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: The new triazoles and caspofungin have excellent in vitro activity against a very large collection of recent clinical isolates of Aspergillus spp.
Abstract: We examined the in vitro activity of caspofungin, posaconazole, voriconazole, ravuconazole, itraconazole, and amphotericin B against 448 recent clinical mold isolates. The endpoint for reading caspofungin was the minimum effective concentration (MEC). Among the triazoles, posaconazole was most active, inhibiting 95% of isolates at ≤1 μg/ml, followed by ravuconazole (91%), voriconazole (90%), and itraconazole (79%). Caspofungin and amphotericin B inhibited 93% and 89% of isolates at ≤1 μg/ml, respectively, with caspofungin demonstrating an MEC 90 of 0.12 μg/ml. All three new triazoles and caspofungin inhibited >95% of Aspergillus spp. at ≤1 μg/ml compared to 83% for itraconazole and 91% for amphotericin B. Amphotericin B inhibited only 38% of Aspergillus terreus isolates at ≤1 μg/ml, whereas the three new triazoles and caspofungin inhibited all A. terreus at ≤0.5 μg/ml. The new triazoles and caspofungin have excellent in vitro activity against a very large collection of recent clinical isolates of Aspergillus spp., and some in vitro activity against selected other filamentous fungi.
347 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...solani complex are significantly less susceptible to voriconazole and posaconazole compared to the other species [61,62]....
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TL;DR: The variability in species distribution in these centers underscores the importance of local epidemiology in guiding the selection of antifungal therapy and expands the current knowledge of the epidemiology and outcomes of invasive candidiasis caused by non-albicans species of Candida in North America.
Abstract: This analysis describes the epidemiology and outcomes of invasive candidiasis caused by non-albicans species of Candida in patients enrolled in the Prospective Antifungal Therapy Alliance (PATH Alliance) registry from 2004 to 2008 A total of 2,496 patients with non-albicans species of Candida isolates were identified The identified species were C glabrata (464%), C parapsilosis (247%), C tropicalis (139%), C krusei (55%), C lusitaniae (16%), C dubliniensis (15%) and C guilliermondii (04%); 111 infections involved two or more species of Candida (44%) Non-albicans species accounted for more than 50% of all cases of invasive candidiasis in 15 of the 24 sites (625%) that contributed more than one case to the survey Among solid organ transplant recipients, patients with non-transplant surgery, and patients with solid tumors, the most prevalent non-albicans species was C glabrata at 637%, 480%, and 538%, respectively In 1,883 patients receiving antifungal therapy on day 3, fluconazole (305%) and echinocandins (475%) were the most frequently administered monotherapies Among the 15 reported species, 90-day survival was highest for patients infected with either C parapsilosis (707%) or C lusitaniae (745%) and lowest for patients infected with an unknown species (467%) or two or more species (532%) In conclusion, this study expands the current knowledge of the epidemiology and outcomes of invasive candidiasis caused by non-albicans species of Candida in North America The variability in species distribution in these centers underscores the importance of local epidemiology in guiding the selection of antifungal therapy
338 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...Among the most significant emerging pathogens are yeast such as Candida glabrata [3,4], Trichosporon spp....
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TL;DR: Signs of marked shifts in candidemia epidemiology over the past 2 decades are described, with adults aged ≥65 years replaced infants as the highest incidence group and adjusted incidence has declined significantly in infants.
Abstract: Bloodstream infections (BSIs) caused by Candida species, also known as candidemia, are an important public health problem in the United States. Candida species are among the most common causes of BSIs and are associated with high morbidity and mortality, as well as increases in hospital cost and length of stay [1–5].
Although most reports that describe the epidemiology of candidemia are from individual institutions or among specific patient groups, few reports in the United States have described the epidemiology of candidemia at a population level. Population-based data are important for describing infections in whole populations and across the entire spectrum of healthcare settings, and can be used to determine group-specific incidence rates to monitor and compare rates of infection over time. Previous population-based surveillance performed by the Centers for Disease Control and Prevention (CDC) and partners in Atlanta, Georgia (1992–1993), and Baltimore, Maryland (1998–2000), described incidence rates of 8.7 per 100 000 population in Atlanta and 24 per 100 000 population in Baltimore [6, 7]. During both surveillance periods, drug resistance to fluconazole was low: 3% of isolates were resistant in 1992, and 3.7% were resistant in 1998 [6–8].
Over the past decade, Candida species with reduced fluconazole susceptibility, such as Candida glabrata, have become more prevalent in some patient populations [8–12]. The newest class of antifungal medications, the echinocandins, are considered first-line empiric therapy for candidemia, and there are reports of echinocandin-resistant invasive infection [13, 14].
To evaluate changes in the epidemiology and antifungal drug resistance of candidemia, we conducted population-based prospective surveillance in metropolitan Atlanta, Georgia, and Baltimore City and County, Maryland, 2 areas where prior surveillance had been conducted.
328 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...Among the most significant emerging pathogens are yeast such as Candida glabrata [3,4], Trichosporon spp....
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TL;DR: The close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.
Abstract: Thirteen Candida glabrata strains harboring a range of mutations in hot spot regions of FKS1 and FKS2 were studied. The mutations were linked to an echinocandin reduced susceptibility phenotype. Sequence alignments showed that 11 out of the 13 mutants harbored a mutation in FKS1 or FKS2 not previously implicated in echinocandin reduced susceptibility in C. glabrata. A detailed kinetic characterization demonstrated that amino acid substitutions in Fks1p and Fks2p reduced drug sensitivity in mutant 1,3--D-glucan synthase by 2 to 3 log orders relative to that in wild-type enzyme. These mutations were also found to reduce the catalytic efficiency of the enzyme (Vmax) and to influence the relative expression of FKS genes. In view of the association of FKS mutations and reduced susceptibility of 1,3--D-glucan synthase, an evaluation of the new CLSI echinocandin susceptibility breakpoint was conducted. Only 3 of 13 resistant fks mutants (23%) were considered anidulafungin or micafungin nonsusceptible (MIC > 2 g/ml) by this criterion. In contrast, most fks mutants (92%) exceeded a MIC of >2 g/ml with caspofungin. However, when MIC determinations were performed in the presence of 50% serum, all C. glabrata fks mutants showed MICs of >2 g/ml for the three echinocandin drugs. As has been observed with Candida albicans, the kinetic inhibition parameter 50% inhibitory concentration may be a better predictor of FKS-mediated resistance. Finally, the close association between FKS1/FKS2 hot spot mutations provides a basis for understanding echinocandin resistance in C. glabrata.
278 citations
TL;DR: In this paper, a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification.
Abstract: Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was compared to phenotypic testing for yeast identification MALDI-TOF mass spectrometry yielded 963% and 845% accurate species level identifications (spectral scores, ≥ 18) for 138 common and 103 archived strains of yeast MALDI-TOF mass spectrometry is accurate, rapid (51 min of hands-on time/identification), and cost-effective ($050/sample) for yeast identification in the clinical laboratory
267 citations
"Pushing the Limits of MALDI-TOF Mas..." refers background in this paper
...determined that MALDI-TOF MS operating costs were lower than those of most conventional and molecular testing systems in terms of reagent costs and hands-on time required for sample processing [46]....
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