Pushing the Limits of MALDI-TOF Mass Spectrometry: Beyond Fungal Species Identification
16 Oct 2015-Journal of Fungi (Multidisciplinary Digital Publishing Institute)-Vol. 1, Iss: 3, pp 367-383
TL;DR: In this article, a review of the use of MALDI-TOF in the clinical mycology laboratory is presented, focusing on present and future applications of this versatile analytical tool in the field of bioinformatics.
Abstract: Matrix assisted laser desorption ionization time of flight (MALDI-TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory.
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TL;DR: Current and future molecular technologies used for fungal identification, and some of the problems associated with development and implementation of these technologies in today’s clinical microbiology laboratories are discussed.
Abstract: Diagnosing fungal infections poses a number of unique problems, including a decline in expertise needed for identifying fungi, and a reduced number of instruments and assays specific for fungal identification compared to that of bacteria and viruses.These problems are exacerbated by the fact that patients with fungal infections are often immunosuppressed, which predisposes to infections from both commonly and rarely seen fungi. In this review, we discuss current and future molecular technologies used for fungal identification, and some of the problems associated with development and implementation of these technologies in today's clinical microbiology laboratories.
90 citations
TL;DR: This minireview aims to provide an overview of currently available online databases for the taxonomy and identification of human and animal-pathogenic fungi and calls for the establishment of a cloud-based dynamic data network platform.
Abstract: The increase in public online databases dedicated to fungal identification is noteworthy. This can be attributed to improved access to molecular approaches to characterize fungi, as well as to delineate species within specific fungal groups in the last 2 decades, leading to an ever-increasing complexity of taxonomic assortments and nomenclatural reassignments. Thus, well-curated fungal databases with substantial accurate sequence data play a pivotal role for further research and diagnostics in the field of mycology. This minireview aims to provide an overview of currently available online databases for the taxonomy and identification of human and animal-pathogenic fungi and calls for the establishment of a cloud-based dynamic data network platform.
39 citations
Cites background from "Pushing the Limits of MALDI-TOF Mas..."
...complex and requires access to validated purpose-built databases of reference spectra (6, 7)....
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TL;DR: New molecular-based approaches for detecting triazole resistance to Aspergillus, real-time polymerase chain reaction (PCR) to detect mutations to the Cyp51A protein, have been developed which are able to detect mostTriazole-resistant A. fumigatus strains in patients with invasive aspergillosis.
Abstract: The incidence of invasive aspergillosis has increased substantially over the past few decades, accompanied by a change in susceptibility patterns of Aspergillus fumigatus with increasing resistance observed against triazole antifungals, including voriconazole and isavuconazole, the most commonly used antifungal agents for the disease. Culture-based methods for determining triazole resistance are still the gold standard but are time consuming and lack sensitivity. We sought to provide an update on non-culture-based methods for detecting resistance patterns to Aspergillus. New molecular-based approaches for detecting triazole resistance to Aspergillus, real-time polymerase chain reaction (PCR) to detect mutations to the Cyp51A protein, have been developed which are able to detect most triazole-resistant A. fumigatus strains in patients with invasive aspergillosis. Over the last few years, a number of non-culture-based methods for molecular detection of Aspergillus triazole resistance have been developed that may overcome some of the limitations of culture. These molecular methods are therefore of high epidemiological and clinical relevance, mainly in immunocompromised patients with hematological malignancies, where culture has particularly limited sensitivity. These assays are now able to detect most triazole-resistant Aspergillus fumigatus strains. Given that resistance rates vary, clinical utility for these assays still depends on regional resistance patterns.
19 citations
TL;DR: Fast, accurate and inexpensive molecular mass determination and the possibility of automation make MALDI-TOF-MS a real alternative to conventional morphological and molecular methods for AMF identification.
Abstract: Arbuscular mycorrhizal fungi (AMF, Glomeromycota) are mutualistic symbionts associated with majority of land plants. These fungi play an important role in plant growth, but their taxonomic identification remains a challenge for academic research, culture collections and inoculum producers who need to certify their products. Identification of these fungi was traditionally performed based on their spore morphology. DNA sequence data have successfully been used to study the evolutionary relationships of AMF, develop molecular identification tools and assess their diversity in the environment. However, these methods require considerable expertise and are not well-adapted for “routine” quality control of culture collections and inoculum production. Here, we show that Matrix-Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry proteomic-based biotyping is a highly efficient approach for AMF identification. Nineteen isolates belonging to fourteen species, seven genera and five families were clearly differentiated by MALDI biotyping at the species level, and intraspecific differentiation was achieved for the majority. AMF identification by MALDI biotyping could be highly useful, not only for research but also in agricultural and environmental applications. Fast, accurate and inexpensive molecular mass determination and the possibility of automation make MALDI-TOF-MS a real alternative to conventional morphological and molecular methods for AMF identification.
19 citations
TL;DR: The role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods is discussed, and the value of custom-made reference spectra for MalDI biotyping is highlighted.
Abstract: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS; MALDI biotyping) has become a standard tool for the accurate, rapid, and economical identification of pathogens in the clinical diagnostics laboratory. The method is continuously being improved, and new applications for distinguishing strains, identifying metabolites or functional characteristics (e.g., antibiotic resistance), and detecting microbes directly in patient samples have been developed. Adopting these methods in other disciplines than clinical diagnostics, for example, in agriculture, food safety and quality testing, or ecology, will open up new opportunities for diagnostics and research. This review focuses on MALDI-TOF MS approaches for the identification of yeasts and filamentous fungi. In contrast to bacterial diagnostics, MALDI biotyping of fungi is more challenging and less established. We thus start by discussing the role of MALDI-TOF MS as a tool for species identification; in particular with respect to DNA-based identification methods. The review then highlights the value of custom-made reference spectra for MALDI biotyping and points out recent advancements of MALDI-TOF MS, mainly from the field of clinical diagnostics that may be adopted and used for fungal diagnostic challenges. The overview ends with a summary of MALDI-TOF MS studies of yeasts and filamentous fungi of agricultural relevance.
16 citations
Cites background from "Pushing the Limits of MALDI-TOF Mas..."
...Consequently, the majority of MALDI-TOF analyses of fungi so far have dealt with clinical isolates [33]....
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References
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TL;DR: In this paper, a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify Fusarium molds.
Abstract: The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp belonging to different species complexes can cause infection Conventional species identification in the clinical laboratory is time-consuming and prone to errors We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 828% of the identifications were correct to the species level This success rate was increased to 91% by lowering the cutoff for identification Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97% The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity
54 citations
15 Jun 2010
TL;DR: The SARAMIS system described herein sequentially extracts core stable mass ions from analyses of multiple individual strains of a particular species to yield a ‘SuperSpectrum’, a list of biomarkers that are weighted according to their specificity from family to (sub)species levels, which has been used successfully to identify microorganisms from diverse phylogenetic lines of bacteria and fungi.
Abstract: Immediately after the first MALDI-TOF instrument became available, microbiologists have been investigating its potential as a platform for high throughput identification of microorganisms. A remarkable finding very early in these investigations was that despite the dynamic nature of the bacterial cell, components of the mass spectral profile were sufficiently stable and remained unchanged in spite of changes in environmental parameters. Further, mass spectral patterns have been found to be taxon specific, consequently, numerous methods were reported that purported to provide an alternative to current identification systems. The SARAMIS system described herein sequentially extracts core stable mass ions from analyses of multiple individual strains of a particular species to yield a ‘SuperSpectrum’, a list of biomarkers that are weighted according to their specificity from family to (sub)species levels. This approach has been used successfully to identify microorganisms from diverse phylogenetic lines of bacteria and fungi with considerable success. The protocol described has evolved of over years of experimental work to yield a robust system that can be readily applied for microbiological identification in a clinical diagnostic laboratory. Identification of Microorganisms in Clinical Routine A crucial step in the epidemiology and the successful therapy of any infectious disease is the identification of the causative microbe. For more than a century, clinical microbiology has relied on the isolation of the suspected pathogen from various samples such as stools, throat swabs, blood, or urine on selective growth media and an identification procedure that is based on the metabolic capacities of the isolate. An array of carbohydrate fermentation and enzymatic reactions are tested that generally involve a colour change of an indicator when a particular substrate is catabolised. The profile of positive and negative reactions is assumed to be characteristic for a bacterial taxon and is consequently used for identification. Modern microbial identification systems are miniaturized, combining some tens of reactions into a single strip or card to allow for high throughput analysis. The major shortcomings of these systems are the need to incubate isolates for several hours to obtain pure cultures, and a required pre-selection of tests. Although this is still the most commonly used method in clinical diagnostic laboratories, microbiologists have been seeking alternative methods for the identification of pathogens for decades. A new era has dawned with the arrival of molecular methods such as the polymerase chain reaction (PCR) and nucleotide sequence analysis. In diagnostic and systematic microbiology today, analysis of genomic sequences is rapidly displacing biochemical tests for the provision of new characters for the circumscription of taxa. For example, a prerequisite for the description of a new species is the inclusion of the sequence of the 16S rRNA gene which now plays a pivotal role in microbial phylogeny. However, despite the widespread use of PCR and sequencing in all fields of microbiology, the technology is still lagging behind in clinical microbiology and is Shah, Gharbia, Encheva (Eds.) Mass spectrometry for microbial proteomics largely restricted to research applications. On the other hand, the high sensitivity and specificity of molecular methods make them indispensable in modern microbiological laboratories, as for example in the detection of methicillin resistant Staphylococcus aureus (MRSA) by real-time PCR assays, or the identification of atypical or very rare pathogens. Mass Spectrometry and Microbiology The application of chemical analyses (referred to as chemotaxonomy) for the identification and classification of microorganisms has been explored extensively prior to molecular analysis. These were based on the characterisation of polar (eg. phospholipids) and non polar lipids such as respiratory quinones (eg. ubiquinones and menaquinones) and long-chained cellular fatty acids . The structure of these lipids were challenging and ushered in a period of intense mass spectral analysis to characterise the vast array of lipids present in the microbial kingdom. While these methods provided characters at the genus and species levels, pyrrolysis mass spectrometry was introduced as a means of typing bacterial isolates . Such approaches were motivated by the need for a rapid method to identify pathogens in only a fraction of the time required for biochemical tests. However, because of the limitations of the technology at that time, mass spectral approaches were confined to the detection of organic molecules in a mass range up to 1,500 Da . Detection of larger molecules was hitherto only possible with techniques such as plasma desorption mass spectrometry . But this was about to change dramatically within a few years with the invention of Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and its success in many fields of life sciences has been phenomenal. The laser desorption and mass spectral analysis of large biomolecules was developed simultaneously by two research groups in Japan and Germany. While the group of Tanaka could successfully detect proteins up to m/z 100,000 Da by direct laser ionization , Karas and Hillenkamp 6 relied on a light absorbing matrix, and achieved similar results by this method. A matrix effect on the desorption rate was observed earlier for smaller molecules 7 and further studies quickly directed the search for matrix candidates to a few small organic molecules that are still the primarily used ones in MALDI-TOF MS applications today, viz. cinnamic acid derivatives 8 and 2,5dihydro benzoic acid . Accuracy and resolution could be significantly improved by the introduction of delayed ion extraction , which compensates for the variability in initial ion velocity . The sensitivity of MALDI-TOF MS for the detection of large proteins was rapidly increased to the femtomolar range 12 and further, aided by sophisticated handling procedures to the zeptomolar range . Within less then a decade, MALDI-TOF MS developed into a widely applied methodology in diverse fields of life sciences , promoted by a number of major advantages it had compared to other mass spectral technologies. These included the possibility to detect unfragmented large molecules, the speed with which a full scan over a wide mass range can be achieved, and the simplicity of sample preparation. For the analysis of whole cells and crude extracts, an outstanding advantage of MALDI compared to other ionization techniques such as electrospray ionization (ESI) or fast atom bombardment (FAB), is the fact that in MALDITOF MS predominantly singly charged ions are detected 15 which simplified the interpretation and treatment of mass spectral data considerably . Mass Spectral ‘Fingerprints’ of Whole Cells The possibility of introducing whole cells into a mass spectrometer and detecting biomolecules in a mass range extending to several kilodaltons was immediately recognised by microbiologists. Shortly after the first MALDI-TOF MS was commercially available (by Vestec and Kratos) the first reports on intact cell mass spectrometry of bacteria were published, some of which highlighted its potential for microbial diagnostics . Essentially these studies showed that mass spectra of whole cells of bacterial strains revealed patterns of mass signals that were reproducible and specific for strains or species. Because cells could be analysed after minimal processing and preparation, required only minute biomass, either as a cell suspension or cells placed directly on a target plate, the implications for diagnostic microbiology were immediately evident and numerous studies ensued. The mass ranges that were selected in early studies varied from m/z 500-2200 18 to m/z 200020000 . In the lower mass range, constituents of the cell wall are detected, and also, but to a lesser degree protein components . The lower mass range was also used for the typing of potentially toxic cyanobacteria 24 and proved to be suitable for the metabolic typing of sub-specific taxonomic units in natural populations . The possibility that mass fingerprints could be used to classify and eventually identify Kallow et al: MALDI-TOF MS for Microbial Identification 3 unknown microbial isolates was pursued in the following years by a number of groups. Most studies focused on the detection of proteins in mass ranges spanning from above m/z 2000 to below m/z 25,000. The identity of proteins detected at this time was not very clear because only a few genomic sequences of microorganisms were available. It was generally assumed that the proteins desorbed from whole cells, that were not subjected to typical mechanical or chemical lysis, were attached to the cell surface . However, studies on membrane-associated proteins of E. coli K12, revealed that the molecular mass of most of these proteins exceeded 20 kDa 26 and were likely to be intracellular proteins. Comparative MALDI-TOF mass spectral studies of isolated ribosomal subunits with those of whole cell mass spectra of E. coli K12 revealed that they shared a large number of mass signals that are commensurate with ribosomal proteins 27 (Figure 1). This large number of ionised ribosomal proteins is in accord with the high level of these proteins (ca. 30% of total proteins) of a cell in its exponential growth phase. Furthermore, studies on isolated ribosomes also revealed that many proteins were modified posttranslationally, and that the observed mass signals were in agreement with the general rules for methionine cleavage . Such studies emphasised that direct ‘translation’ of genomic data in mass patterns is not straightforward. Further microbial taxa have been studied in detail to reveal the identity of proteins detected by whole cell mass spectrometry. A high level of observed peaks in mass spect
53 citations
TL;DR: The low number of polymorphic AFLP bands obtained for C. parapsilosis isolates is in agreement with the limited sequence variability described for this species, but when band intensity was included in the analysis, geographical clustering was observed.
Abstract: Background: Candida parapsilosis is known to show limited genetic variability, despite different karyotypes and phenotypes have been described. To further investigate this aspect, a collection of 62 sensu strictu C. parapsilosis independent isolates from 4 geographic regions (Italy, n = 19; New Zealand, n = 15; Argentina, n = 14; and Hungary, n = 14) and different body sites (superficial and deep seated) were analysed for their genetic and phenotypic traits. Amplification fragment length polymorphism (AFLP) analysis was used to confirm species identification and to evaluate intraspecific genetic variability. Phenotypic characterisation included clinically relevant traits, such as drug susceptibility, in vitro biofilm formation and aspartyl protease secretion. Results: AFLP genotyping showed little variation among isolates, when the presence/absence of bands was considered. However, when AFLP profiles were compared by relative intensity for each fragment, a significant level of variation and geographical clustering was observed. All isolates were found to be susceptible to commonly used antifungals, although a reduced susceptibility to echinocandins was observed in all isolates. C. parapsilosis isolates from different geographic origins varied in the number of biofilm producers, with a higher prevalence of producers isolated in Hungary and Argentina. The frequency of secreted proteinase producers also varied in isolates obtained from different areas, with a higher number of proteinase producers found in Italy and New Zealand. Interestingly, biofilm production and proteinase secretion were negatively correlated. This finding could be explained by assuming that proteinase activity plays a role in detachment and release from a mature biofilm, via degradation of C. parapsilosis adhesins and/or extracellular matrix components, as observed for other microorganisms. Conclusions: The low number of polymorphic AFLP bands (18 out of 80) obtained for C. parapsilosis isolates is in agreement with the limited sequence variability described for this species. However, when band intensity was included in the analysis, geographical clustering was observed. Expression of virulence factors varied among strains isolated from different geographical regions, with biofilm and proteinase producers more frequently isolated from Hungary and Italy, respectively.
53 citations
TL;DR: This optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level.
Abstract: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) has been proved to be a powerful tool for the identification and characterization of microorganisms based on their surface peptide/protein pattern. Because of the complexity of microorganisms, there are no standardized protocols to acquire reproducible peptide/protein profiles for a broad range of microorganisms and for fungi in particular. Small variations during MALDI MS sample preparation affect the quality of mass spectra quite often. In this study, we were aiming to develop a sample preparation method for the analysis of colored, a quite often observed phenomenon, and mycotoxin-producing Fusarium conidia spores using MALDI–TOF MS. Different washing solvent systems for light- and deep-colored (from slightly orange to red-brown) conidia spores and connected sample deposition techniques were evaluated based on MS reproducibility and number and intensities of peaks. As a method of choice for generation of reproducible and characteristic MALDI–TOF mass spectra, the use of a washing process for colored Fusarium conidia spores with acetonitrile/0.5% formic acid (7/3) was found and subsequently combined with two-layer volume technique (spores/matrix (ferulic acid) solution was deposited onto a MALDI target, and after solvent evaporation, a second matrix layer was deposited). With the application of this sample preparation method, for deep-colored Fusarium species, 19 abundant molecular ions in the m/z range 2,000–10,000 were always detected with an S/N ratio of 3:1 or better. Finally this optimized sample preparation for the first time provided mass spectrometric fingerprints of strongly colored Fusarium conidia spores resulting in the possibility of differentiation of such spores at the species level.
49 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...Suppressing pigment formation by growing the fungus in liquid culture [49] or by performing pre-analytical washing steps [50] can help overcome this problem....
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...In addition, the choice of the preparation protocol, of the matrix and solvent, and of the pre-analytical steps can introduce a further degree of variation in number and identity of mass peaks obtained [50,51]....
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..., the conidial melanin pigment inhibited analyte ionization [50,53]....
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TL;DR: These results demonstrate that MALDI-TOF MS may be used to simultaneously determine the Candida species and classification as susceptible or resistant to triazole antifungals and improve the reproducibility of this assay.
Abstract: MALDI-TOF MS can rapidly identify microorganisms to the species level and may be able to detect antimicrobial resistance. We evaluated the ability of this technology to detect triazole resistance in Candida species.35 C. albicans, 35 C. glabrata, and 37 C. tropicalis strains were exposed to fluconazole, voriconazole, or posaconazole at two different concentrations plus a drug-free control: a midrange concentration (CLSI clinical breakpoint or epidemiologic cut-off value), and a high concentration (fluconazole 64 μg/ml, voriconazole & posaconazole 16 μg/ml). The MALDI-TOF MS spectra at these concentrations were used to create the individual composite correlation index (CCI) matrices for each isolate. When the CCI of the midrange/highest concentration was lower than that of the midrange/null concentration, the strain was classified as resistant. These results were then compared to the classifications for susceptible or resistant obtained by measuring the MICs according to the CLSI M27-A3 antifungal susceptibility testing (AFST) method.The MALDI-TOF MS assay was able to classify triazole susceptibility against all strains. Overall, essential agreement between MALDI-TOF MS and AFST varied between 54% and 97%, and was highest for posaconazole against C. glabrata. The reproducibility of the MALDI-TOF MS assay varied between 54.3 and 82.9% and was best for fluconazole against C. albicans and posaconazole against C. glabrata. Reproducibility was also higher for C. glabrata isolates compared to C. albicans and C. tropicalis.These results demonstrate that MALDI-TOF MS may be used to simultaneously determine the Candida species and classification as susceptible or resistant to triazole antifungals. Further studies are needed to refine the methodology and improve the reproducibility of this assay.
46 citations
"Pushing the Limits of MALDI-TOF Mas..." refers methods in this paper
...tropicalis strains, thus indicating that further analysis and protocol optimization are still required prior to applying this technique as an alternative to conventional antifungal susceptibility testing [85]....
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