MALDI-TOF Wat is het en betekenis voor kliniek Johan W. Mouton MD PhD FIDSA JWM Moscow 20-05- 2011
MALDI-TOF MS M atrix A ssisted L aser D esorption I onisation T ime O f F light Massa Spectroscopie JWM Zeist 14-06-2011
MALDI : Sample preparation / deflection ionisation TOF : Separation MS : Detection and analysis
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 14-06-2011
JWM Zeist 14-06-2011
Matrix Saturated solution of a-cyano-4-hydroxycinnamic acid in: 50% acetonitrile, 2.5% tri-fluoracetic-acid
MALDI
TOF Time Of Flight
TOF MS t k m z JWM Zeist 14-06-2011
MS - Identification
MALDI-TOF Chemical treatment - Matrix Spectra Pattern recognition IDENTIFICATION JWM Zeist 14-06-2011
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
MALDITOF MS JWM Zeist 14-06-2011
MALDITOF MS
Veen et al JCM 2010 JWM Zeist 14-06-2011
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 14-06-2011
Enterokokken Onderzoeksopzet 2004-2009 (6 jaar) Enterococcus faecium, amoxicilline S 45 stammen Terug te vinden 40 stammen (89%) Rentenaar 2011 JWM Zeist 14-06-2011
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 14-06-2011
Identification much more reliable with the MALDI TOF than with any of the classical techniques, including 16s PCR Rentenaar 2011 JWM Zeist 14-06-2011
Processing in the lab and Reporting JWM Zeist 14-06-2011
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
Day 1
Colony Suspicious For.triggers subsequent tests to be performed JWM Zeist 14-06-2011
Rule : Insertion of 10 tests to be formed if Suspicious for Pseudomonas
The Second Day.
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
MALDI-TOF does not allow?
First Day : Order Entry Clinical Information
Depending on Request, The plates are added following rules as desribed in SOP
Incubation period
Handwork, Most of It, Requiring Specialized Technicians Colony Regonition JWM Zeist 14-06-2011
MALDI-TOF challenges Reporting Susceptibility testing interpretative rules Database accuracies
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
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
EUCAST Rules -Intrinsic Resistances G-
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