Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE

ABSTRACT Mycoplasma bovis is a leading cause of pneumonia in modern calf rearing. Fast identification is essential to ensure appropriate antimicrobial therapy. Therefore, the objective of this study was to develop a protocol to identify M. bovis from bronchoalveolar lavage fluid (BALf) with matrix-assisted laser desorption ionization–time of flight mass spectrometry MALDI-TOF MS and to determine the diagnostic accuracy in comparison with other techniques. BALf was obtained from 104 cattle, and the presence of M. bovis was determined in the following three ways: (i) rapid identification of M. bovis with MALDI-TOF MS (RIMM) (BALf was enriched and after 24, 48, and 72 h of incubation and was analyzed using MALDI-TOF MS), (ii) triplex real-time PCR for M. bovis, Mycoplasma bovirhinis, and Mycoplasma dispar, and (iii) 10-day incubation on selective-indicative agar. The diagnostic accuracy of the three tests was determined with Bayesian latent class modeling (BLCM). After 24 h of enrichment, M. bovis was identified with MALDI-TOF MS in 3 out of 104 BALf samples. After 48 and 72 h of enrichment, 32/104 and 38/100 samples, respectively, were M. bovis positive. Lipase-positive Mycoplasma-like colonies were seen in 28 of 104 samples. Real-time PCR resulted in 28/104 positive and 12/104 doubtful results for M. bovis. The BLCM showed a sensitivity (Se) and specificity (Sp) of 86.6% (95% credible interval [CI], 69.4% to 97.6%) and 86.4% (CI, 76.1 to 93.8) for RIMM. For real-time PCR, Se was 94.8% (CI, 89.9 to 97.9) and Sp was 88.9% (CI, 78.0 to 97.4). For selective-indicative agar, Se and Sp were 70.5% (CI, 52.1 to 87.1) and 93.9% (CI, 85.9 to 98.4), respectively. These results suggest that rapid identification of M. bovis with MALDI-TOF MS after an enrichment procedure is a promising test for routine diagnostics in veterinary laboratories.

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Real‐time PCR versus MALDI‐TOF MS and culture‐based techniques for diagnosis of bloodstream and pyogenic infections in humans and animals

Journal of Applied Microbiology ◽

10.1111/jam.14862 ◽

2020 ◽

Author(s):

N.K. Abd El‐Aziz ◽

A.A Gharib ◽

E.A.A. Mohamed ◽

A.H. Hussein

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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MALDI-TOF MS platform combined with machine learning to establish a model for rapid identification of methicillin-resistant Staphylococcus aureus

Journal of Microbiological Methods ◽

10.1016/j.mimet.2020.106109 ◽

2021 ◽

Vol 180 ◽

pp. 106109

Author(s):

Kewen Tang ◽

Dongling Tang ◽

Qianyu Wang ◽

Congrong Li

Keyword(s):

Machine Learning ◽

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Rapid Identification ◽

Methicillin Resistant ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Effects of inactivation methods on the analysis ofBacillus atrophaeusendospores using real-time PCR and MALDI-TOF-MS

Engineering in Life Sciences ◽

10.1002/elsc.200800078 ◽

2010 ◽

pp. NA-NA ◽

Cited By ~ 1

Author(s):

Steven R. Talbot ◽

Heiko Russmann ◽

Stefan Köhne ◽

Bärbel Niederwöhrmeier ◽

Gudrun Grote ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Simultaneous Detection of Seven Human Coronaviruses by Multiplex PCR and MALDI-TOF MS

COVID ◽

10.3390/covid2010002 ◽

2021 ◽

Vol 2 (1) ◽

pp. 5-17

Author(s):

Tingting Liu ◽

Lin Kang ◽

Yanwei Li ◽

Jing Huang ◽

Zishuo Guo ◽

...

Keyword(s):

Multiplex Pcr ◽

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Detection Sensitivity ◽

Clinical Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Human Coronaviruses ◽

Tof Ms

Human coronaviruses (HCoVs) are associated with a range of respiratory symptoms. The discovery of severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome, and SARS-CoV-2 pose a significant threat to human health. In this study, we developed a method (HCoV-MS) that combines multiplex PCR with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), to detect and differentiate seven HCoVs simultaneously. The HCoV-MS method had high specificity and sensitivity, with a 1–5 copies/reaction detection limit. To validate the HCoV-MS method, we tested 163 clinical samples, and the results showed good concordance with real-time PCR. Additionally, the detection sensitivity of HCoV-MS and real-time PCR was comparable. The HCoV-MS method is a sensitive assay, requiring only 1 μL of a sample. Moreover, it is a high-throughput method, allowing 384 samples to be processed simultaneously in 30 min. We propose that this method be used to complement real-time PCR for large-scale screening studies.

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Investigation of Fluconazole Susceptibility to Candida Albicans by MALDI-TOF MS and Real-Time PCR for CDR1, CDR2, MDR1 and ERG11

10.21203/rs.3.rs-658761/v1 ◽

2021 ◽

Author(s):

Chanika Maenchantrarath ◽

Pradchama Khumdee ◽

Seksun Samosornsuk ◽

Narissara Mungkornkaew ◽

Worada Samosornsuk

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Spectral Intensity ◽

Spontaneous Mutant ◽

Major Peak ◽

Maldi Tof Ms ◽

Clinical Strains ◽

Mass Spectral ◽

Maldi Tof ◽

Tof Ms

Abstract Background C. albicans is the most important yeast that caused the infection in humans; the trend of resistance to fluconazole (FLC) was also increased, while the FLC susceptibility by conventional method takes time causing the treatment failure. To investigate FLC susceptibility to C. albicans using MALDI-TOF MS and Real-time PCR for CDR1, CDR2, MDR1 and ERG11, overall, 32 C. albicans strains included 4 reference strains (3 FLC susceptible (S) and 1 FLC resistant (R), 1 spontaneous mutant strain (FLC susceptible-dose dependent, SDD) and 27 clinical strains obtained from 2 Thai University Hospitals were performed FLC susceptibility testing by Sensititre YeastOne and broth microdilution method, FLC resistant expression mechanism by Real-time PCR and the major peak determination by MALDI-TOF MS.Results The change of CDR1 and CDR2 mRNA expression were only significantly observed in SDD and R strains. Using MALDI-TOF MS, the change of mass spectral intensity at range 3376-3382 m/z (major peak) was significantly related to FLC susceptibility as SDD (decreased at 4 µg/ml and increase at 8 µg/ml), S (all increased), and R (all slightly decreased or no change) after incubation for 6 h. All 27 clinical strains showed FLC MIC susceptible (MIC range 0.25-2 µg/ml), no change in CDR1 and CDR2 expression and S major peak type. The FLC resistance C. albicans with CDR1 and CDR2 expression may possibly effect the change of mass spectral intensity at range 3376-3382 m/z. Conclusions The MALDI-TOF MS may be used to simultaneously classify and predict FLC resistant C. albicans strains associated with CDR1 and CDR2 expression. Further studies are essential to clarify the methodology and improve the reliability of this assay for routine diagnosis.

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