CTC-5: A novel digital pathology approach to characterise circulating tumour cell biodiversity.

Metastatic progression and tumor evolution complicates the clinical management of cancer patients. Circulating tumor cell (CTC) characterization is a growing discipline that aims to elucidate tumor metastasis and evolution processes. CTCs offer the clinical potential to monitor cancer patients for therapy response, disease relapse, and screen 'at risk' groups for the onset of malignancy. However, such clinical utility is currently limited to breast, prostate, and colorectal cancer patients. Further understanding of the basic CTC biology of other malignancies is required to progress them towards clinical utility. Unfortunately, such basic clinical research is often limited by restrictive characterization methods and high-cost barrier to entry for CTC isolation and imaging infrastructure. As experimental clinical results on applications of CTC are accumulating, it is becoming clear that a two-tier system of CTC isolation and characterization is required. The first tier is to facilitate basic research into CTC characterization. This basic research then informs a second tier specialised in clinical prognostic and diagnostic testing. This study presented in this manuscript describes the development and application of a low-cost, CTC isolation and characterization pipeline; CTC-5. This approach uses an established 'isolation by size' approach (ScreenCell Cyto) and combines histochemical morphology stains and multiparametric immunofluorescence on the same isolated CTCs. This enables capture and characterization of CTCs independent of biomarker-based pre-selection and accommodates both single CTCs and clusters of CTCs. Additionally, the developed open-source software is provided to facilitate the synchronization of microscopy data from multiple sources (https://github.com/CTC5/). This enables high parameter histochemical and immunofluorescent analysis of CTCs with existing microscopy infrastructure without investment in CTC specific imaging hardware. Our approach confirmed by the number of successful tests represents a potential major advance towards highly accessible low-cost technology aiming at the basic research tier of CTC isolation and characterization. The biomarker independent approach facilitates closing the gap between malignancies with poorly, and well-defined CTC phenotypes. As is currently the case for some of the most commonly occurring breast, prostate and colorectal cancers, such advances will ultimately benefit the patient, as early detection of relapse or onset of malignancy strongly correlates with their prognosis.

The biofilm eradication activity of acetic acid in the management of periprosthetic joint infection.

OBJECTIVES: Periprosthetic joint infection following joint arthroplasty surgery is one of the most feared complications. The key to successful revision surgery for periprosthetic joint infections, regardless of treatment strategy, is a thorough deep debridement. In an attempt to limit antimicrobial and disinfectant use, there has been increasing interest in the use of acetic acid as an adjunct to debridement in the management of periprosthetic joint infections. However, its effectiveness in the eradication of established biofilms following clinically relevant treatment times has not been established. Using an in vitro biofilm model, this study aimed to establish the minimum biofilm eradication concentration (MBEC) of acetic acid following a clinically relevant treatment time. MATERIALS AND METHODS: Using a methicillin-sensitive Staphylococcus aureus (MSSA) reference strain and the dissolvable bead assay, biofilms were challenged by 0% to 20% acetic acid (pH 4.7) for ten minutes, 20 minutes, 180 minutes, and 24 hours. RESULTS: The MBEC of acetic acid was found to be: 15%, 11%, 3.2%, and 0.8% following a ten-minute, 20-minute, 180-minute, and 24-hour treatment, respectively. CONCLUSION: This study found that the MBEC of acetic acid following a 10- or 20-minute treatment time exceeded its safety threshold, making these concentrations unsuitable as a topical debridement adjunct. However, a clinically acceptable concentration (5%) was still found to eliminate 96.1% of biofilm-associated MSSA following a 20-minute treatment time.Cite this article: S. T. J. Tsang, P. J. Gwynne, M. P. Gallagher, A. H. R. W. Simpson. The biofilm eradication activity of acetic acid in the management of periprosthetic joint infection. Bone Joint Res 2018;7:517-523. DOI: 10.1302/2046-3758.78.BJR-2018-0045.R1.

The dissolvable bead: A novel in vitro biofilm model for evaluating antimicrobial resistance.

In vitro biofilm assays are a vital first step in the assessment of therapeutic effectiveness. Current biofilm models have been found to be limited by throughput, reproducibility, and cost. We present a novel in vitro biofilm model, utilising a sodium alginate substratum for surface biofilm colony formation, which can be readily dissolved for accurate evaluation of viable organisms. The dissolving bead biofilm assay was evaluated using a range of clinically relevant strains. The reproducibility and responsiveness of the assay to an antimicrobial challenge was assessed using standardised methods. Cryo-scanning electron microscopy was used to image biofilm colonies. Biofilms were grown for 20h prior to testing. The model provides a reproducible and responsive assay to clinically-relevant antimicrobial challenges, as defined by established guidelines. Moreover cryo-scanning electron microscopy demonstrates that biofilm formation is localised exclusively to the alginate bead surface. Our results suggest that this simple model provides a robust and adaptable assay for the investigation of bacterial biofilms.

Gaps and barriers in health-care provision for co-morbid diabetes and chronic kidney disease: a cross-sectional study.

BACKGROUND: Patients with diabetes and chronic kidney disease (CKD) are a complex subset of the growing number of patients with diabetes, due to multi-morbidity. Gaps between recommended and received care for diabetes and chronic kidney disease (CKD) are evident despite promulgation of guidelines. Here, we document gaps in tertiary health-care, and the commonest patient-reported barriers to health-care, before exploring the association between these gaps and barriers. METHODS: This cross-sectional study recruited patients with diabetes and CKD (eGFR < 60 mL/min/1.73 m2) across 4 large hospitals. For each patient, questionnaires were completed examining clinical data, recommended care, and patient-reported barriers limiting health-care. Descriptive statistics, subgroup analyses by CKD stage and hospital, and analyses examining the relationship between health-care gaps and barriers were performed. RESULTS: 308 patients, of mean age 66.9 (SD 11.0) years, and mostly male (69.5%) and having type 2 diabetes (88.0%), participated. 49.1% had stage 3, 24.7% stage 4 and 26.3% stage 5 CKD. Gaps between recommended versus received care were evident: 31.9% of patients had an HbA1c ≥ 8%, and 39.3% had a measured blood pressure ≥ 140/90 mmHg. The commonest barriers were poor continuity of care (49.3%), inadequate understanding/education about CKD (43.5%), and feeling unwell (42.6%). However, barriers associated with a failure to receive items of recommended care were inadequate support from family and friends, conflicting advice from and poor communication amongst specialists, the effect of co-morbidities on self-management and feeling unmotivated (all p < 0.05). CONCLUSIONS: Barriers to health-care varied across CKD stages and hospitals. Barriers associated with a deviation from recommended care were different for different items of care, suggesting that specific interventions targeting each item of care are required.

Paediatric intraocular lens implants: accuracy of lens power calculations.

PurposeThis study aims to evaluate the accuracy of lens prediction formulae on a paediatric population.MethodsA retrospective case-note review was undertaken of patients under 8 years old who underwent cataract surgery with primary lens implantation in a regional referral centre for paediatric ophthalmology, excluding those whose procedure was secondary to trauma. Biometric and refractive data were analysed for 43 eyes, including prediction errors (PE). Statistical measures used included mean absolute error (MAE), median absolute error (MedAE), Student's t-test and Lin's correlation coefficient.ResultsThe mean PE using the SRK-II formula was +0.96 D (range -2.47D to +2.41 D, SD 1.33 D, MAE 1.38 D, MedAE 1.55, n=15). The mean PE was smaller using SRK/T (-0.18 D, range -3.25 D to +3.95 D, SD 1.70 D, MAE 1.30 D, MedAE 1.24, n=27). We performed an analysis of the biometry data using four different formula (Hoffer Q, Holladay 1, SRK-II and SRK/T). Hoffer Q showed a smaller MedAE than other formulae but also a myopic bias.ConclusionOur clinical data suggest SRK/T was more accurate in predicting post-operative refraction in this cohort of paediatric patients undergoing cataract surgery. Hoffer Q may have improved accuracy further.

Angle closure glaucoma in the Northern Ireland Diabetic Retinopathy Screening Programme.

PurposeTo ascertain the risk of angle closure glaucoma associated with mydriasis in the Northern Ireland Diabetic Retinopathy Screening Programme.MethodsA retrospective case note review was carried out, cross referencing hospital records with those of the screening programme, to identify episodes of angle closure glaucoma, which occurred within 14 days of a retinopathy screening episode involving pharmacological mydriasis.ResultsThree cases of angle closure following mydriasis for screening were identified. The incidence of angle closure within the screening programme was calculated to be 1 in 31 755 patients dilated or 0.75 patients per year.ConclusionAngle closure is a rare complication of mydriasis used in photographic screening for diabetic retinopathy. We advocate the provision of clear instructions to patients in screening programmes on when and how to access emergency ophthalmic care following dilation to prevent loss of vision in this rare event.

A randomized pilot study of dietary treatments for polycystic ovary syndrome in adolescents.

BACKGROUND: Evidence is lacking to recommend one diet over another when treating polycystic ovary syndrome (PCOS). OBJECTIVES: To obtain preliminary data, comparing the impact of a low-glycaemic load (LGL) vs. low-fat (LF) diet on biochemical hyperandrogenism in overweight and obese adolescents with PCOS. To ascertain feasibility of recruiting study participants, in partnership with an adolescent clinic, and implementing dietary interventions. METHODS: Randomized controlled trial of 19 overweight and obese adolescents with PCOS and not using hormonal contraceptives (HCs). Interventions comprised nutrition education, dietary counselling and cooking workshops to foster adherence to a LGL (45% carbohydrate, 35% fat, 20% protein) or LF (55% carbohydrate, 25% fat, 20% protein) diet over 6 months. Serum bioavailable testosterone was the primary outcome. RESULTS: Sixteen (LGL, n = 7; LF, n = 9) participants completed the study. Body fat percentage decreased (P < 0.05) in response to the interventions, with no difference between the LGL and LF groups (-1.2% vs. -2.2%; P = 0.16). Bioavailable testosterone did not change for either group (-0.4 vs. -1.8 ng dL(-1) ; P = 0.35). Regarding feasibility, recruiting adolescents posed a challenge, and use of HCs was a main reason for ineligibility. Participants attended 5.9 of 6 in-person visits and 2.6 of 3 cooking workshops, completed 4.9 of 6 telephone counselling calls, and reported high satisfaction with the diets and cooking workshops (≥8 on a 10-cm scale). CONCLUSIONS: Dietary interventions were beneficial for weight control but did not attenuate biochemical hyperandrogenism. Innovative strategies are needed to recruit adolescents for studies aimed at assessing independent effects of diet on features of PCOS.

Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots.

BACKGROUND: Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies. STUDY DESIGN: Whatman(®) Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR). RESULTS: For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods. CONCLUSION: The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date.

The impact of the European Working Time Regulations on Ophthalmic Specialist Training--a national trainee survey.

SUMMARY AND INTRODUCTION: To assess ophthalmic trainees' perspective of the impact of the European Working Time Regulations (EWTR) on their training. METHODS: All trainees in ophthalmology in the UK were emailed a link to an electronic survey asking about their experiences of the EWTR. RESULTS: 324 trainees (46% of those invited) responded to the survey. 44.4% of trainees reported that their posts were compliant with the EWTR. 40.7% felt that training had been adversely affected. 49.1% thought that ophthalmic trainees should opt out of the EWTR to work more than 48 h per week, with 57 the mean number of hours suggested appropriate. DISCUSSION: Many ophthalmic trainees in the United Kingdom are working in rotas which are not compliant with the European Working Time Directive. Many trainees feel that implementation of the EWTD has had a negative effect on training and feel it would be acceptable to work a higher number of hours per week.

Persistent Rhodococcus equi infection in a renal transplant patient: case report and review of the literature.

Rhodococcus equi is a pathogen that mainly causes infection in immunocompromised hosts. We report a case of relapsing R. equi pulmonary infection in a 57-year-old male renal transplant recipient who was treated with 12 months of antibiotics, adjunctive surgery, and a reduction in his immunosuppression. He suffered from relapsing disease, treatment-related complications, and ultimately died of Pneumocystis pneumonia. Case reports in the literature portray a good cure rate for transplant-related R. equi infections, with shorter durations of antibiotics. The case illustrates the difficulties in the management of R. equi infections. Forty cases from the literature were reviewed and compared in terms of epidemiology, location of infection, transplant type, immunosuppression used, treatment used, outcomes, and possible exposures.

Ketorolac therapy for the prevention of acute pseudophakic cystoid macular edema: a systematic review.

To assess the effectiveness of ketorolac vs control for prevention of acute pseudophakic cystoid macular edema (CME). The following databases were searched: Medline (1950-June 11, 2011), The Cochrane Library (Issue 2, 2011), and the TRIP Database (up to 11 June 2011), using no language or other limits. Randomized controlled clinical trials (RCTs) were included that consisted of patients with acute pseudophakic cystoid macular edema, those comparing ketorolac with control, and those having at least a minimum follow-up of 28 days. In the four RCTs evaluating ketorolac vs control, treatment with ketorolac significantly reduced the risk of CME development at the end of treatment (≈ 4 weeks) compared to control (P=0.008; 95% confidence interval (0.03-0.58)). When analyzed individually, each individual study was statistically nonsignificant in its findings with the exception of one study. When the pooled relative risk was calculated, the large sample size of this systematic review led to overall statistical significance, which is attributable to the review's large sample size and not to the individual studies themselves. In this systematic review of four RCTs, two of which compared ketorolac with no treatment and two of which evaluated ketorolac vs placebo drops, treatment with ketorolac significantly reduced the risk of developing CME at the end of ≈ 4 weeks of treatment compared with controls. These results, however, should be interpreted with caution considering the paucity of large randomized clinical trials in the literature.

Nitrogen management is essential to prevent tropical oil palm plantations from causing ground-level ozone pollution.

More than half the world's rainforest has been lost to agriculture since the Industrial Revolution. Among the most widespread tropical crops is oil palm (Elaeis guineensis): global production now exceeds 35 million tonnes per year. In Malaysia, for example, 13% of land area is now oil palm plantation, compared with 1% in 1974. There are enormous pressures to increase palm oil production for food, domestic products, and, especially, biofuels. Greater use of palm oil for biofuel production is predicated on the assumption that palm oil is an "environmentally friendly" fuel feedstock. Here we show, using measurements and models, that oil palm plantations in Malaysia directly emit more oxides of nitrogen and volatile organic compounds than rainforest. These compounds lead to the production of ground-level ozone (O(3)), an air pollutant that damages human health, plants, and materials, reduces crop productivity, and has effects on the Earth's climate. Our measurements show that, at present, O(3) concentrations do not differ significantly over rainforest and adjacent oil palm plantation landscapes. However, our model calculations predict that if concentrations of oxides of nitrogen in Borneo are allowed to reach those currently seen over rural North America and Europe, ground-level O(3) concentrations will reach 100 parts per billion (10(9)) volume (ppbv) and exceed levels known to be harmful to human health. Our study provides an early warning of the urgent need to develop policies that manage nitrogen emissions if the detrimental effects of palm oil production on air quality and climate are to be avoided.

Primary intraocular lymphoma: another great masquerader.

PURPOSE: To describe diverse and atypical presentations of the most common masquerader in neoplastic masquerade syndromes. METHODS: Retrospective interventional case series. The authors identified three patients who presented with atypical and diagnostically challenging masquerading manifestations. These patients were eventually found to have primary intraocular lymphoma (PIOL). Their case histories, presenting signs and symptoms, diagnostic tests, and treatments are described. RESULTS: Patient 1 masqueraded as viral retinitis and branch retinal vein occlusion but was resistant to 5 weeks of oral and intravenous acyclovir. Patient 2 presented with choroidal infiltrates and vision loss. This patient had had breast carcinoma for the last 25 years and secondary metastasis was suspected. Patient 3 had chronic uveo-retinitis and a chronic Propionibacterium acnes infection was suspected. All three patients were diagnosed with PIOL. CONCLUSIONS: PIOL is an aggressive masquerader and not only presents clinical diagnostic difficulties but also requires expert tissue handling and analysis, so that early diagnosis can be made and therapy can be instituted.

The characteristic features of optical coherence tomography in posterior uveitis.

AIM: To describe the different retinal morphological characteristics that can present on optical coherence topography (OCT) in a spectrum of uveitic diseases. METHODS: We reviewed the literature and our own OCT image archive for characteristic features that may be suggestive of a particular disease process. RESULTS: OCT demonstrates a variety of characteristic morphological changes, some that may point towards a specific disease process. We describe the various forms of macular oedema found in uveitis as well as OCT features typically found in multifocal choroiditis, serpiginous chorioretinitis, toxoplasma chorioretinitis, Vogt-Koyanagi-Harada, sympathetic ophthalmia and the vitreomacular traction syndrome. CONCLUSION: Ophthalmologists should be aware of the variety of retinal morphological characteristics that can present on OCT in uveitic disease. Recognition may aid in the diagnostic process, which is complementary to conventional fundal photography and fluorescein angiography. This can facilitate earlier diagnosis and, more importantly, the initiation of specific treatment.

ANCA associated pauci-immune retinal vasculitis.

BACKGROUND: Antinuclear cytoplasmic antibodies (ANCA) are useful diagnostic serological markers for the most common forms of necrotising vasculitis. ANCA associated vasculitides represent distinctive clinicopathological categories--for example, Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis, and idiopathic necrotising crescentic glomerulonephritis, collectively known as the small vessel pauci-immune vasculitides. METHOD: Three cases of ANCA associated pauci-immune retinal vasculitis are described. Their systemic features are described and the clinical significance of ANCA as a diagnostic test in relation to retinal vasculitis discussed. RESULTS: These three cases represent a spectrum of clinical features associated with retinal vasculitis. Two cases have evolved into clinical recognisable entities as microscopic polyangiitis. Adherence to the international consensus statement on testing and reporting of ANCA is recommended and the authors speculate that the incidence of microscopic polyangiitis may be underestimated because of the under-recognition of systemic involvement in patients with retinal vasculitis. CONCLUSION: The receipt of a positive ANCA result should always raise the suspicion of a pauci-immune systemic vasculitis and prompt appropriate investigation. The authors emphasise the importance of the evaluation of systemic features in these patients with retinal vasculitis, enabling earlier recognition and thereby preventing significant morbidity and mortality.

Evidence for genetic predisposition to desmoid tumours in familial adenomatous polyposis independent of the germline APC mutation.

BACKGROUND: Many patients with familial adenomatous polyposis (FAP) die from desmoid tumours which can arise spontaneously but often appear to be surgically induced by prophylactic colectomy. FAP results from germline adenomatous polyposis coli (APC) gene mutations and desmoids arise following biallelic APC mutation, with one change usually occurring distal to the second beta-catenin binding/degradation repeat of the gene (3' to codon 1399). We have suggested that because families with germline mutations in this region already have the requisite change, they are more likely to develop desmoids. However, there are families with 5' germline mutations where desmoids are common. PATIENTS AND METHODS: We examined desmoid risk dependent on germline APC mutation, sex, history of abdominal surgery, and family history in FAP patients from the St Mark's Hospital Polyposis Registry. RESULTS: Overall desmoid prevalence was 15%. Desmoids tended to cluster in susceptible individuals, irrespective of the germline APC mutation. Independent predictors of increased desmoid risk were: germline mutation distal to codon 1399; any family history of disease; and a strong family history of desmoids. A family history of multiple desmoids (>1) increased an individual's own risk of multiplicity. Females had twice the odds of developing desmoids compared with males. There was no significant interaction between any of the three explanatory variables. CONCLUSIONS: Our results indicate the influence of unknown genetic factors independent of APC in susceptibility to desmoid tumours in FAP. The data have implications in terms of clinical management of FAP patients and assessing the balance between chemoprevention and prophylactic colectomy.