Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory.
ABSTRACT: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is increasingly used for the identification of bacteria and fungi in the diagnostic laboratory. We evaluated the mold database of Bruker Daltonik (Bremen, Germany), the Filamentous Fungi Library 1.0. First, we studied 83 phenotypically and molecularly well-characterized, nondermatophyte, nondematiaceous molds from a clinical strain collection. Using the manufacturer-recommended interpretation criteria, genus and species identification rates were 78.3% and 54.2%, respectively. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification to 71.1% without increasing misidentifications. In a subsequent prospective study, 200 consecutive clinical mold isolates were identified by the MALDI Biotyper and our conventional identification algorithm. Discrepancies were resolved by ribosomal DNA (rDNA) internal transcribed spacer region sequence analysis. For the MALDI Biotyper, genus and species identification rates were 83.5% and 79.0%, respectively, when using a species cutoff of 1.7. Not identified were 16.5% of the isolates. Concordant genus and species assignments of MALDI-TOF MS and the conventional identification algorithm were observed for 98.2% and 64.2% of the isolates, respectively. Four erroneous species assignments were observed using the MALDI Biotyper. The MALDI Biotyper seems highly reliable for the identification of molds when using the Filamentous Fungi Library 1.0 and a species cutoff of 1.7. However, expansion of the database is required to reduce the number of nonidentified isolates.
Project description:Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has emerged as a reliable technique to identify molds involved in human diseases, including dermatophytes, provided that exhaustive reference databases are available. This study assessed an online identification application based on original algorithms and an extensive in-house reference database comprising 11,851 spectra (938 fungal species and 246 fungal genera). Validation criteria were established using an initial panel of 422 molds, including dermatophytes, previously identified via DNA sequencing (126 species). The application was further assessed using a separate panel of 501 cultured clinical isolates (88 mold taxa including dermatophytes) derived from five hospital laboratories. A total of 438 (87.35%) isolates were correctly identified at the species level, while 26 (5.22%) were assigned to the correct genus but the wrong species and 37 (7.43%) were not identified, since the defined threshold of 20 was not reached. The use of the Bruker Daltonics database included in the MALDI Biotyper software resulted in a much higher rate of unidentified isolates (39.76 and 74.30% using the score thresholds 1.7 and 2.0, respectively). Moreover, the identification delay of the online application remained compatible with real-time online queries (0.15 s per spectrum), and the application was faster than identifications using the MALDI Biotyper software. This is the first study to assess an online identification system based on MALDI-TOF spectrum analysis. We have successfully applied this approach to identify molds, including dermatophytes, for which diversity is insufficiently represented in commercial databases. This free-access application is available to medical mycologists to improve fungal identification.
Project description:We evaluated the performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the MALDI Bruker Biotyper system (microflex LT; Bruker Daltonik GmbH, Bremen, Germany), on the identification of 50 isolates of clinically encountered molds, including Penicillium marneffei (n = 28), Paecilomyces species (n = 12), Fusarium solani (n = 6), Rhizopus species (n = 3), and Pseudallescheria boydii (n = 1). The isolates were identified to species levels by sequence analysis of the internal transcribed spacer (ITS) regions using primers ITS1 and ITS4. None of the 28 genetically well characterized isolates of P. marneffei were identified as P. marneffei by MALDI-TOF MS, because P. marneffei was not present in either Bruker general library (DB 5627) or Bruker filamentous fungi library V1.0. However, the rate of accurate identification as P. marneffei (score value ? 2.000) was 85.7% based on newly created database from one P. marneffei strain (NTUH-3370) by MALDI Biotyper system. Sequencing analysis of these 22 non-P. marneffei isolates of molds revealed seven Paecilomyces variotii, six F. solani, four Paecilomyces lilacinus, and one each of Paecilomyces sinensis, Rhizopus arrhizus, R. oryzae, R. microspores, and P. boydii. Although all the seven P. variotii isolates, four of the six F. solani, two of the four P. lilacinus, and two of the three isolates of Rhizopus species, and the P. boydii isolate had concordant identification results between MALDI-TOF MS and sequencing analysis, the score values of these isolates were all of <1.700. This study indicated that the MALDI Bruker Biotyper is ineffective for identifying P. marneffei and other unusual molds because of the current database limitations. Therefore, it is necessary to continuously update the MALDI-TOF MS databases.
Project description:We evaluated the accuracy of the Bruker Biotyper matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) system at identifying clinical isolates of <i>Aspergillus</i> species that were grown on agar media. A total of 381 non-duplicate <i>Aspergillus</i> isolates representing 21 different <i>Aspergillus</i> species identified by molecular analysis were included in this study. The Bruker Biotyper MALDI-TOF MS system was able to identify 30.2% (115/381) of the isolates to the species level (score values of ?2.000) and 49.3% to the genus level (score values of 1.700-1.999). When the identification cutoff value was lowered from ?2.000 to ?1.700, the species-level identification rate increased to 79.5% with a slight rise of false identification from 2.6 to 5.0%. From another aspect, a correct species-level identification rate of 89% could be reached by the Bruker Biotyper MALDI-TOF MS system regardless of the score values obtained. The Bruker Biotyper MALDI-TOF MS system had a moderate performance in identification of <i>Aspergillus</i> directly inoculated on solid agar media. Continued expansion of the Bruker Biotyper MALDI-TOF MS database and adoption of alternative cutoff values for interpretation are required to improve the performance of the system for identifying highly diverse species of clinically encountered <i>Aspergillus</i> isolates.
Project description:Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has entered clinical laboratories, facilitating identification of bacteria. Here, we evaluated the MALDI Biotyper (Bruker Daltonics) for the identification of fastidious Gram-negative rods (GNR). Three sample preparation methods, direct colony transfer, direct transfer plus on-target formic acid preparation, and ethanol-formic acid extraction, were analyzed for 151 clinical isolates. Direct colony transfer applied with the manufacturer's interpretation criteria resulted in overall species and genus identification rates of 43.0% and 32.5%, respectively; 23.2% of the isolates were not identified, and two misidentifications (1.3%) were observed. The species identification rates increased to 46.4% and 53.7% for direct transfer plus formic acid preparation and ethanol-formic acid extraction, respectively. In addition, we evaluated score value cutoff alterations. The identification rates hardly increased by reducing the genus cutoff, while reducing the 2.0 species cutoff to 1.9 and to 1.8 increased the identification rates to up to 66.2% without increasing the rate of misidentifications. This study shows that fastidious GNR can reliably be identified using the MALDI Biotyper. However, the identification rates do not reach those of nonfastidious GNR such as the Enterobacteriaceae. In addition, two approaches optimizing the identification of fastidious GNR by the MALDI Biotyper were demonstrated: formic acid-based on-target sample treatment and reductions in cutoff scores to increase the species identification rates.
Project description:Reported matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing.
Project description:Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid and highly accurate identification of clinical pathogens but has not been utilized extensively in clinical mycology due to challenges in developing an effective protein extraction method and the limited databases available. Here, we developed an alternate extraction procedure and constructed a highly stringent database comprising 294 individual isolates representing 76 genera and 152 species. To our knowledge, this is the most comprehensive clinically relevant mold database developed to date. When challenged with 421 blinded clinical isolates from our institution, by use of the BioTyper software, accurate species-level (score of ? 2.0) and genus-level (score of ? 1.7) identifications were obtained for 370 (88.9%) and 18 (4.3%) isolates, respectively. No isolates were misidentified. Of the 33 isolates (7.8%) for which there was no identification (score of <1.7), 25 were basidiomycetes not associated with clinical disease and 8 were Penicillium species that were not represented in the database. Our library clearly outperformed the manufacturer's database that was obtained with the instrument, which identified only 3 (0.7%) and 26 (6.2%) isolates at species and genus levels, respectively. Identification was not affected by different culture conditions. Implementation into our routine workflow has revolutionized our mycology laboratory efficiency, with improved accuracy and decreased time for mold identification, eliminating reliance on traditional phenotypic features.
Project description:Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species- and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications.
Project description:Non-Aspergillus molds including Mucorales, Fusarium, and Scedosporium, etc. are emerging pathogens leading to higher mortality in immunocompromised patients. Fifty-two isolates of genetically confirmed non-Aspergillus molds representing 16 species from 8 genera were collected to evaluate the performance of the Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in identification of non-Aspergillus molds. Antifungal susceptibilities were determined through the Clinical & Laboratory Standards Institute (CLSI) M38-A2 broth microdilution method and the Sensititre YeastOne colorimetric method. Bruker MALDI-TOF MS identified 57.7% (30/52) of isolates cultured in broth and 15.4% (8/52) of isolates cultured on solid agar media to the species level, respectively, according to standard interpretation criteria. Lowering the species level cut-off value (COV) from ?2.0 to ?1.7 could improve the MALDI-TOF MS species-level identification rate to 67.3% (38/52) for isolates cultured on solid media, with a slight increase of false identification rate of 2.6% (1/38). Amphotericin B was the most in vitro fungistatic-active agent for 98.1% (51/52) of the tested non-Aspergillus molds, with minimum inhibitory concentrations (MICs) of ?2 ?g/mL. The susceptibilities to triazoles varied, with MICs of 0.12 to >16 ?g/mL among different species of non-Aspergillus molds. The correlation between the CLSI method and Sensititre YeastOne on antifungal susceptibility testing of non-Aspergillus molds was good, with essential agreement (EA) rates of >90% for triazoles and echinocandins except amphotericin B, which had a lower EA rate of 84.6%. In conclusion, a favorable performance of the Bruker MALDI-TOF MS in identification of clinical non-Aspergillus isolates directly inoculated on solid agar media could be achieved with the adoption of alternative interpretation criteria. Antifungal susceptibility testing is important for non-Aspergillus molds, especially when information on triazole susceptibility is required, and the Sensititre YeastOne is a practical and reliable method to determine antifungal susceptibilities of non-Aspergillus molds.
Project description:Although some Weissella species play beneficial roles in food fermentation and in probiotic products, others such as Weissella confusa are emerging Gram-positive pathogens in immunocompromised hosts. Weissella species are difficult to identify by conventional biochemical methods and commercial automated systems and are easily misidentified as Lactobacillus and Leuconostoc species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly being used for bacterial identification. Little, however, is known about the effectiveness of MALDI-TOF MS in identifying clinical isolates of Weissella to the species level. In this study, we evaluated whether the MALDI-TOF MS Bruker Biotyper system could accurately identify a total of 20 W. confusa and 2 W. cibaria blood isolates that had been confirmed by 16s rRNA sequencing analysis. The MALDI-TOF Biotyper system yielded no reliable identification results based on the current reference spectra for the two species (all score values <1.7). New W. confusa spectra were created by randomly selecting 3 W. confusa isolates and external validation was performed by testing the remaining 17 W. confusa isolates using the new spectra. The new main spectra projection (MSP) yielded reliable score values of >2 for all isolates with the exception of one (score value, 1.963). Our results showed that the MSPs in the current database are not sufficient for correctly identifying W. confusa or W. cibaria. Further studies including more Weissella isolates are warranted to further validate the performance of MALDI-TOF in identifying Weissella species.
Project description:An interlaboratory study using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts (n = 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases. Logistic regression analysis was performed to analyze the association between the number of spectra in the database and the percentage of strains that were correctly identified. A total of 5,460 MALDI-TOF MS results were obtained. Using all results, the area under the ROC curve was 0.95 (95% confidence interval [CI], 0.94 to 0.96). With a sensitivity of 0.84 and a specificity of 0.97, a cutoff value of 1.7 was considered optimal. The overall percentage of correct identifications (formic acid-ethanol extraction method, score ? 1.7) was 61.5% when the commercial Bruker Daltonics database (BDAL) was used, and it increased to 86.8% by using an extended BDAL supplemented with a Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre in-house database (BDAL+CBS in-house). A greater number of main spectra (MSP) in the database was associated with a higher percentage of correct identifications (odds ratio [OR], 1.10; 95% CI, 1.05 to 1.15; P < 0.01). The results from the direct transfer method ranged from 0% to 82.9% correct identifications, with the results of the top four centers ranging from 71.4% to 82.9% correct identifications. This study supports the use of a cutoff value of 1.7 for the identification of yeasts using MALDI-TOF MS. The inclusion of enough isolates of the same species in the database can enhance the proportion of correctly identified strains. Further optimization of the preparation methods, especially of the direct transfer method, may contribute to improved diagnosis of yeast-related infections.