A rapid, standardized protein extraction method using adaptive focused acoustics for identification of mycobacteria by MALDI-ToF MS

Adams, La'Tonzia L.; Dionne, Kim Yi; Fisher, Stefanie; Parrish, Nicole

doi:10.1016/j.diagmicrobio.2016.06.001

Cited by 8 publications

(13 citation statements)

References 19 publications

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“…A similar prospective study on Vitek MS would be helpful to define real performances of IVD v3.0 from primary cultures of respiratory samples. Several authors recently described new extraction protocols (12,18,40,41), which could increase the ratio of mycobacterial proteins to patients' proteins and probably improve the identification rates at the time of detection.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

et al. 2017

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During the last decade, many investigators have studied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification of mycobacteria. Diverse and contradictory results indicated that optimal level for routine testing has not been reached yet. This work aimed to assess Vitek MS through two distinct versions, Saramis v4.12 RUO and the IVD v3.0, under conditions close to routine laboratory practice. Overall, 111 mycobacterial isolates were subjected to protein extraction and same spectra were matched against both databases. The IVD v3.0 database proved to be superior to Saramis v4.12 and its identification rates remarkably increased, from 67% to 94% for isolates grown on Middlebrook 7H10 solid medium and from 62% to 91% for isolates grown on mycobacterial growth indicator tube (MGIT) liquid medium. With this new version, IVD v3.0, MALDI-TOF MS might be integrated into routine clinical diagnostics, although molecular techniques remain mandatory in some cases.

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Section: Discussionmentioning

confidence: 99%

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

et al. 2017

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“…We compared three pretreatment methods: the current conventional method (Bruker Daltonics®, Bremen, Germany), ultrasonication (Covaris®, MA) with heat pretreatment (95°C, 30 minutes) to inactivate mycobacteria, and ultrasonication without heat pretreatment [9]. Three different methods for protein extraction were designed: ultrasonication with heat preparation ( Figure 1A), ultrasonication with no-heat preparation ( Figure 1B), and the current method ( Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

“…For pretreatment, an early version of protein extraction by the conventional method (Bruker Daltonics ®, Bremen, Germany) required many steps to inactivate mycobacteria, had a high infection risk, and took about 40 minutes to prepare for MALDI-TOF MS (Figure 1). Adams et al recently developed the use of Adaptive Focused Acoustics (AFATM) and ultrasonication, and optimized the ultrasonication protocol for clinical use in identification at the species level of mycobacteria grown in solid media, shortening the needed time compared to conventional extraction/inactivation methods [9]. They performed ultrasonication at 18 C using 1-3 minutes of irradiation at 40 W peak incidence power (PIP) and 50% duty factor (DF), and it safely provided a higher average log confidence score [9].…”

Section: Discussionmentioning

confidence: 99%

“…The sonicator was an Acousticsorbilizer (Adaptive Focused AcousticsTM M220 ultrasonicator, Covaris®, MA) with microtubes (microTUBETM, MA) containing silica glass beads. Ultrasonication was performed at 18˚C, using 1 minute of irradiation at 40 watts PIP and 50% DF [9].…”

Section: Methodsmentioning

confidence: 99%

“…Adams et al recently developed a new protein extraction method using ultrasonication [9] that saves time. In the current study, we compared the ultrasonic method and the current method, and moreover compared ultrasonication with and without heat pretreatment for mycobacterial inactivation.…”

Section: Introductionmentioning

confidence: 99%

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Use of Ultrasonication as a Rapid Pretreatment Method for MALDI-TOF MS of Mycobacterial Samples

Niitsuma¹,

Koshiba²,

Saitou³

et al. 2018

Mycobact Dis

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Background:In Japan, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is used as a simple and accurate method for mycobacterial identification, but existing pretreatment techniques are time-consuming and laborious. Objective:We characterized a new pretreatment technique that extracts proteins using ultrasonic disruption. Methods:We compared this new technique with the current pretreatment method and examined the usefulness of ultrasonication, including its use in mycobacterial inactivation. A total of 174 mycobacterial isolates were tested, including 50 Mycobacterium tuberculosis complex strains, 57 Mycobacterium avium strains, 55 Mycobacterium intracellulare strains, and 12 Mycobacterium kansasii strains. The ultrasonic pretreatment method was performed with or without heat-pretreatment at 95°C for 30 minutes, in parallel with the current conventional method. For all tested strains, the mycobacterial identification agreed when comparing the new and conventional methods. Results:Samples prepared by ultrasonication without heat pretreatment exhibited multiple significant differences when compared with samples prepared by ultrasonication with heat pretreatment or by conventional methods. However, most scores were over 2.0, and the lowest score exceeded 1.7. Furthermore, the new techniques could be performed in only 10 minutes for diagnosis. In addition, we confirmed that 1 minute of ultrasonic pretreatment yielded complete inactivation of M. tuberculosis complex strains. Conclusion:The more rapid technique of protein extraction/inactivation by ultrasonication without heat pretreatment is expected to be highly useful in clinical laboratory settings.

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Mycobacterium : Clinical and Laboratory Characteristics of Rapidly Growing Mycobacteria

Brown‐Elliott,

Harrington,

Morimoto

et al. 2023

ClinMicroNow

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Rapidly growing mycobacteria (RGM) contain long‐chain fatty acids, known as mycolic acids, that can be quantitated with chromatographic techniques such as high‐performance liquid chromatography (HPLC). Prior to the molecular era, HPLC was used for identification of RGM species in most major reference laboratories. The RGM are opportunistic pathogens that cause disease in a variety of clinical settings. RGM may also cause bone and joint infections. Chronic lung infections with RGM, most often in nonsmoking older women with bronchiectasis, are sometimes associated with the Mycobacterium avium complex. The use of mycobacterial smears is a rapid and reasonably sensitive step in the diagnosis of RGM disease. PCR amplification and sequencing has been used to detect and identify nontuberculous mycobacteria in specimens from sterile sites, but these tests are generally limited to larger, reference laboratories. The adjunctive nonmolecular tests, including antimicrobial susceptibility tests, have been utilized for preliminary identification of the RGM.

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A rapid, standardized protein extraction method using adaptive focused acoustics for identification of mycobacteria by MALDI-ToF MS

Cited by 8 publications

References 19 publications

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

Comparison of Saramis 4.12 and IVD 3.0 Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Mycobacteria from Solid and Liquid Culture Media

Use of Ultrasonication as a Rapid Pretreatment Method for MALDI-TOF MS of Mycobacterial Samples

Mycobacterium : Clinical and Laboratory Characteristics of Rapidly Growing Mycobacteria

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