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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.

Therapeutic drug monitoring (TDM) of antibiotics has been practiced for more than half a century, but it is still not widely applied for infected patients. It has a traditional focus on limiting toxicity of specific classes of antibiotics such as aminoglycosides and vancomycin. With more patients in critical care with higher levels of sickness severity and immunosuppression as well as an increasingly obese and ageing population, an increasing risk of suboptimal antibiotic exposure continues to escalate. As such, the value of TDM continues to expand, especially for beta-lactams which constitute the most frequently used antibiotic class. To date, the minimum inhibitory concentration (MIC) of infectious microbes rather than classification in terms of susceptible and resistant can be reported. In parallel, increasingly sophisticated TDM technology is becoming available ensuring that TDM is feasible and can deliver personalized antibiotic dosing schemes. There is an obvious need for extensive studies that will quantify the improvements in clinical outcome of individual TDM-guided dosing. We suggest that a broad diagnostic and medical investigation of the TDM arena, including market analyses and analytical technology assessment, is a current priority.

Background In patients with severe sepsis, no randomized clinical trial has tested the concept of de-escalation of empirical antimicrobial therapy. This study aimed to compare the de-escalation strategy with the continuation of an appropriate empirical treatment in those patients. Methods This was a multicenter non-blinded randomized noninferiority trial of patients with severe sepsis who were randomly assigned to de-escalation or continuation of empirical antimicrobial treatment. Recruitment began in February 2012 and ended in April 2013 in nine intensive care units (ICUs) in France. Patients with severe sepsis were assigned to de-escalation ( n  = 59) or continuation of empirical antimicrobial treatment ( n  = 57). The primary outcome was to measure the duration of ICU stay. We defined a noninferiority margin of 2 days. If the lower boundary of the 95 % confidence interval (CI) for the difference in patients assigned to the de-escalation group was less than 2 days, as compared with that of patients assigned to the continuation group, de-escalation was considered to be noninferior to the continuation strategy. Secondary outcomes included mortality at 90 days, occurrence of organ failure, number of superinfections, and number of days with antibiotics during the ICU stay. Results The median duration of ICU stay was 9 [interquartile range (IQR) 5–22] days in the de-escalation group and 8 [IQR 4–15] days in the continuation group, respectively ( P  = 0.71). The mean difference was 3.4 (95 % CI −1.7 to 8.5). A superinfection occurred in 16 (27 %) patients in the de-escalation group and six (11 %) patients in the continuation group ( P  = 0.03). The numbers of antibiotic days were 9 [7–15] and 7.5 [6–13] in the de-escalation group and continuation group, respectively ( P  = 0.03). Mortality was similar in both groups. Conclusion As compared to the continuation of the empirical antimicrobial treatment, a strategy based on de-escalation of antibiotics resulted in prolonged duration of ICU stay. However, it did not affect the mortality rate.

Patients that suffer from sepsis exhibit an early hyper-inflammatory immune response which can lead to organ failure and death. In our study, we assessed the immune modulation in the human in vivo endotoxemia model and compared it to ex vivo LPS stimulation using 38 transcriptomic markers. Blood was collected before and after 4 hours of LPS challenge and tested with the Immune Profiling Panel (IPP) using the FilmArray system. The use of IPP showed that markers from the innate immunity dominated the response to LPS in vivo , mainly markers related to monocytes and neutrophils. Comparing the two models, in vivo and ex vivo , revealed that most of the markers were modulated in a similar pattern (68%). Some cytokine markers such as TNF , IFN-γ and IL-1β were under-expressed ex vivo compared to in vivo . T-cell markers were either unchanged or up-modulated ex vivo , compared to a down-modulation in vivo . Interestingly, markers related to neutrophils were expressed in opposite directions, which might be due to the presence of cell recruitment and feedback loops in vivo . The IPP tool was able to capture the early immune response in both the human in vivo endotoxemia model, a translational model mimicking the immune response observed in septic patients.

The immune response is a dynamic system that maintains the integrity of the body, and more specifically fight against infections. However, an unbalanced host immune response is highlighted in many diseases. Exacerbated responses lead to autoimmune and allergic diseases, whereas, low or inefficient responses favor opportunistic infections and viral reactivations. Conflicting situations may also occur, such as in sepsis where inflammation and compensatory immunosuppression make it difficult to deploy the appropriate drug treatment. Until the current day, assessing the immune profile of patients remains a challenge. This is especially due to the inter-individual variability-a key feature of the immune system-which hinders precise diagnosis, prognosis, and therapeutic stratification. Our incapacity to practically interpret the host response may contribute to a high morbidity and mortality, such as the annual 6 million worldwide deaths in sepsis alone. Therefore, there is a high and increasing demand to assess patient immune function in routine clinical practice, currently met by Immune Functional Assays. Immune Functional Assays (IFA) hold a plethora of potentials that include the precise diagnosis of infections, as well as prediction of secondary and latent infections. Current available products are devoted to indirect pathogen detection such as Mycobacteria tuberculosis interferon gamma release assays (IGRA). In addition, identifying the status and the underlying factors of immune dysfunction (e.g., in septic patients) may guide immune targeted therapies. Tools to monitor and stratify the immune status are currently being studied but they still have many limitations such as technical standardization, biomarkers relevance, systematic interpretation and need to be simplified, in order to set the boundaries of \"healthy,\" \"ill,\" and \"critically ill\" responses. Thus, the design of new tools that give a comprehensive insight into the immune functionality, at the bedside, and in a timely manner represents a leap toward immunoprofiling of patients.

See related research by Mirouse et al., https://ccforum.biomedcentral.com/articles/10.1186/s13054-017-1731-0 This comment refers to the article available at: https://doi.org/10.1186/s13054-017-1731-0.

Acute immuno-depression syndrome (AIDs) had been observed in many life-threatening conditions leading to the Intensive Care Unit. and is associated with recurrent secondary infections. We report one COVID-19 patient with a severe ARDS, demonstrating acute immunodepression syndrome lasting for several weeks. The occurrence of secondary infections despite long treatment by antibiotics led to combined interferon γ (IFNγ) as reported previously. The response to IFNγ was evaluated by the flowcytometry HLA-DR expression on circulating monocytes, which was repeated from time to time. The severe COVID-19 patients responded well to IFNγ without adverse events.

Background The first FDA-approved test to assess risk for acute kidney injury (AKI), [TIMP-2]•[IGFBP7], is clinically available in many parts of the world, including the USA and Europe. We sought to understand how the test is currently being used clinically. Methods We invited a group of experts knowledgeable on the utility of this test for kidney injury to a panel discussion regarding the appropriate use of the test. Specifically, we wanted to identify which patients would be appropriate for testing, how the results are interpreted, and what actions would be taken based on the results of the test. We used a modified Delphi method to prioritize specific populations for testing and actions based on biomarker test results. No attempt was made to evaluate the evidence in support of various actions however. Results Our results indicate that clinical experts have developed similar practice patterns for use of the [TIMP-2]•[IGFBP7] test in Europe and North America. Patients undergoing major surgery (both cardiac and non-cardiac), those who were hemodynamically unstable, or those with sepsis appear to be priority patient populations for testing kidney stress. It was agreed that, in patients who tested positive, management of potentially nephrotoxic drugs and fluids would be a priority. Patients who tested negative may be candidates for “fast-track” protocols. Conclusion In the experience of our expert panel, biomarker testing has been a priority after major surgery, hemodynamic instability, or sepsis. Our panel members reported that a positive test prompts management of nephrotoxic drugs as well as fluids, while patients with negative results are considered to be excellent candidates for “fast-track” protocols.

Background Controversies exist on the nature of COVID-19 related acute respiratory distress syndrome (ARDS) in particular on the static compliance of the respiratory system (Crs). We aimed to analyze the association of Crs with outcome in COVID-19-associated ARDS, to ascertain its determinants and to describe its evolution at day-14. Methods In this observational multicenter cohort of patients with moderate to severe Covid-19 ARDS, Crs was measured at day-1 and day-14. Association between Crs or Crs/ideal body weight (IBW) and breathing without assistance at day-28 was analyzed with multivariable logistic regression. Determinants were ascertained by multivariable linear regression. Day-14 Crs was compared to day-1 Crs with paired t-test in patients still under controlled mechanical ventilation. Results The mean Crs in 372 patients was 37.6 ± 13 mL/cmH 2 O, similar to as in ARDS of other causes. Multivariate linear regression identified chronic hypertension, low PaO 2 /FiO 2 ratio, low PEEP, and low tidal volume as associated with lower Crs/IBW. After adjustment on confounders, nor Crs [OR 1.0 (CI 95% 0.98–1.02)] neither Crs/IBW [OR 0.63 (CI 95% 0.13–3.1)] were associated with the chance of breathing without assistance at day-28 whereas plateau pressure was [OR 0.93 (CI 95% 0.88–0.99)]. In a subset of 108 patients, day-14 Crs decreased compared to day-1 Crs (31.2 ± 14.4 mL/cmH 2 O vs 37.8 ± 11.4 mL/cmH 2 O, p  < 0.001). The decrease in Crs was not associated with day-28 outcome. Conclusion In a large multicenter cohort of moderate to severe COVID-19 ARDS, mean Crs was decreased below 40 mL/cmH 2 O and was not associated with day-28 outcome. Crs decreased between day-1 and day-14 but the decrease was not associated with day-28 outcome.