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1.
Rev Bras Parasitol Vet ; 29(3): e012420, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32756775

RESUMO

Piroplasm species were analyzed by molecular tools in total 31 blood samples from positive dogs, previously checked by stained slides, stored until DNA extraction between 2016 to 2018 in the laboratory Clinical Analyzes in Niterói, Rio de Janeiro. The piroplasms were identified by PCR, targeting the 18S rRNA gene and sequencing. From the total number of samples only 24 (77.4%) were positive and show adequate nucleotide sequences for interpretation with identity between 93%-100% with Babesia vogeli in compared to the sequences isolated of infected dogs from other states in Brazil deposited on GenBank. Most of dogs infected with B. vogeli had anemia (62.5%) and thrombocytopenia (95.8%). The findings of this study are compatible with previous reports in the literature and highlight B. vogeli as the most incriminated species in canine piroplasmosis in Brazil, and thrombocytopenia the hematological alteration most frequently identified in this infection. It is important to note that this is the first study involving the molecular characterization of piroplasms in the metropolitan region of Rio de Janeiro, based on PCR followed by sequencing.


Assuntos
Babesia , Babesiose , Sangue , Doenças do Cão , Manejo de Espécimes , Animais , Babesia/genética , Babesiose/sangue , Sangue/parasitologia , Análise Química do Sangue , Brasil , Doenças do Cão/sangue , Doenças do Cão/parasitologia , Cães , RNA Ribossômico 18S/genética , Manejo de Espécimes/veterinária
2.
PLoS One ; 15(7): e0236046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678844

RESUMO

Defining genetic diversity of viral infections directly from patient specimens is the ultimate goal of surveillance. Simple tools that can provide full-length sequence information on blood borne viral hepatitis viruses: hepatitis C, hepatitis B and hepatitis D viruses (HCV, HBV and HDV) remain elusive. Here, an unbiased metagenomic next generation sequencing approach (mNGS) was used for molecular characterization of HCV infections (n = 99) from Israel which yielded full-length HCV sequences in 89% of samples, with 7 partial sequences sufficient for classification. HCV genotypes were primarily 1b (68%) and 1a (19%), with minor representation of genotypes 2c (1%) and 3a (8%). HBV/HDV coinfections were characterized by suppressed HBV viral loads, resulting in sparse mNGS coverage. A probe-based enrichment approach (xGen) aiming to increase HBV and HDV coverage was validated on a panel of diverse genotypes, geography and titers. The method extended HBV genome coverage a median 61% (range 8-84%) and provided orders of magnitude boosts in reads and sequence depth for both viruses. When HBV-xGen was applied to Israeli samples, coverage was improved by 28-73% in 4 samples and identified HBV genotype A1, A2, D1 specimens and a dual B/D infection. Abundant HDV reads in mNGS libraries yielded 18/26 (69%) full genomes and 8 partial sequences, with HDV-xGen only providing minimal extension (3-11%) of what were all genotype 1 genomes. Advanced molecular approaches coupled to virus-specific capture probes promise to enhance surveillance of viral infections and aid in monitoring the spread of local subtypes.


Assuntos
Sangue/virologia , Vírus de Hepatite/genética , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Estudos de Coortes , Genótipo , Vírus de Hepatite/isolamento & purificação , Humanos
3.
J Zoo Wildl Med ; 51(2): 391-397, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32549570

RESUMO

Systemic isosporosis (formerly atoxoplasmosis), is a protozoal infection that causes death in nestling and fledgling passerine birds impacting ex situ breeding and reintroduction programs. Because current antemortem diagnostic tests lack sensitivity, a qPCR was developed for detection of Isospora spp. using primers and a fluorescent-tagged MGB probe targeting the large subunit (28s) ribosomal RNA gene (assay efficiency = >100%; sensitivity = <1 dsDNA copy). The assay was used to screen postmortem frozen or formalin-fixed paraffin-embedded tissue samples from passerine birds (n = 24; 12 with confirmed systemic isosporosis), whole blood and feces (n = 38) from live passerines, and other tissues infected with phylogenetically similar protozoa. The qPCR identified Isospora sp. DNA in tissues from 21/24 birds including 12/12 birds with cytologically-histologically confirmed infection (100% sensitivity) and 9/12 birds lacking microscopic organisms. The assay also amplified Eimeria sp. DNA; however, sequence analysis ruled out infection in the passerine cases. Blood and/or feces were positive in 30/38 birds, and in only 7/38 birds, blood and feces both contained Isospora sp. DNA. Finally, the qPCR was utilized to screen 30 consecutive daily fecal samples from live passerines (n = 20) to determine optimal sampling protocols. One or more of the daily fecal samples were positive in all 20 birds. In individual birds, the interval between positive qPCR amplification results ranged from 0 to 23 days, with an average of 5.85 days. Simulated application of 13 potential sample collection schedules was used to identify the sensitivity of repeated testing for identification of infected birds. Increased sampling days resulted in higher sensitivity but increased both cost and animal handling requirements. Based on statistical analysis and clinical considerations, the testing recommendation for detection of fecal shedding was collection and assay of five consecutive daily fecal samples, which had an average diagnostic sensitivity of 0.86.


Assuntos
Doenças das Aves/diagnóstico , Isospora/isolamento & purificação , Isosporíase/veterinária , Técnicas de Diagnóstico Molecular/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Aves Canoras , Animais , Doenças das Aves/parasitologia , Sangue/parasitologia , Fezes/parasitologia , Isosporíase/diagnóstico , Isosporíase/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas
4.
Nat Commun ; 11(1): 3169, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576825

RESUMO

Understanding tumor metabolism holds the promise of new insights into cancer biology, diagnosis and treatment. To assess human cancer metabolism, here we report a method to collect intra-operative samples of blood from an artery directly upstream and a vein directly downstream of a brain tumor, as well as samples from dorsal pedal veins of the same patients. After performing targeted metabolomic analysis, we characterize the metabolites consumed and produced by gliomas in vivo by comparing the arterial supply and venous drainage. N-acetylornithine, D-glucose, putrescine, and L-acetylcarnitine are consumed in relatively large amounts by gliomas. Conversely, L-glutamine, agmatine, and uridine 5-monophosphate are produced in relatively large amounts by gliomas. Further we verify that D-2-hydroxyglutarate (D-2HG) is high in venous plasma from patients with isocitrate dehydrogenases1 (IDH1) mutations. Through these paired comparisons, we can exclude the interpatient variation that is present in plasma samples usually taken from the cubital vein.


Assuntos
Biomarcadores Tumorais/sangue , Vasos Sanguíneos/metabolismo , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/metabolismo , Glioma/sangue , Glioma/metabolismo , Metabolômica , Acetilcarnitina/sangue , Adulto , Idoso , Agmatina/sangue , Sangue , Análise Química do Sangue , Glicemia , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Feminino , Glioma/diagnóstico por imagem , Glioma/genética , Glucose , Glutamina/sangue , Glutaratos/sangue , Humanos , Isocitrato Desidrogenase/sangue , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Ornitina/análogos & derivados , Ornitina/sangue , Putrescina/sangue , Uridina Monofosfato/sangue , Adulto Jovem
6.
PLoS One ; 15(6): e0228234, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32589639

RESUMO

A major issue in the surveillance of antimicrobial resistance (AMR) is "de-duplication" or removal of repeated isolates, for which there exist multiple methods. The World Health Organization (WHO) Global Antimicrobial Resistance Surveillance System (GLASS) requires de-duplication by selecting only the first isolate of a given bacterial species per patient per surveillance period per specimen type per age group, gender, and infection origin stratification. However, no study on the comparative application of this method has been reported. The objective of this study was to evaluate differences in data tabulation between the WHO GLASS and the Japan Nosocomial Infections Surveillance (JANIS) system, which counts both patients and isolates after removing repeated isolates of the same bacterial species isolated from a patient within 30 days, regardless of specimen type, but distinguishing isolates with change of antimicrobial resistance phenotype. All bacterial data, consisting of approximately 8 million samples from 1795 Japanese hospitals in 2017 were exported from the JANIS database, and were tabulated using either the de-duplication algorithm of GLASS, or JANIS. We compared the tabulated results of the total number of patients whose blood and urine cultures were taken and of the percentage of resistant isolates of Escherichia coli for each priority antibiotic. The number of patients per specimen type tabulated by the JANIS method was always smaller than that of GLASS. There was a small (< 3%) difference in the percentage of resistance of E. coli for any antibiotic between the two methods in both out- and inpatient settings and blood and urine isolates. The two tabulation methods did not show considerable differences in terms of the tabulated percentages of resistance for E. coli. We further discuss how the use of GLASS tabulations to create a public software and website that could help to facilitate the understanding of and treatment against AMR.


Assuntos
Infecção Hospitalar/epidemiologia , Farmacorresistência Bacteriana , Organização Mundial da Saúde , Adolescente , Adulto , Sangue/microbiologia , Infecção Hospitalar/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Feminino , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Urina/microbiologia , Adulto Jovem
7.
PLoS One ; 15(6): e0231061, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525960

RESUMO

Monitoring the presence and spread of pathogens in the environment is of critical importance. Rapid detection of infectious disease outbreaks and prediction of their spread can facilitate early responses of health agencies and reduce the severity of outbreaks. Current sampling methods are sorely limited by available personnel and throughput. For instance, xenosurveillance utilizes captured arthropod vectors, such as mosquitoes, as sampling tools to access blood from a wide variety of vertebrate hosts. Next generation sequencing (NGS) of nucleic acid from individual blooded mosquitoes can be used to identify mosquito and host species, and microorganisms including pathogens circulating within either host. However, there are practical challenges to collecting and processing mosquitoes for xenosurveillance, such as the rapid metabolization or decay of microorganisms within the mosquito midgut. This particularly affects pathogens that do not replicate in mosquitoes, preventing their detection by NGS or other methods. Accordingly, we performed a series of experiments to establish the windows of detection for DNA or RNA from human blood and/or viruses present in mosquito blood meals. Our results will contribute to the development of xenosurveillance techniques with respect to optimal timing of sample collection and NGS processing and will also aid trap design by demonstrating the stabilizing effect of temperature control on viral genome recovery from blood-fed mosquitoes.


Assuntos
Sangue , Culicidae/virologia , DNA Viral/análise , RNA Viral/análise , Animais , DNA Viral/genética , Monitoramento Ambiental , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real
8.
Nat Commun ; 11(1): 2081, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350278

RESUMO

The blood-cerebrospinal fluid barrier (BCSFB) is a highly dynamic transport interface that serves brain homeostasis. To date, however, understanding of its role in brain development and pathology has been hindered by the absence of a non-invasive technique for functional assessment. Here we describe a method for non-invasive measurement of BSCFB function by using tracer-free MRI to quantify rates of water delivery from arterial blood to ventricular cerebrospinal fluid. Using this method, we record a 36% decrease in BCSFB function in aged mice, compared to a 13% decrease in parenchymal blood flow, itself a leading candidate biomarker of early neurodegenerative processes. We then apply the method to explore the relationship between BCSFB function and ventricular morphology. Finally, we provide proof of application to the human brain. Our findings position the BCSFB as a promising new diagnostic and therapeutic target, the function of which can now be safely quantified using non-invasive MRI.


Assuntos
Sangue/diagnóstico por imagem , Líquido Cefalorraquidiano/diagnóstico por imagem , Imagem por Ressonância Magnética , Adulto , Envelhecimento/fisiologia , Animais , Artérias/diagnóstico por imagem , Encéfalo/diagnóstico por imagem , Plexo Corióideo/fisiologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Reprodutibilidade dos Testes
9.
Nature ; 581(7809): 465-469, 2020 05.
Artigo em Inglês | MEDLINE | ID: covidwho-23868

RESUMO

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.


Assuntos
Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Hospitalização , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Soroconversão , Replicação Viral , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Sangue/virologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Fezes/química , Fezes/virologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina M/análise , Imunoglobulina M/imunologia , Pulmão/virologia , Pandemias , Faringe/virologia , Pneumonia Viral/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , RNA Viral/análise , Escarro/virologia , Urina/virologia , Proteínas do Envelope Viral/genética , Carga Viral/imunologia , Eliminação de Partículas Virais
10.
Lancet Infect Dis ; 20(6): 697-706, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-17918

RESUMO

BACKGROUND: On Dec 31, 2019, China reported a cluster of cases of pneumonia in people at Wuhan, Hubei Province. The responsible pathogen is a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the relevant features of the first cases in Europe of confirmed infection, named coronavirus disease 2019 (COVID-19), with the first patient diagnosed with the disease on Jan 24, 2020. METHODS: In this case series, we followed five patients admitted to Bichat-Claude Bernard University Hospital (Paris, France) and Pellegrin University Hospital (Bordeaux, France) and diagnosed with COVID-19 by semi-quantitative RT-PCR on nasopharyngeal swabs. We assessed patterns of clinical disease and viral load from different samples (nasopharyngeal and blood, urine, and stool samples), which were obtained once daily for 3 days from hospital admission, and once every 2 or 3 days until patient discharge. All samples were refrigerated and shipped to laboratories in the National Reference Center for Respiratory Viruses (The Institut Pasteur, Paris, and Hospices Civils de Lyon, Lyon, France), where RNA extraction, real-time RT-PCR, and virus isolation and titration procedures were done. FINDINGS: The patients were three men (aged 31 years, 48 years, and 80 years) and two women (aged 30 years and 46 years), all of Chinese origin, who had travelled to France from China around mid-January, 2020. Three different clinical evolutions are described: (1) two paucisymptomatic women diagnosed within a day of exhibiting symptoms, with high nasopharyngeal titres of SARS-CoV-2 within the first 24 h of the illness onset (5·2 and 7·4 log10 copies per 1000 cells, respectively) and viral RNA detection in stools; (2) a two-step disease progression in two young men, with a secondary worsening around 10 days after disease onset despite a decreasing viral load in nasopharyngeal samples; and (3) an 80-year-old man with a rapid evolution towards multiple organ failure and a persistent high viral load in lower and upper respiratory tract with systemic virus dissemination and virus detection in plasma. The 80-year-old patient died on day 14 of illness (Feb 14, 2020); all other patients had recovered and been discharged by Feb 19, 2020. INTERPRETATION: We illustrated three different clinical and biological types of evolution in five patients infected with SARS-CoV-2 with detailed and comprehensive viral sampling strategy. We believe that these findings will contribute to a better understanding of the natural history of the disease and will contribute to advances in the implementation of more efficient infection control strategies. FUNDING: REACTing (Research & Action Emerging Infectious Diseases).


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Adulto , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Sangue/virologia , China , Infecções por Coronavirus/virologia , Fezes/virologia , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , Viagem , Urina/virologia , Carga Viral
11.
PLoS Negl Trop Dis ; 14(3): e0008156, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226028

RESUMO

Surveillance of Usutu virus is crucial to prevent future outbreaks both in Europe and in other countries currently naïve to the infection, such as the Americas. This goal remains difficult to achieve, notably because of the lack of large-scale cohort studies and the absence of commercially available diagnostic reagents for USUV. This work started with the first identification of USUV in a blood donor in the Friuli Venezia Giulia (FVG) Region in Northern-Eastern Italy, which is endemic for West Nile virus. Considering that only one IgG ELISA is commercially available, but none for IgM, a novel NS1 antigen based IgG/M ELISA has been developed. This assay tested successfully for the detection of Usutu virus in blood donors with the identification of a second case of transmission and high levels of exposure. Furthermore, two pan-flavivirus antiviral drugs, that we previously characterized to be inhibitors of other flavivirus infectivity, were successfully tested for inhibition of Usutu virus with inhibitory concentrations in the low micromolar range. To conclude, this work identifies North-Eastern Italy as endemic for Usutu virus with implications for the screening of transfusion blood. A novel NS1-based ELISA test has been implemented for the detection of IgM/G that will be of importance as a tool for the diagnosis and surveillance of Usutu virus infection. Finally, Usutu virus is shown to be sensitive to a class of promising pan-flavivirus drugs.


Assuntos
Anticorpos Antivirais/sangue , Antivirais/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Infecções por Flavivirus/diagnóstico , Flavivirus/isolamento & purificação , Testes Sorológicos/métodos , Proteínas não Estruturais Virais/imunologia , Sangue/virologia , Doadores de Sangue , Feminino , Flavivirus/efeitos dos fármacos , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Itália , Testes de Sensibilidade Microbiana , Testes de Neutralização/métodos
12.
Chem Biol Interact ; 324: 109084, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32289290

RESUMO

INTRODUCTION: An imbalance between oxidants and antioxidants in favour of oxidants, potentially leading to damage, is termed oxidative stress. Antioxidants (AO), either enzymatic or non-enzymatic, are the ones that can reduce diverse effects of pro-oxidants such as DNA, proteins and lipids damage. Chalcones (1,3-diaryl-2-propen-1-ones) are open chain flavonoids that are widely biosynthesized in plants. Aim of this study was to test antioxidative potency of 15 chalcones (Chs) in in vitro model in serum (native conditions), so as with exogenously induced oxidative stress. MATERIAL AND METHODS: Oxidative stress was induced in serum samples of healthy individuals with 0.25 mmol/L terc-buthyl-hydroperoxide (TBH), and then we monitored the effects of various concentrations of chalcones on oxidative stress parameters: total antioxidative status (TAS), total oxidative status (TOS), total concentration of sulfhydryl group (SHG) and prooxidative-antioxidative balance (PAB), and calculated prooxidative score, antioxidative score, and oxy score (OS). Nonparametric repeated measures ANOVA (Friedman's test) was used for comparison of antioxidative potency of samples with 15 different chalcones, in a native state and upon TBH influence. Spearman's nonparametric correlation analysis was used for estimation of relation between different parameters. RESULTS: Negative Oxy Score (OS) values for Chs11-15 showed significantly stronger antioxidative potency compared to other investigated chalcones (p < 0.05). Ch11, Ch13 and Ch14 remained with negative OS even after TBH addition, whereas OS of Ch12 and Ch15 became positive, with small nominal values. Samples with Ch11 and Ch13 showed significant negative correlation between TAS and TOS (p < 0.05 for both), but in Ch14 sample the negative correlation existed between TAS and PAB (p < 0.05). CONCLUSION: Lower value of OS (i.e. better antioxidative potency) was noticed in samples with Ch11-Ch15. Electron-donor effects of substituent groups as a structural part of these chalcones could explain its antioxidative capability. Phenolic and methyl groups are responsible for antioxidative ability enhancement of five chalcones with the best activity.


Assuntos
Antioxidantes/farmacologia , Sangue/metabolismo , Chalconas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/síntese química , Antioxidantes/química , Sangue/efeitos dos fármacos , Chalconas/síntese química , Chalconas/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , terc-Butil Hidroperóxido/farmacologia
13.
Nature ; 581(7809): 465-469, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32235945

RESUMO

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.


Assuntos
Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Hospitalização , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Soroconversão , Replicação Viral , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Sangue/virologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Fezes/química , Fezes/virologia , Humanos , Imunoglobulina G/análise , Imunoglobulina G/imunologia , Imunoglobulina M/análise , Imunoglobulina M/imunologia , Pulmão/virologia , Pandemias , Faringe/virologia , Pneumonia Viral/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , RNA Viral/análise , Escarro/virologia , Urina/virologia , Proteínas do Envelope Viral/genética , Carga Viral/imunologia , Eliminação de Partículas Virais
14.
Avian Dis ; 64(1): 69-79, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32267127

RESUMO

The development of immunocompetence in chicks after hatching is not fully understood. However, detailed knowledge of immunocompetence and maturation processes in day-old chicks (DOCs) and juvenile chickens (Gallus gallus domesticus) is necessary to implement enhanced immunization strategies. For viral diseases, this especially includes the development of cellular immunity focusing on T-cell-dependent responses. In the current study, we investigated T-cell subsets in blood and lymphoid tissues of 1-to-21-day-old chickens concerning their cellular composition and localization. We detected an increase of T-cell frequencies in blood and spleen and a shift of the CD8α dimer expression toward a CD8αß expression on the surface of T cells with increasing age. A relocalization of lymphocytes into antigen presentation structures within the spleen was affirmed. In addition, changes in basal messenger RNA (mRNA) level, with increasing IL2 and IFNγ mRNA levels at different ages were measured. These detected changes suggest an improved T-cell-dependent antiviral response with increasing age in chickens. To confirm this finding on a functional level, we conducted a transfer experiment: adult and, as a negative control, neonatal naïve lymphocytes were transferred into DOCs. Afterward, the protection induced by these transferred cells was verified by a sublethal infection by using a highly pathogenic avian influenza virus with neuraminidase deletion, H5Ndel. Previous experiments have shown that adult animals survive infection with this virus strain, while naïve DOCs show severe symptoms or even die. As a result, the transfer of adult, but not neonatal lymphocytes, confers protection to DOCs against the infection, demonstrating functional differences in lymphocytes from chicks of different ages. Collectively, these data reveal the inability of chicks to mount an effective, cellular antiviral response in the first 3 wk of life. Therefore, we propose that the observed maturation of both the innate and the adaptive arms of the immune system early in development is mandatory for controlling influenza infection in chickens, as well as for an effective vaccination with replication-competent viral vaccine strains.


Assuntos
Sangue/imunologia , Galinhas/imunologia , Imunidade Celular , Imunocompetência , Virus da Influenza A Subtipo H5N1/imunologia , Tecido Linfoide/imunologia , Linfócitos T/fisiologia , Fatores Etários , Animais , Feminino , Masculino
15.
Lancet Infect Dis ; 20(6): 697-706, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32224310

RESUMO

BACKGROUND: On Dec 31, 2019, China reported a cluster of cases of pneumonia in people at Wuhan, Hubei Province. The responsible pathogen is a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the relevant features of the first cases in Europe of confirmed infection, named coronavirus disease 2019 (COVID-19), with the first patient diagnosed with the disease on Jan 24, 2020. METHODS: In this case series, we followed five patients admitted to Bichat-Claude Bernard University Hospital (Paris, France) and Pellegrin University Hospital (Bordeaux, France) and diagnosed with COVID-19 by semi-quantitative RT-PCR on nasopharyngeal swabs. We assessed patterns of clinical disease and viral load from different samples (nasopharyngeal and blood, urine, and stool samples), which were obtained once daily for 3 days from hospital admission, and once every 2 or 3 days until patient discharge. All samples were refrigerated and shipped to laboratories in the National Reference Center for Respiratory Viruses (The Institut Pasteur, Paris, and Hospices Civils de Lyon, Lyon, France), where RNA extraction, real-time RT-PCR, and virus isolation and titration procedures were done. FINDINGS: The patients were three men (aged 31 years, 48 years, and 80 years) and two women (aged 30 years and 46 years), all of Chinese origin, who had travelled to France from China around mid-January, 2020. Three different clinical evolutions are described: (1) two paucisymptomatic women diagnosed within a day of exhibiting symptoms, with high nasopharyngeal titres of SARS-CoV-2 within the first 24 h of the illness onset (5·2 and 7·4 log10 copies per 1000 cells, respectively) and viral RNA detection in stools; (2) a two-step disease progression in two young men, with a secondary worsening around 10 days after disease onset despite a decreasing viral load in nasopharyngeal samples; and (3) an 80-year-old man with a rapid evolution towards multiple organ failure and a persistent high viral load in lower and upper respiratory tract with systemic virus dissemination and virus detection in plasma. The 80-year-old patient died on day 14 of illness (Feb 14, 2020); all other patients had recovered and been discharged by Feb 19, 2020. INTERPRETATION: We illustrated three different clinical and biological types of evolution in five patients infected with SARS-CoV-2 with detailed and comprehensive viral sampling strategy. We believe that these findings will contribute to a better understanding of the natural history of the disease and will contribute to advances in the implementation of more efficient infection control strategies. FUNDING: REACTing (Research & Action Emerging Infectious Diseases).


Assuntos
Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Adulto , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Sangue/virologia , China , Infecções por Coronavirus/virologia , Fezes/virologia , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , RNA Viral/isolamento & purificação , Viagem , Urina/virologia , Carga Viral
16.
PLoS Negl Trop Dis ; 14(4): e0007737, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32255793

RESUMO

BACKGROUND: Large-scale control of sleeping sickness has led to a decline in the number of cases of Gambian human African trypanosomiasis (g-HAT) to <2000/year. However, achieving complete and lasting interruption of transmission may be difficult because animals may act as reservoir hosts for T. b. gambiense. Our study aims to update our understanding of T. b. gambiense in local vectors and domestic animals of N.W. Uganda. METHODS: We collected blood from 2896 cattle and 400 pigs and In addition, 6664 tsetse underwent microscopical examination for the presence of trypanosomes. Trypanosoma species were identified in tsetse from a subsample of 2184 using PCR. Primers specific for T. brucei s.l. and for T. brucei sub-species were used to screen cattle, pig and tsetse samples. RESULTS: In total, 39/2,088 (1.9%; 95% CI = 1.9-2.5) cattle, 25/400 (6.3%; 95% CI = 4.1-9.1) pigs and 40/2,184 (1.8%; 95% CI = 1.3-2.5) tsetse, were positive for T. brucei s.l.. Of these samples 24 cattle (61.5%), 15 pig (60%) and 25 tsetse (62.5%) samples had sufficient DNA to be screened using the T. brucei sub-species PCR. Further analysis found no cattle or pigs positive for T. b. gambiense, however, 17/40 of the tsetse samples produced a band suggestive of T. b. gambiense. When three of these 17 PCR products were sequenced the sequences were markedly different to T. b. gambiense, indicating that these flies were not infected with T. b. gambiense. CONCLUSION: The lack of T. b. gambiense positives in cattle, pigs and tsetse accords with the low prevalence of g-HAT in the human population. We found no evidence that livestock are acting as reservoir hosts. However, this study highlights the limitations of current methods of detecting and identifying T. b. gambiense which relies on a single copy-gene to discriminate between the different sub-species of T. brucei s.l.


Assuntos
Animais Domésticos/parasitologia , Reservatórios de Doenças/parasitologia , Topografia Médica , Trypanosoma brucei gambiense/isolamento & purificação , Tripanossomíase Africana/epidemiologia , Tripanossomíase Africana/veterinária , Moscas Tsé-Tsé/parasitologia , Animais , Sangue/parasitologia , Bovinos , Humanos , Microscopia , Reação em Cadeia da Polimerase , Prevalência , Suínos , Trypanosoma brucei gambiense/genética , Uganda/epidemiologia
17.
Int J Syst Evol Microbiol ; 70(5): 3167-3178, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32302276

RESUMO

The Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella (HACEK) group genus Eikenella contained a single species, Eikenella corrodens, for many years. In November 2019, Eikenella exigua was described after recovery from a brain abscess and blood culture in Norway. Coincidentally, characterization of 22 Gram-negative bacteria resembling Eikenella from 17 Canadian patients had been underway. Seven isolates from five patients were conclusively identifiable as E. corrodens. One (NML 120819) was deemed to represent a species of the genus Eikenella most closely related to E. corrodens. Fourteen isolates had 97.6 to 98.8% similarities to E. corrodens by 16S rRNA gene sequencing, forming three distinct groups by genome analyses. The largest contained ten anaerobic isolates from eight patients recovered from blood, brain, bone and other abscesses; upon re-evaluation, this group was found to be most consistent with E. exigua. A second facultatively anaerobic clade consisted of two ocular isolates from one patient and a sinus isolate from a second patient. The third taxon consisted of a single strictly anaerobic blood culture isolate. The novel taxa, like E. corrodens, were poorly reactive biochemically and difficult to discern from each other phenotypically and chemotaxonomically, including by cellular fatty acids. MALDI-TOF (Bruker) and whole-genome sequencing were used to further characterize isolates. Draft genomes for the strains had similar DNA G+C contents (55.38-58.53 mol%) while sizes varied from 1.82 Mb to 2.54 Mb. We propose here emendations of the genus Eikenella and the species Eikenella exigua, as well as describing Eikenella halliae sp. nov. NML 130454T (=LMG 30894T=NCTC 14180T) and Eikenella longinqua sp. nov. NML 02-A-017T (=LMG 30896T=NCTC 14179T), on the basis of these findings.


Assuntos
Sangue/microbiologia , Eikenella/classificação , Filogenia , Bactérias Anaeróbias/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , Hemocultura , Canadá , DNA Bacteriano/genética , Eikenella/isolamento & purificação , Ácidos Graxos/química , Humanos , Noruega , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
18.
Parasit Vectors ; 13(1): 188, 2020 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-32276649

RESUMO

BACKGROUND: Vector-borne diseases are a major public health concern and cause significant morbidity and mortality. Zika virus (ZIKV) is the etiologic agent of a massive outbreak in the Americas that originated in Brazil in 2015 and shows a strong association with congenital ZIKV syndrome in newborns. Cache Valley virus (CVV) is a bunyavirus that causes mild to severe illness in humans and ruminants. In this study, we investigated the vector competence of Virginia mosquitoes for ZIKV and CVV to explore their abilities to contribute to potential outbreaks. METHODS: To determine vector competence, mosquitoes were fed a blood meal comprised of defibrinated sheep blood and virus. The presence of midgut or salivary gland barriers to ZIKV infection were determined by intrathoracic inoculation vs oral infection. After 14-days post-exposure, individual mosquitoes were separated into bodies, legs and wings, and saliva expectorant. Virus presence was detected by plaque assay to determine midgut infection, dissemination, and transmission rates. RESULTS: Transmission rates for Ae. albopictus orally infected (24%) and intrathoracically inoculated (63%) with ZIKV was similar to Ae. aegypti (48% and 71%, respectively). Transmission rates of ZIKV in Ae. japonicus were low, and showed evidence of a midgut infection barrier demonstrated by low midgut infection and dissemination rates from oral infection (3%), but increased transmission rates after intrathoracic inoculation (19%). Aedes triseriatus was unable to transmit ZIKV following oral infection or intrathoracic inoculation. CVV transmission was dose-dependent where mosquitoes fed high titer (ht) virus blood meals developed higher rates of midgut infection, dissemination, and transmission compared to low titer (lt) virus blood meals. CVV was detected in the saliva of Ae. albopictus (ht: 68%, lt: 24%), Ae. triseriatus (ht: 52%, lt: 7%), Ae. japonicus (ht: 22%, lt: 0%) and Ae. aegypti (ht: 10%; lt: 7%). Culex pipiens and Cx. restuans were not competent for ZIKV or CVV. CONCLUSIONS: This laboratory transmission study provided further understanding of potential ZIKV and CVV transmission cycles with Aedes mosquitoes from Virginia. The ability for these mosquitoes to transmit ZIKV and CVV make them a public health concern and suggest targeted control programs by mosquito and vector abatement districts.


Assuntos
Vírus Bunyamwera/isolamento & purificação , Mosquitos Vetores/virologia , Zika virus/isolamento & purificação , Aedes/virologia , Animais , Bioensaio , Sangue/virologia , Infecções por Bunyaviridae/transmissão , Culex/virologia , Vetores de Doenças , Humanos , Intestinos/virologia , Saliva/virologia , Estados Unidos , Carga Viral , Virginia , Infecção por Zika virus/transmissão
19.
PLoS Genet ; 16(4): e1008555, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271760

RESUMO

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Homeostase , Animais , Sangue , Reagentes para Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endonucleases/deficiência , Endonucleases/genética , Feminino , Formaldeído/farmacologia , Humanos , Rim/enzimologia , Fígado/enzimologia , Masculino , Camundongos
20.
Sex Transm Infect ; 96(5): 337-341, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32245779

RESUMO

OBJECTIVE: To provide insight on viral kinetics and genetic diversity of HIV in seminal plasma at baseline and 1 month after initiating antiretroviral therapy (ART). PATIENTS AND METHODS: Blood and seminal samples from patients with newly diagnosed HIV were obtained before ART initiation (T0) and 1 month after ART initiation (T1). HIV env genetic diversity was studied using deep sequencing Nextera and V3 chemistry in a MiSeq Illumina platform. The number of viral quasispecies (5% cut-off) and Shannon Index were used to analyse diversity. RESULTS: Forty-seven ART-naive patients were recruited between September 2016 and November 2018. At enrolment, the number of quasispecies in blood (median 4 (IQR 2-5)) was lower than in the seminal compartment (median 6, (IQR 4-8)) (p<0.01); the Shannon Index was also higher (p<0.001) in the seminal compartment than in blood (1.77 vs 0.64). At T1, for the 13 patients with detectable HIV in both blood/seminal plasma, viral diversity remained higher (p=0.139) in seminal plasma (median 2 (IQR 1-4.5)) than in blood (median 1 (IQR 1-1.5)) Integrase inhibitors (INI)-based regimens achieved higher levels of undetectability and led more frequently to lower variability (p<0.001) than protease inhibitors (PI) or non-nucleoside reverse transcriptase inhibitors (NNRTI). CONCLUSION: We provide here further evidence of a larger genetic diversity in seminal plasma, both at diagnosis and short term after ART initiation. Our results strengthen previous findings on HIV diversity in seminal plasma. In addition, INIs decrease variability more rapidly than PI and NNRTI in both blood and seminal plasma.


Assuntos
Antirretrovirais/uso terapêutico , Sangue/virologia , Variação Genética , Infecções por HIV/tratamento farmacológico , HIV/genética , Sêmen/virologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Adulto , Infecções por HIV/sangue , Infecções por HIV/metabolismo , Inibidores de Integrase de HIV/uso terapêutico , Inibidores da Protease de HIV/uso terapêutico , Humanos , Masculino , Inibidores da Transcriptase Reversa/uso terapêutico
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