Wound samples: moving towards a standardised method of collection and analysis.

Chronic wounds, including diabetic foot ulcers, pressure ulcers and venous leg ulcers, impact the lives of millions of people worldwide. These types of wounds represent a significant physical, social and financial burden to both patients and health care systems. Wound care has made great progress in recent years as a result of the critical research performed in academic, clinical and industrial settings. However, there has been relatively little translation of basic research discoveries into novel and effective treatments. One underlying reason for this paucity may be inconsistency in the methods of wound analysis and sample collection, resulting in the inability of researchers to accurately characterise the healing process and compare results from different studies. This review examines the various types of analytical methods being used in wound research today with emphasis on sampling techniques, processing and storage, and the findings call forth the wound care research community to standardise its approach to wound analysis in order to yield more robust and comparable data sets.

Inclusion of the severe and enduring anorexia nervosa phenotype in genetics research: a scoping review.

BACKGROUND: Anorexia nervosa has one of the highest mortality rates of all mental illnesses. For those who survive, less than 70% fully recover, with many going on to develop a more severe and enduring phenotype. Research now suggests that genetics plays a role in the development and persistence of anorexia nervosa. Inclusion of participants with more severe and enduring illness in genetics studies of anorexia nervosa is critical. OBJECTIVE: The primary goal of this review was to assess the inclusion of participants meeting the criteria for the severe enduring anorexia nervosa phenotype in genetics research by (1) identifying the most widely used defining criteria for severe enduring anorexia nervosa and (2) performing a review of the genetics literature to assess the inclusion of participants meeting the identified criteria. METHODS: Searches of the genetics literature from 2012 to 2023 were performed in the PubMed, PsycINFO, and Web of Science databases. Publications were selected per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). The criteria used to define the severe and enduring anorexia nervosa phenotype were derived by how often they were used in the literature since 2017. The publications identified through the literature search were then assessed for inclusion of participants meeting these criteria. RESULTS: most prevalent criteria used to define severe enduring anorexia nervosa in the literature were an illness duration of ≥ 7 years, lack of positive response to at least two previous evidence-based treatments, a body mass index meeting the Diagnostic and Statistical Manual of Mental Disorders-5 for extreme anorexia nervosa, and an assessment of psychological and/or behavioral severity indicating a significant impact on quality of life. There was a lack of consistent identification and inclusion of those meeting the criteria for severe enduring anorexia nervosa in the genetics literature. DISCUSSION: This lack of consistent identification and inclusion of patients with severe enduring anorexia nervosa in genetics research has the potential to hamper the isolation of risk loci and the development of new, more effective treatment options for patients with anorexia nervosa.

Anorexia nervosa (AN) is a serious illness with a high death rate. Many of those with AN do not recover and have continuing severe psychological and physical symptoms that greatly impact their quality of life. Research has shown that genetics plays an important role, along with environment, in the development and persistence of AN. This review highlights the continued lack of consensus on defining criteria for severe and enduring AN in the literature and the continued focus on younger females with shorter illness durations in AN genetics research. Greater efforts are needed to include older participants with severe AN of longer duration in genetics research in hopes of developing more effective treatments for this underrepresented group.

Ancillary study management systems: a review of needs.

BACKGROUND: The valuable clinical data, specimens, and assay results collected during a primary clinical trial or observational study can enable researchers to answer additional, pressing questions with relatively small investments in new measurements. However, management of such follow-on, "ancillary" studies is complex. It requires coordinating across institutions, sites, repositories, and approval boards, as well as distributing, integrating, and analyzing diverse data types. General-purpose software systems that simplify the management of ancillary studies have not yet been explored in the research literature. METHODS: We have identified requirements for ancillary study management primarily as part of our ongoing work with a number of large research consortia. These organizations include the Center for HIV/AIDS Vaccine Immunology (CHAVI), the Immune Tolerance Network (ITN), the HIV Vaccine Trials Network (HVTN), the U.S. Military HIV Research Program (MHRP), and the Network for Pancreatic Organ Donors with Diabetes (nPOD). We also consulted with researchers at a range of other disease research organizations regarding their workflows and data management strategies. Lastly, to enhance breadth, we reviewed process documents for ancillary study management from other organizations. RESULTS: By exploring characteristics of ancillary studies, we identify differentiating requirements and scenarios for ancillary study management systems (ASMSs). Distinguishing characteristics of ancillary studies may include the collection of additional measurements (particularly new analyses of existing specimens); the initiation of studies by investigators unaffiliated with the original study; cross-protocol data pooling and analysis; pre-existing participant consent; and pre-existing data context and provenance. For an ASMS to address these characteristics, it would need to address both operational requirements (e.g., allocating existing specimens) and data management requirements (e.g., securely distributing and integrating primary and ancillary data). CONCLUSIONS: The scenarios and requirements we describe can help guide the development of systems that make conducting ancillary studies easier, less expensive, and less error-prone. Given the relatively consistent characteristics and challenges of ancillary study management, general-purpose ASMSs are likely to be useful to a wide range of organizations. Using the requirements identified in this paper, we are currently developing an open-source, general-purpose ASMS based on LabKey Server (http://www.labkey.org) in collaboration with CHAVI, the ITN and nPOD.

Increasing the presence of biofilm and healing delay in a porcine model of MRSA-infected wounds.

Data supporting the concept that microbial biofilms are a major cause of non-healing ulcers remain limited. A porcine model was established where delayed healing resulted from methicillin-resistant Staphylococcus aureus (MRSA) infection in full-thickness wounds. At the end of one study a wound remaining open was sampled and a MRSA strain was isolated. This pig-passaged strain was used as the inoculating strain in several subsequent studies. The resulting MRSA wound infections exhibited a greater, more stable tissue bioburden than seen in studies using the parent strain. Furthermore, wounds infected with the passaged strain experienced a greater delay in healing. To understand whether these changes corresponded to an increased biofilm character of the wound infection, wound biopsy samples from studies using either the parent or passaged MRSA strains were examined microscopically. Evidence of biofilm was observed for both strains, as most samples at a minimum had multiple isolated, dense microcolonies of bacteria. However, the passaged MRSA resulted in bacterial colonies of greater frequency and size that occurred more often in concatenated fashion to generate extended sections of biofilm. These results provide a model case in which increasing biofilm character of a wound infection corresponded with a greater delay in wound healing.

In vitro and in vivo studies on matrix metalloproteinases interacting with small intestine submucosa wound matrix.

Small intestine submucosa (SIS), a bioactive extracellular matrix (ECM) containing critical components of the ECM including collagens, proteoglycans, and glycosaminoglycans, has been widely used for wound healing. The purpose of this study was to investigate the interaction between SIS and matrix metalloproteinases (MMPs). MMP-1, MMP-2, and MMP-9 displayed different binding affinities, indicated by a loss in activity in solution upon incubation with SIS at 53·8%, 85·9%, and 36·9% over 24 hours, respectively. A cell migration study was conducted to evaluate the effects of MMPs and SIS on keratinocytes. The results indicated that MMPs inhibit keratinocyte migration in vitro, and that the inhibition can be significantly reduced by pre-incubating the MMP solution with SIS. To evaluate activity in vivo a diabetic mouse wound healing study was conducted. Biopsy samples were collected on different days for analysis of MMP levels by gelatin zymography. MMP activity was found to be attenuated by SIS treatment on day 3 after wounding. On day 7, the attenuation became less significant indicating that the MMP binding ability of SIS had become saturated. SIS was able to reduce MMP activity immediately, and may reduce the inhibitory effects of MMPs on keratinocyte migration.

LabKey Server: an open source platform for scientific data integration, analysis and collaboration.

BACKGROUND: Broad-based collaborations are becoming increasingly common among disease researchers. For example, the Global HIV Enterprise has united cross-disciplinary consortia to speed progress towards HIV vaccines through coordinated research across the boundaries of institutions, continents and specialties. New, end-to-end software tools for data and specimen management are necessary to achieve the ambitious goals of such alliances. These tools must enable researchers to organize and integrate heterogeneous data early in the discovery process, standardize processes, gain new insights into pooled data and collaborate securely. RESULTS: To meet these needs, we enhanced the LabKey Server platform, formerly known as CPAS. This freely available, open source software is maintained by professional engineers who use commercially proven practices for software development and maintenance. Recent enhancements support: (i) Submitting specimens requests across collaborating organizations (ii) Graphically defining new experimental data types, metadata and wizards for data collection (iii) Transitioning experimental results from a multiplicity of spreadsheets to custom tables in a shared database (iv) Securely organizing, integrating, analyzing, visualizing and sharing diverse data types, from clinical records to specimens to complex assays (v) Interacting dynamically with external data sources (vi) Tracking study participants and cohorts over time (vii) Developing custom interfaces using client libraries (viii) Authoring custom visualizations in a built-in R scripting environment. Diverse research organizations have adopted and adapted LabKey Server, including consortia within the Global HIV Enterprise. Atlas is an installation of LabKey Server that has been tailored to serve these consortia. It is in production use and demonstrates the core capabilities of LabKey Server. Atlas now has over 2,800 active user accounts originating from approximately 36 countries and 350 organizations. It tracks roughly 27,000 assay runs, 860,000 specimen vials and 1,300,000 vial transfers. CONCLUSIONS: Sharing data, analysis tools and infrastructure can speed the efforts of large research consortia by enhancing efficiency and enabling new insights. The Atlas installation of LabKey Server demonstrates the utility of the LabKey platform for collaborative research. Stable, supported builds of LabKey Server are freely available for download at http://www.labkey.org. Documentation and source code are available under the Apache License 2.0.

LabKey Server NAb: a tool for analyzing, visualizing and sharing results from neutralizing antibody assays.

BACKGROUND: Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. RESULTS: We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb) assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. CONCLUSIONS: Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. Many members of the HIV research community can also access the LabKey Server NAb tool without installing the software by using the Atlas Science Portal (https://atlas.scharp.org). Atlas is an installation of LabKey Server.

pH in the bacteria-contaminated wound and its impact on clostridium histolyticum collagenase activity: implications for the use of collagenase wound debridement agents.

PURPOSE: We sought to understand the influence of pH on Clostridium collagenase activity, using both in vitro and in vivo, and to understand the influence of bacterial contamination on pH in vivo. DESIGN: Artificial wound eschar was used for the assessment of debridement efficacy in vitro and the wound fluid of a contaminated pig wound model was used for examining pH during healing using in vivo techniques. METHODS: We used a collagen-based artificial wound eschar to test collagenase activity in the collagenase product under various pH conditions. We evaluated bacterial contaminated wounds, using a pig wound model with a bacterial load including Pseudomonas aeruginosa, coagulase-negative staphylococci, and Fusobacterium sp to track the pH of wounds in relation to bacterial load. RESULTS: The pH levels in the wound fluid were all above neutral. They varied from 9.2 on day 1 to 8.3 on day 10. Collagenase achieved its highest activity around a pH of 8.5 when tested in Tris-buffered saline. Using artificial wound eschar, the optimal pH range for C collagenase was determined to be more than a pH of 6. The total initial microbial load (day 0) was higher than levels at any other time during the study. The levels of P aeruginosa began to decrease on day 1, but by day 4 the total pseudomonas population had rebounded to near day 0 levels. This was followed by a distinct decrease and by day 21 levels were lower than those on day 0. The coagulase negative staphylococci (CNS) population behaved differently than the pseudomonas population, decreasing and remaining decreased relative to day 0 through day 14. However, by day 21 the CNS population had increased to near day 0 levels. This greatly influenced the increase in total bioburden by day 21, indicating that it was primarily due to the increase in CNS. CONCLUSION: The data demonstrate that wound pH in a model of contaminated pig wounds is alkaline during the first 10 days of healing. As healing progresses, pH decreases with no significant change in the level of bacterial bioburden. C collagenase exhibited robust activity in the pH range found in this contaminated pig wound model, suggesting it can effectively debride necrotic tissue in the environment found in most chronic wounds in humans.

The prevalence of suspected ventilator-associated pneumonia in Scottish intensive care units.

Ventilator-associated pneumonia is the most common healthcare-associated infection in mechanically ventilated patients. Despite this, accurate diagnosis of ventilator-associated pneumonia is difficult owing to the variety of criteria that exist. In this prospective national audit, we aim to quantify the existence of patients with suspected ventilator-associated pneumonia that would not be detected by our standard healthcare-associated infection screening process. Furthermore, we aim to assess the impact of tracheostomy insertion, subglottic drainage endotracheal tubes and chlorhexidine gel on ventilator-associated pneumonia rate. Of the 227 patients recruited, suspected ventilator-associated pneumonia occurred in 32 of these patients. Using the HELICS definition, 13/32 (40.6%) patients were diagnosed with ventilator-associated pneumonia (H-posVAP). Suspected ventilator-associated pneumonia rate was increased in our tracheostomy population, decreased in the subglottic drainage endotracheal tube group and unchanged in the chlorhexidine group. The diagnosis of ventilator-associated pneumonia remains a contentious issue. The formalisation of the HELICS criteria by the European CDC should allow standardised data collection throughout Europe, which will enable more consistent data collection and meaningful data comparison in the future. Our data add weight to the argument against routine oral chlorhexidine. The use of subglottic drainage endotracheal tubes in preventing ventilator-associated pneumonia is interesting and requires further investigation.

Anti-inflammatory Effects of Clostridial Collagenase Results from In Vitro and Clinical Studies.

BACKGROUND: Digestion of collagen with clostridial collagenase (CC) produces peptides that can induce cellular responses consistent with wound healing in vivo. However, nonhealing human wounds are typically in a state of chronic inflammation. We evaluated the effects of CC on markers of inflammation in cell culture and wound fluid from diabetic patients. METHODS: Lipopolysaccharide-induced release of tumor necrosis factor-α and interleukin-6 from interferon-γ-activated THP-1 monocytes was measured in the presence or absence of CC or CC collagen digests. In the clinical study, 17 individuals with mildly inflamed diabetic foot ulcers were randomized to receive CC ointment (CCO) or hydrogel. Weekly assessments included wound appearance and measurements. Wound exudate was collected at baseline and at 2 and 4 weeks of treatment. A multiplex assay was used to measure levels of analytes, including those associated with inflammation and with inflammation resolution. RESULTS: Lower levels of tumor necrosis factor-α and interleukin-6 were found in media of cells cultured with CC or CC digests of collagen type I or III than for untreated lipopolysaccharide controls (P < .05). Clinically, CCO and hydrogel resulted in improvement in wound appearance and a decrease in mean wound area. The CCO, but not the hydrogel, was found to increase the level of analytes associated with resolution of inflammation while decreasing those associated with inflammation. There was a general correlation between resolution of inflammation and healing. CONCLUSIONS: These results support a hypothesis that debridement with CCO is associated with decreased inflammation and greater progress toward healing.

Validation of the multiple organ dysfunction (MOD) score in critically ill medical and surgical patients.

OBJECTIVE: To validate the Multiple Organ Dysfunction (MOD) score externally. DESIGN: Prospective observational cohort study. SETTING: Mixed medical/surgical ICU in a tertiary referral university hospital. PATIENTS AND PARTICIPANTS: Thousand eight hundred and nine patients admitted to ICU for more than 24 h over a 3-year period. INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The MOD score was calculated daily for all patients. The criterion validity of the individual organ scores, the maximal MOD score and the change in MOD score were assessed by examining the relationship between increasing scores and ICU mortality. Increased maximal MOD scores and each of the six individual organ scores, and change in MOD scores were associated with increased mortality. CONCLUSIONS: Maximal and individual organ scores have criterion validity when tested in a different ICU from that in which the scores were derived, indicating that the scoring systems are reproducible. The association of change in MOD score with mortality indicates that the score is responsive. These data, combined with previous data establishing concept and content validity, indicate that the MOD score is a valid measure of multi-organ dysfunction.

The effect of adsorption, filter material and point of dilution on antibiotic elimination by haemofiltration an in vitro study of levofloxacin.

We studied an in vitro model of continuous venous-venous haemofiltration (CVVH), into which levofloxacin 100 mg was infused, to determine levofloxacin adsorption and to determine the effect of filter material and point of dilution (pre- or post-filter) on sieving coefficient. Mean (standard deviation; S.D.) adsorption was 18.7 (5.3) mg for the polyamide filter and 40.2 (2.0) mg for the polyacrylonitrile (PAN) filter (P < 0.001). Post-dilution resulted in a minor, but statistically significant, decrease in sieving coefficient (pre-dilution 0.96 (S.D. 0.10), post-dilution 0.88 (S.D. 0.11) with the PAN filter. These data indicate that the variability in published values for levofloxacin sieving coefficient are not due to variation in point of dilution or membrane type (PAN or polyamide). Significant adsorption of levofloxacin onto PAN filters occurs.