Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry

Huang, Tung Po; Lee, Chia Chien; Tu, Hui Zin; Lee, Susan Shin Jung

doi:10.1371/journal.pone.0192291

Cited by 28 publications

(21 citation statements)

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“…The sensitivity and specificity for isolates for which a spectrum was identifiable was 96.7% (204/211) and 91.7% (22/24), respectively. Such percentages are higher than those found when using a protein-based approach [ 32 , [37] , [38] , [39] , [40] ], suggesting that the lipid-based approach is a very promising tool for the identification of mycobacteria by MALDI-ToF.…”

Section: Discussionmentioning

confidence: 73%

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Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections

Gonzalo

Broda

Drobniewski

et al. 2021

Clinical Microbiology and Infection

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Objectives: Bacterial diagnosis of mycobacteria is often challenging because of the variability of the sensitivity and specificity of the assay used, and it can be expensive to perform accurately. Although matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become the workhorse of clinical laboratories, the current MALDI methodology (which is based on cytosolic protein profiling) for mycobacteria is still challenging due to the number of steps involved (up to seven) and potential biosafety concerns. Knowing that mycobacteria produce surface-exposed species-specific lipids, we here hypothesized that the detection of those molecules could offer a rapid, reproducible and robust method for mycobacterial identification. Methods: We evaluated the performance of an alternative methodology based on characterized speciesspecific lipid profiling of intact bacteria, without any sample preparation, by MALDI MS; it uses MALDItime-of-flight (ToF) MS combined with a specific matrix (super-2,5-dihydroxybenzoic acid solubilized in an apolar solvent system) to analyse lipids of intact heat-inactivated mycobacteria. Cultured mycobacteria are heat-inactivated and loaded directly onto the MALDI target followed by addition of the matrix. Acquisition of the data is done in both positive and negative ion modes. Blinded studies were performed using 273 mycobacterial strains comprising both the Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria (NTMs) subcultured in Middlebrook 7H9 media supplemented with 10% OADC (oleic acid/dextrose/catalase) growth supplement and incubated for up to 2 weeks at 37 C. Results: The method we have developed is fast (<10 mins) and highly sensitive (<1000 bacteria required); 96.7% of the Mtb complex strains (204/211) were correctly assigned as MTB complex and 91.7% (22/24) NTM species were correctly assigned based only on intact bacteria species-specific lipid profiling by MALDI-ToF MS. Conclusions: Intact bacterial lipid profiling provides a biosafe and unique route for rapid and accurate mycobacterial identification.

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Section: Discussionmentioning

confidence: 73%

“…A total of 273 mycobacterial isolates archived at Imperial College, London, UK were cultured from frozen aliquots into Middlebrook 7H9 broth supplemented with OACD (oleic acid/dextrose/ catalase) without glycerol (Sigma-Aldrich Company Ltd, Gillingham, Dorset, UK) and incubated for 4e6 weeks at 37…”

Section: Clinical Mycobacterium Tuberculosis Isolatesmentioning

confidence: 99%

Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections

Gonzalo

Broda

Drobniewski

et al. 2021

Clinical Microbiology and Infection

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“…Another method for the identification of M. tuberculosi s from MGIT positive bottles using the VITEK MS (bioMérieux, Marcy l’Étoile, France) MALDI-TOF system has been described. The rates of identification are lower than the method describe using the Bruker Mycobacteria library and the method is more laborious involving centrifugation and lysis 8 .…”

Section: Discussionmentioning

confidence: 94%

An improved simple method for the identification of Mycobacteria by MALDI-TOF MS (Matrix-Assisted Laser Desorption- Ionization mass spectrometry)

Alcolea-Medina

Fernández

Montiel

et al. 2019

Sci Rep

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The aim of this study was to establish a simple method for the rapid identification of Mycobacteria species by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass spectrometry) using the Bruker MALDI-TOF Biotyper system (Bruker Daltonik, Bremen, Germany). A multicentre, prospective, and single blind study was performed in three European Hospitals, two Spanish and one UK hospital from May to August 2018. The BD BACTEC MGIT (Becton Dickinson, Berks, UK) liquid culture system was used in all three centres for the growth of Mycobacteria. When signal positive, tubes were removed from the analyser and in addition to standard laboratory procedures were subcultured on blood agar plates for MALDI-TOF analysis. Plates were incubated aerobically for 1 to 7 days at 37 °C and inspected every day. Once any growth was visible, it was transferred to the steel target plate, overlaid with 1 μl of neat formic acid and 1 μl HCCA matrix (alpha hydroxyl 4 cinnamic acid), and analysed in a Bruker Biotyper MALDI-TOF. Results given by MALDI-TOF were compared with the reference methods used for identification in the different centres. At two Spanish hospitals, identification by MALDI-TOF was only attempted on presumptive non-tuberculosis mycobacteria (NTM) and the results were initially compared with the results obtained by a commercial reverse hybridisation assay, GenoType CM/AS (Hain Lifescience, Tübingen, Germany). At the UK Hospital, identification of any presumptive mycobacteria was attempted and compared with the results obtained by whole genome sequencing (WGS). Overall in 142/167 (85%) of cases the identifications obtained were concordant; all Mycobacterium tuberculosis (MTB) isolates 43/43 (100%), 57/76 (75%) of the rapid growing nontuberculous mycobacteria (NTM), and 42/48 (85%) slow growing NTM tested were identified correctly. We report a new, easy, cheap and quick method for isolation and identification of Mycobacterium spp. without the need for additional steps or equipment and this method is in routine used in all three centres.

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“…MALDI-TOF MS is now widely used to identify clinical microorganisms because of lowered cost and faster turn-around times [13,14]. In recent years, four approaches have been reported for detection of certain drug resistant bacteria [15,16] based on the postulation that any or combination of the polypeptides related to drug resistance could possibly alter the spectra obtained by MALDI-TOF MS and possibly enable differentiation from the drug-susceptible bacteria.…”

Section: Introductionmentioning

confidence: 99%

Detection of carbapenem-resistant Klebsiella pneumoniae on the basis of matrix-assisted laser desorption ionization time-of-flight mass spectrometry by using supervised machine learning approach

Huang

Lee

et al. 2020

PLoS ONE

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Background Carbapenem-resistant Klebsiella pneumoniae (CRKP) is emerging as a significant pathogen causing healthcare-associated infections. Matrix-assisted laser desorption/ionisation mass spectrometry time-of-flight mass spectrometry (MALDI-TOF MS) is used by clinical microbiology laboratories to address the need for rapid, cost-effective and accurate identification of microorganisms. We evaluated application of machine learning methods for differentiation of drug resistant bacteria from susceptible ones directly using the profile spectra of whole cells MALDI-TOF MS in 46 CRKP and 49 CSKP isolates. Methods We developed a two-step strategy for data preprocessing consisting of peak matching and a feature selection step before supervised machine learning analysis. Subsequently, five machine learning algorithms were used for classification. Results Random forest (RF) outperformed other four algorithms. Using RF algorithm, we correctly identified 93% of the CRKP and 100% of the CSKP isolates with an overall classification accuracy rate of 97% when 80 peaks were selected as input features. Conclusions We conclude that CRKPs can be differentiated from CSKPs through RF analysis. We used direct colony method, and only one spectrum for an isolate for analysis, without modification

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Rapid identification of mycobacteria from positive MGIT broths of primary cultures by MALDI-TOF mass spectrometry

Cited by 28 publications

References 50 publications

Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections

Performance of lipid fingerprint-based MALDI-ToF for the diagnosis of mycobacterial infections

An improved simple method for the identification of Mycobacteria by MALDI-TOF MS (Matrix-Assisted Laser Desorption- Ionization mass spectrometry)

Detection of carbapenem-resistant Klebsiella pneumoniae on the basis of matrix-assisted laser desorption ionization time-of-flight mass spectrometry by using supervised machine learning approach

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