Tandem MS in Microbiology. Chris Doern, PhD D(ABMM) UT Southwestern Medical Center Dallas, TX

Transcription

1 Tandem MS in Microbiology Chris Doern, PhD D(ABMM) UT Southwestern Medical Center Dallas, TX

2 Learning Objectives After the presentation you should be able to: 1. Understand the mass spectrometry methodology that is being used in clinical microbiology 2. Understand MALDI-TOF work flow for organism identification 3. Understand how to interpret the data provided by the MALDI-TOF 4. Understand why MALDI-TOF is revolutionizing microbiology

3 Diagnosing Bacterial Infection Specimen collection Specimen Processing Transport ~ 2 hrs ~ hrs ~ hrs Evaluation Analysis

4 Microbiology in the Clinical Continuum Specimen collection Transport Processing Incubation Analysis Hours Days Days Specimen stain result Preliminary, presumptive identification Where are treatment decisions made? Final Report: Definitive ID and Susceptibility

5 Microbiology in the Clinical Continuum Specimen collection Transport Processing Incubation Analysis Hours Days Days Specimen stain result Preliminary, presumptive identification Where are treatment decisions made? Final Report: Definitive ID and Susceptibility

6 Why is microbiology so slow? Incubation required prior to analysis. Isolated and pure isolates required prior to analysis. Identification and susceptibility still growth based. No direct from specimen analysis.

7 MALDI-TOF Day 1 Day 1 Day 2 Day 3

8 MALDI-TOF for Bacterial Identification: The Options Bruker Microflex biomerieux Vitek MS

9 Bruker Daltronics How it works

10 Commonly used MALDI matrix substances: Peptides: 4-Hydroxy-α-cyanocinnamic acid (HCCA) Proteins: 2,5-Dihydroxyacetophenone (DHAP) Sinapinic acid (SA) 2,5-Dihydroxybenzoic acid (DHB) Glycans: Nucleic acids: 2,5-Dihydroxybenzoic acid (DHB) 3-Hydroxypicolinic acid (HPA) 2,4,6-Trihydroxyacetophenone (THAP) Why different matrices for different types of sample? It s all about - the amount of energy needed to ionize a particular sample compound (individual matrices show specific energy threshold ) - the stability of a particular sample compound (too hot matrix may lead to non-desired fragmentation of sample compounds) Courtesy of Gongyi Shi

11 MALDI-TOF Procedure

12 MALDI-TOF Procedure

13 MALDI-TOF Procedure

14 MALDI-TOF Procedure

15 MALDI-TOF Procedure

16 What MALDI-TOF can do now Identification of bacteria Aerobic Gram positive and Gram negative organisms Gram positive bacilli! Cystic fibrosis isolates! Anaerobes Identification of yeast Identification of mycobacteria Identification from positive blood cultures

17 Other Benefits of MALDI-TOF Very reliable Gram stain morphology determination. MALDI-TOF doesn t get WRONG answers Confirmation of good identifications not necessary MALDI-TOF can learn

18 Limitations of MALDI-TOF Cannot distinguish between Streptococcus pneumoniae and Streptococcus mitis/oralis group Shigella spp. not in data base Cannot do susceptibility testing Cannot identify mixed specimens

19 What we hope MALDI-TOF will be able to do in the future Identification of filamentous fungi Strain typing Limited susceptibility testing

20 Nuts and Bolts of MALDI MALDI measures proteins between 2,000 and 20,000 daltons. Not a direct from specimen technique. Limit of detection is about 100,000 bugs. May have some utility for high burden monomicrobic specimens such as CSF and Urine. Measures ribosomal housekeeping proteins Conserved and specific to each organism Not FDA cleared See next slide for validation

21 Important Components to MALDI-TOF Spotting technique What media can you spot from? Spot lag time Validation by organism category Compare to routine laboratory method Resolve discrepant results with 16S sequencing

22 Spotting Method

23 Media Performance Evaluation Test subcultures of previously characterized, relevant organisms from selective media after O/N growth Threshold of 70% identification necessary to justify attempt. Exceeded in all cases.

24 Spot Lag Time Experiments 1. Spot plates twice in triplicate 2. Store at RT and test at 24, 48 and 72 hours. Important for streamlined workflow.

25 What we did to evaluate our database Gram negative validation Enterobacteriaceae, Glucose Non-Fermenting, Fastidious Gram negative bacilli and diplococci Minimum 300 isolates No more than 20 of an individual species No more than 30 isolates of an individual genus Supplement with organisms of interest GC, B. cepacia, HACEK, etc Gram positive validation Cocci and bacilli Minimum 200 isolates No more than 30 isolates from an individual species No more than 50 isolates from an individual genus Supplement with organisms of interest Corynebacterium spp., Nocardia

26 What we did in our laboratory Anaerobes Gram positive and negative Minimum 100 isolates No more than 20 of an individual species Validation must include at least 5 species Yeast Minimum 100 isolates No more than 30 isolates from an individual species

27 Formic Acid Overlay vs. Direct Smear: Gram negatives No advantage to FA Most species reliably identified Very reliable for Burkholderia. 0 of 5 Pantoea agglomerans identified

28 Formic Acid Overlay vs Direct Smear vs Full Extraction: Gram negative No difference between direct smear and FA Full extraction of mucoid isolates produced excellent results.

29 Gram negative Summary All but one isolate correctly identified (N=350) Formic Acid overlay not necessary for reliable generation of peaks Relatively higher no peaks rate with mucoid isolates >350 isolates tested 34 Genus tested 1 incorrect result = (99.7% accuracy) 16 with no result provided 15 no peaks (95.4% identified) 1 no reliable identification (99.7%)

30 Formic Acid Overlay vs. Direct Smear: High failure rate without FA 80% failure** without FA 5% failure** with FA 100% correct identification 100% correct to species Avg Score with FA = 2.36 Gram positive **FAILURE = No peaks or no reliable identification.

31 Formic Acid Overlay vs. Direct Smear: High failure rate without FA 20% failure** without FA 3% failure** with FA 100% correct identification 100% correct to species Avg Score with FA = 2.23 Gram positive **FAILURE = No peaks or no reliable identification.

32 Gram positive Summary Nearly 100% correct identification Formic Acid overlay critical for reliable generation of peaks Particularly important for Enterococci and Streptococci. 227 isolates tested 11 genus tested 86.8% produced identification 24 No peaks 5 No reliable information

33 Formic Acid necessary ~8% identification rate without FA. No incorrect identifications above 1.7 Reduce manufacturer recommendation Yeast Identification

34 Now the interesting part: Interpreting and trouble shooting the data Problem # 1 The complex MALDI-TOF is capable of identifying many organisms to the species level within complexes. Here is an example of how this can cause a problem

35 MALDI-TOF identification of Complexes Enterobacter cloacae complex 36 Enterobacter spp. isolates tested 17 - E. cloacae** 7 - E. aerogenes 8 - E. asburiae** 2 - E. gergoviae** 1 - E. ludwigii** 1 - E. kobei** 1 - Cronobacter sakazaki* Burkholderia cepacia complex 15 Burkholderia cepacia complex isolates tested 11 - B. multivorans 4 - B. cepacia ** All members of the E. cloacae complex. * Formerly Enterobacter sakazaki

36 Now the interesting part: Interpreting and trouble shooting the data Problem # 2 The twofer Most labs will run MALDI-TOF identifications at least in duplicate Here is an example of how this can cause a problem

37 Two Correct Answers: Do you trust it? ISOLATE PRIMARY ID SCORE SECONDARY ID SCORE 1 H N. gonorrhoeae N. gonorrhoeae L N. gonorrhoeae N. gonorrhoeae H F N. gonorrhoeae N. gonorrhoeae L F N. gonorrhoeae N. meningitidis H N. gonorrhoeae N. gonorrhoeae L N. gonorrhoeae N. meningitidis H F N. gonorrhoeae N. meningitidis L F N. gonorrhoeae N. meningitidis 2.001

38 Two Correct Answers: What do you do with this? ISOLATE PRIMARY ID SCORE SECONDARY ID SCORE 1 H A. xylosoxidans A. xylosoxidans L A. ruhlandii A. xylosoxidans H F A. xylosoxidans A. xylosoxidans L F A. xylosoxidans A. xylosoxidans Best out of Take the highest value Retest Alternative method

39 Now the interesting part: Interpreting and trouble shooting the data Problem # 3 MALDI FAILURE What do you do when MALDI cannot identify an isolate?

40 When MALDI Fails For >300 Gram negative isolates There 16 instances where the traditional system produced an ID and the MALDI failed to produce a correct identification no peaks that will be retested with extraction Mostly mucoid isolates (PA s, Klebs, Inquilinus) 1 - No reliable identification - Chryseobacterium At worst this is a 5% failure rate.

41 Ok.so how much does the big laser cost?

42 Lasers are expensive: Can we afford a MALDI-TOF? Based on 88% high confidence identifications $4,616 vs. $1, hr vs. 3 hr Cherkaoui et al JCM

43 Current methods Versus MALDI-TOF

44 Current Conditions: Current Methods Versus MALDI-TOF

45 Self-Assessment Questions 1. Which matrix is used for MALDI- TOF organism identification a. 4-Hydroxy-α-cyanocinnamic acid (HCCA) b. 2,5-Dihydroxyacetophenone (DH) c. 2,5-Dihydroxybenzoic acid (DHB) AP) d. 3-Hydroxypicolinic acid (HPA) 3. What is the peptide size range analyzed for organism identification? a daltons b ,000 daltons c. 2,000 20,000 daltons d. 20, ,000 daltons 2. What is the component of organism being analyzed for identification? a. Protein b. Glycan c. Nucleic acid d. Peptides