Filamentous fungi MALDI-TOF identification Stéphane Ranque Parasitologie & Mycologie AP-HM Timone Marseille, France stephane.ranque@ap-hm.fr
Aspergillus flavus Aspergillus ochraceus Conidia 3 to 6 µ Conidia 3 µ Conidiophore finely echinulate Conidiophore very echinulate
Laboratory equipement
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 on MALDI-TOF identification results: Culture medium Incubation time Pretreatment procedure Type of instrument Spectra comparison algorithm Reference database
Practical laboratory issues Culture medium effect on MALDI-TOF identification results
Cycloheximide Effect Species N Mean LS (SD ) SGC SGCc Microsporum audouinii 10 2.077 (±0.264) 2.084 (0.266 ) Microsporum canis 3 2.405 (±0.140) 2.004 (0.181) Microsporum gypseum 4 2.606 (±0.069) 2.343 (0.317) Trichophyton mentagrophytes 4 2.534 (0.22) 2.585 (0.080) Trichophyton rubrum 3 2.564 (0.142) 2.417 (0.145) Trichophyton tonsurans 2 2.005 (0.335) 2.261(0.299)
6192 clinical yeast isolates
Culture medium effect Yes, but no significant impact on MALDI- TOF identification results
Practical laboratory issues Incubation time effect on MALDI-TOF identification results
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 Species Mean LS (SD ) Incubation time 3 days 15 days M. audouinii strain 169 2.096 (0.197) 1.924 (0.06) M. canis strain 249D 2.539 (0.078) 2.147 (0.195) T. mentagrophytes strain 214 2.632 (0.120) 2.428 (0.102) T. rubrum strain 60D 2.665 (0.045) 2.273 (0.036) T. soudanense strain 243D 2.632 (0.120) 2.050 (0.182) T. tonsurans strain 90D 2.705 (0.023) 2.386 (0.06)
Incubation time effect Yes, but the sooner the better from a clinical point of view
Practical laboratory issues Pretreatment procedure effect on MALDI-TOF identification results
FA extraction + bead-beating + heating FA extraction + bead-beating FA extraction + heating FA extraction + washing FA extraction
Smear or Direct deposit Fast FA extraction Complete extraction I Complete extraction II
Pre-treatment procedures Complete extraction procedures perform better than fast procedures
Pretreatment procedure effect Complete extraction procedures: enhance identification results robust to unskilled operators suited to batch processing yet reference library is key
Practical laboratory issues Type of instrument effect on MALDI-TOF identification results
MALDI-TOF identification systems Instruments Bruker Daltonics (Bremen, Germany) Série FLEX : Microflex LT Scientific Analysis Instrument (Manchester, UK) Commercialized systems MaldiBiotyper system Bruker Daltonics Andromas System Andromas SAS, Paris Axima@Saramis Shimadzu (Tokyo, Japan) séries AXIMA Agnostec/bioMérieux VitekMS biomérieux
Mass spectrometer Our experience is limited to three machines by Brüker: Autoflex I Ultraflex 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 Probably not significant
Practical laboratory issues Spectra comparison algorithm effects on MALDI-TOF identification results
Spectra comparison algorithms The three commercialized systems Andromas Vitek MS BioTyper Have distinct proprietary spectra comparison algorithms That perform similarly well for the identification of microorganisms Not evaluated on filamentous fungi
Spectra comparison algorithms effect Probably not significant
Practical laboratory issues 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 in the Marseille and BCCM/IHEM MALDI-TOF reference spectra database
Fungal diversity in the reference database, March 2014
Reference database effect Highly significant
Perspectives Impact of routine MALDI-TOF mold identification in the clinical laboratory
31/12/2011 01/01/2012 Culture Culture Macroscopic and microscopic characterisation After 4 to 7 culture days DNA sequencing for: Scedosporium Fusarium uncharacteristic fungal spp. MALDI-TOF identification After 3 to 4 culture days Macroscopic and microscopic characterisation After 3 to 7 culture days DNA sequencing for : Maldi BIOTYPER LS <1.9 Fusarium Scedosporium uncharacteritic Aspergillus spp.
Culture Routine Identification Procedure Microscopic Examination Fungal Colony Concordant with microscopy YES NO YES MALDI-ToF 4 spots Concordant spots 3 and 1 LS value 1.9 Sequencing ITS2 28S D1-D2 YES Identification to species validated NO YES NO MALDI-ToF 4 spots Concordant spots 3 and 1 LS value 1.9
140 Number of filamentous fungi taxa identified at the laboratory 120 121 100 97 80 60 40 20 0 1 70 42 29 26 22 14 15 12 11 9 10 9 10 9 7 4 2 2009 2010 2011 2012 Fusarium sp. Dermatophytes Penicillium sp. Aspergillus sp. Total
Positive Subcultures : 247 Initial identification (2011) January to April 2011 (302 samples subcultured) Identification : 66.8% 16 distinct species Yes 165 Identification : 97.6% No 82 MALDI-TOF MS-based identification (2013 database) MALDI-TOF MS-based identification (2013 database) Concordant identification 150 Initial misidentification* 15 Identification 76 MALDI-TOF MS-based identification failure 6 DNA sequence-based Identification Total identification : 98,8% 42 distinct species Yes 3 Penicillium madriti P. aurantiogriseum Tilletiopsis albescens No 3 Penicillium sp Phoma sp Scedosporium sp.
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 flavus Mucor circinelloides Aspergillus flavus Mucor circinelloides Aspergillus hortai Penicillium allii Aspergillus fumigatus Penicillium chrysogenum Aspergillus fumigatus Penicillium chrysogenum Aspergillus lentulus Penicillium aurantiogriseum Aspergillus nidulans Penicillium glabrum Aspergillus nidulans Penicillium glabrum Aspergillus puniceus Penicillium citrinum Aspergillus niger Radulidium subulatum Aspergillus niger Radulidium subulatum Aspergillus sclerotiorum Penicillium crustosum Aspergillus niveus Scedosporium apiospermum Aspergillus niveus Scedosporium apiospermum Aspergillus tubingensis Penicillium funiculosum Aspergillus ochraceus Scopulariopsis brevicaulis Aspergillus ochraceus Scopulariopsis brevicaulis Penicillium purpurogenum Penicillium griseofulvum Aspergillus sydowii Tilletiopsis albescens Aspergillus sydowii Alternaria malorum Fusarium. oxysporum Penicillium madriti Aspergillus terreus Aspergillus terreus Phaeoacremonium parasticum Fusarium proliferatum Penicillium nalgiovense Aspergillus ustus Aspergillus versicolor Ulocladium atrum Fusarium solani Penicillium piceum 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