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MALDI-TOF MS for the rapid identification and drug susceptibility testing of filamentous fungi.

ABSTRACT: The present study aimed to evaluate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying filamentous fungi and assessing the in vitro activities of common antifungal drugs against different kinds of filamentous fungi that are commonly encountered in a clinical setting. A total of 123 strains of filamentous fungi (24 species) were submitted for identification by MALDI-TOF MS, and the findings were compared with those obtained by conventional methods. The discrepancies were further investigated by internal transcribed spacer (ITS) sequence analysis. Then, 79 strains were randomly selected for further testing by the minimum inhibitory concentration Etest method. MALDI-TOF MS correctly identified 114 (92.70%) of the 123 filamentous fungi and failed to identify six isolates (4.9%). By contrast, the conventional identification methods made 113 (91.9%) correct identifications. In addition, 15 isolates of filamentous fungi were further identified by ribosomal DNA-ITS sequencing. In the in vitro antifungal susceptibility test, voriconazole showed the strongest antifungal activity among the tested drugs against a broad range of filamentous fungi. Caspofungin showed a better in vitro antifungal activity than fluconazole, itraconazole, and amphotericin B. MALDI-TOF MS offers a cost/time-saving, high-throughput and accurate working protocol for identifying filamentous fungi. Voriconazole could still serve as the first-line drug for treating serious infections caused by filamentous fungi, while caspofungin may be another treatment option for fungal infections.

SUBMITTER: Peng Y

PROVIDER: S-EPMC6895777 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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MALDI-TOF MS for the rapid identification and drug susceptibility testing of filamentous fungi.

Peng Yang Y Zhang Qin Q Xu Chao C Shi Weifeng W

Experimental and therapeutic medicine 20191022 6

The present study aimed to evaluate the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying filamentous fungi and assessing the <i>in vitro</i> activities of common antifungal drugs against different kinds of filamentous fungi that are commonly encountered in a clinical setting. A total of 123 strains of filamentous fungi (24 species) were submitted for identification by MALDI-TOF MS, and the findings were compared with tho ...[more]

PMID: 31819764

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Project description: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.

Project description:BackgroundIn the last decade, an innovative approach has emerged for arthropod identification based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Increasing interest in applying the original technique for arthropod identification has led to the development of a variety of procedures for sample preparation and selection of body parts, among others. However, the absence of a consensual strategy hampers direct inter-study comparisons. Moreover, these different procedures are confusing to new users. Establishing optimized procedures and standardized protocols for mosquito identification by MALDI-TOF MS is therefore a necessity, and would notably enable the sharing of reference MS databases. Here, we assess the optimal conditions for mosquito identification using MALDI-TOF MS profiling.MethodsThree homogenization methods, two of which were manual and one automatic, were used on three distinct body parts (legs, thorax, head) of two mosquito laboratory strains, Anopheles coluzzii and Aedes aegypti, and the results evaluated. The reproducibility of MS profiles, identification rate with relevant scores and the suitability of procedures for high-throughput analyses were the main criteria for establishing optimized guidelines. Additionally, the consequences of blood-feeding and geographical origin were evaluated using both laboratory strains and field-collected mosquitoes.ResultsRelevant score values for mosquito identification were obtained for all the three body parts assayed using MALDI-TOF MS profiling; however, the thorax and legs were the most suitable specimens, independently of homogenization method or species. Although the manual homogenization methods were associated with a high rate of identification on the three body parts, this homogenization mode is not adaptable to the processing of a large number of samples. Therefore, the automatic homogenization procedure was selected as the reference homogenization method. Blood-feeding status did not hamper the identification of mosquito species, despite the presence of MS peaks from original blood in the MS profiles of the three body parts tested from both species. Finally, a significant improvement in identification scores was obtained for field-collected specimens when MS spectra of species from the same geographical area were added to the database.ConclusionThe results of the current study establish guidelines for the selection of mosquito anatomic parts and modality of sample preparation (e.g. homogenization) for future specimen identification by MALDI-TOF MS profiling. These standardized operational protocols could be used as references for creating an international MS database.

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MALDI-TOF MS for the rapid identification and drug susceptibility testing of filamentous fungi.

Publications

MALDI-TOF MS for the rapid identification and drug susceptibility testing of filamentous fungi.

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