Blood RNA Biomarkers Identify Bacterial and Biofilm Coinfections in COVID-19 Intensive Care Patients.

Purpose: Secondary opportunistic coinfections are a significant contributor to morbidity and mortality in intensive care unit (ICU) patients, but can be difficult to identify. Presently, new blood RNA biomarkers were tested in ICU patients to diagnose viral, bacterial, and biofilm coinfections. Methods: COVID-19 ICU patients had whole blood drawn in RNA preservative and stored at -80°C. Controls and subclinical infections were also studied. Droplet digital polymerase chain reaction (ddPCR) quantified 6 RNA biomarkers of host neutrophil activation to bacterial (DEFA1), biofilm (alkaline phosphatase [ALPL], IL8RB/CXCR2), and viral infections (IFI27, RSAD2). Viral titer in blood was measured by ddPCR for SARS-CoV2 (SCV2). Results: RNA biomarkers were elevated in ICU patients relative to controls. DEFA1 and ALPL RNA were significantly higher in severe versus incidental/moderate cases. SOFA score was correlated with white blood cell count (0.42), platelet count (-0.41), creatinine (0.38), and lactate dehydrogenase (0.31). ALPL RNA (0.59) showed the best correlation with SOFA score. IFI27 (0.52) and RSAD2 (0.38) were positively correlated with SCV2 viral titer. Overall, 57.8% of COVID-19 patients had a positive RNA biomarker for bacterial or biofilm infection. Conclusions: RNA biomarkers of host neutrophil activation indicate the presence of bacterial and biofilm coinfections in most COVID-19 patients. Recognizing coinfections may help to guide the treatment of ICU patients.

In Vitro Analysis of Platelet Adhesion, Aggregation, and Surface GP1bα Expression in Stored Refrigerated Whole Blood: A Pilot Study.

BACKGROUND: Warm, fresh whole blood (WB) has been used by the US military to treat casualties in Iraq and Afghanistan. Based on data in that setting, cold-stored WB has been used to treat hemorrhagic shock and severe bleeding in civilian trauma patients in the United States. In an exploratory study, we performed serial measurements of WB's composition and platelet function during cold storage. Our hypothesis was that in vitro platelet adhesion and aggregation would decrease over time. METHODS: WB samples were analyzed on storage days 5, 12, and 19. Hemoglobin, platelet count, blood gas parameters (pH, Po2, Pco2, and Spo2), and lactate were measured at each timepoint. Platelet adhesion and aggregation under high shear were assessed with a platelet function analyzer. Platelet aggregation under low shear was assessed using a lumi-aggregometer. Platelet activation was assessed by measuring dense granule release in response to high-dose thrombin. Platelet GP1bα levels were measured with flow cytometry, as a surrogate for adhesive capacity. Results at the 3 study timepoints were compared using repeat measures analysis of variance and post hoc Tukey tests. RESULTS: Measurable platelet count decreased from a mean of (163 + 53) × 109 platelets per liter at timepoint 1 to (107 + 32) × 109 at timepoint 3 (P = .02). Mean closure time on the platelet function analyzer (PFA)-100 adenosine diphosphate (ADP)/collagen test increased from 208.7 + 91.5 seconds at timepoint 1 to 390.0 + 148.3 at timepoint 3 (P = .04). Mean peak granule release in response to thrombin decreased significantly from 0.7 + 0.3 nmol at timepoint 1 to 0.4 + 0.3 at timepoint 3 (P = .05). Mean GP1bα surface expression decreased from 232,552.8 + 32,887.0 relative fluorescence units at timepoint 1 to 95,133.3 + 20,759.2 at timepoint 3 (P < .001). CONCLUSIONS: Our study demonstrated significant decreases in measurable platelet count, platelet adhesion, and aggregation under high shear, platelet activation, and surface GP1bα expression between cold-storage days 5 and 19. Further studies are needed to understand the significance of our findings and to what degree in vivo platelet function recovers after WB transfusion.

In Vitro Treatment of Extracorporeal Membrane Oxygenation Coagulopathy with Recombinant von Willebrand Factor or Lyophilized Platelets.

OBJECTIVES: The objective was to compare primary hemostasis between adult ECMO patients and cardiac surgical patients before heparinization and cardiopulmonary bypass. Furthermore, the authors explored whether in vitro treatment of ECMO patient blood samples with recombinant von Willebrand Factor (vWF) or lyophilized platelets improved primary hemostasis in vitro. DESIGN: Prospective cohort study. SETTING: Single academic medical center. PARTICIPANTS: Ten cardiac surgical patients and 8 adult ECMO patients. INTERVENTIONS: Cardiac surgical patients and ECMO patients had blood samples collected, and in vitro platelet thrombus formation was assessed using the ATLAS PST device. The ECMO patients had platelet thrombus formation evaluated at baseline and after in vitro treatment with recombinant vWF or lyophilized platelets, whereas cardiac surgical patients had a single blood sample obtained before heparinization and cardiopulmonary bypass run. MEASUREMENTS AND MAIN RESULTS: Median maximum force (39.7 v 260.2 nN) and thrombus area (0.05 v 0.11) at 5 minutes were lower in untreated ECMO patient samples compared with cardiac surgical patients (p = 0.008 and p < 0.001, respectively). The ECMO patient samples treated with recombinant vWF demonstrated an increase in both platelet maximum force (median value of 222.1 v 39.7 nN) (p = 0.01) and platelet thrombus area (median value of 0.16 v 0.05; p = 0.001). The ECMO patient samples treated with lyophilized platelets demonstrated no increase in platelet maximum force (median value of 193.3 v 39.7 nN; p = 0.18); however, there was a significant increase in platelet thrombus area (median value of 0.13 v 0.05; p = 0.04). CONCLUSIONS: Recombinant vWF and lyophilized platelets may help to restore primary hemostasis in ECMO patients. Future studies should further evaluate the safety and efficacy of these potential therapeutics in ECMO patients.

In vitro comparison of spatiotemporal fibrin clot formation dynamics in plasma treated with different protamine-heparin ratios.

INTRODUCTION: Our study aim was to explore how different protamine-heparin ratios impacted enzymatic coagulation and acellular fibrin clot growth in plasma using an in vitro model. We hypothesized that a low protamine-heparin ratio would be associated with superior fibrin clot growth dynamics. METHODS: We performed an in vitro study using 15 plasma samples from a commercial supplier. Different protamine-heparin ratios were added to each donor plasma sample: low ratio (0.7-1), traditional ratio (1-1), and high ratio (1.3-1) and clot formation dynamics were evaluated using a Thrombodynamics analyzer. Study outcomes were initial clot growth velocity and clot size at 30 min. RESULTS: Plasma samples treated with a one-to-one protamine-heparin ratio had significantly lower mean initial clot growth velocity compared to samples treated with a low protamine-heparin ratio; mean difference -2.3 µm/min (95% CI = -4.0 to -0.7, p = .004). Plasma samples treated with a one-to-one protamine-heparin ratio also had significantly smaller mean clot size at 30 min compared to samples treated with a low protamine-heparin ratio; mean difference -54.0 µm (95% CI = -107.6 to -0.4, p = .048). There were no significant differences in mean initial clot growth velocity or clot size at 30 min between plasma samples treated with a high protamine-heparin ratio and those treated with a one-to-one or low protamine-heparin ratio (all p > .05). CONCLUSIONS: Plasma samples treated with a low protamine-heparin ratio had superior clot growth velocity and larger clot size at 30 min compared to a one-to-one ratio, supporting the notion that a low protamine-heparin ratio may optimize enzymatic coagulation after cardiopulmonary bypass.

COVID-19 point-of-care tests can identify low-antibody individuals: In-depth immunoanalysis of boosting benefits in a healthy cohort.

The recommended COVID-19 booster vaccine uptake is low. At-home lateral flow assay (LFA) antigen tests are widely accepted for detecting infection during the pandemic. Here, we present the feasibility and potential benefits of using LFA-based antibody tests as a means for individuals to detect inadequate immunity and make informed decisions about COVID-19 booster immunization. In a health care provider cohort, we investigated the changes in the breadth and depth of humoral and T cell immune responses following mRNA vaccination and boosting in LFA-positive and LFA-negative antibody groups. We show that negative LFA antibody tests closely reflect the lack of functional humoral immunity observed in a battery of sophisticated immune assays, while positive results do not necessarily reflect adequate immunity. After booster vaccination, both groups gain depth and breadth of systemic antibodies against evolving SARS-CoV-2 and related viruses. Our findings show that LFA-based antibody tests can alert individuals about inadequate immunity against COVID-19, thereby increasing booster shots and promoting herd immunity.

Diagnostic accuracy of novel mRNA blood biomarkers of infection to predict outcomes in emergency department patients with undifferentiated abdominal pain.

Abdominal pain represents greater than 20% of US Emergency Department (ED) visits due to a wide range of illnesses. There are currently no reliable blood biomarkers to predict serious outcomes in patients with abdominal pain. Our previous studies have identified three mRNA transcripts related to innate immune activation: alkaline phosphatase (ALPL), interleukin-8 receptor-ß (IL8RB), and defensin-1 (DEFA1) as promising candidates to detect an intra-abdominal infection. The objective of this study was to evaluate the accuracy of these mRNA biomarkers to predict likely infection, hospitalization and surgery in Emergency Department patients with undifferentiated abdominal pain. We prospectively enrolled Emergency Department patients with undifferentiated abdominal pain who received an abdominal CT scan as part of their evaluation. Clinical outcomes were abstracted from the CT scan and medical records. mRNA biomarker levels were calculated independent of the clinical outcomes and their accuracy was assessed to predict infectious diagnoses, surgery and hospital admission. 89 patients were enrolled; 21 underwent surgery; 47 underwent hospital admission; and, no deaths were observed within 30 days. In identifying which cases were likely infectious, mRNA biomarkers' AUC values were: ALPL, 0.83; DEFA1 0.51; IL8RB, 0.74; and ALPL + IL8RB, 0.79. In predicting which Emergency Department patients would receive surgery, the AUC values were: ALPL, 0.75; DEFA1, 0.58; IL8RB, 0.75; and ALPL + IL8RB, 0.76. In predicting hospital admission, the AUC values were: ALPL, 0.78; DEFA1, 0.52; IL8RB, 0.74; and, ALPL + IL8RB, 0.77. For predicting surgery, ALPL + IL8RB's positive likelihood ratio (LR) was 3.97; negative LR (NLR) was 0.70. For predicting hospital admission, the same marker's positive LR was 2.80 with an NLR of 0.45. Where the primary cause for admission was a potentially infectious disorder, 33 of 34 cases (97%) had positive RNA scores. In a pragmatic, prospective diagnostic accuracy trial in Emergency Department patients with undifferentiated abdominal pain, mRNA biomarkers showed good accuracy to identify patients with potential infection, as well as those needing surgery or hospital admission.

RNAseq profiling of blood from patients with coronary artery disease: Signature of a T cell imbalance.

Background: Cardiovascular disease had a global prevalence of 523 million cases and 18.6 million deaths in 2019. The current standard for diagnosing coronary artery disease (CAD) is coronary angiography either by invasive catheterization (ICA) or computed tomography (CTA). Prior studies employed single-molecule, amplification-independent RNA sequencing of whole blood to identify an RNA signature in patients with angiographically confirmed CAD. The present studies employed Illumina RNAseq and network co-expression analysis to identify systematic changes underlying CAD. Methods: Whole blood RNA was depleted of ribosomal RNA (rRNA) and analyzed by Illumina total RNA sequencing (RNAseq) to identify transcripts associated with CAD in 177 patients presenting for elective invasive coronary catheterization. The resulting transcript counts were compared between groups to identify differentially expressed genes (DEGs) and to identify patterns of changes through whole genome co-expression network analysis (WGCNA). Results: The correlation between Illumina amplified RNAseq and the prior SeqLL unamplified RNAseq was quite strong (r = 0.87), but there was only 9 % overlap in the DEGs identified. Consistent with the prior RNAseq, the majority (93 %) of DEGs were down-regulated ~1.7-fold in patients with moderate to severe CAD (>20 % stenosis). DEGs were predominantly related to T cells, consistent with known reductions in Tregs in CAD. Network analysis did not identify pre-existing modules with a strong association with CAD, but patterns of T cell dysregulation were evident. DEGs were enriched for transcripts associated with ciliary and synaptic transcripts, consistent with changes in the immune synapse of developing T cells. Conclusions: These studies confirm and extend a novel mRNA signature of a Treg-like defect in CAD. The pattern of changes is consistent with stress-related changes in the maturation of T and Treg cells, possibly due to changes in the immune synapse.

Longitudinal Analysis of Humoral and Cellular Immune Response Following SARS-CoV-2 Vaccination Supports Utilizing Point-Of-Care Tests to Enhance COVID-19 Booster Uptake.

Individuals with weaker neutralizing responses show reduced protection with SARS-CoV-2 variants. Booster vaccines are recommended for vaccinated individuals, but the uptake is low. We present the feasibility of utilizing point-of-care tests (POCT) to support evidence-based decision-making around COVID-19 booster vaccinations. Using infectious virus neutralization, ACE2 blocking, spike binding, and TCR sequencing assays, we investigated the dynamics of changes in the breadth and depth of blood and salivary antibodies as well as T-cell clonal response following mRNA vaccination in a cohort of healthcare providers. We evaluated the accuracy of two POCTs utilizing either blood or saliva to identify those in whom humoral immunity was inadequate. >4 months after two doses of mRNA vaccine, SARS-CoV-2 binding and neutralizing Abs (nAbs) and T-cell clones declined 40-80%, and 2/3rd lacked Omicron nAbs. After the third mRNA booster, binding and neutralizing Abs increased overall in the systemic compartment; notably, individuals with previously weak nAbs gained sharply. The third dose failed to stimulate secretory IgA, but salivary IgG closely tracked systemic IgG levels. Vaccine boosting increased Ab breadth against a divergent bat sarbecovirus, SHC014, although the TCR-beta sequence breadth was unchanged. Post 3rd booster dose, Ab avidity increased for the Wuhan and Delta strains, while avidity against Omicron and SHC014 increased to levels seen for Wuhan after the second dose. Negative results on POCTs strongly correlated with a lack of functional humoral immunity. The third booster dose helps vaccinees gain depth and breadth of systemic Abs against evolving SARS-CoV-2 and related viruses. Our findings show that POCTs are useful and easy-to-access tools to inform inadequate humoral immunity accurately. POCTs designed to match the circulating variants can help individuals with booster vaccine decisions and could serve as a population-level screening platform to preserve herd immunity. One Sentence Summary: SARS-CoV-2 point-of-care antibody tests are valuable and easy-to-access tools to inform inadequate humoral immunity and to support informed decision-making regarding the current and future booster vaccination.