Multicenter evaluation of three different MALDI-TOF MS systems for identification of clinically relevant filamentous fungi

Abstract Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) holds promise as a potential tool for clinical identification of filamentous fungi. However, due to the lack of an appropriate extraction protocol and the difficulty of database building, the identification power of each system differs. In this study, we selected 126 clinical mould isolates comprising 28 species identified using internal transcribed spacer (ITS) sequencing as the reference method to evaluate three MALDI-TOF MS systems. When using cultures and sample preparation as recommended by the respective vendors, of the 126 strains tested, VITEK MS identified 121 (96.0%) to species-level and 124 (98.4%) to genus-level; Biotyper identified 53 (42.1%) to species-level and 54 (42.9%) to genus-level; Autof identified 74 (58.7%) to species-level and 76 (60.3%) to genus-level. For the Autof system, the tube extraction method recommended by the vendor performed better (59%) than the on-plate lysis (51%). Our study demonstrates that MALDI-TOF MS systems can successfully identify most clinically relevant fungi, while performance is still highly dependent on the database and sample preparation protocol.

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Matrix-assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a Reliable Tool to Identify Species of Catalase-negative Gram-positive Cocci not Belonging to the Streptococcus Genus

The Open Microbiology Journal ◽

10.2174/1874285801610010202 ◽

2016 ◽

Vol 10 (1) ◽

pp. 202-208 ◽

Cited By ~ 7

Author(s):

Marisa Almuzara ◽

Claudia Barberis ◽

Viviana Rojas Velázquez ◽

Maria Soledad Ramirez ◽

Angela Famiglietti ◽

...

Keyword(s):

Extraction Methods ◽

Species Level ◽

Ionization Time ◽

Maldi Tof Ms ◽

Genus Level ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Level Identification ◽

Gram Positive Cocci ◽

Tof Ms

Objective:To evaluate the performance of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) by using 190 Catalase-negative Gram-Positive Cocci (GPC) clinical isolates.Methods:All isolates were identified by conventional phenotypic tests following the proposed scheme by Ruoff and Christensen and MALDI-TOF MS (Bruker Daltonics, BD, Bremen, Germany). Two different extraction methods (direct transfer formic acid method on spot and ethanol formic acid extraction method) and different cut-offs for genus/specie level identification were used. The score cut-offs recommended by the manufacturer (≥ 2.000 for species-level, 1.700 to 1.999 for genus level and <1.700 no reliable identification) and lower cut-off scores (≥1.500 for genus level, ≥ 1.700 for species-level and score <1.500 no reliable identification) were considered for identification. A minimum difference of 10% between the top and next closest score was required for a different genus or species.MALDI-TOF MS identification was considered correct when the result obtained from MS database agreed with the phenotypic identification result.When both methods gave discordant results, the 16S rDNA orsodAgenes sequencing was considered as the gold standard identification method. The results obtained by MS concordant with genes sequencing, although discordant with conventional phenotyping, were considered correct. MS results discordant with 16S orsodA identification were considered incorrect.Results:Using the score cut-offs recommended by the manufacturer, 97.37% and 81.05% were correctly identified to genus and species level, respectively. On the other hand, using lower cut-off scores for identification, 97.89% and 94.21% isolates were correctly identified to genus and species level respectively by MALDI-TOF MS and no significant differences between the results obtained with two extraction methods were obtained.Conclusion:The results obtained suggest that MALDI-TOF MS has the potential of being an accurate tool for Catalase-negative GPC identification even for those species with difficult diagnosis asHelcococcus,Abiotrophia,Granulicatella, among others. Nevertheless, expansion of the library, especially including more strains with different spectra on the same species might overcome potential “intraspecies” variability problems. Moreover, a decrease of the identification scores for species and genus-level identification must be considered since it may improve the MALDI-TOF MS accuracy.

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Identification of Nocardia species using Autof MS 1000 and Bruker MALDI-TOF MS systems: A comparison

10.1101/2021.01.11.426310 ◽

2021 ◽

Author(s):

Bing Ma ◽

Yunqi Tian ◽

Yungang Han ◽

Lifang Ban ◽

Junwen Yang ◽

...

Keyword(s):

Species Identification ◽

Protein Extraction ◽

Reference Method ◽

Invasive Disease ◽

Species Level ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Significant Difference ◽

Tof Ms

ABSTRACTNocardia is an important cause of clinically invasive disease, but for most clinical laboratories, identification of these isolates to the species level is challenging. Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used for identification of most bacterial and fungal isolates. In this multicenter study, we evaluated the identification of Nocardia isolates using Autof MS1000 and Bruker Biotyper. A total of 86 non-duplicate Nocardia isolates from 7 hospital laboratories were evaluated. Further, we carried out sequence analysis of 16S rRNA, gyrB, secA1, hsp65, and rpoB genes as a reference method for Nocardia species identification. The 86 isolates were directly spotted on the target plate and plate protein extraction was performed. Data were analyzed by SPSS 19.0. In total, 72 (83.7%) strains (score ≥ 9.0) and 70 (81.4%) strains (score ≥ 2.0) were correctly identified by the Autof MS1000 and Bruker Biotyper systems, respectively, at the species level. There was no significant difference (P > 0.05) between the two systems using the same protein extraction method. In conclusion, the Autof MS 1000 and Bruker MALDI-TOF systems showed no difference in identification of Nocardia spp. to the species level and could meet the most important clinical requirement for species identification.

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Development and Validation of an In-House Library for Filamentous Fungi Identification by MALDI-TOF MS in a Clinical Laboratory in Medellin (Colombia)

Microorganisms ◽

10.3390/microorganisms8091362 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1362

Author(s):

Juan C. Gómez-Velásquez ◽

Natalia Loaiza-Díaz ◽

Gilma Norela Hernández ◽

Nelson Lima ◽

Ana C. Mesa-Arango

Keyword(s):

Filamentous Fungi ◽

Clinical Laboratory ◽

Species Level ◽

Clinical Isolates ◽

Correct Identification ◽

Maldi Tof Ms ◽

Genus Level ◽

Maldi Tof ◽

Fungal Identification ◽

Tof Ms

Identification of filamentous fungi by conventional phenotypic methods are time-consuming, and a correct identification at the species level is prone to errors. Therefore, a more accurate and faster time-to-results, and cost-effective technique, is required, such as the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). In this study, we describe the development of an in-house spectra library for the identification of filamentous fungi frequently isolated from patients with infections. An in-house spectra library was constructed using 14 reference strains grown in solid medium. Clinical isolates were identified either by the in-house spectra library or the Biotyper commercial library from Bruker Daltonics. Fungal identification was carried following the Biotyper’s established scores: ≤1.699: not reliably identified (NRI); 1.700–1.999: genus-level; ≥2.000: species-level. Clinical isolates were identified, with the in-house library, at species- and genus-level at 88.70% (55) and 3.22% (2), respectively. While 4.80% (3) was NRI and 3.22% (2) was discrepant concerning sequencing. On the contrary, identification up to species and genus-level with the commercial library was 44.44% (16) and 22.22% (8), respectively. NRI and the discrepancy was 30.55% (11) and 2.77% (1), respectively. For the reaming 26 isolates, 16 from Neoscytalidium dimidiatum and 10 from Sporothrix spp., respectively, the absence of spectrum and the specific spectra within the Sporothrix complex in the commercial library resulted in the inability to obtain an identification. In conclusion, the current results advocate the importance that each clinical microbiological laboratory needs to develop an ad hoc library associated with the MALDI-TOF MS fungal identification to overcome the limitations of the available commercial libraries.

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Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Clinically Relevant Filamentous Fungi

Journal of Clinical Microbiology ◽

10.1128/jcm.00825-16 ◽

2016 ◽

Vol 54 (8) ◽

pp. 2068-2073 ◽

Cited By ~ 36

Author(s):

Allison R. McMullen ◽

Meghan A. Wallace ◽

David H. Pincus ◽

Kathy Wilkey ◽

Carey-Ann D. Burnham

Keyword(s):

Filamentous Fungi ◽

Species Level ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Content Type ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Vitek Ms ◽

Tof Ms

Invasive fungal infections have a high rate of morbidity and mortality, and accurate identification is necessary to guide appropriate antifungal therapy. With the increasing incidence of invasive disease attributed to filamentous fungi, rapid and accurate species-level identification of these pathogens is necessary. Traditional methods for identification of filamentous fungi can be slow and may lack resolution. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid and accurate method for identification of bacteria and yeasts, but a paucity of data exists on the performance characteristics of this method for identification of filamentous fungi. The objective of our study was to evaluate the accuracy of the Vitek MS for mold identification. A total of 319 mold isolates representing 43 genera recovered from clinical specimens were evaluated. Of these isolates, 213 (66.8%) were correctly identified using the Vitek MS Knowledge Base, version 3.0 database. When a modified SARAMIS (Spectral Archive and Microbial Identification System) database was used to augment the version 3.0 Knowledge Base, 245 (76.8%) isolates were correctly identified. Unidentified isolates were subcultured for repeat testing; 71/319 (22.3%) remained unidentified. Of the unidentified isolates, 69 were not in the database. Only 3 (0.9%) isolates were misidentified by MALDI-TOF MS (includingAspergillus amoenus[n= 2] andAspergillus calidoustus[n= 1]) although 10 (3.1%) of the original phenotypic identifications were not correct. In addition, this methodology was able to accurately identify 133/144 (93.6%)Aspergillussp. isolates to the species level. MALDI-TOF MS has the potential to expedite mold identification, and misidentifications are rare.

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Identification of the ‘Streptococcus anginosus group’ by matrix-assisted laser desorption ionization – time-of-flight mass spectrometry

Journal of Medical Microbiology ◽

10.1099/jmm.0.076653-0 ◽

2014 ◽

Vol 63 (9) ◽

pp. 1143-1147 ◽

Cited By ~ 12

Author(s):

Katherine Woods ◽

David Beighton ◽

John L. Klein

Keyword(s):

Species Level ◽

Direct Transfer ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Streptococcus Anginosus ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Transfer Method ◽

Tof Ms

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid, accurate and cost-effective identification of a range of bacteria and is rapidly changing the face of routine diagnostic microbiology. However, certain groups of bacteria, for example streptococci (in particular viridans or non-haemolytic streptococci), are less reliably identified by this method. We studied the performance of MALDI-TOF MS for identification of the ‘Streptococcus anginosus group’ (SAG) to species level. In total, 116 stored bacteraemia isolates identified by conventional methods as belonging to the SAG were analysed by MALDI-TOF MS. Partial 16S rRNA gene sequencing, supplemented with sialidase activity testing, was performed on all isolates to provide ‘gold standard’ identification against which to compare MALDI-TOF MS performance. Overall, 100 % of isolates were correctly identified to the genus level and 93.1 % to the species level by MALDI-TOF MS. However, only 77.6 % were correctly identified to the genus level and 59.5 % to the species level by a MALDI-TOF MS direct transfer method alone. Use of a rapid in situ extraction method significantly improved identification rates when compared with the direct transfer method (P<0.001). We recommend routine use of this method to reduce the number of time-consuming full extractions required for identification of this group of bacteria by MALDI-TOF MS in the routine diagnostic laboratory. Only 22 % (1/9) of Streptococcus intermedius isolates were reliably identified by MALDI-TOF MS to the species level, even after full extraction. MALDI-TOF MS reliably identifies S. anginosus and Streptococcus constellatus to the species level but does not reliably identify S. intermedius.

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Establishment and Application of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for Detection of Shewanella Genus

Frontiers in Microbiology ◽

10.3389/fmicb.2021.625821 ◽

2021 ◽

Vol 12 ◽

Author(s):

Keyi Yu ◽

Zhenzhou Huang ◽

Ying Li ◽

Qingbo Fu ◽

Lirong Lin ◽

...

Keyword(s):

Laser Desorption ◽

Rapid Identification ◽

Species Level ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Type Strains ◽

Tof Ms

Shewanella species are widely distributed in the aquatic environment and aquatic organisms. They are opportunistic human pathogens with increasing clinical infections reported in recent years. However, there is a lack of a rapid and accurate method to identify Shewanella species. We evaluated here matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid identification of Shewanella. A peptide mass reference spectra (PMRS) database was constructed for the type strains of 36 Shewanella species. The main spectrum projection (MSP) cluster dendrogram showed that the type strains of Shewanella species can be effectively distinguished according to the different MS fingerprinting. The PMRS database was validated using 125 Shewanella test strains isolated from various sources and periods; 92.8% (n = 116) of the strains were correctly identified at the species level, compared with the results of multilocus sequence analysis (MLSA), which was previously shown to be a method for identifying Shewanella at the species level. The misidentified strains (n = 9) by MALDI-TOF MS involved five species of two groups, i.e., Shewanella algae–Shewanella chilikensis–Shewanella indica and Shewanella seohaensis–Shewanella xiamenensis. We then identified and defined species-specific biomarker peaks of the 36 species using the type strains and validated these selected biomarkers using 125 test strains. Our study demonstrated that MALDI-TOF MS was a reliable and powerful tool for the rapid identification of Shewanella strains at the species level.

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MALDI-TOF MS and its requirements for fungal identification.

Trends in the systematics of bacteria and fungi ◽

10.1079/9781789244984.0119 ◽

2021 ◽

pp. 119-140

Author(s):

Cledir Santos ◽

Paula Galeano ◽

Reginaldo Lima Neto ◽

Manoel Marques Evangelista Oliveira ◽

Nelson Lima

Keyword(s):

Sample Preparation ◽

Black Box ◽

Data Sets ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Fungal Identification ◽

Identification Of Fungi ◽

Tof Ms

Abstract Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is now used as a routine technique for the fast and reliable identification of fungi at the species level and, currently, it represents an important phenotypic methodology based on proteomic profiles. The main limitations to MALDI-TOF MS for fungal identification are related to sample quality (e.g. quality of biological material such as rigidity or pigmentation of cell walls), sample preparation (e.g. the myriad of sample preparation methodologies that deliver different data sets to different MALDI-TOF MS databases) and the databases themselves (e.g. the 'black-box' commercial databases). This chapter presents an overview and discussion of the use of MALDI-TOF MS for fungal identification. The major known limitations of the technique for fungal taxonomy, and how to overcome these, are also discussed.

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Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry in diagnosis of clinical Nocardia species

Journal of Laboratory Medicine ◽

10.1515/labmed-2018-0180 ◽

2019 ◽

Vol 43 (3) ◽

pp. 157-162

Author(s):

Gülşen Hasçelik ◽

Markus Kostrzewa ◽

Hamit Kaan Müştak ◽

Celalettin Uner ◽

Kadir Serdar Diker

Keyword(s):

Species Level ◽

Rrna Gene ◽

Laser Desorption Ionization ◽

Ionization Time ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Maldi Biotyper ◽

Rrna Gene Sequencing ◽

Tof Ms

Abstract Background The routine identification to the species level of Nocardia genus by conventional methods is a fastidious and time-consuming process owing to the limited biochemical reactivity of these microorganisms, often requiring 1 or more days to complete identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a new technology for definitive and rapid species identification. Methods We evaluated the MALDI-TOF MS for the identification of 44 clinical isolates of Nocardia species in comparison to 16S ribosomal RNA (rRNA) gene sequencing. Nocardia isolates were identified by microbiological examination, phenotypical tests and MALDI-TOF MS and the results were compared by 16S rRNA gene sequencing. Results Of the 44 Nocardia strains, the identification of 28 isolates was determined with MALDI Biotyper database. According to this, 16 isolates (57.1%) of the strain log scores were ≥2. Two (7.1%) were identified to the species level (log scores of ≥2) as Nocardia otitidiscaviarum. The addition of a newly established Nocardia database (16 new Nocardia strains included to the original database) did significantly improve the scores. The results were 43 (97.7%) correct identification to the species level (log scores of ≥2). Conclusions This study showed that the identification of clinical Nocardia isolates by the Bruker MALDI Biotyper is highly reliable, whereas identification rates are generally lower than those for some Gram-negative bacteria and Gram-positive cocci. Based on our data, the identification rates can be improved by validated new database entries and the results can be confirmed with nucleic acid sequence analysis.

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MALDI-TOF Mass Spectrometry and Specific Biomarkers: Potential New Key for Swift Identification of Antimicrobial Resistance in Foodborne Pathogens

Microorganisms ◽

10.3390/microorganisms7120593 ◽

2019 ◽

Vol 7 (12) ◽

pp. 593 ◽

Cited By ~ 2

Author(s):

Maureen Feucherolles ◽

Henry-Michel Cauchie ◽

Christian Penny

Keyword(s):

Mass Spectrometry ◽

Antimicrobial Resistance ◽

Foodborne Pathogens ◽

Reference Method ◽

Multi Drug Resistance ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

The Future ◽

Tof Ms

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is today the reference method for direct identification of microorganisms in diagnostic laboratories, as it is notably time- and cost-efficient. In the context of increasing cases of enteric diseases with emerging multi-drug resistance patterns, there is an urgent need to adopt an efficient workflow to characterize antimicrobial resistance (AMR). Current approaches, such as antibiograms, are time-consuming and directly impact the “patient-physician” workflow. Through this mini-review, we summarize how the detection of specific patterns by MALDI-TOF MS, as well as bioinformatics, become more and more essential in research, and how these approaches will help diagnostics in the future. Along the same lines, the idea to export more precise biomarker identification steps by MALDI-TOF(/TOF) MS data towards AMR identification pipelines is discussed. The study also critically points out that there is currently still a lack of research data and knowledge on different foodborne pathogens as well as several antibiotics families such as macrolides and quinolones, and many questions are still remaining. Finally, the innovative combination of whole-genome sequencing and MALDI-TOF MS could be soon the future for diagnosis of antimicrobial resistance in foodborne pathogens.

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Accuracy of conventional identification methods used for Enterobacteriaceae isolates in three Nigerian hospitals

PeerJ ◽

10.7717/peerj.2511 ◽

2016 ◽

Vol 4 ◽

pp. e2511 ◽

Cited By ~ 4

Author(s):

Christiana Jesumirhewe ◽

Peter Oladejo Ogunlowo ◽

Mitsan Olley ◽

Burkhard Springer ◽

Franz Allerberger ◽

...

Keyword(s):

Multidrug Resistant ◽

Species Level ◽

Accurate Identification ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Maldi Biotyper ◽

Identification Of Bacteria ◽

Identification Techniques ◽

Tof Ms

BackgroundEnterobacteriaceae are ubiquitously present in nature and can be found in the intestinal tract of humans and animals as commensal flora. Multidrug-resistant Enterobacteriaceae are increasingly reported and are a threat to public health implicating a need for accurate identification of the isolates to species level. In developing countries, identification of bacteria basically depends on conventional methods: culture and phenotypic methods that hamper the accurate identification of bacteria. In this study, matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) technique was compared to conventional identification techniques.Materials and MethodsIn total, 147 Enterobacteriaceae isolates were collected from March to May 2015 from three medical microbiology laboratories of hospitals in Edo state, Nigeria, after being tested according to the individual laboratories standard operating procedures. All isolates were stored at −20°C until tested centrally by MALDI-TOF MS.ResultsOne hundred and forty five (98.6%) isolates had a MALDI Biotyper best score > or =2.0, indicating a secure genus and probable species identification; and 2(1.36%) isolates had a best score <2.0 indicating probable genus identification. Isolates with best scores of > or =2.0 comprised nine genera and 10 species, respectively. A total of 57.2% and 33.1% of isolates identified had agreement between MALDI-TOF MS and conventional techniques for identification at genus and species level, respectively, when analyzing bacteria with MALDI Biotyper best scores > or =2.0.ConclusionThe results of our study show that the applied conventional identification techniques for Enterobacteriaceae in the investigated Nigerian hospitals are not very accurate. Use of state-of-the-art identification technologies for microorganisms is necessary to guarantee comparability of bacteriological results.

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