MALDI-TOF Wat is het en betekenis voor kliniek

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1 MALDI-TOF Wat is het en betekenis voor kliniek Johan W. Mouton MD PhD FIDSA JWM Moscow

2 MALDI-TOF MS M atrix A ssisted L aser D esorption I onisation T ime O f F light Massa Spectroscopie JWM Zeist

3 MALDI : Sample preparation / deflection ionisation TOF : Separation MS : Detection and analysis

4 MALDI Matrix required to retain structure Matrix assisted laser desorption ionisation Soft laser desorption First described by Tanaka in 1985 (Nobel Prize 2002) and Hillenkamp and Karas in 1985 First used for proteins in 1988 JWM Zeist

described by Tanaka in 1985 (Nobel Prize 2002) and Hillenkamp

5 JWM Zeist

6 Matrix Saturated solution of a-cyano-4-hydroxycinnamic acid in: 50% acetonitrile, 2.5% tri-fluoracetic-acid

7 MALDI

8 TOF Time Of Flight

9 TOF MS t k m z JWM Zeist

10 MS - Identification

11 MALDI-TOF Chemical treatment - Matrix Spectra Pattern recognition IDENTIFICATION JWM Zeist

12 Ionendetector. Meet tijdsgespatieerd intensiteit van ionen die erop vallen Buis met een vaste lengte d Laserstraal van een Zgn zachte laser (337 nm Stikstoflaser) Ionen versneller Met voltage U

13 MALDITOF MS JWM Zeist

14 MALDITOF MS

15 Veen et al JCM 2010 JWM Zeist

16 Enterokokken: Achtergrond In klinische praktijk een Enterococcus faecium gekweekt uit bloed, die amoxicilline gevoelig was, Bij E-test amoxicilline: alsnog resistent. Vraag: hoe vaak is de Phoenix uitslag onbetrouwbaar voor amoxicilline gevoelige E. faecium stammen. Rentenaar 2011 JWM Zeist

17 Enterokokken Onderzoeksopzet (6 jaar) Enterococcus faecium, amoxicilline S 45 stammen Terug te vinden 40 stammen (89%) Rentenaar 2011 JWM Zeist

18 Enterokokken Resultaten na Malditof Determinatie N (%) N (%) analyse E. faecium 26/40 (65%) E. faecium amoxi S 20/40 (50%) 20/26 (77%) E. faecium amoxi R 6/40 (15%) 6/26 (23%) GEEN E. faecium 14/40 (..%) Streptococcus agalactiae 3/40 (7,5%) E. faecalis 1/40 (2.5%) Enterococcus species (geen E. faecium of E. faecalis) 1/40 (2.5%) Streptococcus gallolyticus subspecies gallolyticus 1/40 (2,5%) Streptococcus salivarius 6/40 (15%) Streptococcus anginosus 1/40 (2,5%) Streptococcus species 1/40 (2,5%) Rentenaar 2011 JWM Zeist

.%) Streptococcus agalactiae 3/40 (7,5%) E. faecalis 1/40 (2.5%) Enterococcus species (geen E. faecium of E. faecalis) 1/40 (2.

19 Identification much more reliable with the MALDI TOF than with any of the classical techniques, including 16s PCR Rentenaar 2011 JWM Zeist

20 Processing in the lab and Reporting JWM Zeist

21 Processing of Microbiology Specimens Day 0 : plating, overnight incubation Day 1 : reading, colony processing overnight incubation Day 2 : reading, colony processing overnight incubation Day 3 : reading, colony processing overnight incubation Sampling Arrival at lab Administration Processing Result Report numbering, identification test orders, requests rule based rule based autovalidation and alerts Mouton JW, Las Vegas 2008

22 Day 1

23 Colony Suspicious For.triggers subsequent tests to be performed JWM Zeist

24 Rule : Insertion of 10 tests to be formed if Suspicious for Pseudomonas

25 The Second Day.

26 MALDI-TOF allows Identification on the same day Processing times < one hour -laboratory logistics to be redesigned -susceptibility interpretation depends on identification!! Less tests More accuracy

27 MALDI-TOF does not allow?

28 First Day : Order Entry Clinical Information

29 Depending on Request, The plates are added following rules as desribed in SOP

30 Incubation period

31 Handwork, Most of It, Requiring Specialized Technicians Colony Regonition JWM Zeist

32 MALDI-TOF challenges Reporting Susceptibility testing interpretative rules Database accuracies

33 Identification Rules in Susceptibility Testing Until recently, around 50 species were considered medically important Many strains were misidentified because of a lack of appropriate tests It is now recognized mainly because of the advent of Molecular Biology - that the diversity is much larger than previously thought. The Dutch Semantic Standard contains 2500 entries and growing steadily MIC interpretation depends on species identification

34 Rules in Susceptibility Testing Testresult Quantitative, e.g. MIC Identification If species intrinsically resistant then report as Resistant Else. Species specific Breakpoints Report as Susceptible / Intermediate / Resistant

35 EUCAST Rules -Intrinsic Resistances G-

36 MALDI-TOF MS allows Identification on the same day Processing times < one hour -laboratory logistics to be redesigned Less tests More accuracy Recently direct identification on positive blood-cultures Many challenges, in particular with respect to susceptibility

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