CLINICAL, MOLECULAR, AND EXOSOMAL MECHANISMS OF CARDIAC AND BRAIN DYSFUNCTION IN SEPSIS.

ABSTRACT: Sepsis is a complex disease resulting from a dysregulated inflammatory response to an infection. Initiation of sepsis occurs from a localized infection that disseminates to the bloodstream placing all organ systems at risk. Septic shock is classically observed to manifest itself as systemic hypotension with hyporesponsiveness to vasopressor agents. Myocardial dysfunction occurs resulting in an inability to perfuse major organ systems throughout the body. Most importantly, the brain is hypoperfused creating an ischemic and inflammatory state resulting in the clinical observation of acute mental status changes and cognitive dysfunction commonly known as sepsis-associated encephalopathy. This short review describes the inflammatory molecular mechanisms of myocardial dysfunction, discusses the evidence of the dual roles of the microglia resulting in blood-brain barrier disruption, and suggests that septic-derived exosomes, endosome-derived lipid bilayer spheroids released from living cells, influence cardiac and neurological cellular function.

Building a Comprehensive Rural Anti-Reflux Center.

BACKGROUND: Gastroesophageal disease (GERD) is a highly prevalent gastrointestinal disease. In rural areas, general surgeons perform esophagogastroduodenoscopy (EGD) despite its low diagnostic yield. When EGD findings are equivocal, GERD patients are usually referred to tertiary hospitals for further workup. We envisaged establishing a comprehensive anti-reflux program with diagnostic and therapeutic capabilities in a rural setting. STUDY DESIGN: This is an IRB approved retrospective chart review of patients who presented with GERD symptoms to a rural anti-reflux clinic between August 2015 and February 2021. Standardized workup included upper gastrointestinal study and EGD with concomitant wireless pH placement. High resolution impedance manometry and gastric emptying scans were selectively utilized initially, then were performed routinely. We used endoFLIP impedance planimetry system starting in February 2019. RESULTS: A total of 830 patients were evaluated. There were 537 (64.6%) females and 293 (35.4%) males. The average age was 57.7 ± 15.2 years. The average BMI was 30.8 ± 6.7 kg/m2. Approximately one-third of these patients were referred by the primary care provider (PCP) within our health system and a comparable percentage from external PCPs. Self referral was noted in 15.4% and 19.2% were referred by different specialties such as pulmonary (10.7%), surgical for large hiatal hernia (5.8%), inpatient and emergency room (2%), and gastroenterology (0.7%). CONCLUSION: Rural surgeons with appropriate endoscopic and laparoscopic training can establish a comprehensive anti-reflux program with diagnostic and therapeutic capabilities. It meets the high community need and can expand to be a regional center. The revenues generated are critical for the financial survival of rural hospitals.

Prospective Validation of a Rapid Host Gene Expression Test to Discriminate Bacterial From Viral Respiratory Infection.

Importance: Bacterial and viral causes of acute respiratory illness (ARI) are difficult to clinically distinguish, resulting in the inappropriate use of antibacterial therapy. The use of a host gene expression-based test that is able to discriminate bacterial from viral infection in less than 1 hour may improve care and antimicrobial stewardship. Objective: To validate the host response bacterial/viral (HR-B/V) test and assess its ability to accurately differentiate bacterial from viral infection among patients with ARI. Design, Setting, and Participants: This prospective multicenter diagnostic study enrolled 755 children and adults with febrile ARI of 7 or fewer days' duration from 10 US emergency departments. Participants were enrolled from October 3, 2014, to September 1, 2019, followed by additional enrollment of patients with COVID-19 from March 20 to December 3, 2020. Clinical adjudication of enrolled participants identified 616 individuals as having bacterial or viral infection. The primary analysis cohort included 334 participants with high-confidence reference adjudications (based on adjudicator concordance and the presence of an identified pathogen confirmed by microbiological testing). A secondary analysis of the entire cohort of 616 participants included cases with low-confidence reference adjudications (based on adjudicator discordance or the absence of an identified pathogen in microbiological testing). Thirty-three participants with COVID-19 were included post hoc. Interventions: The HR-B/V test quantified the expression of 45 host messenger RNAs in approximately 45 minutes to derive a probability of bacterial infection. Main Outcomes and Measures: Performance characteristics for the HR-B/V test compared with clinical adjudication were reported as either bacterial or viral infection or categorized into 4 likelihood groups (viral very likely [probability score <0.19], viral likely [probability score of 0.19-0.40], bacterial likely [probability score of 0.41-0.73], and bacterial very likely [probability score >0.73]) and compared with procalcitonin measurement. Results: Among 755 enrolled participants, the median age was 26 years (IQR, 16-52 years); 360 participants (47.7%) were female, and 395 (52.3%) were male. A total of 13 participants (1.7%) were American Indian, 13 (1.7%) were Asian, 368 (48.7%) were Black, 131 (17.4%) were Hispanic, 3 (0.4%) were Native Hawaiian or Pacific Islander, 297 (39.3%) were White, and 60 (7.9%) were of unspecified race and/or ethnicity. In the primary analysis involving 334 participants, the HR-B/V test had sensitivity of 89.8% (95% CI, 77.8%-96.2%), specificity of 82.1% (95% CI, 77.4%-86.6%), and a negative predictive value (NPV) of 97.9% (95% CI, 95.3%-99.1%) for bacterial infection. In comparison, the sensitivity of procalcitonin measurement was 28.6% (95% CI, 16.2%-40.9%; P < .001), the specificity was 87.0% (95% CI, 82.7%-90.7%; P = .006), and the NPV was 87.6% (95% CI, 85.5%-89.5%; P < .001). When stratified into likelihood groups, the HR-B/V test had an NPV of 98.9% (95% CI, 96.1%-100%) for bacterial infection in the viral very likely group and a positive predictive value of 63.4% (95% CI, 47.2%-77.9%) for bacterial infection in the bacterial very likely group. The HR-B/V test correctly identified 30 of 33 participants (90.9%) with acute COVID-19 as having a viral infection. Conclusions and Relevance: In this study, the HR-B/V test accurately discriminated bacterial from viral infection among patients with febrile ARI and was superior to procalcitonin measurement. The findings suggest that an accurate point-of-need host response test with high NPV may offer an opportunity to improve antibiotic stewardship and patient outcomes.

Comparing the Diagnostic Accuracy of Clinician Judgment to a Novel Host Response Diagnostic for Acute Respiratory Illness.

BACKGROUND: Difficulty discriminating bacterial from viral infections drives antibacterial misuse. Host gene expression tests discriminate bacterial and viral etiologies, but their clinical utility has not been evaluated. METHODS: Host gene expression and procalcitonin levels were measured in 582 emergency department participants with suspected infection. We also recorded clinician diagnosis and clinician-recommended treatment. These 4 diagnostic strategies were compared with clinical adjudication as the reference. To estimate the clinical impact of host gene expression, we calculated the change in overall Net Benefit (∆NB; the difference in Net Benefit comparing 1 diagnostic strategy with a reference) across a range of prevalence estimates while factoring in the clinical significance of false-positive and -negative errors. RESULTS: Gene expression correctly classified bacterial, viral, or noninfectious illness in 74.1% of subjects, similar to the other strategies. Clinical diagnosis and clinician-recommended treatment revealed a bias toward overdiagnosis of bacterial infection resulting in high sensitivity (92.6% and 94.5%, respectively) but poor specificity (67.2% and 58.8%, respectively), resulting in a 33.3% rate of inappropriate antibacterial use. Gene expression offered a more balanced sensitivity (79.0%) and specificity (80.7%), which corresponded to a statistically significant improvement in average weighted accuracy (79.9% vs 71.5% for procalcitonin and 76.3% for clinician-recommended treatment; P<.0001 for both). Consequently, host gene expression had greater Net Benefit in diagnosing bacterial infection than clinician-recommended treatment (∆NB=6.4%) and procalcitonin (∆NB=17.4%). CONCLUSIONS: Host gene expression-based tests to distinguish bacterial and viral infection can facilitate appropriate treatment, improving patient outcomes and mitigating the antibacterial resistance crisis.

Discriminating Bacterial and Viral Infection Using a Rapid Host Gene Expression Test.

OBJECTIVES: Host gene expression signatures discriminate bacterial and viral infection but have not been translated to a clinical test platform. This study enrolled an independent cohort of patients to describe and validate a first-in-class host response bacterial/viral test. DESIGN: Subjects were recruited from 2006 to 2016. Enrollment blood samples were collected in an RNA preservative and banked for later testing. The reference standard was an expert panel clinical adjudication, which was blinded to gene expression and procalcitonin results. SETTING: Four U.S. emergency departments. PATIENTS: Six-hundred twenty-three subjects with acute respiratory illness or suspected sepsis. INTERVENTIONS: Forty-five-transcript signature measured on the BioFire FilmArray System (BioFire Diagnostics, Salt Lake City, UT) in ~45 minutes. MEASUREMENTS AND MAIN RESULTS: Host response bacterial/viral test performance characteristics were evaluated in 623 participants (mean age 46 yr; 45% male) with bacterial infection, viral infection, coinfection, or noninfectious illness. Performance of the host response bacterial/viral test was compared with procalcitonin. The test provided independent probabilities of bacterial and viral infection in ~45 minutes. In the 213-subject training cohort, the host response bacterial/viral test had an area under the curve for bacterial infection of 0.90 (95% CI, 0.84-0.94) and 0.92 (95% CI, 0.87-0.95) for viral infection. Independent validation in 209 subjects revealed similar performance with an area under the curve of 0.85 (95% CI, 0.78-0.90) for bacterial infection and 0.91 (95% CI, 0.85-0.94) for viral infection. The test had 80.1% (95% CI, 73.7-85.4%) average weighted accuracy for bacterial infection and 86.8% (95% CI, 81.8-90.8%) for viral infection in this validation cohort. This was significantly better than 68.7% (95% CI, 62.4-75.4%) observed for procalcitonin (p < 0.001). An additional cohort of 201 subjects with indeterminate phenotypes (coinfection or microbiology-negative infections) revealed similar performance. CONCLUSIONS: The host response bacterial/viral measured using the BioFire System rapidly and accurately discriminated bacterial and viral infection better than procalcitonin, which can help support more appropriate antibiotic use.

Impact of Participation of Surgeons in Diagnostic Studies of Gastroesophageal Reflux Disease on Completion of Workup and Utilization of Antireflux Surgery.

Introduction. Patients presenting with symptoms of gastroesophageal reflux disease (GERD) are usually evaluated by gastroenterologists who perform the diagnostic workup and determine when to refer for surgical consideration. The multiple diagnostic studies can be overwhelming, and this leads to dropouts. In a rural setting, without gastroenterology services, the surgeon can diagnose GERD and perform antireflux procedures. This study aimed to assess the completion of the required diagnostic studies and progression to surgical intervention. Methods. This is a retrospective chart review of patients who presented with GERD symptoms between August 2015 and January 2018. Standardized workup included the upper gastrointestinal study and esophagogastroduodenoscopy with concomitant wireless pH placement. High-resolution impedance manometry and the gastric emptying scan were selectively utilized. Results. 429 patients were evaluated. Proton pump inhibitors were used by 82.2% of patients. The required diagnostic workup was completed by 92.7% of all patients. Nearly 75% were suitable candidates for antireflux surgery. Approximately 2/3 of these patients proceeded with antireflux surgery. Discussion. The lack of gastroenterology services in rural hospitals provides a unique opportunity for general surgeons to diagnose and treat GERD patients locally. This avoids fragmentation of care and enables the surgeon to evaluate the entire spectrum of GERD. This structured approach results in increased completion of multiple diagnostic studies. Moreover, surgical candidates are likely to proceed with surgical intervention. Conclusion. A surgical antireflux program with diagnostic and therapeutic capabilities results in increased completion of diagnostic workup and utilization of antireflux surgery.

Proteomic Profiles of Exosomes of Septic Patients Presenting to the Emergency Department Compared to Healthy Controls.

BACKGROUND: Septic Emergency Department (ED) patients provide a unique opportunity to investigate early sepsis. Recent work focuses on exosomes, nanoparticle-sized lipid vesicles (30-130 nm) that are released into the bloodstream to transfer its contents (RNA, miRNA, DNA, protein) to other cells. Little is known about how early changes related to exosomes may contribute to the dysregulated inflammatory septic response that leads to multi-organ dysfunction. We aimed to evaluate proteomic profiles of plasma derived exosomes obtained from septic ED patients and healthy controls. METHODS: This is a prospective observational pilot study evaluating a plasma proteomic exosome profile at an urban tertiary care hospital ED using a single venipuncture blood draw, collecting 40 cc Ethylenediaminetetraacetic acid (EDTA) blood. MEASUREMENTS: We recruited seven patients in the ED within 6 h of their presentation and five healthy controls. Plasma exosomes were isolated using the Invitrogen Total Exosome Isolation Kit. Exosome proteomic profiles were analyzed using fusion mass spectroscopy and Proteome Discoverer. Principal component analysis (PCA) and differential expression analysis (DEA) for sepsis versus control was performed. RESULTS: PCA of 261 proteins demonstrated septic patients and healthy controls were distributed in two groups. DEA revealed that 62 (23.8%) proteins differed between the exosomes of septic patients and healthy controls, p-value < 0.05. Adjustments using the False Discovery Rate (FDR) showed 23 proteins remained significantly different (FDR < 0.05) between sepsis and controls. Septic patients and controls were classified into two distinct groups by hierarchical clustering using the 62 nominally DE proteins. After adjustment multiple comparisons, three acute phase proteins remained significantly different between patients and controls: Serum amyloid A-1, C-reactive protein and Serum Amyloid A-2. Inflammatory response proteins immunoglobulin heavy constant Δ and Fc-fragment of IgG binding protein were increased. CONCLUSION: Exosome proteomic profiles of septic ED patients differ from their healthy counterparts with regard to acute phase response and inflammation.

A Physiologic Approach to Hemodynamic Monitoring and Optimizing Oxygen Delivery in Shock Resuscitation.

The approach to shock resuscitation focuses on all components of oxygen delivery, including preload, afterload, contractility, hemoglobin, and oxygen saturation. Resuscitation focused solely on preload and fluid responsiveness minimizes other key elements, resulting in suboptimal patient care. This review will provide a physiologic and practical approach for the optimization of oxygen delivery utilizing available hemodynamic monitoring technologies. Venous oxygen saturation (SvO2) and lactate will be discussed as indicators of shock states and endpoints of resuscitation within the framework of resolving oxygen deficit and oxygen debt.

The Benefits of Nonablative Radiofrequency Treatment of the Lower Esophageal Sphincter After Transoral Incisionless Fundoplication.

Non-ablative radiofrequency treatment to the lower esophageal sphincter (Stretta) has been shown to be beneficial after failed Nissen fundoplication. To our knowledge, this is the first report of Stretta after transoral incisionless fundoplication (TIF). This patient is a 17-year-old female who had gastroesophageal reflux disease (GERD) symptoms for 9 years. She presented with heartburn, regurgitation, and epigastric discomfort. She used omeprazole for 9 years. Esophagogastroduodenoscopy (EGD) showed a 2 cm sliding hiatal hernia and DeMeester score of 25. The GERD Health-Related Quality of Life (GERD-HRQL) score on omeprazole was 14. Patient underwent a TIF procedure, which was uneventful. Her symptoms resolved, and she discontinued omeprazole. Six months later, she had episodes of repeated violent vomiting followed by recurrence of regurgitation, nausea, bloating, and dysphagia. She resumed omeprazole. Diagnostic workup included gastric emptying scan, which was normal. EGD showed no hiatal hernia and partial disruption of the TIF valve. DeMeester score was 36.3. Esophageal manometry with impedance showed intact peristalsis and normal relaxation of the lower esophageal sphincter. The patient underwent Stretta, which was uneventful. The previous TIF did not increase the complexity of the procedure. There were no immediate or postoperative complications. The patient reported gradual improvement of her symptoms with complete resolution 2 months postoperatively. She discontinued omeprazole. The GERD-HRQL score 17 months post-Stretta was 0. This case highlights the feasibility, safety, and efficacy of performing Stretta following TIF. It provides an endoluminal alternative to complex revisional antireflux surgery. Prospective studies with longer follow-up are required to validate this concept.

F-Actin is associated with a worsening qSOFA score and intensive care unit admission in emergency department patients at risk for sepsis.

Objective: We previously demonstrated that plasma levels of F-actin and Thymosin Beta 4 differs among patients with septic shock, non-infectious systemic inflammatory syndrome and healthy controls and may serve as biomarkers for the diagnosis of sepsis. The current study aims to determine if these proteins are associated with or predictive of illness severity in patients at risk for sepsis in the Emergency Department (ED).Methods: Prospective, biomarker study enrolling patients (>18 years) who met the Shock Precautions on Triage Sepsis rule placing them at-risk for sepsis.Results: In this study of 203 ED patients, F-actin plasma levels had a linear trend of increase when the quick Sequential Organ Failure Assessment (qSOFA) score increased. F-actin was also increased in patients who were admitted to the Intensive Care Unit (ICU) from the ED, and in those with positive urine cultures. Thymosin Beta 4 was not associated with or predictive of any significant outcome measures.Conclusion: Increased levels of plasma F-actin measured in the ED were associated with incremental illness severity as measured by the qSOFA score and need for ICU admission. F-actin may have utility in risk stratification of undifferentiated patients in the ED presenting with signs and symptoms of sepsis.

Validation of a host response test to distinguish bacterial and viral respiratory infection.

BACKGROUND: Distinguishing bacterial and viral respiratory infections is challenging. Novel diagnostics based on differential host gene expression patterns are promising but have not been translated to a clinical platform nor extensively tested. Here, we validate a microarray-derived host response signature and explore performance in microbiology-negative and coinfection cases. METHODS: Subjects with acute respiratory illness were enrolled in participating emergency departments. Reference standard was an adjudicated diagnosis of bacterial infection, viral infection, both, or neither. An 87-transcript signature for distinguishing bacterial, viral, and noninfectious illness was measured from peripheral blood using RT-PCR. Performance characteristics were evaluated in subjects with confirmed bacterial, viral, or noninfectious illness. Subjects with bacterial-viral coinfection and microbiologically-negative suspected bacterial infection were also evaluated. Performance was compared to procalcitonin. FINDINGS: 151 subjects with microbiologically confirmed, single-etiology illness were tested, yielding AUROCs 0•85-0•89 for bacterial, viral, and noninfectious illness. Accuracy was similar to procalcitonin (88% vs 83%, p = 0•23) for bacterial vs. non-bacterial infection. Whereas procalcitonin cannot distinguish viral from non-infectious illness, the RT-PCR test had 81% accuracy in making this determination. Bacterial-viral coinfection was subdivided. Among 19 subjects with bacterial superinfection, the RT-PCR test identified 95% as bacterial, compared to 68% with procalcitonin (p = 0•13). Among 12 subjects with bacterial infection superimposed on chronic viral infection, the RT-PCR test identified 83% as bacterial, identical to procalcitonin. 39 subjects had suspected bacterial infection; the RT-PCR test identified bacterial infection more frequently than procalcitonin (82% vs 64%, p = 0•02). INTERPRETATION: The RT-PCR test offered similar diagnostic performance to procalcitonin in some subgroups but offered better discrimination in others such as viral vs. non-infectious illness and bacterial/viral coinfection. Gene expression-based tests could impact decision-making for acute respiratory illness as well as a growing number of other infectious and non-infectious diseases.

Evaluating the discriminating capacity of cell death (apoptotic) biomarkers in sepsis.

BACKGROUND: Sepsis biomarker panels that provide diagnostic and prognostic discrimination in sepsis patients would be transformative to patient care. We assessed the mortality prediction and diagnostic discriminatory accuracy of two biomarkers reflective of cell death (apoptosis), circulating cell-free DNA (cfDNA), and nucleosomes. METHODS: The cfDNA and nucleosome levels were assayed in plasma samples acquired in patients admitted from four emergency departments with suspected sepsis. Subjects with non-infectious systemic inflammatory response syndrome (SIRS) served as controls. Samples were acquired at enrollment (T0) and 24 h later (T24). We assessed diagnostic (differentiating SIRS from sepsis) and prognostic (28-day mortality) predictive power. Models incorporating procalcitonin (diagnostic prediction) and APACHE II scores (mortality prediction) were generated. RESULTS: Two hundred three subjects were included (107 provided procalcitonin measurements). Four subjects exhibited uncomplicated sepsis, 127 severe sepsis, 35 septic shock, and 24 had non-infectious SIRS. There were 190-survivors and 13 non-survivors. Mortality prediction models using cfDNA, nucleosomes, or APACHEII yielded AUC values of 0.61, 0.75, and 0.81, respectively. A model combining nucleosomes with the APACHE II score improved the AUC to 0.84. Diagnostic models distinguishing sepsis from SIRS using procalcitonin, cfDNA(T0), or nucleosomes(T0) yielded AUC values of 0.64, 0.65, and 0.63, respectively. The three parameter model yielded an AUC of 0.74. CONCLUSIONS: To our knowledge, this is the first head-to-head comparison of cfDNA and nucleosomes in diagnosing sepsis and predicting sepsis-related mortality. Both cfDNA and nucleosome concentrations demonstrated a modest ability to distinguish sepsis survivors and non-survivors and provided additive diagnostic predictive accuracy in differentiating sepsis from non-infectious SIRS when integrated into a diagnostic prediction model including PCT and APACHE II. A sepsis biomarker strategy incorporating measures of the apoptotic pathway may serve as an important component of a sepsis diagnostic and mortality prediction tool.

Change in Lactate Levels After Hemodialysis in Patients With End-Stage Renal Disease.

STUDY OBJECTIVE: Patients with end-stage renal disease commonly visit the emergency department (ED). The purpose of this investigation is to examine the prevalence of baseline abnormal lactate levels and to evaluate the effects of hemodialysis on serum lactate levels. METHODS: This was a prospective observational cohort study performed at an outpatient dialysis facility at an urban tertiary care hospital. The study consisted of 226 patients with end-stage renal disease who were receiving long-term hemodialysis and were enrolled during a 2-day period at the beginning of December 2015. Blood drawn for lactate levels was immediately analyzed before and after hemodialysis sessions. All patients completed their hemodialysis sessions. RESULTS: The prevalence of an abnormal lactate level (greater than 1.8 mmol/L) before hemodialysis was 17.7% (n=40). Overall, lactate levels decreased by 27% (SD 35%) after hemodialysis, with a decrease of 37% (SD 31%) for subgroups with a lactate level of 1.9 to 2.4 mmol/L, and 62% (SD 14%) with a lactate of 2.5 to 3.9 mmol/L. CONCLUSION: The data presented help providers understand the prevalence of abnormal lactate values in an outpatient end-stage renal disease population. After hemodialysis, lactate levels decreased significantly. This information may help medical providers interpret lactate values when patients with end-stage renal disease present to the ED.