Metatranscriptome sequencing identifies Escherichia are major contributors to pathogenic functions and biofilm formation in diabetes related foot osteomyelitis.

Osteomyelitis in the feet of persons with diabetes is clinically challenging and is associated with high rates of amputation. In this study RNA-sequencing was employed to explore microbial metatranscriptomes with a view to understand the relative activity and functions of the pathogen/s responsible for diabetes foot osteomyelitis (DFO). We obtained 25 intraoperative bone specimens from persons with confirmed DFO, observing that Escherichia spp. (7%), Streptomyces spp. (7%), Staphylococcus spp. (6%), Klebsiella spp. (5%) and Proteus spp. (5%) are the most active taxa on average. Data was then subset to examine functions associated with pathogenesis (virulence and toxins), biofilm formation and antimicrobial/multi-drug resistance. Analysis revealed Escherichia spp. are the most active taxa relative to pathogenic functions with K06218 (mRNA interferase relE), K03699 (membrane damaging toxin tlyC) and K03980 (putative peptidoglycan lipid II flippase murJ), K01114 (membrane damaging toxin plc) and K19168 (toxin cptA) being the most prevalent pathogenic associated transcripts. The most abundant transcripts associated with biofilm pathways included components of the biofilm EPS matrix including glycogen synthesis, cellulose synthesis, colonic acid synthesis and flagella synthesis. We further observed enrichment of a key enzyme involved in the biosynthesis of L-rhamnose (K01710 -dTDP-glucose 4,6-dehydratase rfbB, rmlB, rffG) which was present in all but four patients with DFO.

A metatranscriptomic approach to explore longitudinal tissue specimens from non-healing diabetes related foot ulcers.

Cellular mechanisms and/or microbiological interactions which contribute to chronic diabetes related foot ulcers (DRFUs) were explored using serially collected tissue specimens from chronic DRFUs and control healthy foot skin. Total RNA was isolated for next-generation sequencing. We found differentially expressed genes (DEGs) and enriched hallmark gene ontology biological processes upregulated in chronic DRFUs which primarily functioned in the host immune response including: (i) Inflammatory response; (ii) TNF signalling via NFKB; (iii) IL6 JAK-STAT3 signalling; (iv) IL2 STAT5 signalling and (v) Reactive oxygen species. A temporal analysis identified RN7SL1 signal recognition protein and IGHG4 immunoglobulin protein coding genes as being the most upregulated genes after the onset of treatment. Testing relative temporal changes between healing and non-healing DRFUs identified progressive upregulation in healed wounds of CXCR5 and MS4A1 (CD20), both canonical markers of lymphocytes (follicular B cells/follicular T helper cells and B cells, respectively). Collectively, our RNA-seq data provides insights into chronic DRFU pathogenesis.

Host-microbe metatranscriptome reveals differences between acute and chronic infections in diabetes-related foot ulcers.

Virtually all diabetes-related foot ulcers (DRFUs) will become colonized by microorganisms that may increase the risk of developing an infection. The reasons why some ulcerations develop acute clinical infections (AI-DRFUs) whilst others develop chronic infection (CI-DRFUs) and the preceding host-microbe interactions in vivo remain largely unknown. Establishing that acute and chronic infections are distinct processes requires demonstrating that these are two different strategies employed by microbes when interacting with a host. In this study, dual-RNA seq was employed to differentiate the host-microbe metatranscriptome between DRFUs that had localized chronic infection or acute clinical infection. Comparison of the host metatranscriptome in AI-DRFUs relative to CI-DRFUs identified upregulated differentially expressed genes (DEGs) that functioned as regulators of vascular lymphatic inflammatory responses, T-cell signalling and olfactory receptors. Conversely, CI-DRFUs upregulated DEGs responsible for cellular homeostasis. Gene set enrichment analysis using Hallmark annotations revealed enrichment of immune and inflammatory profiles in CI-DRFUs relative to AI-DRFUs. Analysis of the microbial metatranscriptome identified the DEGs being enriched within AI-DRFUs relative to CI-DRFUs included several toxins, two-component systems, bacterial motility, secretion systems and genes encoding for energy metabolism. Functions relevant to DRFU pathology were further explored, including biofilm and bacterial pathogenesis. This identified that the expression of biofilm-associated genes was higher within CI-DRFUs compared to that of AI-DRFUs, with mucR being the most highly expressed gene. Collectively, these data provide insights into the host-microbe function in two clinically-distinct infective phenotypes that affect DRFUs. The data reveal that bacteria in acutely infected DRFUs prioritize motility over biofilm and demonstrate greater pathogenicity and mechanisms, which likely subvert host cellular and immune pathways to establish infection. Upregulation of genes for key vascular inflammatory mediators in acutely infected ulcers may contribute, in part, to the clinical picture of a red, hot, swollen foot, which differentiates an acutely infected ulcer from that of a chronic infection.

A multiomics approach to identify host-microbe alterations associated with infection severity in diabetic foot infections: a pilot study.

Diabetic foot infections (DFIs) are a major cause of hospitalization and can lead to lower extremity amputation. In this pilot study, we used a multiomics approach to explore the host-microbe complex within DFIs. We observed minimal differences in the overall microbial composition between PEDIS infection severities, however Staphylococcus aureus and Streptococcus genera were abundant and highly active in most mild to moderate DFIs. Further, we identified the significant enrichment of several virulence factors associated with infection pathogenicity belonging to both Staphylococcus aureus and Streptococcus. In severe DFIs, patients demonstrated a greater microbial diversity and differential gene expression demonstrated the enrichment of multispecies virulence genes suggestive of a complex polymicrobial infection. The host response in patients with severe DFIs was also significantly different as compared to mild to moderate DFIs. This was attributed to the enrichment of host genes associated with inflammation, acute phase response, cell stress and broad immune-related responses, while those associated with wound healing and myogenesis were significantly depleted.

Utilisation of the 2019 IWGDF diabetic foot infection guidelines to benchmark practice and improve the delivery of care in persons with diabetic foot infections.

AIMS: To utilise the 2019 International Working Group on the Diabetic Foot (IWGDF) - diabetic foot infection (DFI) guidelines as an audit tool for clinical practice in patients with diabetes attending a High-Risk Foot Service. METHODS: Data from 93 consecutive patients were collected over a 19-month period in patients attending a High-Risk Foot Service. The diagnosis and management of each patient in the sample were compared against the 2019 IWGDF DFI guidelines, grouped into four categories: Diagnosis, Microbiology, Treatment of soft tissue infection, and Surgical treatment and osteomyelitis. Deficits in performance were recorded using the recommendations as a benchmark standard. RESULTS: There were 109 DFI events. Nineteen (63%) of the recommendations were met, 7 (24%) were partially met, and four (13%) recommendations were not met. Fourteen of the sample had no documented requests for full blood counts. Tissue was obtained for culture in 32 (29%) of the sample. No percutaneous bone biopsies were performed. Only 13 (28%) patients had intraoperative bone specimens sent for culture and sensitivities, with no bone specimens sent for histopathology. Modification of antibiotic therapy following available culture results was low, occurring in 12 out of 63 possible occasions (19%). The duration of antibiotic regimens in PEDIS 2 infections and osteomyelitis was greater than that recommended. CONCLUSIONS: Utilising the IWGDF DFI guidelines to benchmark clinical practice is a useful tool to identify gaps in clinical performance or service delivery and may help to improve patient care.

Monitoring wound progression to healing in diabetic foot ulcers using three-dimensional wound imaging.

AIM: 3D wound imaging has provided clinicians with even greater wound measurement options. No data is available to guide clinicians as to which 3D measurements may yield the most reflective marker of wound progression to healing. METHOD: A prospective pilot study was undertaken to assess the accuracy of five 3D wound measurements that best reflect metrics of interest to clinicians. Twenty-one diabetic foot ulcers were enrolled from initial ulcer presentation, through to healing. The relationship between mean wound healing measurement variables was examined using linear regression and Pearsons correlation coefficient, in addition to assessing clinician inter-rater reliability of measurements using Intra-class correlation coefficients (ICC). RESULTS: Statistical analysis demonstrated a linear healing slope for each wound measurement as having a value greater than R 0.70 and a statistical significance of p = 0.0001. This suggests that all five wound measurements are useful prognostic markers of wound progression to healing. Low variability of measurements between users indicates good inter-observer reliability. CONCLUSION: 3D wound measurements demonstrate a linear correlation between the measurement and time to healing. This suggests they could be effective prognostic markers of a wounds progression to healing and closure. It may also provide important early identification of wounds not responding to standard care. Larger studies are required to validate our results.

Effect on total microbial load and community composition with two vs six-week topical Cadexomer Iodine for treating chronic biofilm infections in diabetic foot ulcers.

This study compares two vs six weeks of topical antimicrobial therapy with Cadexomer Iodine in patients with diabetic foot ulcers (DFUs) complicated by chronic biofilm infections. Patients with non-healing DFUs with suspected chronic biofilm infections were eligible for enrolment. Patients were randomised to receive either two or six weeks of treatment with topical Cadexomer Iodine. Tissue biopsies from the ulcers were obtained pre-and-post treatment and underwent DNA sequencing and real-time quantitative polymerase chain reaction (PCR) to determine the total microbial load, community composition, and diversity of bacteria. Scanning electron microscopy confirmed biofilm in all 18 ulcers with suspected chronic biofilm infections. Cadexomer Iodine resulted in 14 of 18 (78%) samples achieving a mean 0.5 log10 reduction in microbial load. Regardless of treatment duration, there was no statistical difference in the reduction of total microbial loads. No difference in the rate of wound healing in the two groups was seen at 6 weeks. Cadexomer Iodine reduces the total microbial load in DFUs with chronic biofilm infections and affects microbial community composition and diversity. All ulcers in both groups showed an initial reduction in wound size with application of Cadexomer Iodine, which might reflect its effect on biofilms.

A Cluster of Failures of Midline Catheters in a Hospital in the Home Program: A Retrospective Analysis.

A cluster of 11 midline catheter failures occurred during a 2-week period in a Hospital in the Home program in an urban tertiary hospital in Australia. These failures prompted a 4-month retrospective audit of patients receiving outpatient antimicrobial therapy between December 1, 2016 and March 1, 2017. Primary outcomes were dwell time and catheter failure. Peripherally inserted central catheters had significantly fewer failures and significantly longer dwell times compared with midline catheters. Women experienced higher rates of midline catheter failure than men. The proportion of patients with midline catheters receiving continuous infusions who experienced a failure was markedly higher than those receiving bolus doses. Suggestions for further related research are discussed.

Analysis of proximal bone margins in diabetic foot osteomyelitis by conventional culture, DNA sequencing and microscopy.

Multiple approaches were employed to detect pathogens from bone margins associated with Diabetic Foot Osteomyelitis (DFO). Intra-operative bone specimens of 14 consecutive subjects with suspected DFO were collected over a six-month study period from Liverpool Hospital. Infected bone and a proximal bone margins presumed to be 'clean/non-infected' were collected. Bone material was subjected to conventional culture, DNA sequencing and microscopy. In total, eight of 14 (57%) proximal bone margins had no growth by conventional culture but were identified in all proximal bone specimens by DNA sequencing. Proximal margins had lower median total microbial counts than infected specimens, but these differences were not statistically significant. Pathogens identified by sequencing in infected specimens were identified in proximal margins and the microbiomes were similar (ANOSIM = 0.02, p = 0.59). Using a combination of SEM and/or PNA-FISH, we visualized the presence of microorganisms in infected bone specimens and their corresponding proximal margins of seven patients (50%) with DFO. We identify that bacteria can still reside in what seems to be proximal 'clean' margins. The significance and implications of clinical outcomes requires further analysis from a larger sample size that incorporates differences in surgical and post-operative approaches, correlating any outcomes back to culture-sequence findings.

Outcomes and cost minimisation associated with outpatient parenteral antimicrobial therapy (OPAT) for foot infections in people with diabetes.

OBJECTIVE: To determine clinical outcomes in patients with diabetic foot infections receiving outpatient parenteral antimicrobial therapy (OPAT), to evaluate cost savings from the use of OPAT and to analyse demographic, clinical and laboratory data that may predict OPAT failure. RESEARCH DESIGN AND METHODOLOGY: A retrospective cohort analysis was conducted between 1 January 2007 and 7 July 2012 at a tertiary referral hospital in metropolitan Sydney. Patients with diabetic foot infection were identified from the outpatient parenteral antimicrobial therapy database. Demographic, clinical, laboratory and operative report data were obtained from patient charts and electronic medical records. Potential cost savings were calculated on the estimated cost of expenditure versus the expected savings. Linear regression was used to explore outcomes associated with outpatient parenteral antimicrobial therapy failure. RESULTS: Fifty-nine patients were identified over the 5-year study period. The outpatient parenteral antimicrobial therapy success rate for diabetic foot infections was 88%. Following the resolution of the primary episode of infection, new infective episodes within the study period were high (n = 26, 44%). Regression analysis of variables for OPAT failure failed to indicate any factors reaching statistical significance. A total of 1569 days were saved by using outpatient parenteral antimicrobial therapy for an estimated total cost saving of $983,645 or $16,672 per patient. CONCLUSION: Outpatient intravenous therapy for diabetic foot infections is an effective mode of treatment that can contribute to significant healthcare savings. High re-infection rates associated with diabetes foot ulceration in this population underline the need for close monitoring and management of these patients in multidisciplinary high-risk foot setting.

Update on the inaugural Sydney Diabetic Foot Conference 2013.

The first International Diabetic Foot Conference in Australia was hosted at Liverpool Hospital in Sydney during May 30-31, 2013. In response to the growing diabetes epidemic globally and more locally to Australia, the conference provided the perfect bridge for interaction between the multidisciplinary team members involved in diabetes care and the opportunity to assimilate the most up-to-date evidence-based medicine from some of the most respected researchers in the field.

Concordance between Sources of Morbidity Reports: Self-Reports and Medical Records.

As part of a 10-year follow-up study of morbidity following spouse bereavement, concordance between subject reports of their illness experience and that given by their doctors' and other medical records has been assessed. Enumeration from medical records involved extensive and careful perusal of general practitioner, specialist, and hospital records while subject reports were aided by a structured questionnaire which helped to prompt subjects' memories. The findings showed generally poor concordance between these two sources of morbidity data. Overall only 22% of disease events were found in both sources: of the diseases that did not match 65% were from the record source and 35% were from the self-report source. Despite finding that concordance rates varied with some subject and disease factors, concordance was always less than might be expected to occur by random chance (the throw of a coin). These findings have serious implications for epidemiological and pharmacoeconomic research involving morbidity history as they suggest that neither the subject nor their medical record can generally be assumed to provide a complete enumeration of morbidity burden. Indeed, irrespective of the significant factors under consideration, the maximum concordance reached in this study was 45.7%.

Subacute casemix classification for stroke rehabilitation in Australia. How well does AN-SNAP v2 explain variance in outcomes?

OBJECTIVE: We sought the best predictors for length of stay, discharge destination and functional improvement for inpatients undergoing rehabilitation following a stroke and compared these predictors against AN-SNAP v2. METHOD: The Oxfordshire classification subgroup, sociodemographic data and functional data were collected for patients admitted between 1997 and 2007, with a diagnosis of recent stroke. The data were factor analysed using Principal Components Analysis for categorical data (CATPCA). Categorical regression analyses was performed to determine the best predictors of length of stay, discharge destination, and functional improvement. RESULTS: A total of 1154 patients were included in the study. Principal components analysis indicated that the data were effectively unidimensional, with length of stay being the most important component. Regression analysis demonstrated that the best predictor was the admission motor FIM score, explaining 38.9% of variance for length of stay, 37.4%.of variance for functional improvement and 16% of variance for discharge destination. CONCLUSION: The best explanatory variable in our inpatient rehabilitation service is the admission motor FIM. AN- SNAP v2 classification is a less effective explanatory variable. This needs to be taken into account when using AN-SNAP v2 classification for clinical or funding purposes.

Representations and coverage of non-English-speaking immigrants and multicultural issues in three major Australian health care publications.

BACKGROUND: No recent Australian studies or literature, provide evidence of the extent of coverage of multicultural health issues in Australian healthcare research. A series of systematic literature reviews in three major Australian healthcare journals were undertaken to discover the level, content, coverage and overall quality of research on multicultural health. Australian healthcare journals selected for the study were The Medical Journal of Australia (MJA), The Australian Health Review (AHR), and The Australian and New Zealand Journal of Public Health (ANZPH). Reviews were undertaken of the last twelve (12) years (1996-August 2008) of journal articles using six standard search terms: 'non-English-speaking', 'ethnic', 'migrant', 'immigrant', 'refugee' and 'multicultural'. RESULTS: In total there were 4,146 articles published in these journals over the 12-year period. A total of 90 or 2.2% of the total articles were articles primarily based on multicultural issues. A further 62 articles contained a major or a moderate level of consideration of multicultural issues, and 107 had a minor mention. CONCLUSIONS: The quantum and range of multicultural health research and evidence required for equity in policy, services, interventions and implementation is limited and uneven. Most of the original multicultural health research articles focused on newly arrived refugees, asylum seekers, Vietnamese or South East Asian communities. While there is some seminal research in respect of these represented groups, there are other communities and health issues that are essentially invisible or unrepresented in research. The limited coverage and representation of multicultural populations in research studies has implications for evidence-based health and human services policy.

How are language barriers bridged in acute hospital care? The tale of two methods of data collection.

OBJECTIVE: To explore language service provision in a pilot hospital study with two methods of data collection. METHODS: This mixed mode study design comprises a multilingual telephone survey followed by a medical records audit, undertaken at Liverpool Hospital in 2004-05. RESULTS: Two hundred and fifty-eight patients responded from 360 patients representing nine language groups. About a third of patients with limited English proficiency had used a professional interpreter in hospital. Concordance between the multilingual telephone survey and the medical records audit was apparent, although the telephone survey mostly showed non-significant, higher rates than the audit. While the methods showed high agreement (76%) for frequency of interpreter usage, kappa indicated only fair agreement (PABAK 0.40). Forty-eight percent of the patients preferred relatives as interpreters and 51% felt that their inability to speak English negatively affected their hospital stay. CONCLUSIONS: Professional interpreter usage is lower than desirable in the hospital, especially in the Emergency Department. Relatives frequently interpret. Under-reporting on the medical record is suggested, implying a need for improved documentation, while possible over-reporting in the telephone survey may relate to recall bias and social acquiescence.

Analysis of draft Australian rehabilitation service standards: comparison with international standards.

BACKGROUND: Following her review of health systems and structures Dwyer 1 suggested that there is a need to evaluate models of care for individuals with chronic diseases. Rehabilitation services aim to optimise the activity and participation of individuals with restrictions due to both acute and chronic conditions. Assessing and optimising the standard of these services is one method of assuring the quality of service delivered to these individuals. Knowledge of baseline standards allows evaluation of the impact of health care reforms in this area of need. The aim of this article is to compare the currently available rehabilitation service standards in Australia with those used in the USA and the UK. RESULTS: The mixed method qualitative analysis performed on the three sets of standards demonstrated repeatability and convergence via the use of triangulation. Australian Faculty of Rehabilitation Medicine (AFRM) standards were found to be consistent and concise, to provide definitions, and to cover the majority of clinically relevant issues to an extent similar to the other rehabilitation service standards. Inclusion of standards for business practices, the rehabilitation process for the person served, and outpatient and community-based rehabilitation services should be considered by the AFRM. CONCLUSION: The AFRM standards are an appropriate way of assessing rehabilitation services in Australia. As suggested by other workers 23 there should be ongoing review and field testing of the standards to maximise the relevance and utilisation of the standards.