Rapid Identification of New Delhi Metallo-β-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS

ABSTRACT There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-β-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/μg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

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Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

Open Medicine ◽

10.2478/s11536-007-0069-4 ◽

2008 ◽

Vol 3 (2) ◽

pp. 157-162

Author(s):

Koray Ergunay ◽

Pinar Yurdakul ◽

Burcin Sener ◽

Ugur Ozcelik ◽

Erdem Karabulut ◽

...

Keyword(s):

Dna Extraction ◽

Burkholderia Cepacia ◽

Limit Of Detection ◽

Direct Detection ◽

Extraction Methods ◽

Rapid Identification ◽

Burkholderia Cepacia Complex ◽

Assay Sensitivity ◽

Spin Column ◽

Significant Difference

AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

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Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

F1000Research ◽

10.12688/f1000research.13964.2 ◽

2018 ◽

Vol 7 ◽

pp. 336 ◽

Cited By ~ 2

Author(s):

Geert A. Van Raemdonck ◽

Kara K. Osbak ◽

Xaveer Van Ostade ◽

Chris R. Kenyon

Keyword(s):

Mass Spectrometer ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Treponema Pallidum ◽

Protein Detection ◽

Reaction Monitoring ◽

Protein Biomarkers ◽

Diagnostic Strategies ◽

Plasma And Urine Samples ◽

Candidate Protein

Background:Current syphilis diagnostic strategies are lacking a sensitive manner of directly detectingTreponema pallidumantigens. A diagnostic test that could directly detectT. pallidumantigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up.Methods:In this study, 11 candidateT. pallidumbiomarker proteins were chosen according to their physiochemical characteristics,T. pallidumspecificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer.Results:No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purifiedT. pallidumwere prepared in PBS. Polyclonal anti-T. pallidumantibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300T. pallidum/ml in PBS.Conclusions:Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration ofT. pallidumproteins. Alternative sample preparation strategies may improve the detectability ofT. pallidumproteins in biofluids.

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Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis

F1000Research ◽

10.12688/f1000research.13964.1 ◽

2018 ◽

Vol 7 ◽

pp. 336

Author(s):

Geert A. Van Raemdonck ◽

Kara K. Osbak ◽

Xaveer Van Ostade ◽

Chris R. Kenyon

Keyword(s):

Mass Spectrometer ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Treponema Pallidum ◽

Protein Detection ◽

Reaction Monitoring ◽

Protein Biomarkers ◽

Diagnostic Strategies ◽

Plasma And Urine Samples ◽

Candidate Protein

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12. Evaluation of Rapid Phenotypic Testing for KPC Carbapenemase Producing klebsiella Pneumoniae Directly from Positive Blood Cultures by Use of “Hot Chocolate” Plates

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa417.011 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S7-S7

Author(s):

Alexander Lawandi ◽

Gleice C Leite ◽

Brigitte Lefebvre ◽

Jean Longtin ◽

Todd C Lee

Keyword(s):

Klebsiella Pneumoniae ◽

Carbapenem Resistance ◽

Empiric Therapy ◽

Turnaround Time ◽

Rapid Identification ◽

Blood Cultures ◽

Minimal Growth ◽

Maldi Tof ◽

Considerable Morbidity ◽

Hot Chocolate

Abstract Background Invasive infections with Carbapenemase Producing Enterobacterales are associated with considerable morbidity and mortality, in part due to the risk of inappropriate empiric therapy. Consequently, the rapid identification of carbapenem resistance is crucial to the management of these infections. We sought to evaluate possible reductions in turnaround time to identification of this resistance in blood cultures growing these organisms by applying rapid phenotypic test kits to growth from “hot chocolate” plates. Methods 30 blood cultures, spiked with carbapenem resistant Klebsiella pneumoniae isolates or susceptible controls, were inoculated onto chocolate agars that had pre-warmed at 37°C. These plates were incubated at 37ºC for 3.5 hours. The resulting minimal growth was then identified using MALDI-TOF and underwent rapid phenotypic testing using three commercially available products (β-lacta and β-carba, from Bio-Rad, Marnes-la-Coquette, France, and Carba-NP, from bioMérieux, Durham, NC). The time to identification of carbapenem resistance using this method was then compared to that of the conventional laboratory workup. Results The identification was 100% accurate to the species level using MALDI-TOF paired to the 3.5 hour growth on the “hot choocolate” plates. The β-lacta kit identified resistance to 3rd generation cephalosporins for all ESBL and carbapenemase producing Klebsiella pneumoniae isolates, while the β-carba and Carba-NP kits identified carbapenem resistance only in the carbapenemase producers. The sensitivity of all assays was 100% (95% CI 0.87–1.0) and the specificity of carbapenemase detection was 100% (97.5% one-sided CI 0.4–1.0). The corresponding sensitivities and specificities of direct disc diffusion for ertapenem resistance detection were 88.5% (95% CI 0.70–0.98) and 100% (95%CI 0.40–1.0) respectively. The turnaround time for the rapid kits coupled to the “hot chocolate” plates was 4.25 to 5.1 hours as compared to 16 hours for the conventional workup. Conclusion Rapid phenotypic tests performed after inoculation of “hot chocolate” plates are highly sensitive for the presence of carbapenemase production and can be incorporated into the laboratory workflow for Klebisella pneumoniae with important reductions in turnaround time. Disclosures All Authors: No reported disclosures

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Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2020-0581 ◽

2021 ◽

Vol 59 (1) ◽

pp. 155-163

Author(s):

Mindy Kohlhagen ◽

Surendra Dasari ◽

Maria Willrich ◽

MeLea Hetrick ◽

Brian Netzel ◽

...

Keyword(s):

Limit Of Detection ◽

Turnaround Time ◽

Automated System ◽

Serum Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Automated Method ◽

Positive Specimen ◽

Specimen Identification ◽

Tof Ms

AbstractObjectivesA matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method (Mass-Fix) as a replacement for gel-based immunofixation (IFE) has been recently described. To utilize Mass-Fix clinically, a validated automated method was required. Our aim was to automate the pre-analytical processing, improve positive specimen identification and ergonomics, reduce paper data storage and increase resource utilization without increasing turnaround time.MethodsSerum samples were batched and loaded onto a liquid handler along with reagents and a barcoded sample plate. The pre-analytical steps included: (1) Plating immunopurification beads. (2) Adding 10 μl of serum. (3) Bead washing. (4) Eluting the immunoglobulins (Igs), and reducing to separate the heavy and light Ig chains. The resulting plate was transferred to a second low-volume liquid handler for MALDI plate spotting. MALDI-TOF mass spectra were collected. Integrated in-house developed software was utilized for sample tracking, driving data acquisition, data analysis, history tracking, and result reporting. A total of 1,029 residual serum samples were run using the automated system and results were compared to prior electrophoretic results.ResultsThe automated Mass-Fix method was capable of meeting the validation requirements of concordance with IFE, limit of detection (LOD), sample stability and reproducibility with a low repeat rate. Automation and integrated software allowed a single user to process 320 samples in an 8 h shift. Software display facilitated identification of monoclonal proteins. Additionally, the process maintains positive specimen identification, reduces manual pipetting, allows for paper free tracking, and does not significantly impact turnaround time (TAT).ConclusionsMass-Fix is ready for implementation in a high-throughput clinical laboratory.

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COVID-19 Biomarkers and Advanced Sensing Technologies for Point-of-Care (POC) Diagnosis

Bioengineering ◽

10.3390/bioengineering8070098 ◽

2021 ◽

Vol 8 (7) ◽

pp. 98

Author(s):

Ernst Emmanuel Etienne ◽

Bharath Babu Nunna ◽

Niladri Talukder ◽

Yudong Wang ◽

Eon Soo Lee

Keyword(s):

Response Times ◽

Rna Virus ◽

Point Of Care ◽

Protein Detection ◽

Turnaround Time ◽

Quantitative Detection ◽

Rt Pcr ◽

Specificity And Sensitivity ◽

Small Market ◽

Multiple Biomarkers

COVID-19, also known as SARS-CoV-2 is a novel, respiratory virus currently plaguing humanity. Genetically, at its core, it is a single-strand positive-sense RNA virus. It is a beta-type Coronavirus and is distinct in its structure and binding mechanism compared to other types of coronaviruses. Testing for the virus remains a challenge due to the small market available for at-home detection. Currently, there are three main types of tests for biomarker detection: viral, antigen and antibody. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) remains the gold standard for viral testing. However, the lack of quantitative detection and turnaround time for results are drawbacks. This manuscript focuses on recent advances in COVID-19 detection that have lower limits of detection and faster response times than RT-PCR testing. The advancements in sensing platforms have amplified the detection levels and provided real-time results for SARS-CoV-2 spike protein detection with limits as low as 1 fg/mL in the Graphene Field Effect Transistor (FET) sensor. Additionally, using multiple biomarkers, detection levels can achieve a specificity and sensitivity level comparable to that of PCR testing. Proper biomarker selection coupled with nano sensing detection platforms are key in the widespread use of Point of Care (POC) diagnosis in COVID-19 detection.

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Progesterone Acts via Progesterone Receptors A and B to Regulate Breast Cancer Resistance Protein Expression: Fig. 1.

Molecular Pharmacology ◽

10.1124/mol.107.044289 ◽

2007 ◽

Vol 73 (3) ◽

pp. 613-615 ◽

Cited By ~ 18

Author(s):

Mary Vore ◽

Markos Leggas

Keyword(s):

Breast Cancer ◽

Protein Expression ◽

Breast Cancer Resistance Protein ◽

Progesterone Receptors ◽

Cancer Resistance ◽

Resistance Protein

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Simultaneous Determination of Four Herbicide and Pesticide Residues in Chinese Green Tea Using QuEChERS Purification Procedure and UPLC-MS/MS Analysis

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.634-638.1586 ◽

2013 ◽

Vol 634-638 ◽

pp. 1586-1590

Author(s):

Su Fang Wang ◽

Shou Jie Zhang ◽

Chun Hong Dong ◽

Guo Qing Wang ◽

Jun Feng Guo ◽

...

Keyword(s):

Formic Acid ◽

Simultaneous Determination ◽

Green Tea ◽

Limit Of Detection ◽

Multiple Reaction Monitoring ◽

Graphitized Carbon Black ◽

Ms Analysis ◽

Relative Standard ◽

Limit Of Quantitation

A method for simultaneous determination of residuals of four herbicides and pesticides, simazine, carboxin, diflubenzuron and rotenone, in Chinese green tea was developed. In the proposed method, the tea powder was placed in a centrifuge tube with a plug, extracted in saturated aqueous sodium chloride solution and acetonitrile, agitated using vortex oscillator, and then centrifuged 5 min at 4000 rpm. The supernatant solution was purified by primary secondary amine (PSA) sorbent, C18 power, and graphitized carbon black powder, respectively. Then the purified extracts were dissolved with acetonitrile:0.1% formic acid aqueous solution (40:60, V/V) and agitated, filtered using a syringe with 0.22 μm nylon filter prior to UPLC-MS/MS analysis. The UPLC analysis was performed on an ACQUITY UPLC® HSS T3 column (2.1 mm×100 mm, 1.8 µm), using acetonitrile-0.1% formic acid as mobile phase with the flow rate as 0.3 mL•min-1. Injection volume was 10 µL. Positive ionization mode was applied, and the ions were monitored in the multiple reaction monitoring (MRM) mode with curtain gas 0.069 MPa, collision gas 0.052 MPa, ESI ion spray voltage 5000 V, temperature 550 °C, nebulizer gas 0.24 MPa, and turbo gas 0.28 MPa. The limit of detection (LOD) and limit of quantitation (LOQ) of the proposed method are 1 μg•kg-1and 5 μg•kg-1, respectively. The average recoveries of the four pesticides at 10, 20, and 50 µg•kg-1spiking levels range from 77.4% to 95.3%. TheSupersSuperscript textcript textrelative standard deviation (RSD) (n=6) range form 11.83% to 4.52%.

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SCA Medium: A New Culture Medium for the Isolation of All Candida auris Clades

Journal of Fungi ◽

10.3390/jof7060433 ◽

2021 ◽

Vol 7 (6) ◽

pp. 433

Author(s):

Ahmad Ibrahim ◽

Lucie Peyclit ◽

Rim Abdallah ◽

Saber Khelaifia ◽

Amanda Chamieh ◽

...

Keyword(s):

Culture Medium ◽

Limit Of Detection ◽

Bacterial Species ◽

Multidrug Resistant ◽

Rapid Identification ◽

Clinical Samples ◽

Candida Auris ◽

Phenotypic Profile ◽

Stool Samples ◽

Selective Culture Medium

Candida auris is an emerging multidrug-resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation are necessary for diagnosis and containing its spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection ranging between 101 and 102 CFU/mL. The 100% specificity of SCA (specific C. auris) medium is confirmed on a set of 135 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, allowing the latter to study its phenotypic profile.

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Detection and Differentiation of Avian Mycoplasmas by Surface-Enhanced Raman Spectroscopy Based on a Silver Nanorod Array

Applied and Environmental Microbiology ◽

10.1128/aem.07419-11 ◽

2011 ◽

Vol 78 (6) ◽

pp. 1930-1935 ◽

Cited By ~ 23

Author(s):

Suzanne L. Hennigan ◽

Jeremy D. Driskell ◽

Naola Ferguson-Noel ◽

Richard A. Dluhy ◽

Yiping Zhao ◽

...

Keyword(s):

Raman Spectroscopy ◽

Sensitivity And Specificity ◽

Limit Of Detection ◽

Direct Detection ◽

Nanorod Array ◽

Surface Enhanced Raman Spectroscopy ◽

Mycoplasma Species ◽

Content Type ◽

Surface Enhanced ◽

Surface Enhanced Raman

ABSTRACTMycoplasma gallisepticumis a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to containM. gallisepticuminfections and ensure mycoplasma-free avian stocks, but several factors make detection ofM. gallisepticumand diagnosis ofM. gallisepticuminfection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array–surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, includingAcholeplasma laidlawii,Mycoplasma gallinarum,Mycoplasma gallinaceum,Mycoplasma synoviae, andM. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection ofM. gallisepticumin choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.

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