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MALDI-TOF Technique 2017 AUG 5-6 SPEAKER MALDI-TOFCONFERENCE, TechniqueOF MMTN ATAriya Chindamporn Department of COPYRIGHTMicrobiology, Faculty of Medicine, Chulalongkorn University Bangkok, Thailand PRESENTED Presented at MMTN Malaysia Conference 5–6 August 2017, Kuala Lumpur, Malaysia Outline 2017 AUG 5-6 o Principle of MALTI-TOF o Yeast identification from pure colony o Yeast identification directly from bloodSPEAKER cultures o Influence of sample preparationCONFERENCE,OF method o Mould identification from pure colony MMTN o Prediction of antifungalAT drug resistance COPYRIGHT o Cost effectiveness Disclosure: no conflict of interest PRESENTED Robin P, Clin Chem January 2015 Principle of MALDI-TOF Bader O. Proteomics 2013 MALDI plate MALDI: Matrix Assisted Laser Desorption/Ionization preparation 2017 Agar of choice (Goyer M. J Clin Microbiol. 2012) AUG Streak cells • Sabouraud dextrose agar 5-6 on the • Columbia blood agar MALDI plate • Chromagar Overlay with the Lysis reagent: matrix disolved in • 25–70% formic acid SPEAKER At this step cell components are released lysis reagents CONFERENCE,OF Crystallized MMTNmatrix serve as Let the mixture the chemicalAT ionizing agent for energy transfer from become the laser toCOPYRIGHT the analyte. crystallized matrix PRESENTED Robin P, Clin Chem January 2015 Principle of MALDI-TOF TOF: Time of Fight Bader O. Proteomics 2013 MALDI plate Protein analysis Result analysis preparation 2017 C. parapsilosis#1 AUG Streak cells on the 5-6 C. parapsilosis#2 MALDI plate C. parapsilosis#3 Overlay with the matrix disolved in SPEAKER lysis reagents CONFERENCE,OF C. glabrata www.shimadzu.com MMTN C. bracarensis Let the mixture AT become COPYRIGHT crystallized matrix • M/Z ratio is generated the peak of spectra. • By laser beam, the analyte is • These spectra can determine the desorbed from the target plate precise species PRESENTEDresulting ions acceleration in o highly reproducible within a an electric field. species • Mass and charge (M/Z) of the o sufficiently different even between analyte are measured. highly related species Yeast identification from pure colony • Due to simple cell structure of yeast compared to mould, much easier of yeast identification is revealed. 2017 • The accuracy of identification depended on instrument AUGtype (database) 5-6 MALDI-TOF Accuracy Study Ref. MALDI Biotyper 95.7% 4059/4247 isolates;16 studies Bader O. 2013 SPEAKER Bader O .2011 CONFERENCE,OF Rosenvinge F et al. 2012 SARAMIS system 98.4% 1463/1487 isolates;4 studies Martinez-Lamas L et al. 2011 Santos C. 2011 MMTN VITEK-MS 98.4%AT 183/184 isolates;1 study Iriart, X et al. 2012 COPYRIGHT Andromas databases 100% 160/160 isolates;1 study Bille E et al. 2012 PRESENTED Yeast identification from pure colony MALDI-TOF can also apply for 2017 • Closely related yeast species which can not be clearly discriminated with common biochemical methods AUG • Candida ortho-/meta-/parapsilosis 5-6 (Quiles-Melero I, J Clin Microbiol Infect Dis, 2012 , Martinez-Lamas L, Infec Microbiol Clin, 2011, Santos C, Diagn, Microbiol Infect Dis, 2011, Hendrickx M, Diagn Microbiol Infect Dis, 2011. Kubesova A, Analyst, 2012) • Candida glabrata/bracarensis/nivariensis (Santos C, Diagn Microbiol Infect Dis, 2011) • C. albicans/dubliniensis SPEAKER (Santos C, Diagn Microbiol Infect Dis, 2011) CONFERENCE,OF • Candida haemulonii group I and II complexes (Cendejas-Bueno E, J Clin Microbiol, 2012) • Phenotypically similar speciesMMTN (Jensen RH, J. Clin. Microbiol, 2011, Castanheira M, J Clin Microbiol, 2013, Desnos-Ollivier M, J Clin Microbiol, 2008) AT • C. palmioleophila COPYRIGHT • C. famata • C. guilliermondii PRESENTED Can MALDI-TOF identify yeast directly from blood cultures ? Factor 1 Highly abundant substances disturb the identification process: o culture media (e.g. charcoal and cations other than H+) 2017 (Szabados F, Clin Microbiol Infect, 2011) o human blood (e.g. hemoglobin or albumin) AUG (Marinach-Patrice C, PLoS One, 2010) 5-6 • These components can generate mass peaks that partially overlap with spectra from yeasts. (Marinach-Patrice C, PLoS One, 2010) • To solve the problem, purification procedure before extraction were developed from manufacturers: SPEAKER • Gel matrices (Loonen A, Eur J Clin Microbiol Infect Dis, 2012, Sparbier K, J Clin Microbiol, 2012) CONFERENCE,OF • Filtration devices (Fothergill A, J. Clin. Microbiol, 2012) • Saponification (Ferroni A, J Clin Microbiol, 2010) MMTN • (Spanu T, J Clin Microbiol, 2012, Ferreira L, Clin.Microbiol, Infect. 2011) Differential centrifugationAT • Washing steps: distilledCOPYRIGHT water / low conc. of detergents ie. SDS or Tween80. (Marinach-Patrice C, PLoS One, 2010, Spanu T, J Clin Microbiol, 2012, Ferreira L, Clin.Microbiol, Infect. 2011) Factor 2 Amount of yeast cells in blood culture (<4% of total sample) • ExtraPRESENTED step: centrifugation (Bader O, Proteomics, 2013) The accuracy of yeast identification directly from blood culture 2017 Database Hemoculture Sample prep. (library AUG Reference version) albicans glabrata parapsilosis 5-6 tropicalis krusei guilliermondii C. C. C. C. C. C. C. Ferroni A et al. BacT/Alert Extraction Andromas 20/20 - - - - - w/o charcoal with TFA* only (unknown) 2010 Differential Biotyper Ferreira L et al. BACTEC 0/8 0/9SPEAKER- 0/1 - - centrifugation (V2.0.4.0) 2011 CONFERENCE,OF Marinach- Mycosis IC/F SD wash step Other 5/5 5/5 5/5 5/5 5/5 - Patrice C et al. 2010 MMTN Biotyper - Yan Y et al. Bactec FX Sepsityper 28/28 - 8/8 5/5 - (V3.1.1.0)AT 2011 COPYRIGHT 10 Sepsityper Schubert S et Biotyper 4/5 2/7 1/2 - - 1/1 Aerobic/anaer (V3.1.1.0) al, 2011 obic/F Tween 80 Biotyper Spanu T et al, Mycosis IC/F 187/195 22/26 65/69 28/32 6/8 6/10 PRESENTEDwash (unknown) 2012 *TFA: Trifluoroacetic acid Yeast identification directly from blood cultures In summary 2017 • Direct identification of yeasts from positive blood culture is possible. AUG • The purity of the analyte have major effect to the result. 5-6 • However, even commercial kit (Sepsityper, Bruker Daltonics) for sample purification is available, protocols for purification used in the literature are not yet standardized. • Research used only is now recommended.SPEAKER CONFERENCE,OF Yeast identification directly from urine samples (Sobel J D, Clin Infect Dis, 2011) • Yeasts are also frequentlyMMTN recovered from urinary samples o catheter colonizersAT COPYRIGHT • Technically, yeast identification directly from urine is possible. • However, so far the study of yeast identification directly from urine samples by MALDI-TOF is very rare and all are in research work. PRESENTED Influence of sample preparation method (Kemptner J, Rapid Commun Mass Spectrom, 2009) • Standard protocol of sample prep. is appropriate for most yeasts.2017 • However, due to the stronger cell walls of some fungal cells resulting not sufficiently release their intracellular contents under theseAUG conditions. • Modified procedures was developed. 5-6 Colonies on agar plate Smear prep. Lyse cells on target + air dry SPEAKER CONFERENCE,OF Ethanolize cells (70% EtOH) (the increased peak On-target-lysis number due to fixing step MMTN was revealed) AT COPYRIGHT Cells extraction Cells extraction with Overlay with matrix + formic acid + Spot supernatant air dry, then ready for acetonitrile (increased onto target MALDI analysis protein solubilisationPRESENTED) Candida glabrata spectra derived from each method of cells prep. Cells extraction • Different method2017 give different pattern of spectra. On-target-lysis • For cells thatAUG still do not lyse efficiently,5-6 other extraction methods ie. Direct smear mechanical disruption in a bead- beater can be applied Another factor need to be concern is “Database” • Best preparation method is the same methodSPEAKER with which the spectra of the database have been made. CONFERENCE,OF • It will lead to the highest concordance between acquired test spectra and reference spectra in the identification database. MMTN • Currently, available yeast database are AT • MALDI Biotyper providedCOPYRIGHT both “On -target-lysis” and “cells extraction”. • SARAMIS, VITEK-MS and Andromas systems provided only “On-target-lysis”. PRESENTED (Bader O, Proteomics, 2013, Amiri-Eliasi B, Anal Chem, 2001, Cassagne C, PLoS One, 2011, Hettick J, Mass Spec-trom, 2008) “On-target-lysis” faster & requires less hands-on-time than “cells extraction” • Application of “On-target-lysis” have been developed for use with the MALDI Biotyper system (Bader O, Proteomics, 2013) 2017 • To overcome the fewer peaks generated from this method, the analysis criteria was modified. • In general, M/Z ratio (log score) of MALDI Biotyper referenceAUG spectra • ≥2.000 “species level” identifications 5-6 • 1.700 - 1.999 “genus level only.” • Current V3.0 MALDI Biotyper, decreasing criteria of log score • Lower log-scores starting from 1.500 can be accepted as species-specific for yeasts if certain criteria are met (Steensels D, Acta Clin Belg, 2011, Goyer M, J. Clin Microbiol, 2012, Sparbier K, J Clin Microbiol, 2012) SPEAKER • The 3 additional criteria : (Steensels D, Acta Clin Belg, 2011, Goyer M, J Clin Microbiol, 2012, Sparbier K, J Clin Microbiol, 2012) (i) encompasses a certain numberCONFERENCE, of OFdatabase hits n > 2 or 3 of a single species at the top (ii) have no other species intermingled (iii) significantly difference of log-score to the next species (ie. 0.200). MMTN Rank (Qualtity) Matched pattern Score value (log score) Answer: AT 1 (-) Candida albicans ATCC 24433 1.5 C. albicans COPYRIGHT 2 (-) Candida albicans ATCC 10231 1.49 3 (-) Candida albicans ATCC 90028
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