Search

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. Therefore, rapid diagnostic tests such as lateral flow can be broadly deployed and are typically well-suited to rapidly diagnose hemorrhagic fever viruses, such as Ebola virus. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect very low amount of virus in blood. Here, we developed and characterized an immunoassay test based on immunochromatography coupled to silver amplification technology to detect the secreted glycoprotein of EBOV. The glycoprotein is among the first viral proteins to be detected in blood. This strategy aims at identifying infected patients early following onset of symptoms by detecting low amount of sGP protein in blood samples. The limit of detection achieved by this sGP-targeted kit is 2.2 x 104 genome copies/ml in plasma as assayed in a monkey analytical cohort. Clinical performance evaluation showed a specificity of 100% and a sensitivity of 85.7% when evaluated with plasma samples from healthy controls and patients infected with Zaire Ebola virus from Macenta, Guinea. This rapid and accurate diagnostic test could therefore be used in endemic countries for early detection of infected individuals in point of care settings. Moreover, it could also support efficient clinical triage in hospitals or clinical centers and thus reducing transmission rates to prevent and better manage future severe outbreaks., Author summary Ebola virus disease is a severe disease caused by Ebola virus, a member of the filovirus family, which occurs in humans and other primates. Ebola is believed to be zoonotic, however the natural reservoir is unknown. Overlapping symptoms with other endemic diseases, such as malaria and cholera, make accurate diagnostic challenging. Outbreaks of Ebola have been widespread as the consequence of the absence of available rapid, sensitive, specific, robust, and affordable licensed diagnostic test in remote areas, where outbreaks usually start. Here we have established and validated a rapid diagnostic test, which is fast, sensitive, specific, efficient, affordable, and user-friendly. Its analytical characteristics make it suitable for clinical management during Ebola virus outbreaks in remote areas. Of interest, this rapid diagnostic test detects the presence of an early viral antigen, the secreted glycoprotein, found in blood of patients shortly after infection, suggesting that it could be used to identify infected patients shortly after onset of symptoms.

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. Results Most of septic shock patients had at least one viremia event during the first week (88%). TTV and herpesviruses were detected in 56% and 53% of septic shock patient, respectively. The two most frequent herpesviruses detected within the first week were EBV (35%) and HSV1 (26%). Different kinetic were observed among herpesviruses, faster for EBV and HSV1 than for CMV and HHV6. Although no association was found between herpes viremia and secondary infections, patients with herpesviridae-related viremia were more severe, e.g., higher SOFA scores and plasma lactate levels. While reactivating only 1 virus was not associated with mortality, patients with multiple viremia events had higher ICU mortality. Surprisingly, EBV + TTV early reactivation seemed associated with a lower D28 mortality. No clear association was observed between viremia and immune biomarkers. Conclusion Applying a semi-automated process of viral DNAemia determination to this cohort of 98 patients with septic shock, we observed that the number of patients with positive viremia increased during the first week in the ICU. Of note, there was no improvement in predicting the outcome when using viremia status. Nevertheless, this pilot study, introducing standardized procedures from extraction to detection, provides the basis for future standardized diagnostic criteria. A prospective longitudinal clinical study using these procedures will enable determination of whether such viremia is due to a lack of a latent virus control by the immune system or a true clinical viral infection. Electronic supplementary material The online version of this article (10.1186/s40635-019-0256-z) contains supplementary material, which is available to authorized users.

International audience; The harmful effect of toxic chemicals on natural ecosystems has led to an increasing demand for early-warning systems to detect those toxicants at very low concentrations levels. Whole-cell biosensors based either on chlorophyll fluorescence or enzyme (phosphatase and esterase) inhibition are constructed for real-time detection and on-line monitoring. Results show that these devices are sensitive to heavy metals and pesticides. The system allows the cells to operate in their natural environment which favours long term stability and reflects the toxic action mechanism providing therefore an ecological interest.

International audience; 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. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimisation. Effects of flow rate, algal density, temperature, and pH on the biosensor response to atrazine were studied. Reversibility and detection limits were determined for DNOC and atrazine, simazine, isoproturon, diuron. Detection of photosystem II (PSII) herbicides was achieved at sub-ppb concentration level.

International audience; 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. Mono-phenol and di-phenol were tested in oxygenated solutions, by amperometric measurements at −200 mV (vs. SCE) in a batch system. The limit of detection of these molecules ranges from 5 to 500 nM. Detection of herbicides was obtained from the inhibition of tyrosinase electrode responses. The limit of detection for atrazine and diuron was 1 and 0.5 mg l−1 respectively. These data suggest that PEDT film is a promising PPO immobilisation method.

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. The protocol typically takes 8–10 h after preparation of microfluidic chips and chambers, which can be done in advance. By contrast, enzyme-linked immunospot (ELISPOT), flow cytometry, time-of-flight mass cytometry (CyTOF), and single-cell sequencing enable only end-point measurements and do not enable direct, quantitative measurement of secreted proteins. We illustrate how this system can be used to profile downregulation of tumor necrosis factor-α (TNF-α) secretion by single monocytes in septic shock patients, to study immune responses by measuring rates of cytokine secretion from single T cells, and to measure affinity of antibodies secreted by single B cells. This protocol describes a microfluidic platform for dynamic high-throughput analysis of the phenotypes of single cells. Cell-surface markers and secreted proteins are quantified and characterized by fluorescence detection using tailored immunoassays.