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: Matrix‐assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF MS) was in this study found to be a powerful tool in fast diagnosis of Gram‐negative bacteria and fungi and to a lesser degree of Gram positives.
Abstract: Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a promising and fast method for identifying fungi and bacteria directly from positive blood cultures. Various pre-treatment methods for MALDI-TOF MS identification have been reported for this purpose. In-house results for identification of bacterial colonies by MALDI-TOF MS using a cut-off score of 1.5 did not reduce the diagnostic accuracy compared with the recommended cut-off score of 1.8. A 3-month consecutive study of positive blood cultures was carried out in our laboratory to evaluate whether the Sepsityper™ Kit (Bruker Daltonics) with Biotyper 2.0 software could be used as a fast diagnostic tool for bacteria and fungi and whether a 1.5 cut-off score could improve species identification compared with the recommended score of 1.8. Two hundred and fifty-six positive blood vials from 210 patients and 19 blood vials spiked with fungi were examined. Using the cut-off score of 1.8, 81% Gram-negative bacteria were identified to the species level compared to 84% using a cut-off score of 1.5. For Gram-positive bacteria 44% were identified to the species level with a cut-off of 1.8 compared to 55% with the value of 1.5. The overall identification rate was 63% (cut-off 1.5) and 54% (cut-off 1.8). Seventy-seven per cent of fungal species were identified with both log scores. MALDI-TOF MS was in this study found to be a powerful tool in fast diagnosis of Gram-negative bacteria and fungi and to a lesser degree of Gram positives. Using 1.5 as cut-off score increased the diagnosis for both Gram-positives and -negatives bacteria.
46 citations
TL;DR: Modifications of the preanalytical steps of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification of yeasts appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories.
Abstract: We report here that modifications of the preanalytical steps of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification of yeasts, with regard to the original protocol provided by the manufacturers, appear to be efficient for the reliable routine identification of clinical yeast isolates in medical laboratories. Indeed, when one colony was sampled instead of five and the protein extraction protocol was modified, the performance of MALDI-TOF MS was superior to that of the API ID 32C method (discrepancies were confirmed by using molecular identification), allowing the correct identification of 94% of the 335 clinical isolates prospectively tested. We then demonstrated that the time for which the primary cultures were preincubated on CHROMagar did not impact the identification of yeasts by MALDI-TOF MS, since 95.1 and 96.2% of the 183 clinical yeast isolates prospectively tested were correctly identified after 48 and 72 h of preincubation, respectively.
45 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background or methods in this paper
...No significant differences were found when using 24, 48, or 72 h cultures [35]....
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...In the “on-target-lysis” technique [35,36], the colony smear is overlaid with 0....
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...Other studies focused on the quantity and the age of cultured yeast colonies; Marklein and colleagues reported that it was necessary to have five colonies isolated from CHROMAgar (CHROMagar Microbiology, Paris, France) to provide an accurate identification of yeast isolates [19], while in a successive publication it was determined that an accurate identification can be also achieved by starting from a single colony [35]....
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TL;DR: Vitek MS showed high rate of accuracy for the identification of GNB in urine specimens (≥105 CFU/mL) and could become a rapid and accurate diagnostic method for urinary tract infection caused by GNB.
Abstract: Background We evaluated the coincidence rate between Vitek MS system (bioMerieux, France) and Vitek 2 in identifying uropathogens directly from urine specimens. Methods Urine specimens submitted to our microbiology laboratory between July and September 2013 for Gram staining and bacterial culture were analyzed. Bacterial identification was performed by using the conventional method. Urine specimens showing a single morphotype by Gram staining were processed by culturing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Of 2,370 urine specimens, 251 showed a single morphotype on Gram staining, and among them, 202 were available for MALDI-TOF MS. Results In these 202 specimens, colony growth was observed in 189 specimens, and 145 specimens had significant growth of single-colony morphotype in culture. One hundred and ten (75.9%) of them had colony counts of ≥10(5) colony-forming units (CFU)/mL and included 71 enteric gram-negative bacteria (GNB), 5 glucose-non-fermenting GNB, 9 gram-positive cocci (GPC), and 25 yeasts. Furthermore, 70 (98.6%), 3 (60.0%), 4 (44.4%), and 5 (20.0%), respectively, of these were correctly identified by Vitek MS. Thirty-one specimens (21.4%; 11 GNB, 7 GPC, 12 yeasts, and 1 gram-positive bacillus) had colony counts of 10(4)-10(5) CFU/mL. Four specimens (2.8%) yielded colony counts of 10(3)-10(4) CFU/mL. Conclusions Vitek MS showed high rate of accuracy for the identification of GNB in urine specimens (≥10(5) CFU/mL). This could become a rapid and accurate diagnostic method for urinary tract infection caused by GNB. However, for the identification of GPC and yeasts, further studies on appropriate pre-treatment are warranted.
42 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...MALDI-TOF MS and conventional biochemical testing in the identification of the most relevant uropathogens directly from urine specimens, including specimens with yeast [78]....
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...In fact, a low identification rate (20%) was observed for yeast, with the Vitek MS showing superior ability of identification of yeasts grown on agar [78]....
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TL;DR: Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping, which supports for cost-effective screening of isolates during outbreak situations.
Abstract: Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.
38 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...The possibility of identifying intra-species clones by MALDI-TOF has been demonstrated for several clinically relevant bacteria, such as methicillin-resistant Staphylococcus aureus strains [26], Haemophilus influenzae Type b isolates [27] or Clostridium difficile [28]....
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TL;DR: While MALDI-TOF MS was found to be a reliable method for species-level identification, further studies are required to assess its value as a fungal typing tool.
Abstract: In this study we compare the capability of amplification fragment-length polymorphism (AFLP) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify and subtype isolates of members of the Candida parapsilosis complex (C. parapsilosis, C. orthopsilosis, C. metapsilosis) and Lodderomyces elongisporus, which cannot be differentiated with biochemical methods. Both techniques correctly identified all isolates included in this study and clustered isolates within the different species. DNA-based and mass spectrum-based dendrograms yielded similar outcomes with regard to phylogenetic distance within C. orthopsilosis and C. parapsilosis species. However, a different clustering was obtained for C. metapsilosis for which AFLP was highly effective in differentiating. While MALDI-TOF MS was found to be a reliable method for species-level identification, further studies are required to assess its value as a fungal typing tool.
37 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background or result in this paper
...It is plausible that the discrepancies observed in strain relatedness obtained with the two techniques reflect the different nature of the approaches used [30]....
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...MALDI-TOF MS spectra from additional reference strains, provided correct identification for all Candida parapsilosis species complex isolates included in the study [30]....
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...In our experience, we found that the original Bruker Daltonics BioTyper library database, encompassing quality-controlled entries for more than 3900 microorganisms, was unable to provide acceptable scores when challenged with Candida metapsilosis and Candida orthopsilosis identification [30]....
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...In fact, several reports indicate poor identification of phenotypically indistinguishable species due to the paucity of representative spectra in the database [30,31] or to the need to modify score thresholds for validated identification [32,33]....
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