Publications by authors named "Stéphanie Simon"

103 Publications

NeuroTorp, a lateral flow test based on toxin-receptor affinity for in-situ early detection of cyclic imine toxins.

Anal Chim Acta 2022 Aug 20;1221:339941. Epub 2022 May 20.

Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour La Santé (DMTS), SIMoS/ Laboratoire Toxines, Récepteurs et Canaux Ioniques, 91191 Gisf-sur-Yvette, France; CNRS, ERL9004, 91191 Gisf-sur-Yvette, France. Electronic address:

The emergent cyclic imine toxins produced by marine dinoflagellates are potent antagonists of nicotinic acetylcholine receptors. Shellfish accumulate cyclic imine toxins following filter-feeding on toxic dinoflagellates vectoring them to humans. Herein is presented a lateral flow test for the detection of cyclic imine toxins based on three new concepts for test strips: i) the immobilization of lipoprotein vesicles in the test-line, ii) the high affinity of neurotoxins for their receptor targets and iii) the use of high porosity glass fiber filter membranes as support for the fabrication of the lateral flow test NeuroTorp (WO2017108115). Purified electrocyte membrane vesicles from Torpedo marmorata were used as a source of receptor and were immobilized in the test-line. Biotin-α-bungarotoxin was used as toxin tracer for the NeuroTorp LFT given its high affinity for nicotinic acetylcholine receptors while neutravidin nanogold particle conjugates enable its visual detection. Herein is reported for the first time the use of GF/C glass fiber membranes as the stationary phase for a lateral flow test. The GF/C filter ensures both: the immobilization of a complex lipoprotein in the test-line and the capillary migration of the mobile phase. Scanning electron microscopy studies shed light into the mechanism by which Torpedo-electrocyte membranes vesicles are immobilized in the GF/C glass microfiber. The electrocyte membrane vesicles anchor in neighboring microfibers randomly disposed in the same plane of the GF/C filter forming stable microfilm structures ensuring the functionality of nicotinic acetylcholine receptors. NeuroTorp is a ready-to-use low-cost early warning device for rapid detection of cyclic imine toxins in shellfish by end-users.
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http://dx.doi.org/10.1016/j.aca.2022.339941DOI Listing
August 2022

The Revolution of Lateral Flow Assay in the Field of AMR Detection.

Diagnostics (Basel) 2022 Jul 19;12(7). Epub 2022 Jul 19.

Département Médicaments et Technologies Pour la Santé (DMTS), Université Paris Saclay, CEA, INRAE, SPI, 91191 Gif-sur-Yvette, France.

The global spread of antimicrobial resistant (AMR) bacteria represents a considerable public health concern, yet their detection and identification of their resistance mechanisms remain challenging. Optimal diagnostic tests should provide rapid results at low cost to enable implementation in any microbiology laboratory. Lateral flow assays (LFA) meet these requirements and have become essential tools to combat AMR. This review presents the versatility of LFA developed for the AMR detection field, with particular attention to those directly triggering β-lactamases, their performances, and specific limitations. It considers how LFA can be modified by detecting not only the enzyme, but also its β-lactamase activity for a broader clinical sensitivity. Moreover, although LFA allow a short time-to-result, they are generally only implemented after fastidious and time-consuming techniques. We present a sample processing device that shortens and simplifies the handling of clinical samples before the use of LFA. Finally, the capacity of LFA to detect amplified genetic determinants of AMR by isothermal PCR will be discussed. LFA are inexpensive, rapid, and efficient tools that are easy to implement in the routine workflow of laboratories as new first-line tests against AMR with bacterial colonies, and in the near future directly with biological media.
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http://dx.doi.org/10.3390/diagnostics12071744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9317642PMC
July 2022

Proof of concept of a two-stage GMR sensor-based lab-on-a-chip for early diagnostic tests.

Lab Chip 2022 Jul 12;22(14):2753-2765. Epub 2022 Jul 12.

Université Paris-Saclay, CEA, CNRS, Service de Physique de l'Etat Condensé (SPEC), 91191 Gif-sur-Yvette, France.

The development of rapid, sensitive, portable and inexpensive early diagnostic techniques is a real challenge in the fields of health, defense and in the environment. The current global pandemic has also shown the need for such tests. The World Health Organization has defined ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users) that field diagnostic tests must fulfill, which proves the real need in terms of public health. Giant magnetoresistance (GMR) sensors, which have flourished in a wide variety of spintronic applications (automobile industry, Information Technology, ), also have real potential in the field of health, particularly for the development of early diagnostic point-of-care devices. This work presents a new type of innovative biochip, consisting of GMR sensors arranged on both sides of a microfluidic channel which allow on the one hand to count magnetic objects one by one but also to better distinguish false positives (aggregates of beads, ) from labelled biological targets of interest by determining their magnetic moment. We present the operating principle of this new tool and its great potential as a versatile diagnostic test.
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http://dx.doi.org/10.1039/d2lc00353hDOI Listing
July 2022

Introduction to the Special Issue: "Antibodies for Toxins: From Detection to Therapeutics".

Toxins (Basel) 2022 05 23;14(5). Epub 2022 May 23.

Département Médicaments et Technologies Pour la Santé (DMTS), Université Paris-Saclay, CEA, INRAE, MetaboHUB, F-91191 Gif sur Yvette, France.

This Special Issue aims to provide an up-to-date investigation and reviews linked to antibody-based technologies for medical countermeasures and detection/diagnosis tools for toxins [...].
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http://dx.doi.org/10.3390/toxins14050363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9146352PMC
May 2022

Deep mutational engineering of broadly-neutralizing nanobodies accommodating SARS-CoV-1 and 2 antigenic drift.

MAbs 2022 Jan-Dec;14(1):2076775

CEA, INRAE, Medicines and Healthcare Technologies Department, SIMoS, Université Paris-Saclay, Gif-sur-Yvette, France.

Here, we report the molecular engineering of nanobodies that bind with picomolar affinity to both SARS-CoV-1 and SARS-CoV-2 receptor-binding domains (RBD) and are highly neutralizing. We applied deep mutational engineering to VHH72, a nanobody initially specific for SARS-CoV-1 RBD with little cross-reactivity to SARS-CoV-2 antigen. We first identified all the individual VHH substitutions that increase binding to SARS-CoV-2 RBD and then screened highly focused combinatorial libraries to isolate engineered nanobodies with improved properties. The corresponding VHH-Fc molecules show high affinities for SARS-CoV-2 antigens from various emerging variants and SARS-CoV-1, block the interaction between ACE2 and RBD, and neutralize the virus with high efficiency. Its rare specificity across sarbecovirus relies on its peculiar epitope outside the immunodominant regions. The engineered nanobodies share a common motif of three amino acids, which contribute to the broad specificity of recognition. Our results show that deep mutational engineering is a very powerful method, especially to rapidly adapt existing antibodies to new variants of pathogens.
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http://dx.doi.org/10.1080/19420862.2022.2076775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9132424PMC
May 2022

Dual Blockade of Misfolded Alpha-Sarcoglycan Degradation by Bortezomib and Givinostat Combination.

Front Pharmacol 2022 27;13:856804. Epub 2022 Apr 27.

CECS, I-Stem, Corbeil-Essonne, France.

Limb-girdle muscular dystrophy type R3 (LGMD R3) is a rare genetic disorder characterized by a progressive proximal muscle weakness and caused by mutations in the gene encoding alpha-sarcoglycan (α-SG). Here, we report the results of a mechanistic screening ascertaining the molecular mechanisms involved in the degradation of the most prevalent misfolded R77C-α-SG protein. We performed a combinatorial study to identify drugs potentializing the effect of a low dose of the proteasome inhibitor bortezomib on the R77C-α-SG degradation inhibition. Analysis of the screening associated to artificial intelligence-based predictive ADMET characterization of the hits led to identification of the HDAC inhibitor givinostat as potential therapeutical candidate. Functional characterization revealed that givinostat effect was related to autophagic pathway inhibition, unveiling new theories concerning degradation pathways of misfolded SG proteins. Beyond the identification of a new therapeutic option for LGMD R3 patients, our results shed light on the potential repurposing of givinostat for the treatment of other genetic diseases sharing similar protein degradation defects such as LGMD R5 and cystic fibrosis.
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http://dx.doi.org/10.3389/fphar.2022.856804DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9093689PMC
April 2022

Multiplex Detection of 24 Staphylococcal Enterotoxins in Culture Supernatant Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry.

Toxins (Basel) 2022 03 31;14(4). Epub 2022 Mar 31.

Laboratory for Food Safety, ANSES, Université Paris-Est, 91191 Maisons-Alfort, France.

Staphylococcal food poisoning outbreaks are caused by the ingestion of food contaminated with staphylococcal enterotoxins (SEs). Among the 27 SEs described in the literature to date, only a few can be detected using immuno-enzymatic-based methods that are strongly dependent on the availability of antibodies. Liquid chromatography, coupled to high-resolution mass spectrometry (LC-HRMS), has, therefore, been put forward as a relevant complementary method, but only for the detection of a limited number of enterotoxins. In this work, LC-HRMS was developed for the detection and quantification of 24 SEs. A database of 93 specific signature peptides and LC-HRMS parameters was optimized using sequences from 24 SEs, including their 162 variants. A label-free quantification protocol was established to overcome the absence of calibration standards. The LC-HRMS method showed high performance in terms of specificity, sensitivity, and accuracy when applied to 49 enterotoxin-producing strains. SE concentrations measured depended on both SE type and the coagulase-positive staphylococci (CPS) strain. This study indicates that LC-MS is a relevant alternative and complementary tool to ELISA methods. The advantages of LC-MS clearly lie in both the multiplex analysis of a large number of SEs, and the automated analysis of a high number of samples.
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http://dx.doi.org/10.3390/toxins14040249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9031063PMC
March 2022

TA100 and TA1535 and WP2 uvrA are highly sensitive to detect the mutagenicity of short Alkyl-N-Nitrosamines in the Bacterial Reverse Mutation Test.

Toxicol Rep 2022 8;9:250-255. Epub 2022 Feb 8.

Merck Healthcare KGaA, Frankfurter Straße 250, 64293, Darmstadt, Germany.

Humans are exposed to low levels of N-nitrosamines via different sources. N-Nitrosamines have recently been detected as impurities in various marketed drugs and they are known mutagenic carcinogens belonging to the cohort of concern as referred to in the ICH M7 guideline. Despite their well-known mutagenic properties, there is ongoing discussion on the suitability of the bacterial reverse mutation assay and using induced rat liver S9 as the external source of metabolism to detect their mutagenic potential. Therefore, we have investigated the mutagenic potential of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, and N-nitrosodibutylamine under various conditions. Our work showed that the bacterial reverse mutation assay applying plate incorporation or preincubation protocols and using strains TA100 and TA1535 and WP2 uvrA is suitable to predict the mutagenicity of n-nitrosamines in the presence of phenobarbital/β-naphthoflavone induced rat liver S9.
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http://dx.doi.org/10.1016/j.toxrep.2022.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8850549PMC
February 2022

Comparison of Three Expanded-Spectrum Cephalosporin Hydrolysis Assays and the NG-Test CTX-M Multi Assay That Detects All CTX-M-Like Enzymes.

Diagnostics (Basel) 2022 Jan 14;12(1). Epub 2022 Jan 14.

Team "Resist" UMR1184 Immunology of Viral, Auto-Immune, Hematological and Bacterial Diseases (IMVA-HB), INSERM, Faculty of Medicine, Université Paris-Saclay, CEA, LabEx LERMIT, 94270 Le Kremlin-Bicêtre, France.

Rapid detection of expanded-spectrum cephalosporins (ESC) hydrolysing enzymes is crucial to implement infection control measures and antibiotic stewardship. Here, we have evaluated three biochemical ESC hydrolysis assays (ESBL NDP test, β-LACTA™ test, LFIA-CTX assay) and the NG-Test CTX-M MULTI that detects CTX-M enzymes, on 93 well-characterized Gram-negative isolates, including 60 , 21 spp. and 12 spp. The performances were good for all three hydrolysis assays, with the LFIA-CTX being slightly more sensitive and specific on the tested panel of isolates especially with , without ambiguous results. This study showed that LFIA-CTX may be used for the detection of ESC hydrolysis as a competitive alternative to already available assays (β-LACTA™ test and ESBL NDP test) without any specific equipment and reduced hands-on-time. The lateral flow immunoassay NG-Test CTX-M MULTI has proven to be a useful, easy, rapid, and reliable confirmatory test in for detection of CTX-M-type ESBLs, which account for most of the resistance mechanisms leading to ESC resistance in , but it misses rare ESC hydrolysing β-lactamases (AmpC, minor ESBLs, and carbapenemases). Combining it with the LFIA-CTX assay would yield an assay detecting the most frequently-encountered ESBLs (CTX-M-like β-lactamases) together with ESC hydrolysis.
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http://dx.doi.org/10.3390/diagnostics12010197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8775164PMC
January 2022

Multiplex Lateral Flow Immunoassay for the Detection of Expanded-Spectrum Hydrolysis and CTX-M Enzymes.

Diagnostics (Basel) 2022 Jan 13;12(1). Epub 2022 Jan 13.

Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France.

Background: Early detection of expanded-spectrum cephalosporinase (ESC) hydrolyzing ß-lactamases is essential for antibiotic stewardship. Here we have developed a multiplex lateral flow immunoassay (LFIA) that detects cefotaxime-hydrolyzing activity as well as the most prevalent ESC-hydrolyzing ß-lactamases: the CTX-M-like.

Methods: The Rapid LFIA ESC test was evaluated retrospectively on 188 (139 Enterobacterales, 30 spp. and 14 spp.) agar-grown bacterial isolates with well-characterized ß-lactamase content. One single colony was resuspended in 150 µL extraction buffer containing cefotaxime, incubated at room temperature for 30 min prior to loading on the LFIA for reading within 10 min.

Results: Out of the 188 isolates, all 17 that did not express a β-lactamase hydrolyzing cefotaxime gave negative results, and all 171 isolates expressing a β-lactamase known to hydrolyze cefotaxime, gave a positive test result. In addition, all 86 isolates expressing a CTX-M-variant belonging to one of the five CTX-M-subgroups were correctly identified. The sensitivity and specificity was 100% for both tests.

Conclusions: The results showed that the multiplex LFIA was efficient, fast, low cost and easy to implement in routine laboratory work for the confirmation of ESC hydrolyzing activity and the presence of CTX-M enzymes.
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http://dx.doi.org/10.3390/diagnostics12010190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8775197PMC
January 2022

Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants.

J Proteome Res 2022 02 30;21(2):547-556. Epub 2021 Dec 30.

Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 91191 Gif-sur-Yvette, France.

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.
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http://dx.doi.org/10.1021/acs.jproteome.1c00886DOI Listing
February 2022

Rapid Detection of VanA/B-Producing Vancomycin-Resistant Enterococci Using Lateral Flow Immunoassay.

Diagnostics (Basel) 2021 Sep 29;11(10). Epub 2021 Sep 29.

Bacteriology-Hygiene Unit, Assistance Publique/Hôpitaux de Paris, Service de Bactériologie-Hygiène, Bicêtre Hospital, 94270 Le Kremlin-Bicêtre, France.

Vancomycin-resistant enterococci (VREs) have become one of the most important nosocomial pathogens worldwide, associated with increased treatment costs, prolonged hospital stays and high mortality. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. The lateral flow immunoassay NG-Test VanB (NG Biotech) was evaluated for the rapid detection of VanB-producing vancomycin-resistant enterococci (VanB-VREs) using 104 well-characterized enterococcal isolates. The sensitivity and specificity were both 100% when bacterial cells were grown in the presence of vancomycin used as a VanB inducer. The NG-Test VanB is an efficient, rapid and easy to implement assay in clinical microbiology laboratories for the confirmation of VanB-VREs from colonies. Together with the NG-Test VanA, they could replace the already existing tests available for the confirmation of acquired vancomycin resistance in enterococci, especially from selective media or from antibiograms, with 100% sensitivity and specificity. Rapid detection in less than 15 min will result in more efficient management of carriers and infected patients. In addition, these tests may be used for positive blood cultures, given a 3.5 h sub-culturing step on Chocolate agar PolyViteX in the presence of a 5-µg vancomycin disk, which is routinely performed in many clinical microbiology laboratories for every positive blood culture for subsequent MALDI-TOF identification of the growing bacteria.
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http://dx.doi.org/10.3390/diagnostics11101805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8534553PMC
September 2021

Detection of Borrelia burgdorferi antigens in tissues and plasma during early infection in a mouse model.

Sci Rep 2021 08 30;11(1):17368. Epub 2021 Aug 30.

Paris-Saclay University, CEA, INRAE, Medicines and Healthcare Technologies Department (DMTS), SPI, 91191, Gif-sur-Yvette, France.

Borrelia burgdorferi is the causative agent of Lyme borreliosis, which is the most common tick-borne human disease in Europe and North America. Currently, the diagnosis of Lyme borreliosis is based on serological tests allowing indirect detection of anti-Borrelia antibodies produced by patients. Their main drawback is a lack of sensitivity in the early phase of disease and an incapacity to prove an active infection. Direct diagnostic tests are clearly needed. The objectives of this study were to produce tools allowing sensitive detection of potential circulating Borrelia antigens and to evaluate them in a mouse model. We focused on two potential early bacterial makers, the highly variable OspC protein and the conserved protein FlaB. High-affinity monoclonal antibodies were produced and used to establish various immunoassays and western blot detection. A very good limit of detection for OspC as low as 17 pg/mL of sample was achieved with SPIE-IA. In infected mice, we were able to measure OspC in plasma with a mean value of 10 ng/mL at 7 days post-inoculation. This result suggests that OspC could be a good blood marker for diagnosis of Lyme borreliosis and that the tools developed during this study could be very useful.
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http://dx.doi.org/10.1038/s41598-021-96861-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405660PMC
August 2021

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

Anal Bioanal Chem 2022 Jan 25;414(2):759-789. Epub 2021 Aug 25.

Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (MTS), Gif-sur-Yvette cedex, 91191, France.

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.
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http://dx.doi.org/10.1007/s00216-021-03586-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386160PMC
January 2022

Detection of expanded-spectrum cephalosporin hydrolysis by lateral flow immunoassay.

Microb Biotechnol 2022 02 2;15(2):603-612. Epub 2021 Aug 2.

Département Médicaments et Technologies pour la Santé (DMTS), SPI, Université Paris-Saclay, CEA, INRAE, Gif-sur-Yvette, 91191, France.

Early detection of expanded-spectrum cephalosporin (ESC) resistance is essential not only for an effective therapy but also for the prompt implementation of infection control measures to prevent dissemination in the hospital. We have developed and validated a lateral flow immunoassay (LFIA), called LFIA-CTX test, for the detection of ESC (cefotaxime) hydrolytic activity based on structural discrimination between the intact antibiotic and its hydrolysed product. A single bacterial colony was suspended in an extraction buffer containing cefotaxime. After a 30-min incubation, the solution is loaded on the LFIA for reading within 10 min. A total of 348 well-characterized Gram-negative isolates were tested. Among them, the 38 isolates that did not express any cefotaxime-hydrolysing β-lactamase gave negative results. Of the 310 isolates expressing at least one cefotaxime-hydrolysing β-lactamase, all were tested positive, except three OXA-48-like producers, which were repeatedly detected negative. Therefore, the sensitivity was 99.1% and the specificity was 100%. The LFIA-CTX test is efficient, fast, low-cost and easy to implement in the workflow of a routine microbiology laboratory.
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http://dx.doi.org/10.1111/1751-7915.13892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8867991PMC
February 2022

Small Hsps as Therapeutic Targets of Cystic Fibrosis Transmembrane Conductance Regulator Protein.

Int J Mol Sci 2021 Apr 20;22(8). Epub 2021 Apr 20.

Apoptosis, Cancer and Development Laboratory, Lyon Cancer Research Center, INSERM U1052-CNRS UMR5286, Claude Bernard University Lyon 1, Centre Léon Bérard, F-69008 Lyon, France.

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal and pathological cells. Here, we have reviewed the role played by HspB1, HspB4 and HspB5 in the context of Cystic Fibrosis (CF), a severe monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR) some of which trigger its misfolding and rapid degradation, particularly the most frequent one, F508del-CFTR. While HspB1 and HspB4 favor the degradation of CFTR mutants, HspB5 and particularly one of its phosphorylated forms positively enhance the transport at the plasma membrane, stability and function of the CFTR mutant. Moreover, HspB5 molecules stimulate the cellular efficiency of currently used CF therapeutic molecules. Different strategies are suggested to modulate the level of expression or the activity of these small heat shock proteins in view of potential in vivo therapeutic approaches. We then conclude with other small heat shock proteins that should be tested or further studied to improve our knowledge of CFTR processing.
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http://dx.doi.org/10.3390/ijms22084252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8072646PMC
April 2021

Differentiation, Quantification and Identification of Abrin and Agglutinin.

Toxins (Basel) 2021 04 18;13(4). Epub 2021 Apr 18.

Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute, Seestr. 10, 13353 Berlin, Germany.

Abrin, the toxic lectin from the rosary pea plant has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as agglutinin or the homologous toxin ricin from are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving .
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http://dx.doi.org/10.3390/toxins13040284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073929PMC
April 2021

Innovative and Highly Sensitive Detection of Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Toxins (Basel) 2021 04 8;13(4). Epub 2021 Apr 8.

Biological Toxins, Centre for Biological Threats and Special Pathogens, Robert Koch Institute (RKI), Seestr. 10, 13353 Berlin, Germany.

enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.
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http://dx.doi.org/10.3390/toxins13040266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068247PMC
April 2021

Characterization of a highly neutralizing single monoclonal antibody to botulinum neurotoxin type A.

FASEB J 2021 05;35(5):e21540

Institut Pasteur, Unité des Toxines Bactériennes, UMR CNRS 2001, Paris, France.

Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the H and H subdomains of the BoNT/A1 receptor-binding domain (H ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2- and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to H confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.
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http://dx.doi.org/10.1096/fj.202002492RDOI Listing
May 2021

The Microbiome Meets Nanotechnology: Opportunities and Challenges in Developing New Diagnostic Devices.

Adv Mater 2021 May 14;33(18):e2006104. Epub 2021 Mar 14.

Nanobioelectronics and Biosensors Group, Institut Català de Nanociència i Nanotecnologia (ICN2), UAB Campus, Bellaterra, Barcelona, 08193, Spain.

Monitoring of the human microbiome is an emerging area of diagnostics for personalized medicine. Here, the potential of different nanomaterials and nanobiosensing technologies is reviewed for the development of novel diagnostic devices for the detection and measurement of microbiome-related biomarkers. Moreover, the current and future landscape of microbiome-based diagnostics is defined by exploring the advantages and disadvantages of current nanotechnology-based approaches, especially in the context of developing point-of-care (PoC) devices that would meet the international guidelines known as REASSURED (Real-time connectivity; Ease of specimen collection; Affordability; Sensitivity; Specificity; User-friendliness; Rapid & robust operation; Equipment-free; and Deliverability). Finally, the strategies of the latest international scientific consortia working in this field are analyzed, the current microbiome diagnostics market are reported and the principal ethical, legal, and societal issues related to microbiome R&D and innovation are discussed.
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http://dx.doi.org/10.1002/adma.202006104DOI Listing
May 2021

An antibody targeting type III secretion system induces broad protection against Salmonella and Shigella infections.

PLoS Negl Trop Dis 2021 03 12;15(3):e0009231. Epub 2021 Mar 12.

Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, Gif-sur-Yvette, France.

Salmonella and Shigella bacteria are food- and waterborne pathogens that are responsible for enteric infections in humans and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics requires the development of broadly protective therapies. Recently, the needle tip proteins of the type III secretion system of these bacteria were successfully utilized (SipD for Salmonella and IpaD for Shigella) as vaccine immunogens to provide good prophylactic cross-protection in murine models of infections. From these experiments, we have isolated a cross-protective monoclonal antibody directed against a conserved region of both proteins. Its conformational epitope determined by Deep Mutational Scanning is conserved among needle tip proteins of all pathogenic Shigella species and Salmonella serovars, and are well recognized by this antibody. Our study provides the first in vivo experimental evidence of the importance of this common region in the mechanism of virulence of Salmonella and Shigella and opens the way to the development of cross-protective therapeutic agents.
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http://dx.doi.org/10.1371/journal.pntd.0009231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990167PMC
March 2021

Quantitative Determination of Enterotoxins Types A to I and Variants in Dairy Food Products by Multiplex Immuno-LC-MS/MS.

J Agric Food Chem 2021 Mar 17;69(8):2603-2610. Epub 2021 Feb 17.

Département Médicaments et Technologies pour la Santé (DMTS), SPI, Université Paris-Saclay, CEA, INRAE, 91191 Gif-sur-Yvette, France.

Staphylococcal enterotoxins (SEs) are responsible for frequent food poisoning outbreaks worldwide. Specific identification of SEs is crucial for confirmation of food poisoning, tracking of the incriminated foods or food ingredients, and removal from the food chain. Here, we report on a new food testing protocol addressing the challenge of low abundance of SEs in contaminated food and high sequence heterogeneity. Multiplex ability of targeted high-resolution mass spectrometry was succesfully applied to the simultaneous and quantitative determination of the eight most frequent SEs including sequence variants. In this aim, between three and eight proteotypic peptides of each SE were selected by carefully considering amino acid variations within each type, and sequence homology between types. Quantification of trace levels of SEs directly in food samples was reached by immunoaffinity enrichment and optimized analytical conditions. The assay was validated in dairy food products with a lower limit of quantification down to 0.1 ng/g (in milk), and quantification of SEs was successfully demonstrated in real-life samples collected during staphylococcal food poisoning outbreaks. Importantly, the ability of the method to detect diverse sequence variants was also illustrated. By enabling for the first time the simultaneous quantification of the eight most frequent SEs, the new mass spectrometry-based assay would facilitate the laboratory confirmation of positive samples in situation of food poisoning outbreaks.
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http://dx.doi.org/10.1021/acs.jafc.0c07545DOI Listing
March 2021

Ricin Antibodies' Neutralizing Capacity against Different Ricin Isoforms and Cultivars.

Toxins (Basel) 2021 01 29;13(2). Epub 2021 Jan 29.

Paris-Saclay University, CEA, INRAE, Medicines and Healthcare Technologies Department (DMTS), SPI, 91191 Gif-sur-Yvette, France.

Ricin, a highly toxic protein from , is considered a potential biowarfare agent. Despite the many data available, no specific treatment has yet been approved. Due to their ability to provide immediate protection, antibodies (Abs) are an approach of choice. However, their high specificity might compromise their capacity to protect against the different ricin isoforms (D and E) found in the different cultivars. In previous work, we have shown the neutralizing potential of different Abs (43RCA-G1 (anti ricin A-chain) and RB34 and RB37 (anti ricin B-chain)) against ricin D. In this study, we evaluated their protective capacity against both ricin isoforms. We show that: (i) RB34 and RB37 recognize exclusively ricin D, whereas 43RCA-G1 recognizes both isoforms, (ii) their neutralizing capacity in vitro varies depending on the cultivar, and (iii) there is a synergistic effect when combining RB34 and 43RCA-G1. This effect is also demonstrated in vivo in a mouse model of intranasal intoxication with ricin D/E (1:1), where approximately 60% and 40% of mice treated 0 and 6 h after intoxication, respectively, are protected. Our results highlight the importance of evaluating the effectiveness of the Abs against different ricin isoforms to identify the treatment with the broadest spectrum neutralizing effect.
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http://dx.doi.org/10.3390/toxins13020100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911099PMC
January 2021

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay.

Toxins (Basel) 2021 02 9;13(2). Epub 2021 Feb 9.

CEA, INRAE, Medicines and Healthcare Technologies Department (DMTS), Paris-Saclay University, SPI, 91191 Gif-sur-Yvette, France.

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly . Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of , contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.
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http://dx.doi.org/10.3390/toxins13020130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916246PMC
February 2021

Development and Evaluation of an Immuno-MALDI-TOF Mass Spectrometry Approach for Quantification of the Abrin Toxin in Complex Food Matrices.

Toxins (Basel) 2021 01 13;13(1). Epub 2021 Jan 13.

CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), Université Paris Saclay, SPI, 91191 Gif-sur-Yvette, France.

The toxin abrin found in the seeds of has attracted much attention regarding criminal and terroristic misuse over the past decade. Progress in analytical methods for a rapid and unambiguous identification of low abrin concentrations in complex matrices is essential. Here, we report on the development and evaluation of a MALDI-TOF mass spectrometry approach for the fast, sensitive and robust abrin isolectin identification, differentiation and quantification in complex food matrices. The method combines immunoaffinity-enrichment with specific abrin antibodies, accelerated trypsin digestion and the subsequent MALDI-TOF analysis of abrin peptides using labeled peptides for quantification purposes. Following the optimization of the workflow, common and isoform-specific peptides were detected resulting in a ~38% sequence coverage of abrin when testing ng-amounts of the toxin. The lower limit of detection was established at 40 ng/mL in milk and apple juice. Isotope-labeled versions of abundant peptides with high ionization efficiency were added. The quantitative evaluation demonstrated an assay variability at or below 22% with a linear range up to 800 ng/mL. MALDI-TOF mass spectrometry allows for a simple and fast (<5 min) analysis of abrin peptides, without a time-consuming peptide chromatographic separation, thus constituting a relevant alternative to liquid chromatography-tandem mass spectrometry.
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http://dx.doi.org/10.3390/toxins13010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828309PMC
January 2021

Implementing a standardized screening protocol for parental depression, anxiety, and PTSD symptoms in the Neonatal Intensive Care Unit.

Early Hum Dev 2021 03 16;154:105279. Epub 2020 Nov 16.

Stanford University School of Medicine, Palo Alto, CA, USA. Electronic address:

The aim of this paper is to describe the development of a standardized screening program for parents of infants in the Neonatal Intensive Care Unit (NICU) and to assess its implementation. The standardized screening protocol assessed parental mental health symptoms including depression, anxiety and trauma. Screening began at 14 days post NICU admission and was implemented as part of routine medical care for all caregivers with infants admitted to the NICU at two weeks of age. Screenings were facilitated by pediatric social workers and psychology postdoctoral fellows and included review of critical self-harm items. A total of 158 parents ages 18-42 years (mean = 31.04) were eligible for screening, with 150 completed screenings. Positive screens on any of the three measures resulted in a mental health referral. Approximately 27% of parents had a positive screen that resulted in a mental health referral. The standardized screening protocol was found to be feasible, widely accepted, and effective in establishing referrals for in house mental health services. This model can be used as an example to help other NICUs implement their own universal screening protocols.
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http://dx.doi.org/10.1016/j.earlhumdev.2020.105279DOI Listing
March 2021

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

J Antimicrob Chemother 2021 01;76(1):146-151

Team ReSIST, INSERM U1184, School of Medicine, Université Paris-Saclay, LabEx LERMIT, Le Kremlin-Bicêtre, France.

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.
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http://dx.doi.org/10.1093/jac/dkaa413DOI Listing
January 2021

Prevention of posttraumatic stress disorder in mothers of preterm infants using trauma-focused group therapy: Manual development and evaluation.

Early Hum Dev 2021 03 16;154:105282. Epub 2020 Nov 16.

Division of Child and Adolescent Psychiatry, Stanford University School of Medicine, 401 Quarry Road, Palo Alto, CA 94305, USA. Electronic address:

Background: Preterm birth has been associated with a number of adverse maternal psychological outcomes.

Aims: The current study aims to develop and evaluate the feasibility of a trauma-focused group intervention that is designed to reduce maternal symptoms of anxiety, depression, and posttraumatic stress in a sample of mothers of preterm infants hospitalized in a neonatal intensive care unit (NICU).

Study Design: The study was a one-group pre-/post quasi-experimental design. Participants received a 6-session intervention targeting parental trauma.

Subjects: English-speaking mothers (N = 19) greater than 18 years of age of infants 23-34 weeks gestational age hospitalized in the NICU at Lucile Packard Children's Hospital Stanford.

Outcome Measures: Beck Anxiety Inventory (BAI), Beck Depression Inventory, Second Edition (BDI-II), Davidson Trauma Scale (DTS).

Results: Results from the study indicate that the intervention is feasible, able to be implemented with a high degree of fidelity, is rated as highly satisfactory by participants, and leads to statistically significant reductions in symptoms of anxiety, depression, and posttraumatic stress at 6-week and 6-month follow-ups.

Conclusions: Though encouraging, these findings are preliminary, and future studies should strive to reproduce these findings with a larger sample size and a comparison group.
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http://dx.doi.org/10.1016/j.earlhumdev.2020.105282DOI Listing
March 2021

Methylprednisolone pulse treatment improves ProSP-C trafficking in twins with SFTPC mutation: An isoform story?

Br J Clin Pharmacol 2021 05 4;87(5):2361-2373. Epub 2021 Jan 4.

Université Paris Est Creteil, INSERM, IMRB, F-94010 Creteil, France.

Mutations in the gene encoding surfactant protein C (SP-C) cause interstitial lung disease (ILD), and glucocorticosteroid (GC) treatment is the most recognized therapy in children. We aimed to decipher the mechanisms behind successful GC treatment in twins carrying a BRICHOS c.566G > A (p.Cys189Tyr) mutation in the SP-C gene (SFTPC). METHODS: The twins underwent bronchoscopy before and after GC treatment and immunoblotting analysis of SP-C proprotein (proSP-C) and SP-C mature in bronchoalveolar fluid (BALF). Total RNA was extracted and analysed using quantitative real-time PCR assays. In A549 cells, the processing of mutated protein C189Y was studied by immunofluorescence and immunoblotting after heterologous expression of eukaryotic vectors containing wild type or C189Y mutant cDNA. RESULTS: Before treatment, BALF analysis identified an alteration of the proSP-C maturation process. Functional study of C189Y mutation in alveolar A549 cells showed that pro-SP-C was retained within the endoplasmic reticulum together with ABCA3. After 5 months of GC treatment with clinical benefit, the BALF analysis showed an improvement of proSP-C processing. SFTPC mRNA analysis in twins revealed a decrease in the expression of total SFTPC mRNA and a change in its splicing, leading to the expression of a second shorter proSP-C isoform. In A549 cells, the processing and the stability of this shorter wild-type proSP-C isoform was similar to that of the longer isoform, but the half-life of the mutated shorter isoform was decreased. These results suggest a direct effect of GC on proSP-C metabolism through reducing the SFTPC mRNA level and favouring the expression of a less stable protein isoform.
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http://dx.doi.org/10.1111/bcp.14645DOI Listing
May 2021

A Lateral Flow Immunoassay for the Rapid Identification of CTX-M-Producing Enterobacterales from Culture Plates and Positive Blood Cultures.

Diagnostics (Basel) 2020 Sep 28;10(10). Epub 2020 Sep 28.

Team Resist, UMR1184, School of Medicine of Université Paris-Saclay-INSERM-CEA, LabEx Lermit, 94276 Le Kremlin-Bicêtre, France.

We have developed a lateral flow immunoassay (LFIA), named NG-Test CTX-M MULTI (NG-Test), to detect group 1, 2, 8, 9, 25 CTX-M producers from agar plates and from positive blood cultures in less than 15 min. The NG-Test was validated retrospectively on 113 well-characterized enterobacterial isolates, prospectively on 102 consecutively isolated ESBL-producers from the Bicêtre hospital and on 100 consecutive blood cultures positive with a gram-negative bacilli (GNB). The NG-Test was able to detect all CTX-M producers grown on the different agar plates used in clinical microbiology laboratories. No false positive nor negative results were observed. Among the 102 consecutive ESBL isolates, three hyper mucous isolates showed an incorrect migration leading to invalid results (no control band). Using an adapted protocol, the results could be validated. The NG-Test detected 99/102 ESBLs as being CTX-Ms. Three SHV producers were not detected. Among the 100 positive blood cultures with GNB tested 10/11 ESBL-producers were detected (8 CTX-M-15, 2 CTX-M-27). One SHV-2-producing- was missed. The NG-Test CTX-M MULTI showed 100% sensitivity and specificity with isolates cultured on agar plates and was able to detect 98% of the ESBL-producers identified in our clinical setting either from colonies or from positive blood cultures.
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http://dx.doi.org/10.3390/diagnostics10100764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600033PMC
September 2020
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