FLASH radiotherapy for the treatment of symptomatic bone metastases in the thorax (FAST-02): protocol for a prospective study of a novel radiotherapy approach.

BACKGROUND: FLASH therapy is a treatment technique in which radiation is delivered at ultra-high dose rates (≥ 40 Gy/s). The first-in-human FAST-01 clinical trial demonstrated the clinical feasibility of proton FLASH in the treatment of extremity bone metastases. The objectives of this investigation are to assess the toxicities of treatment and pain relief in study participants with painful thoracic bone metastases treated with FLASH radiotherapy, as well as workflow metrics in a clinical setting. METHODS: This single-arm clinical trial is being conducted under an FDA investigational device exemption (IDE) approved for 10 patients with 1-3 painful bone metastases in the thorax, excluding bone metastases in the spine. Treatment will be 8 Gy in a single fraction administered at ≥ 40 Gy/s on a FLASH-enabled proton therapy system delivering a single transmission proton beam. Primary study endpoints are efficacy (pain relief) and safety. Patient questionnaires evaluating pain flare at the treatment site will be completed for 10 consecutive days post-RT. Pain response and adverse events (AEs) will be evaluated on the day of treatment and on day 7, day 15, months 1, 2, 3, 6, 9, and 12, and every 6 months thereafter. The outcomes for clinical workflow feasibility are the occurrence of any device issues as well as time on the treatment table. DISCUSSION: This prospective clinical trial will provide clinical data for evaluating the efficacy and safety of proton FLASH for palliation of bony metastases in the thorax. Positive findings will support the further exploration of FLASH radiation for other clinical indications including patient populations treated with curative intent. REGISTRATION: ClinicalTrials.gov NCT05524064.

The Association of Incidental Radiation Dose to the Heart Base with Overall Survival and Cardiac Events after Curative-intent Radiotherapy for Non-small Cell Lung Cancer: Results from the NI-HEART Study.

AIMS: Cardiac disease is a dose-limiting toxicity in non-small cell lung cancer radiotherapy. The dose to the heart base has been associated with poor survival in multiple institutional and clinical trial datasets using unsupervised, voxel-based analysis. Validation has not been undertaken in a cohort with individual patient delineations of the cardiac base or for the endpoint of cardiac events. The purpose of this study was to assess the association of heart base radiation dose with overall survival and the risk of cardiac events with individual heart base contours. MATERIALS AND METHODS: Patients treated between 2015 and 2020 were reviewed for baseline patient, tumour and cardiac details and both cancer and cardiac outcomes as part of the NI-HEART study. Three cardiologists verified cardiac events including atrial fibrillation, heart failure and acute coronary syndrome. Cardiac substructure delineations were completed using a validated deep learning-based autosegmentation tool and a composite cardiac base structure was generated. Cox and Fine-Gray regressions were undertaken for the risk of death and cardiac events. RESULTS: Of 478 eligible patients, most received 55 Gy/20 fractions (96%) without chemotherapy (58%), planned with intensity-modulated radiotherapy (71%). Pre-existing cardiovascular morbidity was common (78% two or more risk factors, 46% one or more established disease). The median follow-up was 21.1 months. Dichotomised at the median, a higher heart base Dmax was associated with poorer survival on Kaplan-Meier analysis (20.2 months versus 28.3 months; hazard ratio 1.40, 95% confidence interval 1.14-1.75, P = 0.0017) and statistical significance was retained in multivariate analyses. Furthermore, heart base Dmax was associated with pooled cardiac events in a multivariate analysis (hazard ratio 1.75, 95% confidence interval 1.03-2.97, P = 0.04). CONCLUSIONS: Heart base Dmax was associated with the rate of death and cardiac events after adjusting for patient, tumour and cardiovascular factors in the NI-HEART study. This validates the findings from previous unsupervised analytical approaches. The heart base could be considered as a potential sub-organ at risk towards reducing radiation cardiotoxicity.

Socioeconomic deprivation status predicts both the incidence and nature of Achilles tendon rupture.

PURPOSE: The aim of this study was to describe the epidemiology of Achilles tendon rupture (ATR) and its relationship with socioeconomic deprivation status (SEDS). The hypothesis was that ATR occurs more frequently in socioeconomically deprived patients. Secondary aims were to determine variations in circumstances of injury between more and less deprived patients. METHODS: A 6-year retrospective review of consecutive patients presenting with ATR was undertaken. The health-board population was defined using governmental population data and SEDS was defined using the Scottish Index of Multiple Deprivation. The primary outcome was an epidemiological description and comparison of incidence in more and less deprived cohorts. Secondary outcomes included reporting of the relationship between SEDS and patient and injury characteristics with univariate and binary logistic regression analyses. RESULTS: There were 783 patients (567 male; 216 female) with ATR. Mean incidence for adults (≥ 18 years) was 18.75/100,000 per year (range 16.56-23.57) and for all ages was 15.26/100,000 per year (range 13.51 to 19.07). Incidence in the least deprived population quintiles (4th and 5th quintiles; 18.07 per 100,000/year) was higher than that in the most deprived quintiles (1st and 2nd; 11.32/100,000 per year; OR 1.60, 95%CI 1.35-1.89; p < 0.001). When adjusting for confounding factors, least deprived patients were more likely to be > 50 years old (OR 1.97; 95%CI 1.24-3.12; p = 0.004), to sustain ATR playing sports (OR 1.72, 95%CI 1.11-2.67; p = 0.02) and in the spring (OR 1.65, 95%CI 1.01-2.70; p = 0.045) and to give a history of preceding tendinitis (OR 4.04, 95%CI 1.49-10.95; p = 0.006). They were less likely to sustain low-energy injuries (OR 0.44, 95%CI 0.23-0.87; p = 0.02) and to be obese (OR 0.25-0.41, 95%CI 0.07-0.90; p ≤ 0.03). CONCLUSIONS: The incidence of ATR was higher in less socioeconomically deprived populations and the hypothesis was therefore rejected. Significant variations in patient and predisposing factors, mechanisms of injury and seasonality were demonstrated between most and least deprived groups, suggesting that circumstances and nature of ATR may vary with SEDS and these are not a homogenous group of injuries. LEVEL OF EVIDENCE: Prognostic Study Level III.

The epidemiology of Achilles tendon re-rupture and associated risk factors: male gender, younger age and traditional immobilising rehabilitation are risk factors.

PURPOSE: The aim of this study was to describe the epidemiology of Achilles tendon re-rupture. Secondary aims were to identify factors predisposing to increased Achilles tendon re-rupture risk, at the time of primary Achilles tendon rupture. METHODS: A retrospective review of all patients with primary Achilles tendon rupture and Achilles tendon re-rupture was undertaken. Two separate databases were compiled: the first included all Achilles tendon re-ruptures presenting during the study period and described epidemiology, mechanisms and nature of the re-rupture; the second was a case-control study analysing differences between patients with primary Achilles tendon rupture during the study period, who did, or did not, go on to develop re-rupture, with minimum review period of 1.5 years. RESULTS: Seven hundred and eighty-three patients (567 males, 216 females) attended with primary Achilles tendon rupture and 48 patients (41 males, 7 females) with Achilles tendon re-rupture. Median time to re-rupture was 98.5 days (IQR 82-122.5), but 8/48 re-ruptures occurred late (range 3 to 50 years) after primary Achilles tendon rupture. Males were affected more commonly (OR = 7.40, 95% CI 0.91-60.15; p = 0.034). Mean Achilles tendon re-rupture incidence was 0.94/100,000/year for all ages and 1.16/100,000/year for adults (≥ 18 years). Age distribution was bimodal for both primary Achilles tendon rupture and re-rupture, peaking in the fifth decade, with secondary peaks in older age. Incidence of re-rupture was higher in less socioeconomically deprived sub-populations (OR = 2.01, 95%CI 1.01-3.97, p = 0.04). The majority of re-ruptures were low-energy injuries. Greater risk of re-rupture was noted for patients with primary rupture aged < 45 years [adjusted odds ratio (aOR) 1.96; p = 0.037] and those treated with traditional cast immobilisation (aOR 2.20; p = 0.050). CONCLUSION: The epidemiology of Achilles tendon re-rupture is described and known trends (e.g. male predilection) are confirmed, while other novel findings are described, including incidence of a small but significant number of late re-ruptures, occurring years after the primary injury and an increased incidence of re-rupture in less socioeconomically deprived patients. Younger age and traditional immobilising cast treatment of primary Achilles tendon rupture were independently associated with Achilles tendon re-rupture. LEVEL OF EVIDENCE: III.

Trends and Correlates of Mean Lung Dose in Patients Receiving Breast Radiotherapy in a Single Institution from 2014 to 2018.

AIMS: Higher mean lung dose (MLD) in breast cancer patients has been associated with pneumonitis, pulmonary fibrosis and secondary lung cancer primaries. This study examined MLD in a single institution from 2014 to 18 to assess trends in median MLD (Gy) over time and factors associated with higher MLD to determine best practices for limiting lung toxicity. MATERIALS AND METHODS: General linear regressions were analysed to determine significant change in median MLD over time in patients receiving conventional or hypofractionated schedules for whole breast/chest wall (WB) radiotherapy with or without sequential boost or simultaneous integrated boost, WB tangential radiotherapy only and WB locoregional radiotherapy. Univariate and multivariable linear regression analysed identified factors associated with MLD. RESULTS: In total, 3894 patients were included in the analysis. The total median MLD across all years was 6.8 Gy in patients treated with conventional fractionation and 3.4 Gy in patients treated with hypofractionation. A significant increase in MLD was observed between 2014 and 2018 in patients receiving conventional or hypofractionation, conventional WB treatment with locoregional radiotherapy, conventional WB radiotherapy with simultaneous integrated boost and hypofractionated WB radiotherapy with sequential boost. Increased MLD was significantly correlated with lower lung volume and larger treatment volume due to locoregional radiotherapy, inclusion of a boost, chest wall treatment and reverse decubitus or supine positioning (P < 0.0001). CONCLUSION: A significant increase in MLD was observed over the years in patients receiving conventional and hypofractionated radiotherapy. Techniques such as prone positioning should be considered to lower MLD, particularly for patients with predisposing pulmonary risk.

Outcomes of emergency laparotomy in patients on extracorporeal membrane oxygenation for severe respiratory failure: A retrospective, observational cohort study.

PURPOSE: There is a paucity of literature to support undertaking emergency laparotomy when indicated in patients supported on ECMO. Our study aims to identify the prevalence, outcomes and complications of this high risk surgery at a large ECMO centre. MATERIALS AND METHODS: A single centre, retrospective, observational cohort study of 355 patients admitted to a university teaching hospital Severe Respiratory Failure service between December 2011 and January 2017. RESULTS: The prevalence of emergency laparotomy in patients on ECMO was 3.7%. These patients had significantly higher SOFA and APACHE II scores compared to similar patients not requiring laparotomy. There was no difference in the duration of ECMO or intensive care unit (ICU) stay post decannulation between the two groups. 31% of laparotomy patients survived to hospital discharge. Major haemorrhage was uncommon, however emergency change of ECMO oxygenator was commonly required. CONCLUSION: Survival to hospital discharge is possible following emergency laparotomy on ECMO, however the mortality is higher than for those patients not requiring laparotomy, this likely reflects the severity of underlying organ failure rather than the surgery itself. Our service's collocation with a general surgical service has made this development in care possible. ECMO service planning should consider general surgical provision.

Patient-reported functional outcomes and health-related quality of life following fractures of the talus.

INTRODUCTION: The primary aim of this study was to investigate patient-reported outcomes following talar fractures. Secondary aims were to investigate health-related quality of life and to determine whether it is influenced by functional outcome. MATERIALS AND METHODS: This retrospective study identified 56 talar fractures over eight years. Patients were contacted by post and the Olerud and Molander score (OMS), Manchester-Oxford Foot and Ankle scores (MOXFQ) and Euroqol-5D-3L collected. RESULTS: The mean age was 35.2 years (range 13-78 years). There were four cases (7.1%) of avascular necrosis and one (1.8%) non-union occurred. Data from patient-reported outcome measures were available for 42 patients (75.0%) with a median follow-up of 67.1 months (range 23.2-111.8 months). Mean OMS was 60.0 (standard deviation ± 29.51) and median MOXFQ was 30.33 (interquartile range 47.13). Median Euroqol-5D-3L index was 0.74 (interquartile range 0.213) and median Euroqol-5D-3L visual analogue score was 80 (interquartile range 21). Older age, open fractures, multiple injuries and subsequent avascular necrosis were associated with worse patient-reported outcomes (P < 0.05), with older age, avascular necrosis and open fractures found to be independent predictors of poor OMS, and avascular necrosis and open fractures independently predicting MOXFQ score on regression analysis (P < 0.05). Poor self-reported function, measured by OMS and MOXFQ, correlated with worse health-related quality of life as measured by the Euroqol-5D-3L index (OMS: r = 0.764, P < 0.001; MOXFQ: r = 0.824, P < 0.001) and visual analogue score (OMS: r = 0.450, P = 0.003; MOXFQ: r=0.559, P < 0.001). CONCLUSIONS: Older age, avascular necrosis and open fractures predict poorer functional outcomes following talar fractures. Patients with worse limb-specific functional outcomes are more likely to have a worse perception of health-related quality of life.

Barriers and facilitators to the successful development, implementation and evaluation of care bundles in acute care in hospital: a scoping review.

BACKGROUND: Care bundles are small sets of evidence-based recommendations, designed to support the implementation of evidence-based best clinical practice. However, there is variation in the design and implementation of care bundles, which may impact on the fidelity of delivery and subsequently their clinical effectiveness. METHODS: A scoping review was carried out using the Arksey and O'Malley framework to identify the literature reporting on the design, implementation and evaluation of care bundles. The Embase, CINAHL, Cochrane and Ovid MEDLINE databases were searched for manuscripts published between 2001 and November 2017; hand-searching of references and citations was also undertaken. Data were initially assessed using a quality assessment tool, the Downs and Black checklist, prior to further analysis and narrative synthesis. Implementation strategies were classified using the Expert Recommendations for Implementing Change (ERIC) criteria. RESULTS: Twenty-eight thousand six hundred ninety-two publications were screened and 348 articles retrieved in full text. Ninety-nine peer-reviewed quantitative publications were included for data extraction. These consisted of one randomised crossover trial, one randomised cluster trial, one case-control study, 20 prospective cohort studies and 76 non-parallel cohort studies. Twenty-three percent of studies were classified as poor based on Downs and Black checklist, and reporting of implementation strategies lacked structure. Negative associations were found between the number of elements in a bundle and compliance (Spearman's rho = - 0.47, non-parallel cohort and - 0.65, prospective cohort studies), and between the complexity of elements and compliance (p < 0.001, chi-squared = 23.05). Implementation strategies associated with improved compliance included evaluative and iterative approaches, development of stakeholder relationships and education and training strategies. CONCLUSION: Care bundles with a small number of simple elements have better compliance rates. Standardised reporting of implementation strategies may help to implement care bundles into clinical practice with high fidelity. TRIAL REGISTRATION: This review was registered on the PROSPERO database: CRD 42015029963 in December 2015.

A qualitative exploration of techniques used by expert midwives to preserve the perineum intact.

BACKGROUND: The perineum stretches during birth to allow passage of the baby, but 85% of women sustain some degree of perineal trauma during childbirth, which is painful post-partum. Episiotomy rates vary significantly, with some countries having rates of >60%. Recent Irish and New Zealand studies showed lower severe perineal trauma and episiotomy rates than other countries. AIM: To explore expert Irish and New Zealand midwives' views of the skills that they employ in preserving the perineum intact during spontaneous vaginal birth. METHODS: Following ethical approval a qualitative, descriptive study was undertaken. Semi-structured, recorded, interviews were transcribed and analysed using the constant comparative method. Expert midwives employed in New Zealand and one setting in Ireland, were invited to join the study. "Expert" was defined as achieving, in the preceding 3.5 years, an episiotomy rate for nulliparous women of <11.8%, a 'no suture' rate of 40% or greater, and a severe perineal tear rate of <3.2%. Twenty-one midwives consented to join the study. RESULTS: Four core themes emerged: 'Calm, controlled birth', 'Position and techniques in early second stage', 'Hands on or off?' and 'Slow, blow and breathe the baby out.' Using the techniques described enabled these midwives to achieve rates, in nulliparous women, of 3.91% for episiotomy, 59.24% for 'no sutures', and 1.08% for serious lacerations. CONCLUSIONS: This study provides further understanding of the techniques used by expert midwives at birth. These findings, combined with existing quantitative research, increases the evidence on how to preserve the perineum intact during spontaneous birth.

Transcriptional subtyping and CD8 immunohistochemistry identifies poor prognosis stage II/III colorectal cancer patients who benefit from adjuvant chemotherapy.

PURPOSE: Transcriptomic profiling of colorectal cancer (CRC) has led to identification of four consensus molecular subtypes (CMS1-4), which have prognostic value in stage II/III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors. However, the clinical value of these classifications in predicting response to standard-of-care adjuvant chemotherapy remains unknown. PATIENTS AND METHODS: Using samples from 4 European sites, we assembled a novel stage II/III CRC patient cohort and performed transcriptomic profiling on 156 samples, targeted sequencing and generated a tissue microarray to enable integrated "multi-omics" analyses. We also accessed data from 2 published stage II/III CRC patient cohorts: GSE39582 and GSE14333 (479 and 185 samples respectively). RESULTS: The epithelial-rich CMS2 subtype of CRC benefitted significantly from adjuvant chemotherapy treatment in both stage II and III disease (p=0.02 and p<0.0001 respectively), while the CMS3 subtype significantly benefitted in stage III only (p=0.00073). Following CRIS sub-stratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from adjuvant chemotherapy in stage II and III disease (p=0.0081 and p<0.0001 respectively), while CRIS-D significantly benefitted in stage III only (p=0.0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk of relapse in both stage II and III disease (p=0.0031). CONCLUSION: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying poor prognostic stage II/III patients who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Enteric neural stem cell therapies for enteric neuropathies.

BACKGROUND: Enteric neuropathies exist as a wide range of human disorders which impact on gastrointestinal motility. Current standard therapies for enteric neuropathies are limited to surgical resection or manipulation (eg, myotomy) of affected gut segments or medical management including both therapy (eg, prokinetic pharmacotherapy) and support such as parenteral nutrition. However, such treatments often result in poor prognosis and significant morbidity. The current limitations in treatment options for enteric neuropathies underline the need for alternative approaches to treat these devastating diseases. Recent advances have highlighted the potential of enteric neural stem cells as a possible treatment option for regenerative medicine, in such cases. PURPOSE: The purpose of this review is to provide an up-to-date synopsis of the enteric neural stem cell research field. Here, we review in detail the initial characterization of enteric neural stem cells, early preclinical studies validating their use in murine models through to the most recent findings of therapeutic rescue of diseased gut tissue. We additionally pose a number of questions regarding these recent findings which will need to be addressed prior to clinical translation of this exciting cellular therapeutic.

Correction to: Tram system related cycling injuries.

The author would like to correct the errors in the publication of the original article. The corrected details are given below for your reading.

Tram system related cycling injuries.

INTRODUCTION: Understanding of tram-system related cycling injuries (TSRCI) is poor. The aim of this study was to report the spectrum of injuries, demographics and social deprivation status of patients. Secondary aims included assessment of accident circumstances, effects of TSRCI on patients' confidence cycling, together with time off work and cycling. METHODS: A retrospective review of patients presenting to emergency services across all hospitals in Edinburgh and West Lothian with tram related injuries between May 2009 and April 2016 was undertaken. Medical records and imagining were analysed and patients were contacted by telephone. RESULTS: 191 cyclists (119 males, 72 females) were identified. 63 patients sustained one or more fractures or dislocations. Upper limb fractures/dislocations occurred in 55, lower limb fractures in 8 and facial fractures in 2. Most patients demonstrated low levels of socioeconomic deprivation. In 142 cases, the wheel was caught in tram-tracks, while in 32 it slid on tracks. The latter occurred more commonly in wet conditions (p = 0.028). 151 patients answered detailed questionnaires. Ninety-eight were commuting. 112 patients intended to cross tramlines and 65 accidents occurred at a junction. Eighty patients reported traffic pressures contributed to their accident. 120 stated that their confidence was affected and 24 did not resume cycling. Female gender (p < 0.001) and presence of a fracture/dislocation (p = 0.012) were independent predictors of negative effects on confidence. Patients sustaining a fracture/dislocation spent more time off work (median 5 days vs 1, p < 0.001) and cycling (median 57 days vs 21, p < 0.001). CONCLUSIONS: TSRCI occur predominantly in young to middle-aged adults with low levels of socioeconomic deprivation, most commonly when bicycle wheels get caught in tram-tracks. They result in various injuries, frequently affecting the upper limb. Traffic pressures are commonly implicated. Most patients report negative effects on confidence and a sizeable minority do not resume cycling. TSRCI can result in significant loss of working and cycling days.

In vivo transplantation of fetal human gut-derived enteric neural crest cells.

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75NTR were lentivirally labeled with either GFP or calcium-sensitive GCaMP and transplanted into the hindgut of Rag2- /γc- /C5- -immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut-derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.

A Quality Improvement Approach to Reducing the Caesarean section Surgical Site Infection Rate in a Regional Hospital.

Surgical site infection (SSI) rates are used extensively by hospitals as a basis for quality improvement. A 30-day post-discharge SSI programme for Caesarean section operations has been implemented in Our Lady of Lourdes Hospital since 2011. It has been shown that skin antisepsis and antibiotic prophylaxis are key factors in the prevention of SSI. Using quality improvement methodology, an infection prevention bundle was introduced to address these two factors. Skin antisepsis was changed from povidone-iodine to chlorhexidine-alcohol. Compliance with choice of antibiotic prophylaxis increased from 89.6% in 2014 to 98.5% in 2015. Compliance with timing also improved. The SSI rate of 7.5% was the lowest recorded to date, with the majority of SSIs (64%) diagnosed after hospital discharge. The level of variation was also reduced. However, the continued presence of variation and possibility of lower infection rates from the literature imply that further improvements are required.

Direct Screening of Blood by PCR and Pyrosequencing for a 16S rRNA Gene Target from Emergency Department and Intensive Care Unit Patients Being Evaluated for Bloodstream Infection.

Here we compared the results of PCR/pyrosequencing to those of culture for detecting bacteria directly from blood. DNA was extracted from 1,130 blood samples from 913 patients suspected of bacteremia (enrollment criteria were physician-ordered blood culture and complete blood count [CBC]), and 102 controls (healthy blood donors). Real-time PCR assays for beta-globin and Universal 16S rRNA gene targets were performed on all 1,232 extracts. Specimens identified by Universal 16S rRNA gene PCR/pyrosequencing as containing staphylococci, streptococci, or enteric Gram-negative rods had target-specific PCR/pyrosequencing performed. Amplifiable beta-globin (melting temperature [Tm], 87.2°C ± 0.2°C) occurred in 99.1% (1,120/1,130) of patient extracts and 100% (102/102) of controls. Concordance between PCR/pyrosequencing and culture was 96.9% (1,085/1,120) for Universal 16S rRNA gene targets, with positivity rates of 9.4% (105/1,120) and 11.3% (126/1,120), respectively. Bacteria cultured included staphylococci (59/126, 46.8%), Gram-negative rods (34/126, 27%), streptococci (32/126, 25.4%), and a Gram-positive rod (1/126, 0.8%). All controls screened negative by PCR/pyrosequencing. Clinical performance characteristics (95% confidence interval [CI]) for Universal 16S rRNA gene PCR/pyrosequencing included sensitivity of 77.8% (69.5 to 84.7), specificity of 99.3% (98.6 to 99.7), positive predictive value (PPV) of 93.3% (86.8 to 97.3), and negative predictive value (NPV) of 97.2% (96.0 to 98.2). Bacteria were accurately identified in 77.8% (98/126) of culture-confirmed sepsis samples with Universal 16S PCR/pyrosequencing and in 76.4% (96/126) with follow-up target-specific PCR/pyrosequencing. The initial PCR/pyrosequencing took ∼5.5 h to complete or ∼7.5 h when including target-specific PCR/pyrosequencing compared to 27.9 ± 13.6 h for Gram stain or 81.6 ± 24.0 h for phenotypic identification. In summary, this molecular approach detected the causative bacteria in over three-quarters of all culture-confirmed cases of bacteremia directly from blood in significantly less time than standard culture but cannot be used to rule out infection.

Lentiviral labeling of mouse and human enteric nervous system stem cells for regenerative medicine studies.

BACKGROUND: Reliable methods of labeling human enteric nervous system (ENS) stem cells for use in novel cell replacement therapies for enteric neuropathies are lacking. Here, we explore the possibility of using lentiviral vectors expressing fluorescent reporter genes to transduce, label, and trace mouse and human ENS stem cells following transplantation into mouse gut. METHODS: Enteric nervous system precursors, including ENS stem cells, were isolated from enzymatically dissociated mouse and human gut tissues. Lentivirus containing eGFP or mCherry fluorescent reporter genes was added to gut cell cultures at a multiplicity of infection of 2-5. After fluorescence activated cell sorting for eGFP and subsequent analysis with markers of proliferation and cell phenotype, transduced mouse and human cells were transplanted into the gut of C57BL/6 and immune deficient Rag2-/gamma chain-/C5 mice, respectively and analyzed up to 60 days later. KEY RESULTS: Mouse and human transduced cells survived in vitro, maintained intense eGFP expression, proliferated as shown by BrdU incorporation, and formed characteristic neurospheres. When transplanted into mouse gut in vivo and analyzed up to 2 months later, transduced mouse and human cells survived, strongly expressed eGFP and integrated into endogenous ENS networks. CONCLUSIONS & INFERENCES: Lentiviral vectors expressing fluorescent reporter genes enable efficient, stable, long-term labeling of ENS stem cells when transplanted into in vivo mouse gut. This lentiviral approach not only addresses the need for a reliable fluorescent marker of human ENS stem cells for preclinical studies, but also raises the possibility of using lentiviruses for other applications, such as gene therapy.