665. Clinical and Financial Impact of Next Generation Sequencing (NGS) in addition to Conventional Microbiology Testing in our Urban Referral Health Center

Background Clinical microbiology traditionally relies on culture methodology and serological testing, that have inherent limitations. Newer diagnostic techniques such as Next Generation Sequencing (NGS) have shown promise to improve microbial identification. In select scenarios, we send clinical specimens to reference laboratories for NGS testing in addition to current standard of care (SOC) diagnostics. We wanted to determine how this diagnostic approach has impacted patient care. We also wanted to review the financial burden through cost-benefit analysis for these ‘send-out’ tests. Methods We performed a retrospective chart review of all cases over a 3-year period in which NGS was submitted. Data, including demographics, comorbidities, antimicrobial use, and diagnosis (by SOC and NGS) were gathered. We delineated how often there was concordance or discordance between SOC and NGS. We also obtained information on financial cost (direct and indirect) and turnaround time (TAT) for NGS results. Results A total of 33 clinical specimens from 25 patients were sent for NGS. The majority of specimens comprised joint tissue/fluid, organ tissue and CSF. Concordance occurred between SOC and NGS testing in 75.8% (25/33) of samples; of those, 88% excluded infection. NGS identified a pathogen in 20% (5/25) patients in which concomitant SOC testing was negative. A subsequent change in antimicrobial management occurred in 16% (4/25) of patients. The mean TAT was 14 days and average cost per specimen was &821.52 (range: &573-&1590). Table 1. Pathogens identified by NGS with negative traditional microbiological test results Figure 1. Distribution of specimen site (in %) sent for NGS Conclusion NGS can provide additional diagnostic sensitivity in infectious diseases, which at our institution identified a new pathogen in 20% and a resultant treatment change in 16% of our patients. This testing may also allow physicians to reaffirm the absence of an infection diagnosis. A larger NGS testing population may reveal more significant benefits. While the attributable cost of NGS was substantial, it should be measured against the costs of administration of unnecessary antibiotics, inaccurate diagnosis, and adverse patient outcomes that may result from SOC testing alone. Considering its financial cost and extended TAT, in-house NGS testing may be warranted to facilitate a higher volume of testing. Disclosures All Authors: No reported disclosures

1368. Species Diversity and Co-Occurrence Patterning in Bone and Soft Tissue Infections

Background Polymicrobial results from conventional cultures in bone and soft tissue infections have generally been reported as independent co-occurrences (i.e. assuming no relationship or interaction between species). The objective of this study was to identify non-random (dependent) co-occurrence in bone and soft tissue infections. Methods We used single center Theradoc (Premiere Inc., Charlotte, NC) data on microbiology specimens collected over a decade (4/2010 to 4/2020). We included only tissue, fluid and swab specimens identified as being obtained from bone, skin, soft tissue or a wound during a patient’s first episode of infection. Blood and liver biopsy specimens were excluded. Patterning involving >2 organisms was examined with factor analysis. Analyses were done with R version 3.6.1. Threshold p-values were adjusted for multiple comparisons, often to p< 0.001. Results Polymicrobial culture results were seen much more frequently in the foot (76%) and sacrum (78%) than specimens from other locations (43%). The highest Shannon diversity index was seen in sacral specimens, followed by foot and leg specimens. Pseudomonas aeruginosa and E. coli was found more frequently in sacral bone and soft tissue specimens, while S. aureus was found most often in the spine bone and foot bone and soft tissue. Corynebacterium was found more frequently in foot bone (23%) and foot soft tissue (18%) than in non-foot soft tissue specimens (11%). Analysis of pairwise species co-occurrences in foot specimens (n=765) demonstrated that S. aureus made significantly less frequent the presence of Bacteroides, Enterobacter, and other staphylococcal species (Table 1). Analysis of non-foot soft tissue specimens (n=2,647) confirmed this pairwise associations and suggested three patterns (Table 2): A, polymicrobial without S. aureus; B, Enterococcus + gram negatives; and C, streptococcal. Table 1. Pairwise co-occurrences between species in bone and soft tissue specimens from foot infections (n=765). Factor analysis results demonstrating species associated with three polymicrobial patterns in soft tissue infection specimens. Blue denotes positive association (more often occurring as part of the pattern), red or grey denotes negative associations (less often occurring as part of the pattern). Numbers denote factor weights, a measure of the strength of the association. Conclusion Species incidence and diversity vary by location and tissue type. The many non-random occurrences found suggest complex interrelationships between microbes in soft tissue and bone infections, including organisms often classified as non-pathogens. Further study may further characterize these relationships and aid in antibiotic selection. Disclosures All Authors: No reported disclosures

Lymphatic Clearance of Immune Cells in Cardiovascular Disease

Recent advances in our understanding of the lymphatic system, its function, development, and role in pathophysiology have changed our views on its importance. Historically thought to be solely involved in the transport of tissue fluid, lipids, and immune cells, the lymphatic system displays great heterogeneity and plasticity and is actively involved in immune cell regulation. Interference in any of these processes can be deleterious, both at the developmental and adult level. Preclinical studies into the cardiac lymphatic system have shown that invoking lymphangiogenesis and enhancing immune cell trafficking in ischaemic hearts can reduce myocardial oedema, reduce inflammation, and improve cardiac outcome. Understanding how immune cells and the lymphatic endothelium interact is also vital to understanding how the lymphatic vascular network can be manipulated to improve immune cell clearance. In this Review, we examine the different types of immune cells involved in fibrotic repair following myocardial infarction. We also discuss the development and function of the cardiac lymphatic vasculature and how some immune cells interact with the lymphatic endothelium in the heart. Finally, we establish how promoting lymphangiogenesis is now a prime therapeutic target for reducing immune cell persistence, inflammation, and oedema to restore heart function in ischaemic heart disease.

AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

The lymphatics control tissue fluid homeostasis; its dysfunction contributes to lymphedema. VEGFR3 signaling dictates LEC fate specification and lymphangiogenesis. Cholesterol is essential for cell function and organ development, yet the molecular mechanism by which cholesterol controls lymphangiogenesis is unknown. Here, we show that APOA1 binding protein (AIBP), a secreted protein, enhances LEC specification and increases lymphangiogenesis. Mechanistically, AIBP-mediated cholesterol efflux disrupts LEC caveolae, which abolishes CAV-1-dependent inhibition of VEGFR3 signaling. Loss of Aibp2, the zebrafish paralog of human AIBP, reduces LEC progenitors and impairs lymphangiogenesis; the impairment can be rescued by caveolae disruption. CAV-1 mutant that is deficient in VEGFR3 binding, thereby abolishing its inhibition, enhances VEGFR3 signaling and accelerates lymphatic growth. Furthermore, AIBP expression is reduced in the epidermis of human lymphedema. Administrating recombinant AIBP augments VEGFC-induced lymphangiogenesis and increases secondary tail lymphedema resolution in adult mice. Our studies reveal AIBP and CAV-1 as critical regulators of VEGFR3 signaling and identify previously unidentified therapeutic targets for lymphedema treatment.

Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities

Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements. We introduce here the BAC-transgenic mouse reporter strain Vegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements. Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery, liver and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.

Similar Piperacillin/Tazobactam Target Attainment in Obese versus Nonobese Patients despite Differences in Interstitial Tissue Fluid Pharmacokinetics

Precision dosing of piperacillin/tazobactam in obese patients is compromised by sparse information on target-site exposure. We aimed to evaluate the appropriateness of current and alternative piperacillin/tazobactam dosages in obese and nonobese patients. Based on a prospective, controlled clinical trial in 30 surgery patients (15 obese/15 nonobese; 0.5-h infusion of 4 g/0.5 g piperacillin/tazobactam), piperacillin pharmacokinetics were characterized in plasma and at target-site (interstitial fluid of subcutaneous adipose tissue) via population analysis. Thereafter, multiple 3–4-times daily piperacillin/tazobactam short-term/prolonged (recommended by EUCAST) and continuous infusions were evaluated by simulation. Adequacy of therapy was assessed by probability of pharmacokinetic/pharmacodynamic target-attainment (PTA ≥ 90%) based on time unbound piperacillin concentrations exceed the minimum inhibitory concentration (MIC) during 24 h (%fT>MIC). Lower piperacillin target-site maximum concentrations in obese versus nonobese patients were explained by the impact of lean (approximately two thirds) and fat body mass (approximately one third) on volume of distribution. Simulated steady-state concentrations were 1.43-times, 95%CI = (1.27; 1.61), higher in plasma versus target-site, supporting targets of %fT>2×MIC instead of %fT>4×MIC during continuous infusion to avoid target-site concentrations constantly below MIC. In all obesity and renally impairment/hyperfiltration stages, at MIC = 16 mg/L, adequate PTA required prolonged (thrice-daily 4 g/0.5 g over 3.0 h at %fT>MIC = 50) or continuous infusions (24 g/3 g over 24 h following loading dose at %fT>MIC = 98) of piperacillin/tazobactam.

Designing electrode configuration of electroosmosis based edema treatment as a complement to hyperosmotic therapy

Background Hyperosmotic therapy is a mainstay treatment for cerebral edema. Although often effective, its disadvantages include mainly acting on the normal brain region with limited effectiveness in eliminating excess fluid in the edema region. This study investigates how to configure our previously proposed novel electroosmosis based edema treatment as a complement to hyperosmotic therapy. Methods Three electrode configurations are designed to drive the excess fluid out of the edema region, including 2-electrode, 3-electrode, and 5-electrode designs. The focality and directionality of the induced electroosmotic flow (EOF) are then investigated using the same patient-specific head model with localized edema. Results The 5-electrode design shows improved EOF focality with reduced effect on the normal brain region than the other two designs. Importantly, this design also achieves better directionality driving excess edema tissue fluid to a larger region of surrounding normal brain where hyperosmotic therapy functions better. Thus, the 5-electrode design is suggested to treat edema more efficiently via a synergic effect: the excess fluid is first driven out from the edema to surrounding normal brain via EOF, where it can then be treated with hyperosmotic therapy. Meanwhile, the 5-electrode design drives 2.22 mL excess fluid from the edema region in an hour comparable to the other designs, indicating a similar efficiency of EOF. Conclusions The results show that the promise of our previously proposed novel electroosmosis based edema treatment can be designed to achieve better focality and directionality towards a complement to hyperosmotic therapy.

Lymphatic Malformations: Genetics, Mechanisms and Therapeutic Strategies

Lymphatic vessels maintain tissue fluid homeostasis by returning to blood circulation interstitial fluid that has extravasated from the blood capillaries. They provide a trafficking route for cells of the immune system, thus critically contributing to immune surveillance. Developmental or functional defects in the lymphatic vessels, their obstruction or damage, lead to accumulation of fluid in tissues, resulting in lymphedema. Here we discuss developmental lymphatic anomalies called lymphatic malformations and complex lymphatic anomalies that manifest as localized or multifocal lesions of the lymphatic vasculature, respectively. They are rare diseases that are caused mostly by somatic mutations and can present with variable symptoms based upon the size and location of the lesions composed of fluid-filled cisterns or channels. Substantial progress has been made recently in understanding the molecular basis of their pathogenesis through the identification of their genetic causes, combined with the elucidation of the underlying mechanisms in animal disease models and patient-derived lymphatic endothelial cells. Most of the solitary somatic mutations that cause lymphatic malformations and complex lymphatic anomalies occur in genes that encode components of oncogenic growth factor signal transduction pathways. This has led to successful repurposing of some targeted cancer therapeutics to the treatment of lymphatic malformations and complex lymphatic anomalies. Apart from the mutations that act as lymphatic endothelial cell–autonomous drivers of these anomalies, current evidence points to superimposed paracrine mechanisms that critically contribute to disease pathogenesis and thus provide additional targets for therapeutic intervention. Here, we review these advances and discuss new treatment strategies that are based on the recently identified molecular pathways.