231 research outputs found

    Modelling RT-qPCR cycle-threshold using digital PCR data for implementing SARS-CoV-2 viral load studies

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    Objectives To exploit the features of digital PCR for implementing SARS-CoV-2 observational studies by reliably including the viral load factor expressed as copies/μL. Methods A small cohort of 51 Covid-19 positive samples was assessed by both RT-qPCR and digital PCR assays. A linear regression model was built using a training subset, and its accuracy was assessed in the remaining evaluation subset. The model was then used to convert the stored cycle threshold values of a large dataset of 6208 diagnostic samples into copies/μL of SARSCoV- 2. The calculated viral load was used for a single cohort retrospective study. Finally, the cohort was randomly divided into a training set (n = 3095) and an evaluation set (n = 3113) to establish a logistic regression model for predicting case-fatality and to assess its accuracy. Results The model for converting the Ct values into copies/μL was suitably accurate. The calculated viral load over time in the cohort of Covid-19 positive samples showed very low viral loads during the summer inter-epidemic waves in Italy. The calculated viral load along with gender and age allowed building a predictive model of case-fatality probability which showed high specificity (99.0%) and low sensitivity (21.7%) at the optimal threshold which varied by modifying the threshold (i.e. 75% sensitivity and 83.7% specificity). Alternative models including categorised cVL or raw cycle thresholds obtained by the same diagnostic method also gave the same performance. Conclusion The modelling of the cycle threshold values using digital PCR had the potential of fostering studies addressing issues regarding Sars-CoV-2; furthermore, it may allow setting up predictive tools capable of early identifying those patients at high risk of case-fatality already at diagnosis, irrespective of the diagnostic RT-qPCR platform in use. Depending upon the epidemiological situation, public health authority policies/aims, the resources available and the thresholds used, adequate sensitivity could be achieved with acceptable low specificity

    The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs

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    Background: Gain-of-function mutations in KIT are driver events of oncogenesis in mast cell tumours (MCTs) affecting companion animals. Somatic mutations of KIT determine the constitutive activation of the tyrosine kinase receptor leading to a worse prognosis and a shorter survival time than MCTs harbouring wild-type KIT. However, canine MCTs carrying KIT somatic mutations generally respond well to tyrosine kinase inhibitors; hence their presence represents a predictor of treatment effectiveness, and its detection allows implementing a stratified medical approach. Despite this, veterinary oncologists experience treatment failures, even with targeted therapies whose cause cannot be elucidated. The first case of an MCT-affected dog caused by a secondary mutation in the tyrosine kinase domain responsible for resistance has recently been reported. The knowledge of this and all the other mutations responsible for resistance would allow the effective bedside implementation of a deeply stratified and more effective medical approach. Case presentation: The second case of a canine MCT carrying a different resistance mutation is herein described. The case was characterised by aggressive behaviour and early metastasis unresponsive to both vinblastine- and masitinib-based treatments. Molecular profiling of the tumoural masses revealed two different mutations; other than the already known activating mutation p.Asn508Ile in KIT exon 9, which is tyrosine kinase inhibitor-sensitive, a nearly adjacent secondary missense mutation, p.Ala510Val, which had never before been described, was detected. In vitro transfection experiments showed that the secondary mutation did not cause the constitutive activation by itself but played a role in conferring resistance to masitinib. Conclusions: This study highlighted the importance of the accurate molecular profiling of an MCT in order to improve understanding of the molecular mechanism underlying tumourigenesis and reveal chemoresistance in MCTs for more effective therapies. The detection of the somatic mutations responsible for resistance should be included in the molecular screening of MCTs, and a systematic analysis of all the cases characterised by unexpected refractoriness to therapies should be investigated in depth at both the genetic and the phenotypic level

    A large deletion in the GP9 gene in Cocker Spaniel dogs with Bernard-Soulier syndrome

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    Inherited bleeding disorders including abnormalities of platelet number and function rarely occur in a variety of dog breeds, but are probably underdiagnosed. Genetically characterized canine forms of platelet disorders provide valuable large animal models for understanding similar platelet disorders in people. Breed-specific disease associated genetic variants in only eight different genes are known to cause intrinsic platelet disorders in dogs. However, the causative genetic variant in many dog breeds has until now remained unknown. Four cases of a mild to severe bleeding disorder in Cocker Spaniel dogs are herein presented. The affected dogs showed a platelet adhesion defect characterized by macrothrombocytopenia with variable platelet counts resembling human Bernard-Soulier syndrome (BSS). Furthermore, the lack of functional GPIb-IX-V was demonstrated by immunocytochemistry. Whole genome sequencing of one affected dog and visual inspection of the candidate genes identified a deletion in the glycoprotein IX platelet (GP9) gene. The GP9 gene encodes a subunit of a platelet surface membrane glycoprotein complex; this functions as a receptor for von Willebrand factor, which initiates the maintenance of hemostasis after injury. Variants in human GP9 are associated with Bernard-Soulier syndrome, type C. The deletion spanned 2460 bp, and included a significant part of the single coding exon of the canine GP9 gene on dog chromosome 20. The variant results in a frameshift and premature stop codon which is predicted to truncate almost two-thirds of the encoded protein. PCR-based genotyping confirmed recessive inheritance. The homozygous variant genotype seen in affected dogs did not occur in 98 control Cocker Spaniels. Thus, it was concluded that the structural variant identified in the GP9 gene was most likely causative for the BSS-phenotype in the dogs examined. These findings provide the first large animal GP9 model for this group of inherited platelet disorders and greatly facilitate the diagnosis and identification of affected and/or normal carriers in Cocker Spaniels

    Correlating qrt-pcr, dpcr and viral titration for the identification and quantification of sars-cov-2: A new approach for infection management

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    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays

    Rheolytic Thrombectomy with or without Adjunctive Indwelling Pharmacolysis in Patients Presenting with Acute Pulmonary Embolism Presenting with Right Heart Strain and/or Pulseless Electrical Activity

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    Purpose. To evaluate the safety and efficacy of the Possis rheolytic thrombectomy with or without indwelling catheter-directed pharmacolysis for the treatment of massive pulmonary embolus in patients presenting with right heart strain and/or a pulseless electrical activity (PEA). Materials and Methods. Retrospective review of patients undergoing pulmonary pharmacolysis was performed (07/2004–06/2009). Pre- and posttreatment Miller index scoring weres calculated and compared. Patients were evaluated for tPA doses, ICU stay, hospital stay, and survival by Kaplan-Meier analysis. Results. 11 patients with massive PE were found, with 10/11 presenting with a Miller score of >17 (range: 16–27, mean: 23.2). CTPA and/or echocardiographic evidence of right heart strain was found in 10/11 patients. 3 (27%) patients presented with a PEA event. Two (18%) patients had a contraindication to pharmacolysis and were treated with mechanical thrombectomy alone. The intraprocedural mortality was 9% (n = 1/11). Of the 10 patients who survived the initial treatment, 7 patients underwent standard mechanical thrombectomy initially, while 5 received power pulse spray mechanical thrombectomy. Eight of these 10 patients underwent adjunctive indwelling catheter-directed thrombolysis. The mean catheter-directed infusion duration was 18 hours (range of 12–26 hours). The average intraprocedural, infusion, and total doses of tPA were 7 mg, 19.7 mg, and 26.7 mg, respectively. There was a 91% (10/11) technical success rate. The failure was the single mortality. Average reduction in Miller score was 9.5 or 41% (P = 0.009), obstructive index of 6.4 or 47% (P = 0.03), and perfusion index of 2.7 or 28% (P = 0.05). Average ICU and hospital stay were 7.4 days (range 2–27 days) and 21.3 days (range 6–60 days), respectively. Intent to treat survival was 90% at 6, 12, and 18 months. Conclusion. Rheolytic thrombectomy with or without adjunctive catheter-directed thrombolysis provides a safe and effective method for treatment of acute PE in patients who present with right heart strain and/or a PEA event

    ABCB1 c.-6-180 T > G polymorphism and clinical risk factors in a multi-breed cohort of dogs with refractory idiopathic epilepsy

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    Epilepsy is the most common chronic neurological disorder in dogs. Approximately 20-30% of dogs do not achieve satisfactory seizure control with two or more anti-epileptic drugs at appropriate dosages. This condition, defined as refractory epilepsy, is a multifactorial condition involving both acquired and genetic factors. The P glycoprotein might play and important role in the pathophysiological mechanism and it is encoded by the ABCB1 gene. An association between a single nucleotide variation of the ABCB1 gene (c.-6-180 T > G) and phenobarbital resistance has previously been reported in a Border collie population with idiopathic epilepsy. To date, the presence and relevance of this polymorphism has not been assessed in other breeds. A multicentre retrospective, case-control study was conducted to investigate associations between ABCB1 c.-6-180 T > G, clinical variables, and refractoriness in a multi-breed population of dogs with refractory idiopathic epilepsy. A secondary aim was to evaluate the possible involvement of the ABCB1 c.-6-180 T > G single nucleotide variation this population. Fifty-two refractory and 50 responsive dogs with idiopathic epilepsy were enrolled. Of these, 45 refractory and 50 responsive (control) dogs were genotyped. The G allele was found in several breeds, but there was no evidence of association with refractoriness (P = 0.69). The uncertain role of the c.-6-180T>G variation was further suggested by an association between the T/T genotype with both refractoriness and responsiveness in different breeds. Furthermore, high seizure density (cluster seizure) was the main clinical risk factor for refractory idiopathic epilepsy (P = 0.003)

    Acute lower gastrointestinal bleeding: predictive factors and clinical outcome for the patients who needed first-time mesenteric conventional angiography

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    PURPOSE:We aimed to investigate patients with lower gastrointestinal bleeding who presented to the emergency department requiring initial conventional angiography. We report risk-stratified and mesenteric conventional angiography outcomes.METHODS:We retrospectively reviewed patients with lower gastrointestinal bleeding between 2001 and 2012. We included all consecutive patients with clinical lower gastrointestinal bleeding with a requirement of further angiography and possible embolization. Patients who had prior interventions or surgery were excluded.RESULTS:A total of 88 patients (35 women, 53 men) with a median age of 71 years (range, 23–99 years) were included in the analysis. Conventional angiography was positive and endovascular treatment was intended in 35 patients. Once the source of bleeding was found angiographically, endovascular treatment had a technical success rate of 90.3% and clinical success rate of 71.4%. Overall early rebleeding rate (30 days) was 13.6%.CONCLUSION:Identifying the source of lower gastrointestinal bleeding remains to be a clinical and angiographic challenge. Although we did not observe an association between mortality and clinical success, increased early rebleeding rates were associated with higher mortality rates

    An overview of using small punch testing for mechanical characterization of MCrAlY bond coats

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    Considerable work has been carried out on overlay bond coats in the past several decades because of its excellent oxidation resistance and good adhesion between the top coat and superalloy substrate in the thermal barrier coating systems. Previous studies mainly focus on oxidation and diffusion behavior of these coatings. However, the mechanical behavior and the dominant fracture and deformation mechanisms of the overlay bond coats at different temperatures are still under investigation. Direct comparison between individual studies has not yet been achieved due to the fragmentary data on deposition processes, microstructure and, more apparently, the difficulty in accurately measuring the mechanical properties of thin coatings. One of the miniaturized specimen testing methods, small punch testing, appears to have the potential to provide such mechanical property measurements for thin coatings. The purpose of this paper is to give an overview of using small punch testing to evaluate material properties and to summarize the available mechanical properties that include the ductile-to-brittle transition and creep of MCrAlY bond coat alloys, in an attempt to understand the mechanical behavior of MCrAlY coatings over a broad temperature range

    Powder Compaction: Compression Properties of Cellulose Ethers

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    Effective development of matrix tablets requires a comprehensive understanding of different raw material attributes and their impact on process parameters. Cellulose ethers (CE) are the most commonly used pharmaceutical excipients in the fabrication of hydrophilic matrices. The innate good compression and binding properties of CE enable matrices to be prepared using economical direct compression (DC) techniques. However, DC is sensitive to raw material attributes, thus, impacting the compaction process. This article critically reviews prior knowledge on the mechanism of powder compaction and the compression properties of cellulose ethers, giving timely insight into new developments in this field
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