Bio-Rad (France)

Salmonella contamination in poultry remains a persistent public health challenge, despite ongoing regulatory efforts to reduce its presence. Traditional culture-based enrichment methods commonly employed for detecting Salmonella have inherent limitations, such as biases in bacterial growth and inaccurate quantification of initial contamination levels. This study introduces an enrichment-free approach for quantifying Salmonella in raw poultry, utilizing the Pathotrak system for bacterial separation and concentration, followed by Droplet Digital PCR (ddPCR) for absolute quantification. Our findings highlight that whole-cell preparations within ddPCR droplets yield significantly higher detection efficiency compared to extracted DNA, likely due to the reduced loss of genetic material during processing. The method was validated by correlating theoretical inoculation levels with ddPCR measurements, resulting in a strong linear relationship (R2=0.903) and a slope of 0.99, indicating high accuracy. Residuals from the regression model were normally distributed, confirming its validity. This platform offers a robust, rapid, and precise tool for detecting low levels of Salmonella in poultry products without enrichment, making it a more accurate and efficient approach to bacterial quantification in complex food matrices.

Abstract Background ISO document 14971-Application of Risk Management to Medical Devices and CLSI document EP23-Laboratory Quality Control Based on Risk Management use a Severity of Harm model to designate maximum acceptable probability of patient harm (risk) based on the severity of the consequence to the patient. When designing a risk managed quality control program for the clinical diagnostic laboratory, the starting point is to select Severity of Harm categories for each analyte being tested. As each Severity of Harm Category (Negligible,Minor,Serious,Critical,Catastrophic) can be mapped to a corresponding maximum acceptable probability of patient harm from erroneous results (0.01,0.001,0.0001, 0.00001,0.000001). The laboratory can then design quality control strategies that have a predicted probability of patient harm below this maximum for a risk managed quality control program. Unfortunately, there is little guidance on setting Severity of Harm designations, and they are by nature, subjective. To remedy this, we conducted a survey of laboratory professionals, asking them to rate the Severity of Harm for 20 analytes. Method An international Severity of Harm survey was conducted to solicit Severity of Harm designations for analytes in the clinical diagnostic laboratory. 261 respondents from 42 countries answered the question: “How would you rate the severity of patient harm if a clinician would interpret incorrect reported results from these analytes in the most unfavorable situation?”. The available ratings were described as: Results Conclusion While Severity of Harm designations are specific to the local clinical system, guidance can be provided by collecting the opinion of a wide variety of clinical laboratory professionals. This provides support for the clinical diagnostic community in building risk managed quality control programs.

Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

The Atacama Desert hosts diverse ecosystems including salt flats and shallow Andean lakes. Several heavy metals are found in the Atacama Desert, and microorganisms growing in this environment show varying levels of resistance/tolerance to copper, tellurium, and arsenic, among others. Herein, we report the genome sequence and comparative genomic analysis of a new Exiguobacterium strain, sp. SH31, isolated from an altiplanic shallow athalassohaline lake. Exiguobacterium sp. SH31 belongs to the phylogenetic Group II and its closest relative is Exiguobacterium sp. S17, isolated from the Argentinian Altiplano (95% average nucleotide identity). Strain SH31 encodes a wide repertoire of proteins required for cadmium, copper, mercury, tellurium, chromium, and arsenic resistance. Of the 34 Exiguobacterium genomes that were inspected, only isolates SH31 and S17 encode the arsenic efflux pump Acr3. Strain SH31 was able to grow in up to 10 mM arsenite and 100 mM arsenate, indicating that it is arsenic resistant. Further, expression of the ars operon and acr3 was strongly induced in response to both toxics, suggesting that the arsenic efflux pump Acr3 mediates arsenic resistance in Exiguobacterium sp. SH31.

N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling.

Introduction: The pressing need to determine the KRAS/BRAF mutational status for selecting patients with colorectal cancer (CRC) for anti-EGFR therapy provides a great opportunity to use circulating DNA (ctDNA) as a theranostic tool for personalized medicine. Better understanding of ctDNA origin (necrosis, apoptosis and active release) may increase the reliability of using abnormal ctDNA as biomarker.Areas covered: The authors showed that examining the proportion of ctDNA originating from tumor, microenvironment and normal cells, through size distribution and mutation load may help to discriminate mechanisms of ctDNA release.Expert opinion: Contrary to the literature, it was observed that tumor-derived ctDNA was mostly shorter than 100 bp. Tumor-derived ctDNA from cancer patients exhibited a specific ctDNA size distribution profile and significantly higher ctDNA fragmentation than ctDNA from healthy individuals. Examination of the KRAS and BRAF mutational load in 48 mutated samples revealed very high variation ranging from 0.037 to 68.8%. This suggests either that tumor cells variably release ctDNA compared with tumor-associated stroma cells or normal cells, or that mutant ctDNA analysis may depend on tumor clonality. Detection of point mutation by quantifying the proportion of mutant ctDNA fragments provides a powerful tool for assessing the proportion of ctDNA from different origins.