Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria, including Brucella, and yeasts.

The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has proven to be rapid and accurate for the majority of clinical isolates. Some gaps remain concerning rare, emerging, or highly pathogenic species, showing the need to continuously expand the databases. In this multicenter study, we evaluated the accuracy of the VITEK MS v3.2 database in identifying 1172 unique isolates compared to identification by DNA sequence analysis. A total of 93.6% of the isolates were identified to species or group/complex level. A remaining 5.2% of the isolates were identified to the genus level. Forty tests gave a result of no identification (0.9%) and 12 tests (0.3%) gave a discordant identification compared to the reference identification. VITEK MS is also the first MALDI-TOF MS system that is able to delineate the four members of the Acinetobacter baumannii complex at species level without any specific protocol or special analysis method. These findings demonstrate that the VITEK MS v3.2 database is highly accurate for the identification of bacteria and fungi encountered in the clinical laboratory as well as emerging species like Candida auris and the highly pathogenic Brucella species.

Application of MALDI-TOF MS to species complex differentiation and strain typing of food related fungi: Case studies with Aspergillus section Flavi species and Penicillium roqueforti isolates.

Filamentous fungi are one of the main causes of food losses worldwide and their ability to produce mycotoxins represents a hazard for human health. Their correct and rapid identification is thus crucial to manage food safety. In recent years, MALDI-TOF emerged as a rapid and reliable tool for fungi identification and was applied to typing of bacteria and yeasts, but few studies focused on filamentous fungal species complex differentiation and typing. Therefore, the aim of this study was to evaluate the use of MALDI-TOF to identify species of the Aspergillus section Flavi, and to differentiate Penicillium roqueforti isolates from three distinct genetic populations. Spectra were acquired from 23 Aspergillus species and integrated into a database for which cross-validation led to more than 99% of correctly attributed spectra. For P. roqueforti, spectra were acquired from 63 strains and a two-step calibration procedure was applied before database construction. Cross-validation and external validation respectively led to 94% and 95% of spectra attributed to the right population. Results obtained here suggested very good agreement between spectral and genetic data analysis for both Aspergillus species and P. roqueforti, demonstrating MALDI-TOF applicability as a fast and easy alternative to molecular techniques for species complex differentiation and strain typing of filamentous fungi.

An update on the routine application of MALDI-TOF MS in clinical microbiology.

Introduction: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has entered clinical diagnostics and is today a generally accepted and integral part of the workflow for microbial identification. MALDI-TOF MS identification systems received approval from national and international institutions, such as the USA-FDA, and are continuously improved and adopted to other fields like veterinary and industrial microbiology. The question is whether MALDI-TOF MS also has the potential to replace other conventional and molecular techniques operated in routine diagnostic laboratories. Areas covered: We give an overview of new advancements of mass spectral analysis in the context of microbial diagnostics. In particular, the expansion of databases to increase the range of readily identifiable bacteria and fungi, the refined discrimination of species complexes, subspecies, and types, the testing for antibiotic resistance or susceptibility, progress in sample preparation including automation, and applications of other mass spectrometry techniques are discussed. Expert opinion: Although many new approaches of MALDI-TOF MS are still in the stage of proof of principle, it is expectable that MALDI-TOF MS will expand its role in the clinical microbiology laboratory of the future. New databases, instruments and analytical software modules will continue to be developed to further improve diagnostic efficacy.

Development and application of MALDI-TOF MS for identification of food spoilage fungi.

Filamentous fungi are frequently involved in food spoilage and cause important food losses and substantial economic damage. Their rapid and accurate identification is a key step to better manage food safety and quality. In recent years, MALDI-TOF MS has emerged as a powerful tool to identify microorganisms and has successfully been applied to the identification of filamentous fungi especially in the clinical context. The aim of this study was to implement a spectral database representative of food spoilage molds. To this end, after application of a standardized extraction protocol, 6477 spectra were acquired from 618 fungal strains belonging to 136 species and integrated in the VITEK MS database. The performances of this database were then evaluated by cross-validation and ∼95% of correct identification to the species level was achieved, independently of the cultivation medium and incubation time. The database was also challenged with external isolates belonging to 52 species claimed in the database and 90% were correctly identified to the species level. To our best knowledge, this is the most comprehensive database of food-relevant filamentous fungi developed to date. This study demonstrates that MALDI-TOF MS could be an alternative to conventional techniques for the rapid and reliable identification of spoilage fungi in food and industrial environments.

Comparison of Vitek MS and MALDI Biotyper for Identification of Actinomycetaceae of Clinical Importance.

The Vitek MS in vitro diagnostic (IVD) and MALDI Biotyper IVD systems were evaluated for the identification of 158 strains of Actinomycetaceae. Correct species-level identification rates of 60.7% and 58.2% were obtained with the Vitek MS system after direct deposit and with the MALDI Biotyper system after on-plate formic acid treatment, respectively.

A Simple and Safe Protocol for Preparing Brucella Samples for Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Analysis.

We describe a simple protocol to inactivate the biosafety level 3 (BSL3) pathogens Brucella prior to their analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry. This method is also effective for several other bacterial pathogens and allows storage, and eventually shipping, of inactivated samples; therefore, it might be routinely applied to unidentified bacteria, for the safety of laboratory workers.

Identification and typing of the emerging pathogen Candida auris by matrix-assisted laser desorption ionisation time of flight mass spectrometry.

Candida auris is an emerging antifungal resistant yeast species causing nosocomial and invasive infections, emphasising the need of improved diagnostics and epidemiological typing methods. We show that MALDI-TOF VITEK-MS followed by amplified length polymorphisms allows for accurate species identification and subsequent epidemiological characterisation of strains encountered during potential outbreaks.

Microbial typing by matrix-assisted laser desorption ionization-time of flight mass spectrometry: do we need guidance for data interpretation?

The integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology has revolutionized species identification of bacteria, yeasts, and molds. However, beyond straightforward identification, the method has also been suggested to have the potential for subspecies-level or even type-level epidemiological analyses. This minireview explores MALDI-TOF MS-based typing, which has already been performed on many clinically relevant species. We discuss the limits of the method's resolution and we suggest interpretative criteria allowing valid comparison of strain-specific data. We conclude that guidelines for MALDI-TOF MS-based typing can be developed along the same lines as those used for the interpretation of data from pulsed-field gel electrophoresis (PFGE).

Automatic identification of mixed bacterial species fingerprints in a MALDI-TOF mass-spectrum.

MOTIVATION: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has been broadly adopted by routine clinical microbiology laboratories for bacterial species identification. An isolated colony of the targeted microorganism is the single prerequisite. Currently, MS-based microbial identification directly from clinical specimens can not be routinely performed, as it raises two main challenges: (i) the nature of the sample itself may increase the level of technical variability and bring heterogeneity with respect to the reference database and (ii) the possibility of encountering polymicrobial samples that will yield a 'mixed' MS fingerprint. In this article, we introduce a new method to infer the composition of polymicrobial samples on the basis of a single mass spectrum. Our approach relies on a penalized non-negative linear regression framework making use of species-specific prototypes, which can be derived directly from the routine reference database of pure spectra. RESULTS: A large spectral dataset obtained from in vitro mono- and bi-microbial samples allowed us to evaluate the performance of the method in a comprehensive way. Provided that the reference matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprints were sufficiently distinct for the individual species, the method automatically predicted which bacterial species were present in the sample. Only few samples (5.3%) were misidentified, and bi-microbial samples were correctly identified in up to 61.2% of the cases. This method could be used in routine clinical microbiology practice.

Progress in proteomics for clinical microbiology: MALDI-TOF MS for microbial species identification and more.

Although classical proteomic approaches are still used regularly in routine clinical diagnostic procedures, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) MS has recently moved into diagnostic microbiology laboratories. MALDI-TOF MS is currently replacing phenotypic microbial identification. Many laboratories now use MALDI-TOF MS for its high efficiency, both from a diagnostic and a cost-per-analysis point of view. The US FDA has now cleared two of the commercially available systems for in vitro diagnostics. This will further spark development of MS applications in antimicrobial susceptibility testing and epidemiology. This review summarizes the state of affairs of MALDI-TOF MS in clinical microbiology; however, this is an active field of research subject to rapid evolution. We emphasize assessment of the clinical relevance and studies focusing on data obtained through comparative analyses of different MALDI-TOF MS instrumentation and multicenter validation studies. The future of MALDI-TOF MS, including antimicrobial susceptibility testing and epidemiological typing, is also highlighted.

Rapid inactivation of Mycobacterium and nocardia species before identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the Vitek MS system for rapid and accurate identification of dermatophytes on solid cultures.

The objective of this research was to extend the Vitek MS fungal knowledge base version 2.0.0 to allow the robust identification of clinically relevant dermatophytes, using a variety of strains, incubation times, and growth conditions. First, we established a quick and reliable method for sample preparation to obtain a reliable and reproducible identification independently of the growth conditions. The Vitek MS V2.0.0 fungal knowledge base was then expanded using 134 well-characterized strains belonging to 17 species in the genera Epidermophyton, Microsporum, and Trichophyton. Cluster analysis based on mass spectrum similarity indicated good species discrimination independently of the culture conditions. We achieved a good separation of the subpopulations of the Trichophyton anamorph of Arthroderma benhamiae and of anthropophilic and zoophilic strains of Trichophyton interdigitale. Overall, the 1,130 mass spectra obtained for dermatophytes gave an estimated identification performance of 98.4%. The expanded fungal knowledge base was then validated using 131 clinical isolates of dermatophytes belonging to 13 taxa. For 8 taxa all strains were correctly identified, and for 3 the rate of successful identification was >90%; 75% (6/8) of the M. gypseum strains were correctly identified, whereas only 47% (18/38) of the African T. rubrum population (also called T. soudanense) were recognized accurately, with a large quantity of strains misidentified as T. violaceum, demonstrating the close relationship of these two taxa. The method of sample preparation was fast and efficient and the expanded Vitek MS fungal knowledge base reliable and robust, allowing reproducible dermatophyte identifications in the routine laboratory.

Conformation change in a self-recognizing autotransporter modulates bacterial cell-cell interaction.

Bacteria mostly live as multicellular communities, although they are unicellular organisms, yet the mechanisms that tie individual bacteria together are often poorly understood. The adhesin involved in diffuse adherence (AIDA-I) is an adhesin of diarrheagenic Escherichia coli strains. AIDA-I also mediates bacterial auto-aggregation and biofilm formation and thus could be important for the organization of communities of pathogens. Using purified protein and whole bacteria, we provide direct evidence that AIDA-I promotes auto-aggregation by interacting with itself. Using various biophysical and biochemical techniques, we observed a conformational change in the protein during AIDA-AIDA interactions, strengthening the notion that this is a highly specific interaction. The self-association of AIDA-I is of high affinity but can be modulated by sodium chloride. We observe that a bile salt, sodium deoxycholate, also prevents AIDA-I oligomerization and bacterial auto-aggregation. Thus, we propose that AIDA-I, and most likely other similar autotransporters such as antigen 43 (Ag43) and TibA, organize bacterial communities of pathogens through a self-recognition mechanism that is sensitive to the environment. This could permit bacteria to switch between multicellular and unicellular lifestyles to complete their infection.

Nomenclatural issues concerning cultured yeasts and other fungi: why it is important to avoid unneeded name changes.

The unambiguous application of fungal names is important to communicate scientific findings. Names are critical for (clinical) diagnostics, legal compliance, and regulatory controls, such as biosafety, food security, quarantine regulations, and industrial applications. Consequently, the stability of the taxonomic system and the traceability of nomenclatural changes is crucial for a broad range of users and taxonomists. The unambiguous application of names is assured by the preservation of nomenclatural history and the physical organisms representing a name. Fungi are extremely diverse in terms of ecology, lifestyle, and methods of study. Predominantly unicellular fungi known as yeasts are usually investigated as living cultures. Methods to characterize yeasts include physiological (growth) tests and experiments to induce a sexual morph; both methods require viable cultures. Thus, the preservation and availability of viable reference cultures are important, and cultures representing reference material are cited in species descriptions. Historical surveys revealed drawbacks and inconsistencies between past practices and modern requirements as stated in the International Code of Nomenclature for Algae, Fungi, and Plants (ICNafp). Improper typification of yeasts is a common problem, resulting in a large number invalid yeast species names. With this opinion letter, we address the problem that culturable microorganisms, notably some fungi and algae, require specific provisions under the ICNafp. We use yeasts as a prominent example of fungi known from cultures. But viable type material is important not only for yeasts, but also for other cultivable Fungi that are characterized by particular morphological structures (a specific type of spores), growth properties, and secondary metabolites. We summarize potential proposals which, in our opinion, will improve the stability of fungal names, in particular by protecting those names for which the reference material can be traced back to the original isolate.

A MALDI-TOF MS database with broad genus coverage for species-level identification of Brucella.

Brucella are highly infectious bacterial pathogens responsible for a severely debilitating zoonosis called brucellosis. Half of the human population worldwide is considered to live at risk of exposure, mostly in the poorest rural areas of the world. Prompt diagnosis of brucellosis is essential to prevent complications and to control epidemiology outbreaks, but identification of Brucella isolates may be hampered by the lack of rapid and cost-effective methods. Nowadays, many clinical microbiology laboratories use Matrix-Assisted Laser Desorption Ionization-Time Of Flight mass spectrometry (MALDI-TOF MS) for routine identification. However, lack of reference spectra in the currently commercialized databases does not allow the identification of Brucella isolates. In this work, we constructed a Brucella MALDI-TOF MS reference database using VITEK MS. We generated 590 spectra from 84 different strains (including rare or atypical isolates) to cover this bacterial genus. We then applied a novel biomathematical approach to discriminate different species. This allowed accurate identification of Brucella isolates at the genus level with no misidentifications, in particular as the closely related and less pathogenic Ochrobactrum genus. The main zoonotic species (B. melitensis, B. abortus and B. suis) could also be identified at the species level with an accuracy of 100%, 92.9% and 100%, respectively. This MALDI-TOF reference database will be the first Brucella database validated for diagnostic and accessible to all VITEK MS users in routine. This will improve the diagnosis and control of brucellosis by allowing a rapid identification of these pathogens.

O-linked glycosylation ensures the normal conformation of the autotransporter adhesin involved in diffuse adherence.

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. In this study, we identified several peptides of AIDA-I modified by the addition of heptoses by use of mass spectrometry and N-terminal sequencing of proteolytic fragments of AIDA-I. One threonine and 15 serine residues were identified as bearing heptoses, thus demonstrating for the first time that AIDA-I is O-glycosylated. We observed that unglycosylated AIDA-I is expressed in smaller amounts than its glycosylated counterpart and shows extensive signs of degradation upon heat extraction. We also observed that unglycosylated AIDA-I is more sensitive to proteases and induces important extracytoplasmic stress. Lastly, as was previously shown, we noted that glycosylation is required for AIDA-I to mediate adhesion to cultured epithelial cells, but purified mature AIDA-I fused to GST was found to bind in vitro to cells whether or not it was glycosylated. Taken together, our results suggest that glycosylation is required to ensure a normal conformation of AIDA-I and may be only indirectly necessary for its cell-binding function.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the identification of microbial species in clinical microbiology laboratories. MALDI-TOF-MS has swiftly become the new gold-standard method owing to its key advantages of simplicity and robustness. However, as with all new methods, adoption of the MALDI-TOF MS approach is still not widespread. Optimal sample preparation has not yet been achieved for several applications, and there are continuing discussions on the need for improved database quality and the inclusion of additional microbial species. New applications such as in the field of antimicrobial susceptibility testing have been proposed but not yet translated to the level of ease and reproducibility that one should expect in routine diagnostic systems. Finally, during routine identification testing, unexpected results are regularly obtained, and the best methods for transmitting these results into clinical care are still evolving. We here discuss the success of MALDI-TOF MS in clinical microbiology and highlight fields of application that are still amenable to improvement.

Factors impacting time to acceptance and publication for peer-reviewed publications.

OBJECTIVE: Timely publication of data is important for the medical community and provides a valuable contribution to data disclosure. The objective of this study was to identify and evaluate times to acceptance and publication for peer-reviewed manuscripts, reviews, and letters to the editor. RESEARCH DESIGN AND METHODS: Key publication metrics for published manuscripts, reviews, and letters to the editor were identified by eight Amgen publications professionals. Data for publications submitted between 1 January 2013 and 1 November 2015 were extracted from a proprietary internal publication-tracking database. Variables included department initiating the study, publication type, number of submissions per publication, and the total number of weeks from first submission to acceptance, online publication, and final publication. RESULTS: A total of 337 publications were identified, of which 300 (89%) were manuscripts. Time from submission to acceptance and publication was generally similar between clinical and real-world evidence (e.g. observational and health economics studies) publications. Median (range) time from first submission to acceptance was 23.4 (0.2-226.2) weeks. Median (range) time from first submission to online (early-release) publication was 29.7 (2.4-162.6) weeks. Median (range) time from first submission to final (print) publication was 36.2 (2.8-230.8) weeks. Time from first submission to acceptance, online publication, and final publication increased accordingly with number of submissions required for acceptance, with similar times noted between each subsequent submission. CONCLUSIONS: Analysis of a single-company publication database showed that the median time for manuscripts to be fully published after initial submission was 36.2 weeks, and time to publication increased accordingly with the number of submissions. Causes for multiple submissions and time from clinical trial completion to first submission were not assessed; these were limitations of the study. Nonetheless, publication planners should consider these results when evaluating timelines and identifying potential journals early in the publication planning process.

Routine identification of Nocardia species by MALDI-TOF mass spectrometry.

We here show adequate species identification for bacterial isolates of the genus Nocardia spp. through VITEK mass spectrometry. Application of a specific sample preparation method in combination with a robust matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) database leads to 94% accurate identification to the species level on a set of 164 isolates. The possibility to identify Nocardia spp. using MALDI-TOF MS will be available in the next release of VITEK MS update (IVD Version 3.0).