Background: Nucleocapsid antigenemia in adults has demonstrated high sensitivity and specificity for acute infection, and antigen burden is associated with disease severity. Data regarding SARS-CoV-2 antigenemia in children are limited. Methods: We retrospectively analyzed blood plasma specimens from hospitalized children with COVID-19 or MIS-C. Nucleocapsid and spike were measured using ultrasensitive immunoassays. Results: We detected nucleocapsid antigenemia in 62% (50/81) and spike antigenemia in 27% (21/79) of children with acute COVID-19 but 0% (0/26) and 15% (4/26) with MIS-C from March 2020-March 2021. Higher nucleocapsid levels were associated with radiographic infiltrates and respiratory symptoms in children with COVID-19. Conclusions: Antigenemia lacks the sensitivity to diagnose acute infection in children but is associated with signs and symptoms of lower respiratory tract involvement. Further study into the mechanism of antigenemia, its association with specific organ involvement, and the role of antigenemia in the pathogenesis of COVID-19 is warranted.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α–stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

While microscopy-based cellular assays, including microfluidics, have significantly advanced over the last several decades, there has not been concurrent development of widely-accessible techniques to analyze time-dependent microscopy data incorporating phenomena such as fluid flow and dynamic cell adhesion. As such, experimentalists typically rely on error-prone and time-consuming manual analysis, resulting in lost resolution and missed opportunities for innovative metrics. We present a user-adaptable toolkit packaged into the open-source, standalone Interactive Cellular assay Labeled Observation and Tracking Software (iCLOTS). We benchmark cell adhesion, single-cell tracking, velocity profile, and multiscale microfluidic-centric applications with blood samples, the prototypical biofluid specimen. Moreover, machine learning algorithms characterize previously imperceptible data groupings from numerical outputs. Free to download/use, iCLOTS addresses a need for a field stymied by a lack of analytical tools for innovative, physiologically-relevant assays of any design, democratizing use of well-validated algorithms for all end-user biomedical researchers who would benefit from advanced computational methods.

Introduction: Swab pooling may allow for more efficient use of point-of-care assays for SARS-CoV-2 detection in settings where widespread testing is warranted, but the effects of pooling on assay performance are not well described. Methods: We tested the Thermo-Fisher Accula rapid point-of-care RT-PCR platform with contrived pooled nasal swab specimens. Results: We observed a higher limit of detection of 3,750 copies/swab in pooled specimens compared to 2,250 copies/swab in individual specimens. Assay performance appeared worse in a specimen with visible nasal mucous and debris, although performance was improved when using a standard laboratory mechanical pipette compared to the transfer pipette included in the assay kit. Conclusion: Clinicians and public health officials overseeing mass testing efforts must understand limitations and benefits of swab or sample pooling, including reduced assay performance from pooled specimens. We conclude that the Accula RT-PCR platform remains an attractive candidate assay for pooling strategies owing to the superior analytical sensitivity compared to most home use and point-of-care tests despite the inhibitory effects of pooled specimens we characterized.

Background: As the transmission of endemic respiratory pathogens returns to prepandemic levels, understanding the epidemiology of respiratory coinfections in children with SARS-CoV-2 is of increasing importance. Methods: We performed a retrospective analysis of all pediatric patients 0-21 years of age who had a multiplexed BioFire Respiratory Panel 2.1 test performed at Children's Healthcare of Atlanta, Georgia, from January 1 to December 31, 2021. We determined the proportion of patients with and without SARS-CoV-2 who had respiratory coinfections and performed Poisson regression to determine the likelihood of coinfection and its association with patient age. Results: Of 19,199 respiratory panel tests performed, 1466 (7.64%) were positive for SARS-CoV-2, of which 348 (23.74%) also had coinfection with another pathogen. The most common coinfection was rhino/enterovirus (n = 230, 15.69%), followed by adenovirus (n = 62, 4.23%), and RSV (n = 45, 3.507%). Coinfections with SARS-CoV-2 were most commonly observed in the era of Delta (B.1.617.2) predominance (190, 54.60%), which coincided with periods of peak rhino/enterovirus and RSV transmission. Although coinfections were common among all respiratory pathogens, they were significantly less common with SARS-CoV-2 than other pathogens, with exception of influenza A and B. Children <2 years of age had the highest frequency of coinfection and of detection of any pathogen, including SARS-CoV-2. Among children with SARS-CoV-2, for every 1-year increase in age, the rate of coinfections decreased by 8% (95% CI, 6-9). Conclusions: Respiratory coinfections were common in children with SARS-CoV-2. Factors associated with the specific pathogen, host, and time period influenced the likelihood of coinfection.

Sickle cell disease (SCD) is caused by a single point mutation in the β-globin gene of hemoglobin, which produces an altered sickle hemoglobin (HbS). The ability of HbS to polymerize under deoxygenated conditions gives rise to chronic hemolysis, oxidative stress, inflammation, and vaso-occlusion. Herein, we review recent findings using microfluidic technologies that have elucidated mechanisms of oxygen-dependent and -independent induction of HbS polymerization and how these mechanisms elicit the biophysical and inflammatory consequences in SCD pathophysiology. We also discuss how validation and use of microfluidics in SCD provides the opportunity to advance development of numerous therapeutic strategies, including curative gene therapies.

The 4th Maastricht Consensus Conference on Thrombosis (MCCT), included the following themes: Theme 1: The coagulome as a critical driver of cardiovascular disease Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow and kidney. Four investigators shared their views on these organ-specific topics. Theme 2: Novel mechanisms of thrombosis Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infections associated-coagulopathies perturb the hemostatic balance resulting in thrombosis and/or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies This theme included state of the art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: how to utilize ex vivo models? Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularised organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation (ECMO) associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management Plenary presentations addressed controversial areas, ie thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies and clinically tested factor XI(a) inhibitors,both possibly with reduced bleeding risk. Finally, Covid-19 associated coagulopathy is revisited.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subgenomic RNA (sgRNA) may indicate actively replicating virus, but sgRNA abundance has not been systematically compared between SARS-CoV-2 variants. sgRNA was quantified in 169 clinical samples by real-time reverse-transcription polymerase chain reaction, demonstrating similar relative abundance among known variants. Thus, sgRNA detection can identify individuals with active viral replication regardless of variant.

UNLABELLED: Red blood cell (RBC) disorders affect billions worldwide. While alterations in the physical properties of aberrant RBCs and associated hemodynamic changes are readily observed, in conditions such as sickle cell disease and iron deficiency, RBC disorders can also be associated with vascular dysfunction. The mechanisms of vasculopathy in those diseases remain unclear and scant research has explored whether biophysical alterations of RBCs can directly affect vascular function. Here we hypothesize that the purely physical interactions between aberrant RBCs and endothelial cells, due to the margination of stiff aberrant RBCs, play a key role in this phenomenon for a range of disorders. This hypothesis is tested by direct simulations of a cellular scale computational model of blood flow in sickle cell disease, iron deficiency anemia, COVID-19, and spherocytosis. We characterize cell distributions for normal and aberrant RBC mixtures in straight and curved tubes, the latter to address issues of geometric complexity that arise in the microcirculation. In all cases aberrant RBCs strongly localize near the vessel walls (margination) due to contrasts in cell size, shape, and deformability from the normal cells. In the curved channel, the distribution of marginated cells is very heterogeneous, indicating a key role for vascular geometry. Finally, we characterize the shear stresses on the vessel walls; consistent with our hypothesis, the marginated aberrant cells generate large transient stress fluctuations due to the high velocity gradients induced by their near-wall motions. The anomalous stress fluctuations experienced by endothelial cells may be responsible for the observed vascular inflammation. SIGNIFICANCE STATEMENT: A common and potentially life-threatening complication of blood cell disorders is inflammation and dysfunction of the vascular wall, for reasons that remain unclear. To address this issue, we explore a purely biophysical hypothesis involving red blood cells using detailed computational simulations. Our results show that red blood cells that are pathologically altered in cell shape, size, and stiffness, which occurs in various blood disorders, strongly marginate, residing primarily in the cell-free layer near blood vessel walls, generating large shear stress fluctuations at the vessel wall that may be responsible for endothelial damage and inflammation.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies’ epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.