filamentous fungi maldi-tof identification
TRANSCRIPT
Diapositive [email protected]
Conidiophore finely echinulate Conidiophore very echinulate
Aspergillus flavus Aspergillus ochraceus
MALDI-TOF code bar
Gel and stack views of the MALDI-TOF MS spectra of 8 filamentous fungi
Practical laboratory issues
• Culture medium • Incubation time • Pretreatment procedure • Type of instrument • Spectra comparison algorithm • Reference database
Practical laboratory issues
Cycloheximide Effect
SGC SGCc
Trichophyton tonsurans 2 2.005 (0.335) 2.261(0.299)
6192 clinical yeast isolates
Yes, but no significant impact on MALDI- TOF identification results
Practical laboratory issues
MALDI-ToF spectra reproducibilty
(4 distinct sub-cultures of a T. rubrum isolate)
Incubation time effect Mean LS values of 40 spots of six dermatophyte strains tested against reference spectra aquired
from 3-day-old culture
Incubation time effect
Yes, but the sooner the better from a clinical point of view
Practical laboratory issues
FA extraction
FA extraction + bead-beating + heating
Smear or Direct deposit Fast FA extraction Complete extraction I Complete extraction II
Pre-treatment procedures
Practical laboratory issues
• Autoflex I
• Microflex LT
• Changing from an Autoflex I to a Microflex LT significantly improved identification quality
• No evidence that the Shimadzu and Brüker machines perform differently
Type of instrument effect
Spectra comparison algorithms
• Vitek MS
• That perform similarly well for the identification of microorganisms
• Not evaluated on filamentous fungi
Spectra comparison algorithms effect
Reference database effect on MALDI-TOF identification results
Overall performance of the NIH mold database and the Bruker library when challenged against 421 clinical isolates
Library(ies) No. of isolates (%) with the indicated score
≥2.0 ≤1.99 and ≥1.7 <1.7
Combined NIH and Bruker 370 (88.9) 18 (4.3) 33 (7.8)
NIH alone 370 (88.9) 18 (4.3) 33 (7.8)
Bruker alone 3 (0.7) 26 (6.2) 392 (93.1)
J Clin Microbiol. 2013 Mar; 51(3): 828–834.
Filamentous fungi species implemented
MALDI-TOF reference spectra database
Reference database effect
Impact of routine MALDI-TOF mold identification in the clinical laboratory
Culture
DNA sequencing for:
Macroscopic and microscopic characterisation
DNA sequencing for :
Concordant with microscopy
MALDI-ToF 4 spots
YES
YES
NO
1 4
7 10
Concordant identification
Identification
76
Fungal diversity in the first four months of 2011
initial identification vs MS identification
2011, without MS 2011, with MS
Alternaria alternata Exophiala dermatitidis Alternaria alternata Exophiala dermatitidis Aspergillus
calidoustus
Paecilomyces
lilacinus
Aspergillus fumigatus Penicillium
chrysogenum Aspergillus fumigatus
Aspergillus niger Radulidium subulatum Aspergillus niger Radulidium subulatum Aspergillus
sclerotiorum
Penicillium
crustosum
oxysporum Penicillium madriti
parasticum
Fusarium
proliferatum
Penicillium
nalgiovense
Aspergillus versicolor Tilletiopsis albescens Trichoderma
longibrachiatum
Fusarium
verticillioides
Penicillium
pinophilum
Mold species diversity according to the origin of the sample for 16 months
Strength and pitfalls of MALDI-TOF mass spectrometry
Strength • Fast • Accurate • Simple • Cost • Routine integration
Pitfalls • Colonies included in the agar • Disinterest of industrials for
filamentous fungi identification • Inaccurate and incompletes fungal
reference spectra data bases • Proprietary bench-based systems • No publicly available fungal spectra
database
Conidiophore finely echinulate Conidiophore very echinulate
Aspergillus flavus Aspergillus ochraceus
MALDI-TOF code bar
Gel and stack views of the MALDI-TOF MS spectra of 8 filamentous fungi
Practical laboratory issues
• Culture medium • Incubation time • Pretreatment procedure • Type of instrument • Spectra comparison algorithm • Reference database
Practical laboratory issues
Cycloheximide Effect
SGC SGCc
Trichophyton tonsurans 2 2.005 (0.335) 2.261(0.299)
6192 clinical yeast isolates
Yes, but no significant impact on MALDI- TOF identification results
Practical laboratory issues
MALDI-ToF spectra reproducibilty
(4 distinct sub-cultures of a T. rubrum isolate)
Incubation time effect Mean LS values of 40 spots of six dermatophyte strains tested against reference spectra aquired
from 3-day-old culture
Incubation time effect
Yes, but the sooner the better from a clinical point of view
Practical laboratory issues
FA extraction
FA extraction + bead-beating + heating
Smear or Direct deposit Fast FA extraction Complete extraction I Complete extraction II
Pre-treatment procedures
Practical laboratory issues
• Autoflex I
• Microflex LT
• Changing from an Autoflex I to a Microflex LT significantly improved identification quality
• No evidence that the Shimadzu and Brüker machines perform differently
Type of instrument effect
Spectra comparison algorithms
• Vitek MS
• That perform similarly well for the identification of microorganisms
• Not evaluated on filamentous fungi
Spectra comparison algorithms effect
Reference database effect on MALDI-TOF identification results
Overall performance of the NIH mold database and the Bruker library when challenged against 421 clinical isolates
Library(ies) No. of isolates (%) with the indicated score
≥2.0 ≤1.99 and ≥1.7 <1.7
Combined NIH and Bruker 370 (88.9) 18 (4.3) 33 (7.8)
NIH alone 370 (88.9) 18 (4.3) 33 (7.8)
Bruker alone 3 (0.7) 26 (6.2) 392 (93.1)
J Clin Microbiol. 2013 Mar; 51(3): 828–834.
Filamentous fungi species implemented
MALDI-TOF reference spectra database
Reference database effect
Impact of routine MALDI-TOF mold identification in the clinical laboratory
Culture
DNA sequencing for:
Macroscopic and microscopic characterisation
DNA sequencing for :
Concordant with microscopy
MALDI-ToF 4 spots
YES
YES
NO
1 4
7 10
Concordant identification
Identification
76
Fungal diversity in the first four months of 2011
initial identification vs MS identification
2011, without MS 2011, with MS
Alternaria alternata Exophiala dermatitidis Alternaria alternata Exophiala dermatitidis Aspergillus
calidoustus
Paecilomyces
lilacinus
Aspergillus fumigatus Penicillium
chrysogenum Aspergillus fumigatus
Aspergillus niger Radulidium subulatum Aspergillus niger Radulidium subulatum Aspergillus
sclerotiorum
Penicillium
crustosum
oxysporum Penicillium madriti
parasticum
Fusarium
proliferatum
Penicillium
nalgiovense
Aspergillus versicolor Tilletiopsis albescens Trichoderma
longibrachiatum
Fusarium
verticillioides
Penicillium
pinophilum
Mold species diversity according to the origin of the sample for 16 months
Strength and pitfalls of MALDI-TOF mass spectrometry
Strength • Fast • Accurate • Simple • Cost • Routine integration
Pitfalls • Colonies included in the agar • Disinterest of industrials for
filamentous fungi identification • Inaccurate and incompletes fungal
reference spectra data bases • Proprietary bench-based systems • No publicly available fungal spectra
database