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The journey of translating a molecular discovery into the clinic involves multiple steps and requires planning, time, effort, and money. In this review, we provide a quick guide on the technical and clinical validation parameters that are necessary for successful commercialization of molecular and other markers. We also briefly address the different options for regulatory approvals. Successful clinical implantation depends on rigorous technical and clinical validation, and the ability to develop clear guidelines for the indications for testing (i.e. which patients are eligible to have this test), the frequency of testing, and also a clear interpretation of test results. Successful implementation requires providing evidence that the results of this test can be used to improve patient care. There are currently multiple routes for implementation of clinical molecular tests, which include regulatory agency- approved companion diagnostics, laboratory developed tests, or direct-to-consumer testing. Regulatory approval is considered the gold-standard, but it requires time and resources. There is an ongoing debate about the need for regulatory approval of laboratory developed testing. Ongoing oversight is maintained through lab accreditation and proficiency testing programs, which provide a common approach to ensuring high standards and consistent performance in clinical molecular labs. Before moving into the clinic, confirmation of both the clinical and analytic validity of a new molecular test is essential.
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Digital pathology (DP) has emerged as a cutting-edge technology that promises to revolutionise diagnostics in clinical laboratories. This perspective article explores the implementation planning and considerations of DP in a single multicentre institution in Canada, the University Health Network, discussing benefits, challenges, potential implications and considerations for future adopters. We examine the transition from traditional microscopy to digital slide scanning and its impact on pathology practice, patient care and medical research. Furthermore, we address the regulatory, infrastructure and change management considerations for successful integration into clinical laboratories. By highlighting the advantages and addressing concerns, we aim to shed light on the transformative potential of DP and its role in shaping the future of diagnostics.
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Laboratórios Clínicos , Patologia Clínica , Humanos , Patologia Clínica/métodos , Canadá , Microscopia/métodosRESUMO
Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.
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Ebolavirus/genética , Evolução Molecular , Olho/virologia , Doença pelo Vírus Ebola/fisiopatologia , Doença pelo Vírus Ebola/virologia , Animais , Anticorpos Antivirais/imunologia , Modelos Animais de Doenças , Ebolavirus/imunologia , Feminino , Furões/imunologia , Doença pelo Vírus Ebola/imunologia , Masculino , RNA Viral/genéticaRESUMO
The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.
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Betacoronavirus/efeitos dos fármacos , Indicadores e Reagentes/farmacologia , Inativação de Vírus/efeitos dos fármacos , Animais , Betacoronavirus/isolamento & purificação , COVID-19 , Teste para COVID-19 , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Filtração/instrumentação , Humanos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , SARS-CoV-2 , Células VeroRESUMO
Nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), a homogeneous approach to select DNA aptamers, is among the most efficient partitioning techniques. In contrast with surface-based systematic evolution of ligands by exponential enrichment (SELEX) approaches, the ability of NECEEM to select aptamers to unmodified proteins in solution is preferable for identifying aptamers for eventual in vivo use. The high stringency and low sample volumes of NECEEM, although generally beneficial, can result in binding of very few aptamers, requiring highly efficient amplification to propagate them. When amplified with standard PCR, detectable library enrichment can fail due to the fast conversion of the aptamers into byproducts and preferential amplification of nonbinders. As an alternative, we proposed the use of emulsion PCR (ePCR), which is known to reduce byproduct formation, as a PCR mode for coupling with NECEEM partitioning. For the first time, we tested the advantages of ePCR in NECEEM-based aptamer selection to a medically relevant DNA repair enzyme, ABH2. We report that the combination of ePCR with NECEEM allowed for the selection of aptamers in the first three rounds of SELEX, while SELEX with conventional PCR failed in a number of attempts. Selected aptamers to an unmodified ABH2 protein have potential use in diagnostics and as leads for anticancer cotherapies, used as enhancements of alkylating agents in chemotherapy.
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Aptâmeros de Nucleotídeos/química , Enzimas Reparadoras do DNA/química , Dioxigenases/química , Eletroforese Capilar/métodos , Emulsões/química , Reação em Cadeia da Polimerase/métodos , Técnica de Seleção de Aptâmeros/métodos , Homólogo AlkB 2 da Dioxigenase Dependente de alfa-Cetoglutarato , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Enzimas Reparadoras do DNA/genética , Dioxigenases/genética , Biblioteca Gênica , HumanosRESUMO
The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in regenerative medicine. We report here the proteomic profile of 293T epithelial cells reprogrammed to a pluripotent state using undifferentiated embryonal carcinoma (NCCIT) cellular extracts. 293T cells were reversibly permeabilized with streptolysin O, incubated in an extract of NCCIT cells or a control extract of 293T cells for 1 h, resealed with CaCl(2), and cultured. OCT4 and SOX2 gene expression were up-regulated in NCCIT extract-treated cells relative to control cells, whereas there was no alteration in DNMT3B gene expression. Thirty percent of NCCIT extract-treated cells were positive for SSEA-4, and karyotyping confirmed their 293T origin, excluding the possibility of contamination from NCCIT cells. Two-dimensional PAGE revealed approximately 400 protein spots for each cell type studied. At least 10 protein spots in the proteome of NCCIT extract-treated cells had an expression profile similar to that of NCCIT and remained unaltered in control cells. Using tandem mass spectrometry, we identified these proteins, which include 78-kDa glucose-regulated protein precursor and tropomyosin alpha-3 chain. This investigation provides the first evidence that proteins are altered in a specific manner in NCCIT extract-treated cells. This is the first report on the proteomic characterization of the nuclear reprogramming process.
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Células Epiteliais/metabolismo , Proteômica , Sequência de Bases , Linhagem Celular , Sistema Livre de Células , Cromatografia Líquida , Primers do DNA , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Cariotipagem , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
The development of safe diagnostic protocols for working with SARS-CoV-2 clinical samples at Biosafety Level 2 (BSL2) requires understanding of the effect of heat-treatment on SARS-CoV-2 viability and downstream RT-PCR sensitivity. In this study heating SARS-CoV-2/England/2/2020 to 56 °C and 60 °C for 15, 30 and 60 min reduced the virus titre by between 2.1 and 4.9 log10 pfu/mL (as determined by plaque assay). Complete inactivation did not occur and there was significant variability between replicates. Viable virus was detected by plaque assay after heat-treatment at 80 °C for 15 or 30 min but not 60 or 90 min. After heat-treatment at 80 °C for 60 min infectious virus was only detected by more sensitive virus culture. No viable virus was detected after heating to 80 °C for 90 min or 95 °C for 1 or 5 min. RT-PCR sensitivity was not compromised by heating to 56 °C and 60 °C. However, RT-PCR sensitivity was reduced (≥3 Ct value increase) after heating the virus to 80 °C for 30 min or longer, or 95 °C for 1 or 5 min. In summary we found that the efficacy of heat-inactivation varies greatly depending on temperature and duration. Local validation of heat-inactivation and its effects downstream is therefore essential for molecular testing.
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SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Inativação de Vírus , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19 , Temperatura Alta , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures.
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Antivirais/farmacologia , Teste Sorológico para COVID-19/métodos , SARS-CoV-2/efeitos dos fármacos , Acetamidas , Soluções Tampão , COVID-19/diagnóstico , COVID-19/virologia , Fluoracetatos , Guanidina/efeitos adversos , Humanos , Inativação de Vírus/efeitos dos fármacosRESUMO
Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.
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Sequenciamento de Nucleotídeos em Larga Escala/métodos , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/patogenicidade , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga ViralRESUMO
The use of stem cells for generating cell types suitable for therapy is dependent on understanding the mechanisms, and identifying biomarkers, that control cell fate into different lineages. In this study, we aimed to characterize the nuclear protein dynamics of NTERA-2 cells undergoing retinoic acid-induced differentiation. We focused specifically on the first six days of differentiation, to provide insight into the earliest differentiation events, and employed techniques to specifically monitor the nuclear proteome. Well-characterized gene expression markers were used to precisely stage cell differentiation across the experimental time course. A combination of the novel iTRAQ and ExacTag labeling technologies, together with LC-ESI tandem mass spectrometry, were then used to accurately measure nuclear protein expression changes occurring within these differentiation-staged cells. We report proteins that showed significantly altered expression over the first 6 days of differentiation. Extensive bioinformatic analysis was undertaken, resulting in the construction of a novel interactome network, which revealed the temporal dynamics of the nuclear protein network in the context of neuronal differentiation.
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Núcleo Celular/metabolismo , Células-Tronco de Carcinoma Embrionário/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Análise de Variância , Biomarcadores/metabolismo , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Análise por Conglomerados , Humanos , Marcação por Isótopo , Neurônios/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/classificação , Mapeamento de Interação de Proteínas , Proteoma/efeitos dos fármacos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
During the Ebola outbreak in 2014-2015 in Sierra Leone, residual clinical specimens and accompanying data were collected from routine diagnostic testing in Public Health England (PHE) led laboratories. Most of the samples with all the accompanying data were transferred to PHE laboratories in the UK for curation by PHE. The remainder have been kept securely in Sierra Leone. The biobank holds approximately 9955 samples of which 1108 tested positive for Ebola virus. Researchers from the UK and overseas, from academia, government other research organisations and commercial companies can submit proposals to the biobank to access and use the samples. The Ministry of Health and Sanitation in Sierra Leone (MOHS) retains ownership of the data and materials and is working with PHE and other researchers to develop and conduct a series of research projects that will inform future healthcare and public health strategies relating to Ebola. The Ebola Biobank Governance Group (EBGG) was established to guarantee equality of access to the biobank for the most scientifically valuable research including by researchers from low and middle-income countries. Ensuring benefit to the people of Sierra Leone is an over-arching principle for decisions of the EBGG. Four ongoing research collaborations are based on the first wave of biobank proposals approved by EBGG. Whilst the biobank is a valuable resource its completeness and sample quality are consistent with the outbreak conditions under which they were collected.
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Recent studies have shown that transcriptomic analysis of blood samples taken from patients with acute Ebola virus disease (EVD) during the 2013-2016 West African outbreak was suggestive that a severe inflammatory response took place in acutely ill patients. The significant knowledge gained from studying the Makona variant, a cause of the largest known EVD outbreak, may be applicable to other species of ebolavirus, and other variants of the Ebola virus (EBOV) species. To investigate the ability of Makona to initiate an inflammatory response in human macrophages and characterise the host response in a similar manner to previously characterised EBOV variants, the human monocytic cell line THP-1 was differentiated into macrophage-like cells and infected with Makona. RNA-Seq and quantitative proteomics were used to identify and quantify host mRNA and protein abundance during infection. Data from infection with Reston virus (RESTV) were used as comparators to investigate changes that may be specific to, or enhanced in, Makona infection in relation to a less pathogenic species of ebolavirus.. This study found demonstrable induction of the inflammatory response, and increase in the activation state of THP-1 macrophages infected with Makona. NFκB and inflammation-associated transcripts displayed significant changes in abundance, reflective of what was observed in human patients during the 2013-2016 EBOV outbreak in West Africa, and demonstrated that transcriptomic changes found in Makona-infected cells were similar to that observed in Reston virus infection and that have been described in previous studies of other variants of EBOV.
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Ebolavirus/isolamento & purificação , Ebolavirus/patogenicidade , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doença pelo Vírus Ebola/virologia , Humanos , Interferons/genética , Interferons/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Proteômica , Células THP-1RESUMO
OBJECTIVE: The aim of this study was to determine whether there is an association between ultrasound fetal biometry and amniotic fluid insulin levels at delivery in women with pre-existing diabetes or impaired glucose tolerance in pregnancy. STUDY DESIGN: This retrospective cohort study identified 93 women who had amniotic fluid insulin levels measured at time of delivery. Standardised estimated fetal weight and fetal growth velocity were calculated from serial third trimester fetal ultrasound measurements. RESULTS: Women with pre-existing diabetes had significantly greater mean growth velocity [1.39 (95% CI: 0.43-2.23) versus 0.39 (95% CI: -01.7-0.95); p=0.04], significantly greater mean estimated fetal weight (EFW) Z score prior to delivery [2.36 (95% CI: 1.82-2.9) versus 1.38 (95% CI: 1.02-1.74); p=0.002] and greater mean birthweight centile [82 (95% CI: 0.74-0.89) versus 67 (95% CI: 58-76); p=0.02] than those with GDM/IGT. Amniotic fluid insulin levels demonstrated a similar significant difference between the pre-existing and GDM/IGT groups [20.5 (95% CI: 12.9-28.1) versus 8.5 (95% CI: 5.4-11.7); p=0.001]. An association between fetal growth and size and amniotic fluid insulin was observed in women with pre-existing diabetes. Positive likelihood ratios were 1.67 and 2.08, respectively, for the prediction of liquor insulin greater than the 95th centile in women with pre-existing diabetes. CONCLUSION: Ultrasound measures of fetal size and growth used in this study are not sufficiently accurate to predict those infants likely to be at risk from the adverse effects of fetal hyperinsulinaemia.
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Biometria/métodos , Diabetes Gestacional/sangue , Doenças Fetais/diagnóstico , Hiperinsulinismo/diagnóstico , Gravidez em Diabéticas/sangue , Ultrassonografia Pré-Natal/métodos , Líquido Amniótico/química , Estudos de Coortes , Complicações do Diabetes/sangue , Feminino , Desenvolvimento Fetal , Doenças Fetais/etiologia , Peso Fetal , Humanos , Hiperinsulinismo/etiologia , Recém-Nascido , Insulina/análise , Valor Preditivo dos Testes , Gravidez , Complicações na Gravidez/sangue , Estudos RetrospectivosRESUMO
The Ebola virus (EBOV) variant Makona (which emerged in 2013) was the causative agent of the largest outbreak of Ebola Virus Disease recorded. Differences in virus-host interactions between viral variants have potential consequences for transmission, disease severity and mortality. A detailed profile of the cellular changes induced by the Makona variant compared with other Ebola virus variants was lacking. In this study, A549 cells, a human cell line with a robust innate response, were infected with the Makona variant or with the Ecran variant originating from the 1976 outbreak in Central Africa. The abundance of viral and cellular mRNA transcripts was profiled using RNASeq and differential gene expression analysis performed. Differences in effects of each virus on the expression of interferon-stimulated genes were also investigated in A549 NPro cells where the type 1 interferon response had been attenuated. Cellular transcriptomic changes were compared with those induced by human respiratory syncytial virus (HRSV), a virus with a similar genome organisation and replication strategy to EBOV. Pathway and gene ontology analysis revealed differential expression of functionally important genes; including genes involved in the inflammatory response, cell proliferation, leukocyte extravasation and cholesterol biosynthesis. Whilst there was overlap with HRSV, there was unique commonality to the EBOV variants.
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Ebolavirus/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Transcriptoma , Células A549 , Perfilação da Expressão Gênica , Humanos , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Análise de Sequência de RNARESUMO
The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT-qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log-linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource-limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats.
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In the grieving process, patient, family, and health professionals have the same needs-rest, relaxation, nourishment, a sense of security, trust, hope in the future, and humor among them. Grief, defined as a shared, universal, and natural neuropsychobiologic expression in response to loss, is distinct from mourning, a practice that varies in expression across diverse cultures. To aid in an understanding of grief and its effects, the author looks at the models for grief proposed by Kubler-Ross, Bowlby, Parkes, Worden, and Wolfelt. Addressing patients' concerns requires physicians be empathic, attentive, and respective and have willingness to take time, be present, and listen.
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Pesar , Adaptação Psicológica , Cuidadores , Família , Humanos , Modelos Teóricos , Pacientes , MédicosRESUMO
Lujo virus is an emerging arenavirus circulating in Southern Africa. Although to date there has only been a single outbreak of the novel haemorrhagic disease resulting from human infection with this virus, the case-fatality rate of exposed individuals, including nosocomial transmission, was 80%. The ability to identify viral haemorrhagic fevers accurately, especially those capable of nosocomial transmission, is of critical importance. Timely identification of these diseases allow medical professionals to isolate patients and implement barrier nursing techniques in order to prevent onward transmission of the virus. While rapid diagnostic methods are published for most viral haemorrhagic fevers, at present there are no such virus specific protocols for Lujo haemorrhagic fever. This report details the first set of diagnostic molecular assays designed to identify Lujo viral RNA rapidly, and demonstrates the potential functionality of these assays for use in the clinical setting. Although these assays have been designed and validated against a solitary isolate of Lujo virus, this represents the entirety of strains detected to date, and offer quick, cheap and easy methods for use in diagnostic laboratories.
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Infecções por Arenaviridae/diagnóstico , Febres Hemorrágicas Virais/diagnóstico , Lujo virus/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , África Austral , Animais , Infecções por Arenaviridae/virologia , Custos e Análise de Custo , Febres Hemorrágicas Virais/virologia , Humanos , Lujo virus/genética , Técnicas de Diagnóstico Molecular/economia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Fatores de Tempo , Virologia/economiaRESUMO
Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 106 plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 102 pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 104 pfu gave an infection that was intermediate between those of the 106 pfu and 102 pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (106 pfu) and low (102 pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines.
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Antivirais/uso terapêutico , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/uso terapêutico , Eliminação de Partículas Virais , Animais , Modelos Animais de Doenças , Furões , Resultado do TratamentoRESUMO
Sequential sampling from animals challenged with highly pathogenic organisms, such as haemorrhagic fever viruses, is required for many pharmaceutical studies. Using the guinea pig model of Ebola virus infection, a catheterized system was used which had the benefits of allowing repeated sampling of the same cohort of animals, and also a reduction in the use of sharps at high biological containment. Levels of a PS-targeting antibody (Bavituximab) were measured in Ebola-infected animals and uninfected controls. Data showed that the pharmacokinetics were similar in both groups, therefore Ebola virus infection did not have an observable effect on the half-life of the antibody.