Large scale sequencing of SARS-CoV-2 genomes from one region allows detailed epidemiology and enables local outbreak management

The COVID-19 pandemic has spread rapidly throughout the world. In the UK, the initial peak was in April 2020; in the county of Norfolk (UK) and surrounding areas, which has a stable, low-density population, over 3,200 cases were reported between March and August 2020. As part of the activities of the national COVID-19 Genomics Consortium (COG-UK) we undertook whole genome sequencing of the SARS-CoV-2 genomes present in positive clinical samples from the Norfolk region. These samples were collected by four major hospitals, multiple minor hospitals, care facilities and community organisations within Norfolk and surrounding areas. We combined clinical metadata with the sequencing data from regional SARS-CoV-2 genomes to understand the origins, genetic variation, transmission and expansion (spread) of the virus within the region and provide context nationally. Data were fed back into the national effort for pandemic management, whilst simultaneously being used to assist local outbreak analyses. Overall, 1,565 positive samples (172 per 100,000 population) from 1,376 cases were evaluated; for 140 cases between two and six samples were available providing longitudinal data. This represented 42.6% of all positive samples identified by hospital testing in the region and encompassed those with clinical need, and health and care workers and their families. 1,035 cases had genome sequences of sufficient quality to provide phylogenetic lineages. These genomes belonged to 26 distinct global lineages, indicating that there were multiple separate introductions into the region. Furthermore, 100 genetically-distinct UK lineages were detected demonstrating local evolution, at a rate of [~]2 SNPs per month, and multiple co-occurring lineages as the pandemic progressed. Our analysis: identified a sublineage associated with 6 care facilities; found no evidence of reinfection in longitudinal samples; ruled out a nosocomial outbreak; identified 16 lineages in key workers which were not in patients indicating infection control measures were effective; found the D614G spike protein mutation which is linked to increased transmissibility dominates the samples and rapidly confirmed relatedness of cases in an outbreak at a food processing facility. The large-scale genome sequencing of SARS-CoV-2-positive samples has provided valuable additional data for public health epidemiology in the Norfolk region, and will continue to help identify and untangle hidden transmission chains as the pandemic evolves. Major pointsIn Norfolk and surrounding regions O_LI100 distinct UK lineages were identified. C_LIO_LI16 UK lineages found in key workers were not observed in patients or in community care. C_LIO_LI172 genomes from SARS-CoV-2 positive samples sequenced per 100,000 population representing 42.6% of all positive cases. C_LIO_LISARS-CoV-2 genomes from 1035 cases sequenced to a high quality. C_LIO_LIOnly 5 countries, out of 103, have sequenced more SARS-CoV-2 genomes than have been sequenced in Norfolk for this paper. C_LIO_LISamples covered the entire first wave, March to August 2020. C_LIO_LIStable evolutionary rate of 2 SNPs per month. C_LIO_LID614G mutation is the dominant genotype and associated with increased transmission. C_LIO_LINo evidence of reinfection in 42 cases with longitudinal samples. C_LIO_LIWGS identified a sublineage associated with care facilities. C_LIO_LIWGS ruled out nosocomial outbreaks. C_LIO_LIRapid WGS confirmed the relatedness of cases from an outbreak at a food processing facility. C_LI

Do blood constituents in varicose veins differ from the systemic blood constituents?

OBJECTIVE: Varicose veins represent one of the most frequent vascular diseases and are in most cases benign. However, advanced disease is frequently associated with complications such as chronic venous insufficiency and superficial vein thrombosis. The pathogenic mechanisms are not well understood. Besides increased venous pressure, it is suggested that local blood constituents trigger various mechanisms responsible for the progression of the disease and its complications. DESIGN: The aim of this study was to investigate the changes in the blood in varicose veins and to compare them with the systemic markers of inflammation and endothelial damage. MATERIALS AND METHODS: Forty patients with primary varicose veins were included in the study. Most patients were class C2. Blood samples were taken from the leg from the tortuous and dilated varicose tributaries of the great saphenous vein and from the cubital vein. RESULTS: The values of basic hematologic tests were comparable between blood samples (varicose vs. systemic). In varicose veins, the following parameters were significantly increased in comparison with systemic blood: hsCRP (3.12 ± 2.18 mg/L vs. 2.04 ± 2.21 mg/L, p = .04), IL-6 (3.54 ± 2.59 pg/mL vs. 2.25 ± 1.27 pg/mL, p = .008), vWF (118.4  ±  27% vs. 83.2  ±  22%, p < .05). D-dimer, in samples taken from the leg varicose veins, was also significantly higher than in the systemic blood (104.3 ± 9.3 ng/mL vs. 89.5 ± 8.3 ng/mL, p = .039). CONCLUSIONS: Some inflammatory markers and indicators of endothelial dysfunction are increased in varicose vein blood. This is most probably the consequence of deteriorated blood flow in dilated and tortuous superficial veins, and increased venous pressure. Damage to the venous wall, which causes a chronic inflammatory response, together with the procoagulant properties of local blood may promote further progression of the disease and thrombotic complications.

Elevated endocan levels and its association with clinical severity in Stevens-Johnson Syndrome and toxic epidermal necrolysis.

AIM: The aim of this study was to determine the levels of endocan and other biomarkers of inflammation in the systemic circulation of three groups of patients: 1) biopsy confirmed Stevens Johnson Syndrome, Toxic Epidermal Necrolysis (SJS/TEN) subjects; 2) patients with allergic skin reactions but biopsy negative for SJS/TEN; and 3) normal controls. Besides, this paper aims to investigate the association of endocan levels with the extent of the skin lesions, the presence of purpura, and the degree of acute renal insufficiency, as well as to investigate endocan as a marker of clinical severity by correlating endocan levels with the SCORTEN results (a prognositic score for SJS/TEN). METHODS: Sixteen patients over the age of 18 years who were referred to Loyola University Medical Center with severe allergic skin reactions were recruited over a two-year period from May 2012 to May 2014. A diagnosis of SJS or TEN was confirmed in 7 subjects by skin biopsy. Citrated plasma samples were assayed for endocan, tumor necrosis factor-α (TNFα), vascular endothelial growth factor (VEGF), and C-reactive protein (CRP). The differences between SJS/TEN subjects, biopsy negative subjects, and normal controls (N.=23) were explored using ANOVA and Tukey's post-hoc test. Associations with other clinical variables were identified using linear and logistic regression. RESULTS: Biopsy positive SJS/TEN subjects and biopsy negative subjects had higher endocan levels than normal controls (SJS/TEN: 3.01 ng/mL [IQR: 2.15-8.11]; biopsy negative: 3.96 ng/mL [IQR: 1.54-4.85]; normal controls: 1.79 ng/mL [IQR: 1.67-1.98]; ANOVA P=0.0038). Endocan levels were more strongly associated with SCORTEN in SJS/TEN subjects than in biopsy negative subjects (R2 SJS/TEN=0.5110; biopsy negative=0.0317). SJS/TEN subjects exhibited significantly higher levels of TNF-α compared to normal controls (P=0.0267). The TNF-α levels were significantly lower compared to biopsy negative subjects (P=0.0052). VEGF levels were also elevated among SJS/TEN and biopsy negative subjects compared to normal controls (SJS/TEN: 12.04 pg/mL: [IQR: 7.64-52.7]; biopsy negative: 10.54 pg/mL [IQR: 4.17-6.46]; normal controls: 4.94 pg/mL [IQR: 4.17-6.46]; ANOVA P<0.0001). There was no significant difference in VEGF levels between SJS/TEN and biopsy negative subjects (P=0.7110). Similarly, CRP levels were elevated among SJS/TEN patients and biopsy negative subjects compared to normal controls (SJS/TEN: 32.09 µg/mL [IQR: 31.49-52.08]; biopsy negative: 83.38 µg/mL [IQR: 44.74-145.38]; healthy normal: 1.08 µg/mL [IQR: 0.73-2.03]; ANOVA P<0.0001). There was no significant difference in CRP levels between SJS/TEN and biopsy negative subjects (P=0.2416). CONCLUSION: To our knowledge, this is the first study to evaluate enodcan, a marker of endothelial dysfunction, in the systemic circulation of SJS/TEN patients. Elevated endocan levels were more strongly associated with disease severity among SJS/TEN subjects than among less severe allergic reactions with skin involvement.

Medical management of patients with peripheral arterial disease.

Peripheral arterial disease (PAD) is one of the most frequent manifestations of atherosclerosis and is associated with atherosclerosis in the coronary and carotid arteries, leading to a highly increased incidence of cardiovascular events. Major risk factors of PAD are similar to those that lead to atherosclerosis in other vascular beds. However, there are differences in the power of individual risk factors in the different vascular territories. Cigarette smoking and diabetes mellitus represent the greatest risks of PAD. For prevention of the progression of PAD and accompanying cardiovascular events similar preventative measures are used as in coronary artery disease (CAD). However, recent data indicate that there are some differences in the efficacy of drugs used in the prevention of atherothrombotic events in PAD. Antiplatelet treatment is indicated in virtually all patients with PAD. In spite of the absence of hard evidence- based data on the long term efficacy of aspirin, it is still considered as a first line treatment and clopidogrel as an effective alternative. The new antiplatelet drugs ticagrelol and prasugrel also represent promising options for treatment of PAD. Statin therapy is indicated to achieve the target low density lipoprotein cholesterol level of ≤2.5 mmol/L (100 mg/dL) and there is emerging evidence that lower levels are more effective. Statins may also improve walking capacity. Antihypertensive treatment is indicated to achieve the goal blood pressure (<140/90 mmHg). All classes of antihypertensive drugs including beta-blockers are acceptable for treatment of hypertension in patients with PAD. Diabetic patients with PAD should reduce their glycosylated haemoglobin to ≤7%. As PAD patients represent the group with the highest risk of atherothrombotic events, these patients need the most intensive treatment and elimination of risk factors of atherosclerosis. These measures should be as comprehensive as those in patients with established coronary and cerebrovascular disease.

Pharmacological profile of sulodexide.

Since its introduction, sulodexide has been used on and off for several indications. More recently this agent has become revitalized and tested in newer indications. Sulodexide is composed of glycosaminoglycan that includes a mixture of fast-moving heparin and dermatan sulfate. It exerts its anticoagulant and antithrombotic action through interactions with both AT and HCII. Sulodexide has been proven to have effects on the fibrinolytic system, platelets, endothelial cells, inflammation and more recently metalloproteases. The administration of sulodexide results in the release of lipoprotein lipase and has been shown to reduce the circulating level of lipids. It has also shown to decrease the viscosity of both whole blood and plasma. Sulodexide differs from heparin in its oral bioavailability and longer half-life. There is also less bleeding associated with sulodexide. In addition, oral administration of sulodexide does not interfere with the pharmacologic actions of commonly used agents. Similar to heparin, sulodexide releases TFPI which contributes to its antithrombotic effect and anti-inflammatory properties. Sulodexide has been proven to be effective in peripheral arterial thrombosis and venous thrombosis. It is also clinically active in the treatment of venous leg ulcers and intermittent claudication. More recent data suggest that sulodexide can be used in tinnitus and in vascular vertigo. Additional studies in these indications are required. Sulodexide was generally safe and well tolerated in the clinical trials, without any severe bleeding complications. Therefore sulodexide appears to be a good treatment for all arterial and venous diseases and for the prevention of progression of disease.

Association between serum levels of pro-metalloproteinase 1, tissue inhibitor of metalloproteinases 1 and 2 and prevalent cardiovascular disease in a population-based study.

AIM: The aim of the study was to test the association between circulating levels of matrix prometalloproteinase1 (pro-MMP1) and its tissue inhibitors TIMP1 and TIMP2 with prevalent cardiovascular events. METHODS: Prevalent cardiovascular events were documented in 500 participants of the Cyprus study (46% men) over the age of 40. Serum levels of pro-MMP1, TIMP1 and TIMP2 were measured with ELISA and the association between quartiles of serum levels and presence of cardiovascular disease (CVD) was tested using multivariable binary regression models. RESULTS: Lower serum levels of pro-MMP1 and TIMP1 were strongly associated with presence of CVD at baseline even after adjustment for conventional risk factors (P(for trend)=0.006 and P=0.001, respectively) and inflammatory factors (P(for trend)=0.005 and P=0.002, respectively) with people in the highest quartile of pro-MMP1 having a reduced odds for cardiovascular disease by about 70% compared to the lowest quartile (OR(adjusted)=0.26; 95% CI=0.19 to 0.75; P=0.01), whereas people with TIMP1 levels >1000 ng/mL had a 75% reduced odds for CVD compared to the rest (OR(adjusted)=0.25; 95% CI=0.11 to 0.60; P(for trend)=0.002). TIMP2 levels were not associated with prevalent cardiovascular disease. CONCLUSION: A strong association between lower levels of circulating pro-MMP1 and TIMP1 and risk of prevalent cardiovascular disease in a general population cohort over 40 years is evident, independent from common cardiovascular and inflammatory risk factors. The role of MMP1 and its tissue inhibitors, should be tested further in prospective studies of cardiovascular disease.

Prevention and treatment of venous thromboembolism--International Consensus Statement.

The aim of this document is to provide a clear and concise account of the evidence regarding efficacy or harm for various methods available to prevent and manage venous thromboembolism (VTE).

Thromboelastographic evaluation of blood coagulation in the presence of branded and generic enoxaparins.

AIM: Enoxaparin is the most widely used low-molecular-weight heparin (LMWH) in the USA and has been approved for clinical use in multiple indications. Enoxaparin is a complex biological product with multiple known activities relevant to its antithrombotic effects, and variations in different forms of enoxaparin may have important clinical implications. This study aimed to compare the physiological anticoagulant activity of branded and a generic enoxaparin, using thromboelastography (TEG) to evaluate their effect on the dynamic formation of the blood clot as quantitated by interactions between coagulation factors and inhibitors, fibrinogen, platelets and the fibrinolytic system. METHODS: Whole native (no preservative) blood was obtained from 7 healthy volunteers. Samples were immediately mixed with various concentrations of branded or generic enoxaparin and TEG was performed to assess anticoagulant activity. Five different batches of each enoxaparin (branded and generic) were tested. RESULTS: Generic enoxaparin showed more variation in anticoagulation response with a less predictable concentration-dependent and linear response compared with branded enoxaparin. There was also an apparent batch-to-batch variation for generic enoxaparin. The results demonstrated a lower overall anticoagulant effect (P=0.05; no overlap of 95% confidence intervals) with a wider inter-individual variation for generic enoxaparin in comparison with branded enoxaparin. Some individuals responded with a higher than expected anticoagulant response to the given concentration of the generic enoxaparin. CONCLUSION: The findings of this study suggest that other pre-clinical and clinical studies should be done to validate the clinical interchangeability between branded and generic enoxaparin.

Defibrotide blunts the prothrombotic effect of thalidomide on endothelial cells.

Patients with multiple myeloma (MM) are at relatively high risk of developing thromboembolic events such deep venous thrombosis (DVT) where thalidomide therapy has been identified to increase this risk. Defibrotide (DF), a polydisperse oligonucleotide, showed previously to counteract the alterations in endothelial cells (ECs) induced by lipopolysaccharide. It prompts us to investigate the impact of thalidomide on ECs and whether DF modulates changes in fibrinolysis induced by thalidomide. In this in vitro study, MM by itself alters the profibrinolytic potential of ECs decreasing the tissue plasminogen activator (t-PA) and increasing the plasminogen activator inhibitor 1 (PAI-1) levels which is potentiated by thalidomide. Defibrotide was able to counteract these effects. Additionally, DF upregulated the t-PA and downregulated PAI-1 gene expression modulated by thalidomide. Defibrotide also protects ECs from thalidomide-mediated cell death without interfering with its antitumor effects. These findings support DF clinical use for the prevention of DVT induced by immunomodulatory drugs.

Prophylaxis of venous thromboembolism: low molecular weight heparin compared to the selective anticoagulants rivaroxaban, dabigatran and fondaparinux.

Newer therapeutic options available in the prevention of postoperative thromboembolism, currently focused on fondaparinux, rivaroxaban and dabigatran warrant an overall therapeutic assessment. The constitutive comparisons with enoxaparin are based on a combined outcome measure solely driven by the incidence of "asymptomatic deep vein thrombosis". Its validity as a clinically relevant endpoint is missing if antithrombotics of different classes are compared. This is because they target different phases of thrombogenesis i. e. ahead and beyond the asymptomatic stage of thrombosis. Additional concerns refer to the dosing-regimens and their practical administration: Fondaparinux, rivaroxaban and dabigatran are dosed to achieve maximum effects very close to their limits of tolerance whereas wide dosing spectra for the low molecular weight herparin (LMWH)'s indicate the potential for dose adaptation and increase. The other disadvantage to the control-heparin originates in the timing for the 1st administration which doesn't fit in with the "just-in-time" principle. So the enoxaparin-regimen is lacking in benchmark-quality - with the consequence that the meaning of the Phase III-trials does'nt go beyond a mere technical demarcation from the marketed variant of the product as defined by the stipulations in the package insert. As to tolerance the selective anticoagulants exhibit an increased risk of major and other clinically relevant bleeding, exceeding that of enoxaparin by 30% (P<0.001). The outcome of the meta-analyses on fondaparinux, rivaroxaban and dabigatran is supported by product-specific calculations and assessments of the European Medicine Equivalence Agency (EMEA). Rivaroxaban and dabigatran show significant age-dependent renal accumulation. Because the dose-finding studies were restricted to patients over 60 year old the regimens definitely established are not applicable to younger patients. The reason for the limited therapeutic index of the selective anticoagulants originates in their monovalent activity as such not adequately matching the complexity of thrombogenesis and early thrombus extension. Their class-specific limitations are compensated through more intensive dosage-regimens which result in accentuated bleeding complications. Connotatively the hypothesis emerged that antiXa- and IIa-effects interact synergistically which translates into enhanced efficacy and tolerance. Experimental studies on hirudin with pentasaccharide and hirudin with "lower low molecular weight heparin" (3KDA) support such rationale.

Leukocyte telomere length is associated with measures of subclinical atherosclerosis.

AIMS: Our aim was to test the association of mean leukocyte telomere length (LTL) with ultrasonic measures of subclinical atherosclerosis such as intima-media thickness in the common carotid (IMTcc) and sum of plaque areas (SPA) and with serological markers. METHODS AND RESULTS: Carotid and femoral bifurcations were scanned in 762 general population volunteers (46% men) over 40. Four features were considered: (a) IMTcc, (b) sum plaque areas of carotid plaques (SPAcar), (c) sum plaque area of common femoral plaques (SPAfem) and (d) sum plaque area (SPA--sum of the plaque areas of the largest plaques present in each of both carotid and femoral bifurcations). Mean LTL was determined with a quantitative real-time PCR-based method. IMTcc was strongly associated with mean LTL both before and after correction for traditional risk factors (B=-0.002; 95% CI=-0.004 to -0.00; p=0.014). In sex-specific analysis, the association was stronger in men (p for sex interaction<0.001). SPAfem was associated with LTL in women before and after correction (B=-0.195; 95% CI=-0.38 to -0.01; p=0.037) (p for sex interaction<0.001). LTL was also associated with age and sex-adjusted levels of hsCRP (p=0.012), sCD40L (p=0.042), homocysteine (p=0.006), creatinine (p=0.02), ApoA1 (p=0.01), Lp(a) (p=0.04) and HOMA-IR (p=0.008). CONCLUSIONS: Our results support the telomere hypothesis and highlight potential differences in the biological mechanisms leading to intima-media thickening and/or plaque formation between vascular beds. They may provide insights into a novel treatment of antisenescence to prevent atherosclerosis.

Recommendations on biosimilar low-molecular-weight heparins.

Based on the results of large clinical trials, several low-molecular-weight heparins (LMWHs) have been approved for prophylaxis and the treatment of venous and arterial thromboembolism. As a result of expiration or pending expiration of patent protection of the originator LMWHs, many generic or biosimilar LMWHs have been approved in some countries and more are likely to be approved elsewhere. Their greater availability may reduce the treatment costs. The Working Party on Requirements for Development of Biosimilar LMWHs of the Subcommittee on Control of Anticoagulation, Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis has reached a consensus on recommendations to ensure the quality of biosimilar LMWHs as compared with the originator LMWHs.

Molecular profiling of heparinase-I resistant glycosaminoglycans in contaminated heparins. Comparative studies with uncontaminated heparin and porcine oversulfated chondroitin sulfate.

AIM: Heparin is a widely used anticoagulant which is usually obtained from porcine mucosal tissue. The structure of heparin is comparable to other naturally occurring glycosaminoglycans such as chondroitin sulfate and dermatan sulfate. The commercially available heparin preparations may contain small amounts of dermatan sulfate as a carry-over impurity. More recently (November 2007 to April 2008), an increased incidence of adverse events and deaths associated with the use of heparin alerted regulatory agencies to investigate the composition of heparin. As a result, oversulfated chondroitin sulfate was found to be the main determinant of the observed adverse reactions. This glycosaminoglycan is not usually found in the mammalian tissues. METHODS: This investigation reports on the comparison of contaminant free and contaminated heparins and their digestion by heparinase-I. It also describes the molecular profile of the contaminant isolated from the recalled heparin preparations in comparison to oversulfated chondroitin sulfate. The anticoagulant and anti-Xa activities are also reported. RESULTS: The contaminant is found to be comparable to the synthesized OSCS as both were resistant to heparinase-I digestion. The contaminant and OSCS exhibited weaker anticoagulant activities than heparin and did not have any anti-Xa effects. CONCLUSION: This data strongly suggests that such glycosaminoglycans as chondroitin sulfate can be structurally modified to exhibit anticoagulant activities and their molecular weight can be adjusted to mimic heparin.