Multicenter study to assess the use of BL-DetecTool for the detection of CTX-M-type ESBLs and carbapenemases directly from clinical specimens.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories. IMPORTANCE: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment.

Evaluation of a new rapid immunochromatographic assay for the detection of GES-producing Gram-negative bacteria.

BACKGROUND: As carbapenemase-producing Enterobacterales are increasingly reported worldwide, their rapid detection is crucial to reduce their spread and prevent infections and outbreaks. Lateral flow immunoassays (LFIAs) have become major tools for the detection of carbapenemases. However, as for most commercially available assays, only the five main carbapenemases are targeted. OBJECTIVES: Here, we have developed and evaluated an LFIA prototype for the rapid and reliable detection of the increasingly identified GES-type ß-lactamases. METHODS: The GES LFIA was validated on 103 well-characterized Gram-negative isolates expressing various ß-lactamases grown on Mueller-Hinton (MH) agar, chromogenic, and chromogenic/selective media. RESULTS: The limit of detection of the assay was 106 cfu per test with bacteria grown on MH agar plates. GES LFIA accurately detected GES-type ß-lactamases irrespective of the culture media and the bacterial host. The GES LFIA was not able to distinguish between GES-ESBLs and GES-carbapenemases. Because GES enzymes are still rare, their detection as an ESBL or a carbapenemase remains important, especially because extensive use of carbapenems to treat ESBL infections may select for GES variants capable of hydrolysing carbapenems. CONCLUSIONS: The GES LFIA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of GES-type ß-lactamases. Combining it with immunochromatographic assays targeting the five main carbapenemases (KPC, NDM, VIM, IMP and OXA-48) would improve the overall sensitivity for the most frequently encountered carbapenemases and ESBLs, especially in non-fermenters.

Rapid detection of CTX-M-type ESBLs and carbapenemases directly from biological samples using the BL-DetecTool.

BACKGROUND: Lateral flow immunoassays (LFIA) have shown their usefulness for detecting CTX-M- and carbapenemase-producing Enterobacterales (CPEs) in bacterial cultures. Here, we have developed and validated the BL-DetecTool to detect CTX-M enzymes and carbapenemases directly from clinical samples. METHODS: The BL-DetecTool is an LFIA that integrates an easy sample preparation device named SPID (Sampling, Processing, Incubation and Detection). It was evaluated in three University hospitals on urine, blood culture (BC) and rectal swab (RS) specimens either of clinical origin or on spiked samples. RS evaluation was done directly and after a 24 h enrichment step. RESULTS: The CTX-M BL-DetecTool was tested on 485 samples (154 BC, 150 urines, and 181 RS) and revealed a sensitivity and specificity of 97.04% (95% CI 92.59%-99.19%) and 99.43% (95% CI 97.95%-99.93%), respectively. Similarly, the Carba5 BL-DetecTool was tested on 382 samples (145 BC, 116 urines, and 121 RS) and revealed a sensitivity and specificity of 95.3% (95% CI 89.43%-98.47%) and 100% (95% CI 98.67%-100%), respectively. While with the Carba5 BL-DetecTool five false negatives were observed, mostly in RS samples, with the CTX-M BL-DetecTool, in addition to four false-negatives, two false-positives were also observed. Direct testing of RS samples revealed a sensitivity of 78% and 86% for CTX-M and carbapenemase detection, respectively. CONCLUSIONS: BL-DetecTool showed excellent biological performance, was easy-to-use, rapid, and could be implemented in any microbiology laboratory around the world, without additional equipment, no need for electricity, nor trained personnel. It offers an attractive alternative to costly molecular methods.

Metabolomics for personalized medicine: the input of analytical chemistry from biomarker discovery to point-of-care tests.

Metabolomics refers to the large-scale detection, quantification, and analysis of small molecules (metabolites) in biological media. Although metabolomics, alone or combined with other omics data, has already demonstrated its relevance for patient stratification in the frame of research projects and clinical studies, much remains to be done to move this approach to the clinical practice. This is especially true in the perspective of being applied to personalized/precision medicine, which aims at stratifying patients according to their risk of developing diseases, and tailoring medical treatments of patients according to individual characteristics in order to improve their efficacy and limit their toxicity. In this review article, we discuss the main challenges linked to analytical chemistry that need to be addressed to foster the implementation of metabolomics in the clinics and the use of the data produced by this approach in personalized medicine. First of all, there are already well-known issues related to untargeted metabolomics workflows at the levels of data production (lack of standardization), metabolite identification (small proportion of annotated features and identified metabolites), and data processing (from automatic detection of features to multi-omic data integration) that hamper the inter-operability and reusability of metabolomics data. Furthermore, the outputs of metabolomics workflows are complex molecular signatures of few tens of metabolites, often with small abundance variations, and obtained with expensive laboratory equipment. It is thus necessary to simplify these molecular signatures so that they can be produced and used in the field. This last point, which is still poorly addressed by the metabolomics community, may be crucial in a near future with the increased availability of molecular signatures of medical relevance and the increased societal demand for participatory medicine.

Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci.

BACKGROUND: VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. OBJECTIVES: To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. METHODS: NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. RESULTS: All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. CONCLUSIONS: NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

Improvement of the Immunochromatographic NG-Test Carba 5 Assay for the Detection of IMP Variants Previously Undetected.

Here, we evaluated the immunochromatographic assay NG-Test Carba 5v2 (NG-Biotech), with improved IMP variant detection on 31 IMP producers, representing the different branches of the IMP phylogeny, including 32 OXA-48, 19 KPC, 12 VIM, 14 NDM, and 13 multiple carbapenemase producers (CPs), 13 CPs that were not targeted, and 13 carbapenemase-negative isolates. All tested IMP variants were accurately detected without impairing detection of the other carbapenemases. Thus, NG-Test Carba 5v2 is now well adapted to countries with high IMP prevalence and to the epidemiology of CP-Pseudomonas aeruginosa, where IMPs are most frequently detected.

Development and Multicentric Validation of a Lateral Flow Immunoassay for Rapid Detection of MCR-1-Producing Enterobacteriaceae.

Colistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector. As a consequence, colistin resistance is emerging worldwide. Among the colistin resistance mechanisms, the spread of the plasmid-encoded colistin resistance gene mcr-1 (mostly in Escherichia coli) is of particular concern due to its increased transferability compared to that of chromosome-encoded resistance. The early detection of MCR-1-producing bacteria is essential to prevent further spread and provide appropriate antimicrobial therapy. Lateral flow immunoassays (LFIAs) were manufactured with selected monoclonal antibodies. A collection of 177 human and 121 animal enterobacterial isolates was tested in a multicentric study. One bacterial colony grown on agar plates was suspended in extraction buffer and dispensed on the cassette. Migration was allowed for 15 min, and the results were monitored by the appearance of a specific band. The positive results showed a pink line resulting in an unambiguous interpretation. All MCR-1-producing isolates were found to be positive by the LFIA, and no false-negative results were observed. Three out of four MCR-2-producing isolates were also found to be positive. Our test does not detect MCR-3-, MCR-4-, or MCR-5-producing isolates. LFIA allows the detection of MCR-1 with 100% sensitivity and 98% specificity. This test is fast, sensitive, specific, easy to use, and cost-effective and can therefore be implemented in any microbiology laboratory worldwide. LFIA is a major tool for the rapid detection and monitoring of MCR-1 producers in humans and animals.

A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing Enterobacteriaceae.

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM- and IMP-type and OXA-48-like). Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5. Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3% (retrospectively) to 100% (prospectively) specificity. Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae.

Rapid Detection of Abrin Toxin and Its Isoforms in Complex Matrices by Immuno-Extraction and Quantitative High Resolution Targeted Mass Spectrometry.

Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 µg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.

Development and Validation of a Lateral Flow Immunoassay for Rapid Detection of NDM-Producing Enterobacteriaceae.

The global spread of carbapenemase-producing Enterobacteriaceae (CPE) that are often resistant to most, if not all, classes of antibiotics is a major public health concern. The NDM-1 carbapenemase is among the most worrisome carbapenemases given its rapid worldwide spread. We have developed and evaluated a lateral flow immunoassay (LFIA) (called the NDM LFIA) for the rapid and reliable detection of NDM-like carbapenemase-producing Enterobacteriaceae from culture colonies. We evaluated the NDM LFIA using 175 reference enterobacterial isolates with characterized ß-lactamase gene content and 74 nonduplicate consecutive carbapenem-resistant clinical isolates referred for expertise to the French National Reference Center (NRC) for Antibiotic Resistance during a 1-week period (in June 2016). The reference collection included 55 non-carbapenemase producers and 120 carbapenemase producers, including 27 NDM producers. All 27 NDM-like carbapenemase producers of the reference collection were correctly detected in less than 15 min by the NDM LFIA, including 22 strains producing NDM-1, 2 producing NDM-4, 1 producing NDM-5, 1 producing NDM-7, and 1 producing NDM-9. All non-NDM-1 producers gave a negative result with the NDM LFIA. No cross-reaction was observed with carbapenemases (VIM, IMP, NDM, KPC, and OXA-48-like), extended-spectrum ß-lactamases (ESBLs) (TEM, SHV, and CTX-M), AmpCs (CMY-2, DHA-2, and ACC-1), and oxacillinases (OXA-1, -2, -9, and -10). Similarly, among the 74 referred nonduplicate consecutive clinical isolates, all 7 NDM-like producers were identified. Overall, the sensitivity and specificity of the assay were 100% for NDM-like carbapenemase detection with strains cultured on agar. The NDM LFIA was efficient, rapid, and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of NDM-like carbapenemase-producing Enterobacteriaceae.

Bimodal fluorescence/129Xe NMR probe for molecular imaging and biological inhibition of EGFR in Non-Small Cell Lung Cancer.

Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/129Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized 129Xe NMR. This promising system should find important applications for theranostic use.

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

Phage amplification and immunomagnetic separation combined with targeted mass spectrometry for sensitive detection of viable bacteria in complex food matrices.

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.

Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces.

Detection of Yersinia pestis in environmental and food samples by intact cell immunocapture and liquid chromatography-tandem mass spectrometry.

Yersinia pestis is the causative agent of bubonic and pneumonic plague, an acute and often fatal disease in humans. In addition to the risk of natural exposure to plague, there is also the threat of a bioterrorist act, leading to the deliberate spread of the bacteria in the environment or food. We report here an immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) method for the direct (i.e., without prior culture), sensitive, and specific detection of Y. pestis in such complex samples. In the first step, a bottom-up proteomics approach highlighted three relevant protein markers encoded by the Y. pestis-specific plasmids pFra (murine toxin) and pPla (plasminogen activator and pesticin). Suitable proteotypic peptides were thoroughly selected to monitor the three protein markers by targeted MS using the selected reaction monitoring (SRM) mode. Immunocapture conditions were optimized for the isolation and concentration of intact bacterial cells from complex samples. The immuno-LC-SRM assay has a limit of detection of 2 × 10(4) CFU/mL in milk or tap water, which compares well with those of state-of-the-art immunoassays. Moreover, we report the first direct detection of Y. pestis in soil, which could be extremely useful in confirming Y. pestis persistence in the ground.

Fast and direct extraction of cell-associated hepatotoxins from toxic cyanobacteria.

Microcystins are an important group of toxins produced by cyanobacteria of different genera. An increasing number of water contaminations by this class of toxins have been reported that are susceptible to generate important public health problems. We designed an efficient method for extracting these toxins on-site for a rapid testing of potentially contaminated water. The extraction parameters have been optimized using Microcystis aeruginosa and the technique successfully applied to different laboratory cultures and field samples. The procedure employs a simple and stable reagent mix of propanol, Tween 20, and trifluoroacetic acid. It is directly active on crude cell suspensions without any pre-treatment. Extraction yields measured by immunological quantification were at least equal to the best values obtained with the most commonly used laboratory techniques. An additional simple concentration/extraction step is also described that allows measurements on samples too dilute for direct detection by immunochromatographic techniques.

Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin.

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

A multicentre evaluation of the NG-test DetecTool OXA-23 for the rapid detection of OXA-23 carbapenemase directly from blood cultures.

Objectives: A multicentre study evaluating NG-Test DetecTool OXA-23 for the detection of OXA-23 carbapenemase directly from positive blood cultures (PBCs). Methods: The NG-Test DetecTool OXA-23 is an immunoassay that integrates a sample preparation device. We evaluated NG-Test DetecTool OXA-23 on 189 spiked and 126 clinical PBCs. The clinical samples' standard-of-care procedure consisted of bacterial identification from the first day of positivity by MALDI-TOF MS, conventional culture and antimicrobial susceptibility testing. The immunoassay results were verified molecularly. The strains used for the spiked samples consisted of well-characterized Acinetobacter baumannii and Proteus mirabilis strains. Results: The NG-Test DetecTool OXA-23 was evaluated on 315 PBCs and revealed sensitivity of 100% (95% CI: 98.21%-100.00%) and specificity of 100% (95% CI: 96.73%-100.00%). It provided 204 true-positive results for OXA-23 in 196 bottles with carbapenem-resistant A. baumannii (CRAB) and 8 bottles with carbapenem-resistant P. mirabilis and also provided 111 true-negative results. There were no false-positive and no false-negative results. Among the 315 PBCs studied, 83 clinical blood cultures collected in the ICU of a Greek university hospital, which were tested prospectively, all yielded CRAB, and OXA-23 was correctly detected in all samples from the first day of positivity using the NG-Test DetecTool OXA-23. Conclusions: The NG-Test DetecTool OXA-23 has exhibited excellent sensitivity and specificity for OXA-23 detection in PBCs and can provide valuable information for appropriate selection of antibiotic therapy and early implementation of infection control measures.

Rapid quantification of clostridial epsilon toxin in complex food and biological matrixes by immunopurification and ultraperformance liquid chromatography-tandem mass spectrometry.

Epsilon toxin (ETX) is one of the most lethal toxins produced by Clostridium species and is considered as a potential bioterrorist weapon. Here, we present a rapid mass spectrometry-based method for ETX quantification in complex matrixes. As a prerequisite, naturally occurring prototoxin and toxin species were first structurally characterized by top-down and bottom-up experiments, to identify the most pertinent peptides for quantification. Following selective ETX immunoextraction and trypsin digestion, two proteotypic peptides shared by all the toxin forms were separated by ultraperformance liquid chromatography (UPLC) and monitored by ESI-MS (electrospray ionization-mass spectrometry) operating in the multiple reaction monitoring mode (MRM) with collision-induced dissociation. Thorough protocol optimization, i.e., a 15 min immunocapture, a 2 h enzymatic digestion, and an UPLC-MS/MS detection, allowed the whole quantification process including the calibration curve to be performed in less than 4 h, without compromising assay robustness and sensitivity. The assay sensitivity in milk and serum was estimated at 5 ng·mL(-1) for ETX, making this approach complementary to enzyme linked immunosorbent assay (ELISA) techniques.