureus [MRSA]), it has good potential to guide empirical antimicrobial choice in the treatment of BSIs, yet there remain technical variables that may affect test performance 4/1/2016 Dr.T.V.Rao MD 18

19. APPLICATIONS Microbiology■ It is used for the identification of microorganisms. • Species diagnosis by this procedure is much faster, more accurate & cheaper than other procedures based on biochemical tests. Forensic analysis Environmental analysis • Pesticides on foods • Soil and groundwater contamination 4/1/2016 Dr.T.V.Rao MD 19

20. What it means in Critical care ■ Identifying the etiologic pathogen, followed by antimicrobial susceptibility testing, is critical in the management of BSIs, as delays in effective antimicrobial therapy can adversely affect patient outcomes . MALDI-TOF MS has significant potential over phenotypic methods, as it is able to detect bacterial pathogens directly from blood culture broths reliably and quickly. However, the performance of MALDI-TOF MS is affected by blood culture bottle type, methodology in sample preparation prior to MS analysis, and the interpretive criteria employed 4/1/2016 Dr.T.V.Rao MD 20

21. (MALDI-TOF MS) is a Emerging Technology in Diagnostic Microbiology■ Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a novel method for the direct identification of bacteria from blood culture broths 4/1/2016 Dr.T.V.Rao MD 21

22. MALDI-TOF Vs. Molecular Testing ■ MALDI ■ Rapid, efficient identification from isolated colonies and liquids (MALDI-TOF/MS) ■ Molecular ■ Direct detection from patient specimens (Molecular) ■ Direct detection of resistance genes (MecA, VRE, CTX-M, KPC, NDM) 4/1/2016 Dr.T.V.Rao MD 22

23. MALDI-TOF MS Overview 23 desorption ionization acceleration separation detection m z = 2eU L² t² m: mass z: charge U: acceleration voltage L: path length t: time e: elementary charge ion detector + + + + ++ target matrix/analyte crystals acceleration zone Time of Flight ring electrode Uncharged Drift region Vacuum tube Which protein molecules? Those that are easily desorbed, Like ribosomal proteins Absorbs e from LASER Multiple LASER shots Courtesy of bioMérieux4/1/2016 Dr.T.V.Rao MD

24. Add matrix solution* Air dry for 1-2 min. MALDI TOF Sample Preparation 24 Create Spectra Target Slide 48 wells Step 1 Step 2 Step 3 Step 4 Bacteria, molds, yeasts, mycobacteria Spot target slide with direct colony (can be up to 5 days old). Load target slides • Matrix Solution: (0.5 µl -cyano-4-hydroxycinnamic acid) NOTE: Other sample types: - sediment from positive blood cultures - sediment from certain specimen (e.g. urines) 4/1/2016 Dr.T.V.Rao MD

25. Helps in the Rapid identification of Aetiological agents■ Helps identification of the etiological agents of life- threatening bloodstream infections. An alternative approach for rapid MALDI-TOF based bacterial identification starting from a short culture on agar might yield sufficient bacterial growth in 4 to 6 hours (data not shown). Given the importance of positive blood cultures, this delay may be clinically relevant as compared to the 30 to 45 minutes needed for the ammonium chloride erythrocytes-lysing procedure. 4/1/2016 Dr.T.V.Rao MD 25

26. 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 4/1/2016 Dr.T.V.Rao MD 26

27. Benefits of Rapid Positive Blood Culture Identifications 274/1/2016 Dr.T.V.Rao MD

28. Definitive identifications as fast as current Gram stains!? Direct Detection for Positive Blood Culture Bottles By MALDIPurpose: Separate human and bacterial/yeast ribosomal proteins Methods: Lysis/centrifugation or membrane filtration Journal of Clinical Microbiology 51;805-809, 2013 Journal of Clinical Microbiology 48;1584-1591, 2010 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? Bruker Sepsityper Kit 284/1/2016 Dr.T.V.Rao MD

29. Expectations Vs. reality■ Should be able to decrease the number of secondary identification systems required in Clinical Microbiology ■ Very good technology, but not perfect ■ Experienced technologists still needed ■ Better patient care through faster definitive results ■ Positive effect on workflow??4/1/2016 Dr.T.V.Rao MD 29

30. ■ E. coli Vs. Shigella – Very closely related and cannot be differentiated – Molecular methods ■ Streptococcus pneumoniae Vs. Streptococcus mitis group – Very closely related, new databases can give a definitive ID – Differentiate by Bile solubility or optichin disk ■ Bordetella pertussis Vs. Bordetella bronchioseptica – Very closely related and cannot be differentiated – Rarely cultured No Test Is Perfect 30 4/1/2016 Dr.T.V.Rao MD

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32. • Stenotrophomonas maltophilia Vs. Pseudomonas hibiscola, Ps. gentculata, Ps. betelli ■ Very closely related and cannot be differentiated ■ Biochemical ID required • The Acinetobacter baumanii-calcoaceticus complex (A. baumanii, A. calcoaceticus, A. genospecies 3, A. genospecies : ■ Species differentiation can be difficult. – A. baumanii and A. calcoaceticus can be differentiated, there are several members of the “Genospecies 3” clustering with A. baumanii or A. calcoacteticus, this can lead to “A. genospecies 3” ID result where biochemistry will identify A. baumanii or A. calcoaceticus No Test Is Perfect 324/1/2016 Dr.T.V.Rao MD

33. Limitations with Identification of Streptococcal species ■ The lower yield of valid MALDI-TOF MS results with streptococci and staphylococci might be due: (i) to the close relatedness of the different species of streptococci belonging to the S. mitis group (i.e. S. pneumoniae, S. mitis, S. sanguinis, S. oralis, …), (ii) to some relatedness of different coagulase negative staphylococci, (iii) to the cell wall composition of Gram positive bacteria conferring increased resistance to lysis, and (iv) partially to the possible presence of some residual blood proteins 4/1/2016 Dr.T.V.Rao MD 33

34. Limitations in identification of Staphylococcal species For staphylococci, the major goal is to differentiate S. aureus from coagulase negative staphylococci and this may be accurately done on blood 108 culture bacterial pellets using the MALDI-TOF MS. In the routine practice, the difficulty in identifying S. pneumoniae from other species of the S. mitis group is much more clinically 1relevant and represents a current limitation of the MALDI-TOF MS. The presence of a capsule may also partially explain the low identification rate of S. pneumoniae, H. influenza and K. pneumoniae. Improved extraction protocols specifically designed for encapsulated bacteria are thus warranted. 4/1/2016 Dr.T.V.Rao MD 34

35. 1. Reversal of sensitivity by production of carbapenemases 1. Modified Hodge test 2. Some β-lactamases can be inhibited by specific inhibitors 1. Clavulanic acid for some ESBLs 2. Boronic acid for KPC 3. Chelating agents for MBLs 3. Carbapenemase detection by MALDI-TOF 1. Directly measure changes in M/S by hydrolysis and, in some cases, decarboxylation of the antibiotic Future Uses and on going work on - Detection of Carbapenemases in the Clinical Laboratory 354/1/2016 Dr.T.V.Rao MD

36. Advantages with MALDI-TOF ■ Rapid identification ~ 1min per isolate ■ Consolidation of identification testing onto a single platform – Current Phenotypic methods ■ Gram stain, Vitek 2, Microscan, numerous API methods, disks on media, growth characteristics, selective media, chromogenic media, biochemical tests, serologic tests, enzymatic reactions – Genotypic methods ■ amplified nucleic acid methods, nucleic acid sequencing ■ Reduced cost per test – Cost will be <$1.50 per determination ■ Reduced Hands-on-Time ■ Tech setup time 2-3 minutes ■ Flexibility - each bench get their own target slide ■ High throughput – 192 isolates/run (4 hours) ■ Outbreak strain typing is possible, eventually. May need different matrix – Local strains can be included in a user defined database – Outbreaks can be identified prospectively rather than retospectively 364/1/2016 Dr.T.V.Rao MD

37. Next big change in Clinical Microbiology ■ MALDI-TOF/MS is faster, better, ■ Better than current full identification methods – Modify to fit your laboratory. ■ Use in conjunction with rapid methods ■ RUO Vs. IVD databases – Amount of validation required?? – Same identification expertise on all shifts – Retrospective outbreak evaluations – Identification directly from positive blood culture bottles and other body fluids – Identification not dependent on interaction with biochemicals ■ Limited reference spectra in database for some genera and species – Identifications will get better ■ Can be automated 374/1/2016 Dr.T.V.Rao MD

38. References ■ Identification of Bacteria in Blood Culture Broths Using Matrix- Assisted Laser Desorption-Ionization Sepsityper™ and Time of Flight Mass SpectrometryJen Kok,1,2,* Lee C. Thomas,1 Thomas Olma,1 Sharon C. A. Chen,1 and Jonathan R. Iredell1, PLoS One. 2011; 6(8): e23285. NCBI resource 4/1/2016 Dr.T.V.Rao MD 38

39. ■ Program Created by Dr.T.V.Rao MD for Benefit of Medical and Technical Professionals on newer and emerging technologies in Diagnostic Microbiology with resources from world wide web ■ doctortvrao@gmail.com 4/1/2016 Dr.T.V.Rao MD 39

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