RESUMO
We adopted the reverse-transcriptase-loop-mediated isothermal amplification (RT-LAMP) to detect severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) in patient samples. Two primer sets for genes N and Orf1ab were designed to detect SARS-CoV-2, and one primer set was designed to detect the human gene Actin. We collected prospective 138 nasopharyngeal swabs, 70 oropharyngeal swabs, 69 salivae, and 68 mouth saline wash samples from patients suspected to have severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 to test the RT-LAMP in comparison with the gold standard technique reverse-transcription quantitative polymerase chain reaction (RT-qPCR). The accuracy of diagnosis using both primers, N5 and Orf9, was evaluated. Sensitivity and specificity for diagnosis were 96% (95% confidence interval [CI]: 87-99) and 85% (95% CI: 76-91) in 138 samples, respectively. Accurate diagnosis results were obtained only in nasopharyngeal swabs processed via extraction kit. Accurate and rapid diagnosis could aid coronavirus disease 2019 (COVID-19) pandemic management by identifying, isolating, and treating patients rapidly.
Assuntos
COVID-19 , SARS-CoV-2 , Brasil , COVID-19/diagnóstico , Teste para COVID-19 , Técnicas de Laboratório Clínico/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudos Prospectivos , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
Influenza A viruses (IAVs) cause more than 2 million annual episodes of seasonal acute respiratory infections (ARI) and approximately 500,000 deaths worldwide. Depending on virus strain and host immune status, acute infections by IAV may reach sites other than the respiratory tract. In the present study, IAV RNA and antigens were searched for in tissues of palatine tonsils and adenoids removed from patients without ARI symptoms. A real-time reverse transcriptase PCR (RT-PCR) screening revealed that 8 tissue samples from 7 patients out of 103 were positive for IAV. Positive samples were subjected to next-generation sequencing (NGS) and 3 of 8 tissues yielded complete IAV pH1N1 genomes, whereas in 5 samples, the PB1 gene was not fully assembled. Phylogenetic analysis placed tonsil-derived IAV in clusters clearly segregated from contemporaneous Brazilian viruses. Flow cytometry of dispersed tissue fragments and serial immunohistochemistry of paraffin-embedded sections of naturally infected biopsies indicated that CD20+ B lymphocytes, CD8+ T lymphocytes, and CD11c+ cells are susceptible to IAV infection. We sought to investigate whether these lymphoid tissues could be sites of viral replication and sources of viable virus particles. MDCK cells were inoculated with tissue lysates, enabling recovery of one IAV isolate confirmed by immunofluorescence, reverse transcriptase quantitative PCR (RT-qPCR), and NGS. The data indicate that lymphoid tissues not only harbor expression of IAV proteins but also contain infectious virus. Asymptomatic long-term infection raises the possibility of IAV shedding from tonsils, which may have an impact on host-to-host transmission.IMPORTANCE Influenza A virus (IAV) infections are important threats to human health worldwide. Although extensively studied, some aspects of virus pathogenesis and tissue tropism remain unclear. Here, by different strategies, we describe the asymptomatic infection of human lymphoid organs by IAV in children. Our results indicate that IAV was not only detected and isolated from human tonsils but displayed unique genetic features in comparison with those of contemporaneous IAVs circulating in Brazil and detected in swabs and nasal washes. Inside the tissue microenvironment, immune cells were shown to be carrying IAV antigens, especially B and T CD8+ lymphocytes. Taken together, these results suggest that human lymphoid tissues can be sites of silent IAV infections with possible impact on virus shedding to the population.
Assuntos
Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Tonsilite/virologia , Tonsila Faríngea/patologia , Adolescente , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Pré-Escolar , Estudos Transversais , Cães , Feminino , Humanos , Hipertrofia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Masculino , Tonsila Palatina/patologia , Filogenia , Estudos Prospectivos , Linfócitos T/patologia , Tonsilectomia/métodos , Tonsilite/complicações , Tonsilite/cirurgia , Replicação Viral , Eliminação de Partículas ViraisRESUMO
Cholesteatomas are frequent middle ear benign tumors of unknown etiology. Infectious agents have been considered as possible contributing factors in the pathogenesis of cholesteatomas. Aiming to investigate the presence of respiratory viruses in primary cholesteatoma tissues, 26 formalin-fixed paraffin-embedded primary cholesteatoma tissues obtained from patients seen at the of the Clinical Hospital of the University of São Paulo School of Medicine, in Ribeirão Preto, Brazil were tested by real-time polymerase chain reaction (PCR). Considering the PCR results, 35% of the tissues were positive for human rhinovirus (HRV), 15.3% for human enterovirus (EV), 3.8% for human metapneumovirus (HMPV), and 3.8% for human bocavirus (HBoV). Serial immunohistochemistry for virus antigens and cell surface markers evidenced that the viruses were associated with fibroblasts, dendritic cells, macrophages, B lymphocytes, CD4+ , and CD8+ T lymphocytes. These findings indicate for the first time the presence of active respiratory virus infection in primary cholesteatoma tissues, suggesting that persisting virus infection in the middle could play a role in the pathogenesis and evolution of cholesteatomas.
Assuntos
Colesteatoma/virologia , Enterovirus/isolamento & purificação , Bocavirus Humano/isolamento & purificação , Metapneumovirus/isolamento & purificação , Rhinovirus/isolamento & purificação , Adolescente , Adulto , Idoso , Brasil , Colesteatoma/patologia , Estudos Transversais , Enterovirus/genética , Feminino , Bocavirus Humano/genética , Humanos , Masculino , Metapneumovirus/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Rhinovirus/genética , Adulto JovemRESUMO
Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.
Assuntos
COVID-19 , Armadilhas Extracelulares , Humanos , Animais , Camundongos , COVID-19/genética , COVID-19/patologia , Armadilhas Extracelulares/metabolismo , Tratamento Farmacológico da COVID-19 , SARS-CoV-2/metabolismo , Pulmão/patologia , Complemento C5a/genética , Complemento C5a/metabolismoRESUMO
BACKGROUND: Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis. RESULTS: Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein. CONCLUSION: Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.
Assuntos
Vírus da Dengue/genética , Dengue/virologia , Polimorfismo Genético , Proteínas do Envelope Viral/genética , Substituição de Aminoácidos/genética , Brasil , Análise por Conglomerados , Vírus da Dengue/isolamento & purificação , Genótipo , Humanos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Monoamine oxidase is a flavoenzyme bound to the mitochondrial outer membranes of the cells, which is responsible for the oxidative deamination of neurotransmitter and dietary amines. It has two distinct isozymic forms, designated MAO-A and MAO-B, each displaying different substrate and inhibitor specificities. They are the well-known targets for antidepressant, Parkinson's disease, and neuroprotective drugs. Elucidation of the x-ray crystallographic structure of MAO-B has opened the way for the molecular modeling studies. In this work we have used molecular modeling, density functional theory with correlation, virtual screening, flexible docking, molecular dynamics, ADMET predictions, and molecular interaction field studies in order to design new molecules with potential higher selectivity and enzymatic inhibitory activity over MAO-B.
Assuntos
Simulação por Computador , Desenho de Fármacos , Modelos Moleculares , Inibidores da Monoaminoxidase/síntese química , Monoaminoxidase/química , Monoaminoxidase/metabolismo , Termodinâmica , Interações Medicamentosas , Humanos , Indanos/química , Indanos/farmacologia , Isoenzimas/química , Isoenzimas/metabolismo , Inibidores da Monoaminoxidase/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologiaRESUMO
The aim of this study was to investigate HIV-1 molecular diversity and the epidemiological profile of HIV-1-infected patients from Ribeirão Preto, Brazil. A nested PCR followed by sequencing of a 302-base pair fragment of the env gene (C2-V3 region) was performed in samples from HIV-1-positive patients. A total of 45 sequences were aligned with final manual adjustments. The phylogenetic analyses showed a higher prevalence of HIV-1 subtype B in the studied population (97.8%) with only one sample yielding an F1 subtype. The viral genotyping prediction showed that CCR5 tropism was the most prevalent in the studied cohort. Geno2pheno analysis showed that R5 and CXCR4 prediction were 69% and 31%, respectively. There was no statistical significance, either in viral load or in CD4(+) T cell count when R5 and X4 prediction groups were compared. Moreover, the GPGR tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (34.1%) followed by GWGR, identified in 18.1% of the samples. The high level of B subtype in this Brazilian population reinforces the nature of the HIV epidemic in Brazil, and corroborates previous data obtained in the Brazilian HIV-infected population.