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.

scholarly journals Evaluation of Vitek MS for Differentiation of Cryptococcus neoformans and Cryptococcus gattii Genotypes

2018 ◽  
Vol 57 (1) ◽  
Author(s):  
Lumena P. Machado Siqueira ◽  
Viviane M. Favero Gimenes ◽  
Roseli Santos de Freitas ◽  
Márcia de Souza Carvalho Melhem ◽  
Lucas Xavier Bonfietti ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Cryptococcus Gattii ◽  
Correct Identification ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Mass Spectral ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Vitek Ms ◽  
Tof Ms

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii are the main pathogenic species of invasive cryptococcosis among the Cryptococcus species. Taxonomic studies have shown that these two taxa have different genotypes or molecular types with biological and ecoepidemiological peculiarities. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been proposed as an alternative method for labor-intensive methods for C. neoformans and C. gattii genotype differentiation. However, Vitek MS, one of the commercial MALDI-TOF MS instruments, has not been yet been evaluated for this purpose. Thus, we constructed an in-house database with reference strains belonging to the different C. neoformans (VNI, VNII, VNIII, and VNIV) and C. gattii (VGI, VGII, VGIII, and VGIV) major molecular types by using the software Saramis Premium (bioMérieux, Marcy-l’Etoile, France). Then, this new database was evaluated for discrimination of the different genotypes. Our in-house database provided correct identification for all C. neoformans and C. gattii genotypes; however, due to the intergenotypic mass spectral similarities, a careful postanalytic evaluation is necessary to provide correct genotype identification.

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.

scholarly journals A MALDI-TOF MS database for fast identification of Vibrio spp. potentially pathogenic to marine mollusks

2021 ◽  
Vol 105 (6) ◽  
pp. 2527-2539
Author(s):  
M. Moussa ◽  
E. Cauvin ◽  
A. Le Piouffle ◽  
O. Lucas ◽  
A. Bidault ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Dna Amplification ◽  
Extraction Methods ◽  
Rapid Identification ◽  
Blind Test ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Identification Technique ◽  
Perfect Discrimination ◽  
Tof Ms

Abstract In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120 main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus. Key points • Development of a MALDI-TOF MS database to quickly affiliate Vibrio species. • Increase of the reactivity when faced with Vibrio associated with mollusk diseases. • Validation of MALDI-TOF MS as routine diagnostic tool.

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.

scholarly journals Insufficient Discriminatory Power of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry Dendrograms to Determine the Clonality of Multi-Drug-ResistantAcinetobacter baumanniiIsolates from an Intensive Care Unit

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
John Hoon Rim ◽  
Yangsoon Lee ◽  
Sung Kuk Hong ◽  
Yongjung Park ◽  
MyungSook Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Protein Extraction ◽  
Rank Correlation ◽  
Extraction Methods ◽  
Gold Standard Method ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Clonality Analysis ◽  
Tof Ms

While pulsed-field gel electrophoresis (PFGE) is recognized as the gold standard method for clonality analysis, MALDI-TOF MS has recently been spotlighted as an alternative tool for species identification. Herein, we compared the dendrograms of multi-drug-resistant (MDR)Acinetobacter baumanniiisolates by using MALDI-TOF MS with those by using PFGE. We used direct colony and protein extraction methods for MALDI-TOF MS dendrograms. The isolates with identical PFGE patterns were grouped into different branches in MALDI-TOF MS dendrograms. Among the isolates that were classified as very close isolates in MALDI-TOF MS dendrogram, PFGE band patterns visually showed complete differences. We numeralized similarity among isolates by measuring distance levels. The Spearman rank correlation coefficient values were 0.449 and 0.297 between MALDI-TOF MS dendrogram using direct colony and protein extraction method versus PFGE, respectively. This study is the first paper focusing solely on the dendrogram function of MALDI-TOF MS compared with PFGE. Although MALDI-TOF MS is a promising tool to identify species in a rapid manner, our results showed that MALDI-TOF MS dendrograms could not substitute PFGE for MDRAcinetobacter baumanniiclonality analysis.

Identification of Lice Stored in Alcohol Using MALDI-TOF MS

2020 ◽  
Author(s):  
Hanene Benyahia ◽  
Basma Ouarti ◽  
Adama Zan Diarra ◽  
Mehdi Boucheikhchoukh ◽  
Mohamed Nadir Meguini ◽  
...  
Keyword(s):  
Economic Consequences ◽  
Correct Identification ◽  
Protein Profiles ◽  
Innovative Strategy ◽  
Maldi Tof Ms ◽  
Pediculus Humanus ◽  
Maldi Tof ◽  
Ms Analysis ◽  
Storage Methods ◽  
Tof Ms

Abstract Lice pose major public and veterinary health problems with economic consequences. Their identification is essential and requires the development of an innovative strategy. MALDI-TOF MS has recently been proposed as a quick, inexpensive, and accurate tool for the identification of arthropods. Alcohol is one of the most frequently used storage methods and makes it possible to store samples for long periods at room temperature. Several recent studies have reported that alcohol alters protein profiles resulting from MS analysis. After preliminary studies on frozen lice, the purpose of this research was to evaluate the influence of alcohol preservation on the accuracy of lice identification by MALDI-TOF MS. To this end, lice stored in alcohol for variable periods were submitted for MS analysis and sample preparation protocols were optimized. The reproducibility and specificity of the MS spectra obtained on both these arthropod families allowed us to implement the reference MS spectra database (DB) with protein profiles of seven lice species stored in alcohol. Blind tests revealed a correct identification of 93.9% of Pediculus humanus corporis (Linnaeus, 1758) and 98.4% of the other lice species collected in the field. This study demonstrated that MALDI-TOF MS could be successfully used for the identification of lice stored in alcohol for different lengths of time.

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 Evaluation of ID Fungi Plates Medium for Identification of Molds by MALDI Biotyper

2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Marie Gladys Robert ◽  
Charlotte Romero ◽  
Céline Dard ◽  
Cécile Garnaud ◽  
Odile Cognet ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Susceptibility Testing ◽  
Antifungal Susceptibility ◽  
Antifungal Susceptibility Testing ◽  
Maldi Tof Ms ◽  
Extraction Protocol ◽  
Maldi Tof ◽  
Morphological Aspects ◽  
Routine Identification ◽  
Tof Ms

ABSTRACT MALDI-TOF mass spectrometry (MS) identification of pathogenic filamentous fungi is often impaired by difficulties in harvesting hyphae embedded in the medium and long extraction protocols. The ID Fungi Plate (IDFP) is a novel culture method developed to address such difficulties and improve the identification of filamentous fungi by MALDI-TOF MS. We cultured 64 strains and 11 clinical samples on IDFP, Sabouraud agar-chloramphenicol (SAB), and ChromID Candida agar (CAN2). We then compared the three media for growth, ease of harvest, amount of material picked, and MALDI-TOF identification scores after either rapid direct transfer (DT) or a long ethanol-acetonitrile (EA) extraction protocol. Antifungal susceptibility testing and microscopic morphology after subculture on SAB and IDFP were also compared for ten molds. Growth rates and morphological aspects were similar for the three media. With IDFP, harvesting of fungal material for the extraction procedure was rapid and easy in 92.4% of cases, whereas it was tedious on SAB or CAN2 in 65.2% and 80.3% of cases, respectively. The proportion of scores above 1.7 (defined as acceptable identification) were comparable for both extraction protocols using IDFP (P = 0.256). Moreover, rates of acceptable identification after DT performed on IDFP (93.9%) were significantly higher than those obtained after EA extraction with SAB (69.7%) or CAN2 (71.2%) (P = <0.001 and P = 0.001, respectively). Morphological aspects and antifungal susceptibility testing were similar between IDFP and SAB. IDFP is a culture plate that facilitates and improves the identification of filamentous fungi, allowing accurate routine identification of molds with MALDI-TOF-MS using a rapid-extraction protocol.

scholarly journals Candida auris Clinical Isolates from South Korea: Identification, Antifungal Susceptibility, and Genotyping

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Yong Jun Kwon ◽  
Jong Hee Shin ◽  
Seung A Byun ◽  
Min Ji Choi ◽  
Eun Jeong Won ◽  
...  
Keyword(s):  
Antifungal Susceptibility ◽  
Diagnostic Tools ◽  
Candida Auris ◽  
Content Type ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Fluconazole Resistant ◽  
Vitek 2 ◽  
Vitek Ms ◽  
Tof Ms

ABSTRACT Candida auris is an emerging worldwide fungal pathogen. Over the past 20 years, 61 patient isolates of C. auris (4 blood and 57 ear) have been obtained from 13 hospitals in Korea. Here, we reanalyzed those molecularly identified isolates using two matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, including Biotyper and Vitek MS, followed by antifungal susceptibility testing, sequencing of the ERG11 gene, and genotyping. With a research-use-only (RUO) library, 83.6% and 93.4% of the isolates were correctly identified by Biotyper and Vitek MS, respectively. Using an in vitro diagnostic (IVD) library of Vitek MS, 96.7% of the isolates were correctly identified. Fluconazole-resistant isolates made up 62.3% of the isolates, while echinocandin- or multidrug-resistant isolates were not found. Excellent essential (within two dilutions, 96.7%) and categorical agreements (93.4%) between the Clinical and Laboratory Standards Institute (CLSI) and Vitek 2 (AST-YS07 card) methods were observed for fluconazole. Sequencing ERG11 for all 61 isolates revealed that only 3 fluconazole-resistant isolates showed the Erg11p amino acid substitution K143R. All 61 isolates showed identical multilocus sequence typing (MLST). Pulsed-field gel electrophoresis (PFGE) analyses revealed that both blood and ear isolates had the same or similar patterns. These results show that MALDI-TOF MS and Vitek 2 antifungal susceptibility systems can be reliable diagnostic tools for testing C. auris isolates from Korean hospitals. The Erg11p mutation was seldom found among Korean isolates of C. auris, and multidrug resistance was not found. Both MLST and PFGE analyses suggest that these isolates are genetically similar.

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