Mapping bacterial extracellular vesicle research: insights, best practices and knowledge gaps.

Bacterial extracellular vesicles (BEVs) enable communication between bacteria and their natural habitats, including multicellular organisms such as humans. Consequently, the study of BEVs has rapidly gained attention with recent research raising the prospect of developing BEVs as biomarkers and treatments to manage (mal)functioning of natural habitats. Although diverse technologies are available, the composition of their source, their heterogeneity in biophysical and biochemical features, and their multifaceted cargo composition challenges the analysis of BEVs. To map current practices in BEV research, we analyzed 845 publications released in 2015-2021, reporting 3338 BEV-related experiments. The extracted data are accessible via the publicly available EV-TRACK knowledgebase ( https://evtrack.org/ ). We identify the need for transparent reporting, delineate knowledge gaps, outline available best practices and define areas in need of guidance to ensure advances in BEV research and accelerate BEV applications.

Prospective multi-center evaluation of the incidence of unplanned extubation and its outcomes in French intensive care units. The Safe-ICU study.

BACKGROUND: We aimed to determine the epidemiology and outcomes of unplanned extubation (UE), both accidental and self-extubation, in ICU. METHODS: A multicentre prospective cohort study was conducted in 47 French ICUs. The number of mechanical ventilation (MV) days, and planned and unplanned extubation were recorded in each center over a minimum period of three consecutive months to evaluate UE incidence. Patient characteristics, UE environmental factors, and outcomes were compared based on the UE mechanism (accidental or self-extubation). Self-extubation outcomes were compared with planned extubation using a propensity-matched population. Finally, risk factors for extubation failure (re-intubation before day 7) were determined following self-extubation. RESULTS: During the 12-month inclusion period, we found a pooled UE incidence of 1.0 per 100 MV days. UE accounted for 9% of all endotracheal removals. Of the 605 UE, 88% were self-extubation and 12% were accidental-extubations. The latter had a worse prognosis than self-extubation (34% vs. 8% ICU-mortality, p < 0.001). Self-extubation did not increase mortality compared with planned extubation (8% vs. 11%, p = 0.075). Regardless of the type of extubation, planned or unplanned, extubation failure was independently associated with a poor outcome. Cancer, higher respiratory rate, lower PaO2/FiO2 at the time of extubation, weaning process not-ongoing, and immediate post-extubation respiratory failure were independent predictors of failed self-extubation. CONCLUSION: Unplanned extubation, mostly represented by self-extubation, is common in ICU and accounts for 9% of all endotracheal extubations. While accidental extubations are a serious and infrequent adverse event, self-extubation does not increase mortality compared to planned extubation.

Upper gastrointestinal bleeding on veno-arterial extracorporeal membrane oxygenation support.

INTRODUCTION: Patients on veno-arterial extracorporeal membrane oxygenation (V-A ECMO) support are at a high risk of hemorrhagic complications, including upper gastrointestinal bleeding (UGIB). The objective of this study was to evaluate the incidence and impact of this complication in V-A ECMO patients. MATERIALS AND METHODS: A retrospective single-center study (2013-2017) was conducted on V-A ECMO patients, excluding those who died within 24 h. All patients with suspected UGIB underwent esophagogastroduodenoscopy (EGD) and were analyzed and compared to the remainder of the cohort, from the initiation of ECMO until 5 days after explantation. RESULTS: A total of 150 V-A ECMO cases (65 after cardiac surgery and 85 due to medical etiology) were included. 90% of the patients received prophylactic proton pump inhibitor therapy and enteral nutrition. Thirty-one patients underwent EGD for suspected UGIB, with 16 confirmed cases of UGIB. The incidence was 10.7%, with a median occurrence at 10 [7-17] days. There were no significant differences in clinical or biological characteristics on the day of EGD. However, patients with UGIB had significant increases in packed red blood cells and fresh frozen plasma needs, mechanical ventilation duration and V-A ECMO duration, as well as in length of intensive care unit and hospital stays. There was no significant difference in mortality. The only independent risk factor of UGIB was a history of peptic ulcer (OR = 7.32; 95% CI [1.07-50.01], p = 0.042). CONCLUSION: UGIB occurred in at least 1 out of 10 cases of V-A ECMO patients, with significant consequences on healthcare resources. Enteral nutrition and proton pump inhibitor prophylaxis did not appear to protect V-A ECMO patients. Further studies should assess their real benefits in these patients with high risk of hemorrhage.

Identification and validation of extracellular vesicle reference genes for the normalization of RT-qPCR data.

Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.

Hypnosis during Endovascular Abdominal Aortic Aneurysm Repair.

(1) Background: Endovascular abdominal aneurysm repair (EVAR) is associated with a reduction in early morbidity and mortality compared with open repair. Procedures performed under hypnosis might represent an alternative to further reduce the risks related to general anesthesia (GA). This study aimed to assess the feasibility and safety of hypnosis and local anesthesia during EVAR. (2) Methods: All consecutive patients who underwent EVAR or fenestrated/branched EVAR (f/bEVAR) under hypnosis and local anesthesia (n = 28) between 2017 and 2019 were retrospectively studied and matched to control patients who underwent the same interventions under GA. (3) Results: There was neither a significant difference in the length of ICU stay (p = 0.06), nor in the occurrence of endoleaks, reintervention, and 30-day mortality rate (p = 1.00, 0.73, and 0.24, respectively). The hypnosis group had lower use of norepinephrine (maximum dose 0.04 ± 0.1 vs. 1.2 ± 4.0 mg·h-1, p < 0.001), shorter procedure duration (181.2 ± 71.4 vs. 214.3 ± 79.6 h, p = 0.04), and shorter length of stay (5.4 ± 3.2 vs. 8.4 ± 5.9 days, p = 0.002). (4) Conclusions: In this pioneering study, hypnosis during EVAR appears feasible and safe. It is associated with lower intraoperative use of norepinephrine, as well as procedure duration and length of in-hospital stay.

The impact of extracorporeal membrane oxygenation on antifungal pharmacokinetics: A systematic review.

BACKGROUND AND OBJECTIVE: The use of extracorporeal membrane oxygenation (ECMO) as a cardiocirculatory or respiratory support has tremendously increased in critically ill patients. In the setting of ECMO support, invasive fungal infections are a severe cause of morbidity and mortality. This vulnerable population is at risk of suboptimal antifungal exposure due to an increased volume of distribution (Vd), drug sequestration and decreased clearance. Here, we aimed to summarize ex-vivo and clinical studies on the potential impact of ECMO on the pharmacokinetics (PK) of antifungal agents and dosing requirements. METHODS: A systematic search of the literature within electronic databases PubMed and EMBASE was conducted from database inception to 30 April 2023. Inclusion criteria were as follows: critically ill patients receiving ECMO regardless of age and reporting at least one PK parameter. RESULTS: Thirty-six studies met inclusion criteria, including seven ex-vivo experiments and 29 clinical studies evaluating three classes of antifungals: polyenes, triazoles and echinocandins. Based on the available ex-vivo PK data, we found a significant sequestration of highly lipophilic and protein-bound antifungals within the ECMO circuit such as voriconazole, posaconazole and micafungin but the PK of several antifungals remains to be addressed such as amphotericin B, isavuconazole and anidulafungin. Most clinical studies have shown increased Vd of some antifungals like fluconazole and micafungin, particularly in the pediatric population. Conflicting data exist about caspofungin exposure. CONCLUSIONS: The available literature on the antifungal PK changes in ECMO setting is scarce. Whenever possible, therapeutic drug monitoring is highly advised to personalize antifungal therapy.

Isolating plasma extracellular vesicles from mouse blood using size-exclusion chromatography, density gradient, and ultracentrifugation.

Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).1.

Density-Based Fractionation of Cell-Conditioned Medium to Prepare Proteomics Grade Extracellular Vesicles.

The identification of the molecular composition of extracellular vesicles (EV) by omics approaches, including proteomics, requires the separation of EV from non-EV confounding factors present in the source biofluid. In this protocol, we present the sequential implementation of density gradient ultracentrifugation and size-exclusion chromatography to prepare EV from cell-conditioned medium with high specificity and repeatability. This approach enables the recovery of intact purified EV suited for downstream functional assays and biomarker discovery by omics approaches.

Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles.

Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration.

Long-Term Results of Endoscopic Metal Stenting for Biliary Anastomotic Stricture after Liver Transplantation.

(1) Background: Anastomotic biliary stricture (ABS) is a well-known complication of liver transplantation which can lead to secondary biliary cirrhosis and graft dysfunction. The goal of this study was to evaluate the long-term outcomes of endoscopic metal stenting of ABS in the setting of deceased donor liver transplantation (DDLT). (2) Methods: Consecutive DDLT patients with endoscopic metal stenting for ABS between 2010 and 2015 were screened. Data on diagnosis, treatment and follow-up (until June 2022) were collected. The primary outcome was endoscopic treatment failure defined as the need for surgical refection. (3) Results: Among the 465 patients who underwent LT, 41 developed ABS. It was diagnosed after a mean period of 7.4 months (+/-10.6) following LT. Endoscopic treatment was technically successful in 95.1% of cases. The mean duration of endoscopic treatment was 12.8 months (+/-9.1) and 53.7% of patients completed a 1-year treatment. After a mean follow-up of 6.9 years (+/-2.3), endoscopic treatment failed in nine patients (22%) who required surgical refection. Conclusions: Endoscopic management with metal stenting of ABS after DDLT was technically successful in most cases, and half of the patients had at least one year of indwelling stent. Endoscopic treatment long-term failure rate occurred in one fifth of the patients.

The Relative Contributions of Occupational and Community Risk Factors for COVID-19 among Hospital Workers: The HOP-COVID Cohort Study.

The relative contributions of occupational and community sources of COVID-19 among health-care workers (HCWs) are still subject to debate. In a cohort study at a 2814-bed tertiary medical center (five hospitals) in the Paris area of France, we assessed the proportion of hospital-acquired cases among staff and identified risk factors. Between May 2020 and June 2021, HCWs were invited to complete a questionnaire on their COVID-19 risk factors. RT-PCR and serology test results were retrieved from the virology department. Mixed-effects logistic regression was used to account for clustering by hospital. The prevalence of COVID-19 was 15.6% (n = 213/1369 respondents) overall, 29.7% in the geriatric hospitals, and 56.8% of the infections were hospital-acquired. On multivariable analyses adjusted for COVID-19 incidence and contact in the community, a significantly higher risk was identified for staff providing patient care (especially nursing assistants), staff from radiology/functional assessment units and stretcher services, and staff working on wards with COVID-19 clusters among patients or HCWs. The likelihood of infection was greater in geriatric wards than in intensive care units. The presence of significant occupational risk factors after adjustment for community exposure is suggestive of a high in-hospital risk and emphasizes the need for stronger preventive measures-especially in geriatric settings. Clinicaltrials.gov NCT04386759.

Healthcare trajectory of critically ill patients with necrotizing soft tissue infections: a multicenter retrospective cohort study using the clinical data warehouse of Greater Paris University Hospitals.

BACKGROUND: Necrotizing skin and soft tissue infections (NSTIs) are rare but serious and rapidly progressive infections characterized by necrosis of subcutaneous tissue, fascia and even muscle. The care pathway of patients with NSTIs is poorly understood. A better characterization of the care trajectory of these patients and a better identification of patients at risk of a complicated evolution, requiring prolonged hospitalization, multiple surgical re-interventions, or readmission to the intensive care unit (ICU), is an essential prerequisite to improve their care. The main objective of this study is to obtain large-scale data on the care pathway of these patients. We performed a retrospective multicenter observational cohort study in 13 Great Paris area hospitals, including patients hospitalized between January 1, 2015 and December 31, 2019 in the ICU for surgically confirmed NSTIs. RESULTS: 170 patients were included. The median duration of stay in ICU and hospital was 8 (3-17) and 37 (14-71) days, respectively. The median time from admission to first surgical debridement was 1 (0-2) day but 69.9% of patients were re-operated with a median of 1 (0-3) additional debridement. Inter-hospital transfer was necessary in 52.4% of patients. 80.2% of patients developed organ failures during the course of ICU stay with 51.8% of patients requiring invasive mechanical ventilation, 77.2% needing vasopressor support and 27.7% renal replacement therapy. In-ICU and in-hospital mortality rates were 21.8% and 28.8%, respectively. There was no significant difference between patients with abdomino-perineal NSTIs (n = 33) and others (n = 137) in terms of in-hospital or ICU mortality. Yet, immunocompromised patients (n = 43) showed significantly higher ICU and in-hospital mortality rates than non-immunocompromised patients (n = 127) (37.2% vs. 16.5%, p = 0.009, and 53.5% vs. 20.5%, p < 0.001). Factors associated with a complicated course were the presence of a polymicrobial infection (adjusted odds ratio [aOR = 3.18 (1.37-7.35); p = 0.007], of a bacteremia [aOR = 3.29 (1.14-9.52); p = 0.028] and a higher SAPS II score [aOR = 1.05 (1.02-1.07); p < 0.0001]. 62.3% of patients were re-hospitalized within 6 months. CONCLUSION: In this retrospective multicenter study, we showed that patients with NSTI required complex management and are major consumers of care. Two-thirds of them underwent a complicated hospital course, associated with a higher SAPS II score, a polymicrobial NSTI and a bacteremia.

Vasoplegic Syndrome after Cardiac Surgery for Infective Endocarditis.

Purpose: Post-operative vasoplegic syndrome is a dreaded complication in infective endocarditis (IE). Methods and Results: This retrospective study included 166 consecutive patients referred to cardiac surgery for non-shocked IE. Post-operative vasoplegic syndrome was defined as a persistent hypotension (mean blood pressure < 65 mmHg) refractory to fluid loading and cardiac output restoration. Cardiac surgery was performed 7 (5−12) days after the beginning of antibiotic treatment, 4 (1−9) days after negative blood culture and in 72.3% patients with adapted anti-biotherapy. Timing of cardiac surgery was based on ESC guidelines and operating room availability. Most patients required valve replacement (80%) and cardiopulmonary bypass (CPB) duration was 106 (95−184) min. Multivalvular surgery was performed in 43 patients, 32 had tricuspid valve surgery. Post-operative vasoplegic syndrome was reported in 53/166 patients (31.9%, 95% confidence interval of 24.8−39.0%) of the whole population; only 15.1% (n = 8) of vasoplegic patients had a post-operative documented infection (6 positive blood cultures) and no difference was reported between vasoplegic and non-vasoplegic patients for valve culture and the timing of cardiac surgery. Of the 23 (13.8%) in hospital-deaths, 87.0% (n = 20) occurred in the vasoplegic group and the main causes of death were multiorgan failure (n = 17) and neurological complications (n = 3). Variables independently associated with vasoplegic syndrome were CPB duration (1.82 (1.16−2.88) per tertile) and NTproBNP level (2.11 (1.35−3.30) per tertile). Conclusions: Post-operative vasoplegic syndrome is frequent and is the main cause of death after IE cardiac surgery. Our data suggested that the mechanism of vasoplegic syndrome was more related to inflammatory cardiovascular injury rather than the consequence of ongoing bacteremia.

Potential of Multiplex Polymerase Chain Reaction Performed on Protected Telescope Catheter Samples for Early Adaptation of Antimicrobial Therapy in ARDS Patients.

BACKGROUND: Diagnosis of co/superinfection in patients with Acute Respiratory Distress Syndrome (ARDS) is challenging. The FilmArray Pneumonia plus Panel (bioMérieux, France), a new rapid multiplex Polymerase Chain Reaction (mPCR), has never been assessed on a blinded protected telescope catheter (PTC) samples, a very common diagnostic tool in patients under mechanical ventilation. We evaluated the performance of mPCR on PTC samples compared with conventional culture and its impact on antibiotic stewardship. METHODS: Observational study in two intensive care units, conducted between March and July 2020, during the first wave of the COVID-19 pandemic in France. RESULTS: We performed 125 mPCR on blinded PTC samples of 95 ARDS patients, including 73 (77%) SARS-CoV-2 cases and 28 (29%) requiring extracorporeal membrane oxygenation. Respiratory samples were drawn from mechanically ventilated patients either just after intubation (n = 48; 38%) or later for suspected ventilator-associated pneumonia (VAP) (n = 77; 62%). The sensitivity, specificity, positive, and negative predictive values of mPCR were 93% (95% CI 84-100), 99% (95% CI 99-100), 68% (95% CI 54-83), and 100% (95% CI 100-100), respectively. The overall coefficient of agreement between mPCR and standard culture was 0.80 (95% CI 0.68-0.89). Intensivists changed empirical antimicrobial therapy in only 14% (18/125) of cases. No new antibiotic was initiated in more than half of the CAP/HAP pneumonia-suspected cases (n = 29; 60%) and in more than one-third of those suspected to have VAP without affecting or delaying their antimicrobial therapy. CONCLUSIONS: Rapid mPCR was feasible on blinded PTC with good sensitivity and specificity. New antibiotics were not initiated in more than half of patients and more than one-third of VAP-suspected cases. Further studies are needed to assess mPCR potential in improving antibiotic stewardship.

Population Pharmacokinetics of Amikacin in Patients on Veno-Arterial Extracorporeal Membrane Oxygenation.

Veno-arterial extracorporeal membrane oxygenation (V-A ECMO) support leads to complex pharmacokinetic alterations, whereas adequate drug dosing is paramount for efficacy and absence of toxicity in critically ill patients. Amikacin is a major antibiotic used in nosocomial sepsis, especially for these patients. We aimed to describe amikacin pharmacokinetics on V-A ECMO support and to determine relevant variables to improve its dosing. All critically ill patients requiring empirical antimicrobial therapy, including amikacin for nosocomial sepsis supported or not by V-A ECMO, were included in a prospective population pharmacokinetic study. This population pharmacokinetic analysis was built with a dedicated software, and Monte Carlo simulations were performed to identify doses achieving therapeutic plasma concentrations. Thirty-nine patients were included (control n = 15, V-A ECMO n = 24); 215 plasma assays were performed and used for the modeling process. Patients received 29 (24-33) and 32 (30-35) mg/kg of amikacin in control and ECMO groups, respectively. Data were best described by a two-compartment model with first-order elimination. Inter-individual variabilities were observed on clearance, central compartment volume (V1), and peripherical compartment volume (V2). Three significant covariates explained these variabilities: Kidney Disease Improving Global Outcomes (KDIGO) stage on amikacin clearance, total body weight on V1, and ECMO support on V2. Our simulations showed that the adequate dosage of amikacin was 40 mg/kg in KDIGO stage 0 patients, while 25 mg/kg in KDIGO stage 3 patients was relevant. V-A ECMO support had only a secondary impact on amikacin pharmacokinetics, as compared to acute kidney injury.