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

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.

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

2020 ◽  
Vol 59 (1) ◽  
pp. 81-86
Author(s):  
Yue Sun ◽  
Jian Guo ◽  
Rong Chen ◽  
Liang Hu ◽  
Qihang Xia ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Filamentous Fungi ◽  
Reference Method ◽  
Species Level ◽  
Its Sequencing ◽  
Maldi Tof Ms ◽  
Genus Level ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

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.

scholarly journals 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

2016 ◽  
Vol 10 (1) ◽  
pp. 202-208 ◽  
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.

scholarly journals A Moldy Application of MALDI: MALDI-ToF Mass Spectrometry for Fungal Identification

2019 ◽  
Vol 5 (1) ◽  
pp. 4 ◽  
Author(s):  
Robin Patel
Keyword(s):  
Mass Spectrometry ◽  
Filamentous Fungi ◽  
Bacterial Identification ◽  
Ionization Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Fungal Identification ◽  
Routine Identification ◽  
Tof Ms

As a result of its being inexpensive, easy to perform, fast and accurate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) is quickly becoming the standard means of bacterial identification from cultures in clinical microbiology laboratories. Its adoption for routine identification of yeasts and even dimorphic and filamentous fungi in cultures, while slower, is now being realized, with many of the same benefits as have been recognized on the bacterial side. In this review, the use of MALDI-ToF MS for identification of yeasts, and dimorphic and filamentous fungi grown in culture will be reviewed, with strengths and limitations addressed.

scholarly journals Multicenter Evaluation of Rapid BACpro® II for the Accurate Identification of Microorganisms Directly from Blood Cultures Using MALDI-TOF MS

Diagnostics ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 2251
Author(s):  
Marina Oviaño ◽  
André Ingebretsen ◽  
Anne K. Steffensen ◽  
Antony Croxatto ◽  
Guy Prod’hom ◽  
...  
Keyword(s):  
Blood Cultures ◽  
Species Level ◽  
Correct Identification ◽  
Gram Positive ◽  
Gram Negative ◽  
Maldi Tof Ms ◽  
Gram Positive Bacteria ◽  
Maldi Tof ◽  
Level Identification ◽  
Tof Ms

The identification of microorganisms directly from blood cultures using MALDI-TOF MS has been shown to be the most impacting application of this methodology. In this study, a novel commercial method was evaluated in four clinical microbiology laboratories. Positive blood culture samples (n = 801) were processed using a rapid BACpro® II kit and then compared with the routine gold standard. A subset of monomicrobial BCs (n = 560) were analyzed in parallel with a Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) and compared with the rapid BACpro® II kit. In addition, this kit was also compared with two different in-house methods. Overall, 80.0% of the monomicrobial isolates (609/761; 95% CI 71.5–88.5) were correctly identified by the rapid BACpro® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper® Kit showed that the rapid BACpro® II kit generated higher rates of correct species-level identification for all categories (p > 0.0001), except for yeasts identified with score values > 1.7. It also proved superior to the ammonium chloride method (p > 0.0001), but the differential centrifugation method allowed for higher rates of correct identification for Gram negative bacteria (p > 0.1). The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods.

scholarly journals Evaluation of the rapidBACpro® II kit for the rapid identification of microorganisms directly from blood cultures using MALDI-TOF MS

2021 ◽  
Author(s):  
Marina Oviaño ◽  
André Ingebretsen ◽  
Anne K Steffensen ◽  
Antony Croxatto ◽  
Guy Prod’hom ◽  
...  
Keyword(s):  
Blood Cultures ◽  
Species Level ◽  
Correct Identification ◽  
Gram Positive ◽  
Gram Negative ◽  
Maldi Tof Ms ◽  
Gram Positive Bacteria ◽  
Maldi Tof ◽  
Level Identification ◽  
Tof Ms

AbstractObjectivesIdentification of microorganisms directly from blood cultures (BCs) using MALDI-TOF MS has shown to be the application with most impact in this methodology. In this study, a novel commercial method, the rapidBACpro® II, was evaluated in four clinical microbiology laboratories.MethodsPositive blood culture samples (n=801) were processed using the rapidBACpro® II kit and then compared with routine gold standard. A subset of monomicrobial BCs (n=560) were analyzed in parallel with the Sepsityper® kit (Bruker Daltonics, Bremen, Germany) and compared with the rapidBACpro® II kit. In addition, the rapidBACpro® II kit was also compared with two different in-house methods.ResultsOverall, 80.0% of the monomicrobial isolates (609/761) were correctly identified by the rapidBACpro® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper® kit yielded higher rates of correct species-level identification provided by the rapidBACpro® II kit for all categories (p>0.0001) except for yeasts identified with score values >1.7. It also proved superior to the ammonium chloride method (p>0.0001) but the differential centrifugation method allowed higher rates of correct identification for Gram negative bacteria (p>0.1).ConclusionsThe rapidBACpro® II kit allowed a high rate of microorganisms correctly identified. The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods. This fact was especially interesting in the case of Staphylococcus sp. and Streptococcus sp. in order to elucidate their clinical impact, for example in device-associated bacteremia.

scholarly journals Identification and differentiation of avianMycoplasmaspecies using MALDI-TOF MS

2019 ◽  
Vol 31 (4) ◽  
pp. 620-624
Author(s):  
Liane Baudler ◽  
Sandra Scheufen ◽  
Luisa Ziegler ◽  
Franca Möller Palau-Ribes ◽  
Christa Ewers ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Live Vaccine ◽  
Species Level ◽  
Molecular Methods ◽  
Clinical Isolates ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Ms Analysis ◽  
Mycoplasma Spp ◽  
Tof Ms

The identification of avian Mycoplasma spp. by conventional immunologic, phenotypic, and molecular methods can be demanding and time-consuming. We evaluated MALDI-TOF MS for its suitability to identify avian mycoplasmas at the species level. We generated a mycoplasma spectral database of 36 main spectrum profiles (MSPs) representing 23 avian Mycoplasma spp. using 23 type and reference strains, 1 live vaccine strain, and 8 clinical isolates. We then used 112 avian Mycoplasma clinical isolates of different avian mycoplasmas, 4 Mycoplasma live vaccine strains, and 1 Mycoplasma type strain, previously cultured and identified to the species level by molecular methods, to evaluate the MSP database. Protein extraction and MALDI-TOF MS analysis were performed with a maximum of 3 repetitions per isolate. MALDI-TOF MS resulted in accurate species-level identification with a score of ≥2.0 for 112 of 117 (96%) isolates. The MALDI-TOF MS analysis of 4 of 5 isolates that did not yield a score of ≥2.0 resulted in best-match identifications that were still concordant at species level with the molecular method used for previous identification. Therefore, MALDI-TOF MS is a promising tool for reliable identification of avian Mycoplasma spp.

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

2016 ◽  
Vol 54 (8) ◽  
pp. 2068-2073 ◽  
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.

scholarly journals A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

2022 ◽  
Author(s):  
Husam Salah ◽  
Anna Kolecka ◽  
Anna Rozaliyani ◽  
Retno Wahyuningsih ◽  
Saad J. Taj-Aldeen ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Laser Desorption ◽  
Correct Identification ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Cultivation Method ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

AbstractMatrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

Performance of the new ID-fungi plate using two types of reference libraries (Bruker and MSI) to identify fungi with the Bruker MALDI Biotyper

2020 ◽  
Vol 58 (7) ◽  
pp. 946-957 ◽  
Author(s):  
Laura Heireman ◽  
Sofie Patteet ◽  
Sophia Steyaert
Keyword(s):  
Growth Medium ◽  
Web Application ◽  
Fungal Infections ◽  
Species Level ◽  
Promising Alternative ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Fungal Identification ◽  
Significant Difference ◽  
Tof Ms

Abstract During the last decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized the diagnosis of fungal infections. Recently, a new Conidia ID-fungi plate (IDFP) medium was introduced to facilitate growth and sampling of fungi. This study aimed to evaluate the IDFP for fungal MALDI-TOF MS identification by comparison with a standard fungal growth medium using two reference libraries. A total of 75 filamentous fungal isolates (including 32 dermatophytes) were inoculated on IDFP and Sabouraud-gentamicin-chloramphenicol (SGC) agar and identified by MALDI-TOF MS using formic acid/acetonitrile extraction. Both the commercially available Bruker library (version 2.0) and the public available MSI web application (version 2018) were applied. For 15% of the isolates, a faster growth was noticed on IDFP compared to SGC. IDFP enhanced the performance of fungal identification compared to SGC for both MSI (increase of 16% identifications to genus and 5% to species level) and Bruker library (increase of 22% identifications to genus and 8% to species level). In total, only 73% of the tested isolates were present in the Bruker library compared to 92% for MSI library. No significant difference (P = 0.46) in MALDI score between IDFP and SGC was observed for the MSI library, but scores were significantly (P = 0.03) higher for IDFP when using Bruker library, potentially explained by the prevention of agar contamination by using IDFP since the Bruker database was created from liquid media. IDFP is a promising alternative growth medium for MALDI-TOF MS fungal identification which would strongly benefit from optimizing the Bruker reference library.

scholarly journals Developing Two Rapid Protein Extraction Methods Using Focused-Ultrasonication and Zirconia-Silica Beads for Filamentous Fungi Identification by MALDI-TOF MS

2021 ◽  
Vol 11 ◽  
Author(s):  
Ya-Ting Ning ◽  
Wen-Hang Yang ◽  
Wei Zhang ◽  
Meng Xiao ◽  
Yao Wang ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Protein Extraction ◽  
Extraction Methods ◽  
Correct Identification ◽  
Routine Method ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Silica Beads ◽  
Vitek Ms ◽  
Tof Ms

Filamentous fungi identification by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenging due to the lack of simple and rapid protein extraction methods and insufficient species coverage in the database. In this study, we created two rapid protein extraction methods for filamentous fungi: a one-step zirconia-silica beads method (ZSB) and a focused-ultrasonication method (FUS). The identification accuracy of two methods were evaluated with the VITEK MS, as well as number of spectra peaks and signal-to-noise ratio (S/N) with M-Discover 100 MALDI-TOF MS compared to the routine method. The better method was applied to build a filamentous fungi in-house spectra library for the M-Discover 100 MS, and then another one and routine method were performed in parallel to verify the accuracy and commonality of the in-house library. Using the two optimized methods, the dedicated operating time before MALDI-TOF MS analysis was reduced from 30 min to 7 (ZSB) or 5 (FUS) min per sample, with only a few seconds added for each additional strain. And both two methods identified isolates from most mold types equal to or better than the routine method, and the total correct identification rate using VITEK MS was 79.67, 76.42, and 76.42%, respectively. On the other hand, the two rapid methods generally achieved higher maximum and minimum S/N ratios with these isolates tested as compared to the routine method. Besides, the ZSB method produced overall mean of maximum and minimum S/N ratio higher than that by FUS. An in-house library of M-Discover MS was successfully built from 135 isolates from 42 species belonging to 18 genera using the ZSB method. Analysis of 467 isolates resulted in 97.22% correctly identified isolates to the species level by the ZSB method versus 95.50% by the routine method. The two novel methods are time- and cost-effective and allow efficient identification of filamentous fungi while providing a simplified procedure to build an in-house library. Thus, more clinical laboratories may consider adopting MALDI-TOF MS for filamentous fungi identification in the future.

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