Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

First report of Lyme borreliosis leading to cardiac bradydysrhythmia in two cats.

CASE SERIES SUMMARY: Two cats were presented for investigation of bradyarrhythmia detected by their referring veterinarians during routine examination. Both cats had extensive investigations, including haematology, serum biochemistry with electrolytes and thyroxine concentrations, systolic blood pressure measurement, echocardiography, electrocardiography and infectious disease testing. Infectious disease testing included serology for Toxoplasma gondii, Ehrlichia canis, Anaplasma phagocytophilum and Borrelia burgdorferi, and PCR for B burgdorferi antigen in both cats. Case 1 was also assessed by PCR for Bartonella henselae antigen and case 2 was assessed for Dirofilaria immitis by serology. All infectious disease tests, other than for B burgdorferi, were negative. Case 1 was diagnosed with Lyme carditis based on marked bradydysrhythmia, positive B burgdorferi serology, a structurally normal heart and clinical resolution with appropriate treatment with a 4-year follow-up. Case 2 was diagnosed with Lyme carditis based on marked bradydysrhythmia and positive B burgdorferi PCR; however, this cat had structural heart disease that did not resolve with treatment. RELEVANCE AND NOVEL INFORMATION: This small case series describes two B burgdorferi positive cats presenting with newly diagnosed cardiac abnormalities consistent with those found in humans and dogs with Lyme carditis. Both cats were asymptomatic as perceived by their owners; the arrhythmia was detected by their veterinarians.

Predicting the distribution of Phortica variegata and potential for Thelazia callipaeda transmission in Europe and the United Kingdom.

BACKGROUND: Male fruitflies Phortica variegata (Drosophilidae, Steganinae) are the intermediate host of the zoonotic nematode Thelazia callipaeda (Spirurida, Thelaziidae). More than 10 years ago, when T. callipaeda was confined to remote regions of southern Italy, ecological niche models were used to predict the potential distribution of P. variegata across Europe and the likely risk of the nematode spreading through infected dogs travelling to/from endemic regions. As predicted, over the last 10 years T. callipaeda has spread rapidly across Europe. Recently, we identified the potential for its introduction to the UK through infected dogs travelling to/from endemic regions of mainland Europe. METHODS: Here updated information is used to re-evaluate the model-predicted European, and specifically, UK distribution to determine the likelihood of T. callipaeda becoming established. Additionally, the UK distribution of P. variegata was further investigated through snapshot fly trapping at model-predicted locations. RESULTS: Ecological niche modelling using Genetic Algorithm for Rule-set Prediction (GARP) analysis suggests a European range similar to that described previously, with some indication of potential spread further eastward. Finer scale UK mapping suggested that P. variegata presence was limited mostly to southern England, but highlighted regions where P. variegata has not been documented previously. The arbitrary fly trapping identified activity of P. variegata at two locations where the species has been found previously late in the season. No specimens were collected at model-predicted locations, although habitat suitable for the species was identified. CONCLUSIONS: GARP-model prediction of P. variegata distribution suggests presence of suitable conditions in previously undocumented regions of the UK and Europe and highlight the possibility for further spread of T. callipaeda across Europe, including the UK. Further work to validate the P. variegata UK model with field data will help improve its accuracy in predicting suitable areas, whilst surveillance of sylvatic definitive host species in such locations is advised to monitor for evidence of autochthonous T. callipaeda transmission.

Three cases of imported eyeworm infection in dogs: a new threat for the United Kingdom.

In July 2016 we described the first known case of canine ocular thelaziosis in the UK in a dog recently imported from Romania. Here we confirm our initial diagnosis using PCR followed by sequence analysis, and we report a further two clinical cases in dogs with recent history of travel to Italy and France. In view of the presence in the UK of the vector for Thelazia callipaeda, namely Phortica spp, we discuss the significance of these three cases in the context of the UK government's pet travel scheme, disease control and both animal and public health in the UK.

Angiostrongylus vasorum in the eye: new case reports and a review of the literature.

BACKGROUND: Nematodes of the genus Angiostrongylus are important causes of potentially life-threatening diseases in several animal species and humans. Angiostrongylus vasorum affects the right ventricle of the heart and the pulmonary arteries in dogs, red foxes and other carnivores. The diagnosis of canine angiostrongylosis may be challenging due to the wide spectrum of clinical signs. Ocular manifestations have been seldom reported but have serious implications for patients. METHODS: The clinical history of three cases of infection with A. vasorum in dogs diagnosed in UK, France and Italy, was obtained from clinical records provided by the veterinary surgeons along with information on the diagnostic procedures and treatment. Nematodes collected from the eyes of infected dogs were morphologically identified to the species level and molecularly analysed by the amplification of the nuclear 18S rRNA gene. RESULTS: On admission, the dogs were presented with various degrees of ocular discomfort and hyphema because of the presence of a motile object in the eye. The three patients had ocular surgery during which nematodes were removed and subsequently morphologically and molecularly identified as two adult males and one female of A. vasorum. CONCLUSIONS: Three new cases of canine ocular angiostrongylosis are reported along with a review of other published clinical cases to improve the diagnosis and provide clinical recommendation for this parasitic condition. In addition, the significance of migratory patterns of larvae inside the host body is discussed. Veterinary healthcare workers should include canine angiostrongylosis in the differential diagnosis of ocular diseases.

Robotic radical prostatectomy as the initial step in multimodal therapy for men with high-risk localised prostate cancer: initial experience of 160 men.

OBJECTIVES: • To report the outcome of robotic-assisted laparoscopic radical prostatectomy (RALP) for men with localised high-risk prostate cancer at diagnosis. • Although commonly managed by radiotherapy (RT) with prolonged androgen-deprivation therapy (ADT), we hypothesize that initiation of multimodal therapy with RALP is oncologically efficacious and may allow many men to avoid ADT. PATIENTS AND METHODS: • Between December 2003 and September 2010, 1480 men underwent RALP of whom 160 fulfilled the National Comprehensive Control Network criteria for high-risk disease (prostate-specific antigen (PSA) > 20 ng/mL and/or clinical stage, cT ≥ 3 and/or biopsy Gleason score ≥ 8). • Biochemical recurrence (postoperative PSA ≥ 0.2) was used to assess outcome after RALP monotherapy. • Treatment failure was defined as either a rising PSA level after salvage RT or the initiation of ADT. RESULTS: • The mean age ± standard deviation was 63.1 ± 6.3 years. Median PSA level was 9.95 ng/mL (interquartile range 6.0-21.4). • Analysis of prostatectomy specimen showed Gleason 8-10 cancers in 65 (41%), and extracapsular disease, pT ≥ 3, in 96 (60%) of which seminal vesicle invasion was evident in 36 (23%). Downgrading by prostatectomy occurred in 64 (40% of total group) and five (3%) were downstaged to pT2 disease. By contrast, any upgrading occurred in 29 (18% of total group) and upstaging occurred in 68 (43%). The overall positive surgical margin rate was 38%, correlating with stage pT2 (15%) or pT3 (53%). • With median follow-up of 26.2 months (interquartile range 5.5-37.3), two non-cancer-related deaths have occurred (overall survival 98.8%; cancer-specific survival 100%), and biochemical recurrence has occurred in 53 men (33%). RALP surgery has served as monotherapy (n= 117, 73%), or has been followed by salvage RT (n= 24, 15%) and/or ADT (n= 43, 27%). Overall 2-year and 3-year treatment failure was 31 and 41%, respectively. • Serum PSA level was the only independent predictor of overall treatment failure (hazard ratio [HR] 1.02, P= 0.001) although a strong trend was observed for both clinical stage (HR 1.22, P= 0.058) and the number of positive biopsy cores on transrectal biopsy (HR 1.06, P= 0.057). CONCLUSIONS: • RALP incorporating the use of postoperative RT is a good multimodal management strategy for men with this aggressive variant of prostate cancer. • At median follow-up in excess of 2 years, we found low rates of treatment failure enabling a high proportion of men to remain free of ADT.

Prostate abscess: a rare complication of brachytherapy for prostate cancer.

Brachytherapy involves the therapeutic implantation of a radio-active seed source into, or close to, prostate cancer. We report the rare case of a 76-year-old man who presented with a prostate abscess after months of intractable pelvic pain following prostate cancer treatment with iodine-125 brachytherapy. Despite multiple investigations, the diagnosis was made only once the abscess discharged exudate per-urethra.

Variable histological and ultrasonic characteristics of restenosis after drug-eluting stents.

Bare-metal stents have undergone intense pathological and clinical examination, but histological characterization of drug-eluting stent (DES) restenosis (ISR) remains unknown. We report a series of cases (n=6) with intravascular ultrasound (IVUS) and pathological examinations over 8 months after DES deployment. Tissue samples were obtained using atherectomy devices in 5 cases and a thrombectomy catheter in 1 case. Histology revealed not only smooth muscle cell proliferation, which correlated with homogeneous hypoechoic tissue by IVUS in one case, but also demonstrated delayed healing features such as organized fibrin deposition in 3 cases (one with homogeneous echolucent tissue by IVUS), macrophage and T-lymphocyte infiltration in others. IVUS appearance of ISR components varied from echolucent to echodense images. This report suggests a variable histological and IVUS pattern of ISR after DES implantation. Further investigations are necessary to define the potentially pro-thrombotic histological features of ISR after DES implantation, and the relationship between the molecular mechanisms of thrombosis and DES restenosis.

Quantitative magnetic resonance perfusion imaging detects anatomic and physiologic coronary artery disease as measured by coronary angiography and fractional flow reserve.

OBJECTIVES: To evaluate the ability of quantitative perfusion cardiac magnetic resonance (CMR) to assess the hemodynamic significance of coronary artery disease (CAD) compared with well-established anatomic and physiologic techniques. BACKGROUND: Fractional flow reserve (FFR) is considered by many investigators to be a reliable stenosis-specific method to determine hemodynamically significant CAD. Quantitative perfusion CMR is a promising noninvasive approach to detect CAD but has yet to be validated against FFR. METHODS: This is a prospective study in patients with suspected CAD who underwent coronary angiography, FFR, and CMR assessments. The quantitative myocardial perfusion reserve (MPR) was calculated in 720 myocardial sectors (8 sectors/slice). The MPR was calculated from the ratio between stress and rest myocardial flow based on signal intensity time curves using deconvolution analysis. Stress was simulated with adenosine for both FFR and MPR. The MPR assessments were compared to FFR (n = 44 coronary segments) and quantitative coronary angiography (n = 108 segments) in the corresponding coronary territories. RESULTS: The MPR was 1.54 +/- 0.36 in segments with FFR < or =0.75 (n = 14) and 2.11 +/- 0.68 in those with FFR >0.75 (n = 30; p = 0.0054). An MPR cutoff of 2.04 was 92.9% (95% CI 77.9 to 100.0) sensitive and 56.7% (95% CI 32.8 to 80.6) specific in predicting a coronary segment with FFR < or =0.75. The MPR was 1.54 +/- 0.49 in coronary segments with > or =50% diameter stenosis (DS) (n = 47) and 2.13 +/- 0.80 in segments with <50% DS (n = 61; p < 0.001). An MPR cutoff of 2.04 was 85.1% (95% CI 71.1 to 99.2) sensitive and 49.2% (95% CI 33.6 to 64.8) specific in predicting CAD with > or =50% DS. CONCLUSIONS: Quantitative perfusion CMR is a safe noninvasive test that represents a stenosis-specific alternative to determine the hemodynamic significance of CAD.

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.

PSA testing: are patients aware of what lies ahead?

INTRODUCTION: Screening for prostate cancer with serum prostate specific antigen (PSA) remains a controversial topic. The UK NHS Executive has issued extensive guidance stressing the importance of adequate counselling prior to performing this test. This study aims to assess men's knowledge of the PSA test at the time of their referral and their attitude towards screening. PATIENTS AND METHODS: A total of 219 men referred to urology via the 'fast track' prostate cancer service were recruited into the study. Of these, 191 were referred from primary care and 28 from secondary care. All men completed a questionnaire regarding their knowledge and expectation of the test. RESULTS: The response rate for completed questionnaires was 100%. Overall, 91 (41.5%) men were aware that their PSA had been performed prior to referral and only 79 (36%) men understood why the test was being done. Patients referred from secondary care appeared to be better informed. Despite these figures, 175 (80%) men said they would recommend PSA testing to a friend or colleague, and 196 (89%) men said the test should be broadly publicised. CONCLUSIONS: Nearly two-thirds of the men referred to urology with an elevated PSA were unaware that they had even had their PSA done. Information about the limitations of PSA testing and the consequence of a positive test result had been deficient. Informed counselling for the PSA test should form part of the consultation of any physician intending to undertake this test whether for lower urinary tract symptoms or for prostate cancer screening.

Intravascular ultrasound characterization of the "black hole" phenomenon after drug-eluting stent implantation.

An intraluminal echolucent tissue, dubbed "black hole," has been identified by intravascular ultrasonography after intracoronary brachytherapy. This study reports the characteristics and incidence of the black hole in patients treated with drug-eluting stent implantation using a sirolimus-eluting stent (SES). We included intravascular ultrasound data from the Compassionate Use of Sirolimus-Eluting Stent (SECURE, n = 61 lesions) registry, a study involving patients in whom previous brachytherapy had failed, and the DIABETES trial (n = 165 lesions), a multicenter, randomized study comparing SES versus bare metal stents in diabetic patients. Intravascular ultrasound follow-up was scheduled at 8 months (SECURE trial, post-brachytherapy population) and 9 months (DIABETES trial). In the SECURE population, a black hole was observed in 10 patients (19.6%). Seven black hole segments had significant intimal hyperplasia (> 10%). A black hole accounted for 27% of total intraluminal tissue. In the DIABETES trial, 2 patients (2.5%) in the SES group and none in the bare metal stent group showed echolucent intimal hyperplasia. In conclusion, a black hole occurred frequently after implantation of a SES in patients in whom intracoronary brachytherapy had previously failed. Black holes were also identified in a nonirradiated population, although the incidence was lower than in the post-brachytherapy patients. Bare metal stents were not associated with this phenomenon.

Sirolimus-eluting stents for treatment of complex bypass graft disease: insights from the SECURE registry.

UNLABELLED: Graft disease remains a therapeutic challenge. Procedural complications and unsatisfactory patency rates are realized with both percutaneous intervention and repeat coronary artery bypass graft (CABG) surgery. The efficacy of sirolimus-eluting stents (SES) for the treatment of de novo coronary artery disease has been established, but the use of this technology to treat bypass graft disease remains undefined. The aim of this study was to evaluate the safety and feasibility of SES to treat patients with high-risk bypass graft disease. METHODS: The compassionate use of SES (SECURE) study included patients with recurrent coronary disease and no acceptable alternative available treatment, including brachytherapy or CABG. Outcomes from 76 patients (n = 94 lesions) with graft lesions treated with SES in the U.S. were compared with the outcomes of 176 patients (n = 311 lesions) with only native vessels. IVUS follow-up was performed at 8 months in 14 patients with graft SES treatment. RESULTS: In-hospital outcomes were similar between both groups, with 99% of patients discharged without adverse events. The incidence of target vessel failure including death, MI, and TVR at 12 months in the bypass graft group (55.3%) was similar to that observed in the native vessel group (45.5%, p = 0.17). Intimal hyperplasia (IH) measured by IVUS was 11.8 +/- 16.5%; 50% of patients with graft SES had < 1% IH at 8 months. CONCLUSION: Implantation of SES in high-risk patients with recurrent bypass graft disease is feasible. SES represents a future.

Synergistic use of sirolimus-eluting stents and intravascular ultrasound for the treatment of unprotected left main and vein graft disease.

Treating unprotected left main disease and degenerated saphenous vein grafts by percutaneous intervention remains one of the more challenging situations facing interventional cardiologists. We present two cases showing how the use of sirolimus-eluting stents in combination with other novel techniques might alter treatment paradigms in the future.

Multiple plaque morphologies in a single coronary artery: insights from volumetric intravascular ultrasound.

Intravascular ultrasound provides detailed information on vessel wall and plaque morphology. This report illustrates the use of three-dimensional volumetric IVUS reconstruction images to characterize different patterns of plaque morphology, including vulnerable and ruptured plaques, within a single coronary artery in a patient with unstable angina.

Hemodynamic complications of ventricular septal rupture after acute myocardial infarction.

Ventricular septal rupture (VSR) is a rare but serious complication following acute myocardial infarction (MI). Patients may present with a new murmur associated with a thrill. Right heart catheterization will demonstrate elevated right atrial and pulmonary artery pressures as well as an oxygen step-up at the right ventricular level. Patients with a right ventricular infarction or cardiogenic shock and a ventricular septal rupture have high in-hospital mortality rates. Prompt diagnosis followed by surgical repair is essential for patients with VSR following MI.

Novel method for the isolation and characterisation of the putative prostatic stem cell.

BACKGROUND: Prostate stem cells, responsible for the development, maturation, and function of the prostate, have been implicated in the aetiology of both benign prostate hyperplasia (BPH) and prostate cancer (CaP). However, research has been hampered by the lack of a definitive stem cell marker. We have adapted the protocol for differential Hoechst 33342 uptake by hemopoietic stem cells to enable isolation of putative stem cells from the prostate. METHODS: Prostate epithelial cells isolated from prostate tissue obtained from patients with BPH after transurethral resection of the prostate were stained with Hoechst 33342. The Hoechst 33342 Red/Blue flow cytometry profile was then determined. Hoechst 33342 and Pyronin Y staining was used to determined the cell cycle status. RESULTS: A verapamil-sensitive side population (SP) can be isolated from primary prostate tissue accounting for 1.38% +/- 0.07% of prostate epithelial cells. Cell cycle analysis of this SP population revealed that the majority of SP cells are in either G0 (12.38 +/- 0.31%) or G1 (63.19 +/- 2.13%). CONCLUSIONS: The Hoechst 33342 dye efflux protocol can be adapted for the isolation of a SP from primary prostate tissue.

Debulking does not benefit patients undergoing intracoronary beta-radiation therapy for in-stent restenosis: insights from the START trial.

Intracoronary brachytherapy has become the current treatment of choice for patients with in-stent restenosis (ISR). The aim of the present study was to determine whether plaque extraction using debulking techniques prior to brachytherapy would improve the outcomes of patients with ISR. Patients enrolled into the START (n = 476) and START-40 (n = 205) trials were divided into four subgroups according to their treatment assignments: debulking-radiation, debulking-placebo, balloon angioplasty (BA) radiation, and BA placebo. Patients were further divided according to their ISR lesion length: all lesions, > 15 mm, and > 19 mm. Restenosis rates were higher in placebo, nonradiated lesions undergoing debulking (52.7%) vs. BA alone (38.5%; P = 0.04). Postprocedural minimal lumen diameter (MLD) was similar among the subgroups. Outcomes were similar between debulking and BA within each therapeutic arm. MLD after debulking radiation was greater in patients with ISR > 15 mm (post-MLD was 1.9 vs. 1.7 mm; P = 0.06) but not in the placebo. Debulking radiation patients had greater MLD at follow-up, but restenosis (23.5% after debulking vs. 32.7% BA alone) and late loss (0.3 mm in both subgroups) were not statistically different. There was a trend toward higher mortality among debulked patients (3.7%) compared to BA alone (0.8%). In patients with ISR > 19 mm, four patients died following debulking radiation as compared to no death after BA (P = 0.05). Our results do not support the strategy of plaque extraction prior to intracoronary beta-radiation for ISR.

Intravascular ultrasound catheter entrapment after coronary artery stenting.

Intracoronary ultrasound (IVUS) facilitates optimal stent deployment in the treatment of coronary artery disease, which may favorably improve long-term outcome after stenting. Complications associated with IVUS include coronary vasospasm and rarely more serious adverse events such as vessel perforation or stent deformation. We report an IVUS catheter tip entrapment within a self-deploying nitinol stent.