Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and gram-positive bacteria.

In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain.

A stratification strategy to predict secondary infection in critical illness-induced immune dysfunction: the REALIST score.

Background: Although multiple individual immune parameters have been demonstrated to predict the occurrence of secondary infection after critical illness, significant questions remain with regards to the selection, timing and clinical utility of such immune monitoring tests.

Research Question: As a sub-study of the REALISM study, the REALIST score was developed as a pragmatic approach to help clinicians better identify and stratify patients at high risk for secondary infection, using a simple set of relatively available and technically robust biomarkers.

Study Design And Methods: This is a sub-study of a single-centre prospective cohort study of immune profiling in critically ill adults admitted after severe trauma, major surgery or sepsis/septic shock.

Monocyte Trajectories Endotypes Are Associated With Worsening in Septic Patients.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The immune system plays a key role in sepsis onset and remains dysregulated over time in a heterogeneous manner. Here, we decipher the heterogeneity of the first week evolution of the monocyte HLA-DR (mHLA-DR) surface protein expression in septic patients, a key molecule for adaptive immunity onset.

Immune Profiling Demonstrates a Common Immune Signature of Delayed Acquired Immunodeficiency in Patients With Various Etiologies of Severe Injury.

Objectives: The host response plays a central role in the pathophysiology of sepsis and severe injuries. So far, no study has comprehensively described the overtime changes of the injury-induced immune profile in a large cohort of critically ill patients with different etiologies.

Design: Prospective observational cohort study.

Assessing the Functional Heterogeneity of Monocytes in Human Septic Shock: a Proof-of-Concept Microfluidic Assay of TNFα Secretion.

Objective: The development of advanced single-cell technologies to decipher inter-cellular heterogeneity has enabled the dynamic assessment of individual cells behavior over time, overcoming the limitation of traditional assays. Here, we evaluated the feasibility of an advanced microfluidic assay combined to fluorescence microscopy to address the behavior of circulating monocytes from septic shock patients.

Methods: Seven septic shock patients and ten healthy volunteers were enrolled in the study.

Dynamic single-cell phenotyping of immune cells using the microfluidic platform DropMap.

Characterization of immune responses is currently hampered by the lack of systems enabling quantitative and dynamic phenotypic characterization of individual cells and, in particular, analysis of secreted proteins such as cytokines and antibodies. We recently developed a simple and robust microfluidic platform, DropMap, to measure simultaneously the kinetics of secretion and other cellular characteristics, including endocytosis activity, viability and expression of cell-surface markers, from tens of thousands of single immune cells. Single cells are compartmentalized in 50-pL droplets and analyzed using fluorescence microscopy combined with an immunoassay based on fluorescence relocation to paramagnetic nanoparticles aligned to form beadlines in a magnetic field.

Characterization and analytical validation of a new antigenic rapid diagnostic test for Ebola virus disease detection.

Hemorrhagic fever outbreaks are difficult to diagnose and control in part because of a lack of low-cost and easily accessible diagnostic structures in countries where etiologic agents are present. Furthermore, initial clinical symptoms are common and shared with other endemic diseases such as malaria or typhoid fever. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need, particularly in outbreak settings.

Early herpes and TTV DNAemia in septic shock patients: a pilot study.

Background: Septic shock patients exhibit an increased incidence of viral reactivation. Precise timing of such reactivation-as an early marker of immune suppression, or as a consequence of the later-is not known precisely. Here, using a fully designed nucleic acid extraction automated procedure together with tailored commercial PCR kits, we focused on the description of early reactivation within the first week of ICU admission of several herpes viruses and Torque Teno virus (TTV) in 98 septic shock patients.

Molecularly imprinted conducting polymer based electrochemical sensor for detection of atrazine.

An original electrochemical sensor based on molecularly imprinted conducting polymer (MICP) is developed, which enables the recognition of a small pesticide target molecule, atrazine. The conjugated MICP, poly(3,4-ethylenedioxythiophene-co-thiophene-acetic acid), has been electrochemically synthesized onto a platinum electrode following two steps: (i) polymerization of comonomers in the presence of atrazine, already associated to the acetic acid substituent through hydrogen bonding, and (ii) removal of atrazine from the resulting polymer, which leaves the acetic acid substituents open for association with atrazine. The obtained sensing MICP is highly specific towards newly added atrazine and the recognition can be quantitatively analyzed by the variation of the cyclic voltammogram of MICP.

Amperometric tyrosinase based biosensor using an electrogenerated polythiophene film as an entrapment support.

An amperometric enzyme sensor using tyrosinase, also called polyphenol oxidase (PPO), was constructed for determination of phenolic compounds and herbicides. The enzyme was entrapped in a conducting polymer, poly 3,4-ethylenedioxythiophene (PEDT), electrochemically generated on a glassy carbon electrode. Several experimental parameters in the electropolymerisation process and working conditions were determined to optimise biosensor performances.

Cytometric bead assay of cytokines in sepsis: a clinical evaluation.

Background: The clinical relevancy of an attractive new multiparametric method, cytometric beads assay (CBA), was evaluated for the monitoring of cytokines in sepsis.

Methods: A total of 52 samples (26 patients) were simultaneously tested by CBA and chemiluminescence (IL-6, IL-8, IL-1beta, and TNFalpha) or ELISA (IL-10, IL-12 p40, IL-12 p70, and soluble TNFalpha-RI).

Results: CBA standard curves were linear from 20-5,000 ng/L except for IL-1beta (40-5,000 ng/L).

Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides.

An optical biosensor was designed for determination of herbicides as aquatic contaminants. Detection was obtained with immobilised Chlorella vulgaris microalgae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. The algal chlorophyll fluorescence modified by the presence of herbicides was collected at the tip of an optical fibre bundle and sent to a fluorimeter.

Quantification of transcriptional activities of reporter gene constructs in primary cultures of sympathetic neurons.

Primary cultures of sympathetic neurons provide an attractive cellular model for investigating the mechanisms of neurotransmitter phenotypic plasticity. However, it has not been possible to transfect these neurons by conventional techniques, and this has been a major impediment to molecular investigations of neuronal gene expression in this system. Here, reporter plasmids were transferred into the nuclei of cultured sympathetic neurons by microinjection.