RESUMO
A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.
Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-IdadeRESUMO
Cutaneous squamous cell carcinoma (cSCC) is a neoplasm type often diagnosed in dogs. However, studies focused on further investigating its molecular biology, mainly biomarkers to help implementing new therapies, remain scare in the literature. Thus, immunostaining and the gene expression of epidermal growth factor receptors (HER1 and HER2) in canine cSCC presenting different cell differentiation degrees were herein assessed. Thirty-two (32) canine cSCC were selected, classified based on to their cell differentiation degree and subjected to immunohistochemical study to assess HER1 and HER2 immunostaining intensity and distribution. In addition, HER1 and HER2 gene expression was investigated through real-time PCR. Membranous and cytoplasmic immunostaining were observed in both markers. HER2 prevailed in poorly differentiated cSCC; there was positive protein expression correlation between both markers. Mean HER1 gene expression was higher in moderately differentiated, whereas mean HER2 gene expression was higher in poorly differentiated cSCC. Moreover, there was gene expression correlation between markers, regardless of cell differentiation degree. Thus, HER2 protein immunostaining and gene expression were higher in poorly differentiated canine cSCC and it enabled understanding that increase observed in this epidermal growth factor receptor is proportional to this neoplasm's cell differentiation degree in canine species. Results in the current study helped better understanding canine cSCC's molecular biology; however, it is relevant studying other markers aiming to investigate signaling pathways.
Assuntos
Carcinoma de Células Escamosas , Doenças do Cão , Receptores ErbB , Imuno-Histoquímica , Receptor ErbB-2 , Neoplasias Cutâneas , Animais , Cães , Doenças do Cão/genética , Doenças do Cão/metabolismo , Carcinoma de Células Escamosas/veterinária , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias Cutâneas/veterinária , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Imuno-Histoquímica/veterinária , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Reação em Cadeia da Polimerase em Tempo Real/veterináriaRESUMO
Leishmaniases are a group of neglected diseases of significant public health concern, with Brazil being the primary focus of this disease in the Americas. The municipality of Sobral, in the state of Ceará, is a historical focus of visceral leishmaniasis in both humans and dogs, but data on Leishmania spp. infections in cats are limited. Between April 2021 and February 2022, 205 cats from a referral hospital population were sampled and tested for Leishmania spp. by real-time PCR. Eight cats (3.9%; 95% CI: 1.7-7.5%) tested positive. Among these, three (37.5%) displayed clinical signs compatible with feline leishmaniosis. Non-domiciled cats showed significantly higher positivity compared to domiciled ones (Fisher's exact test, P = 0.0124). Considering their potential role as reservoirs of L. infantum, it is crucial to conduct further studies to understand the Leishmania spp. circulating among cats in Sobral and to implement measures for reducing their exposure to phlebotomine sand fly vectors in this important focus of leishmaniases.
Assuntos
Doenças do Gato , Leishmaniose , Animais , Gatos , Brasil/epidemiologia , Doenças do Gato/epidemiologia , Doenças do Gato/parasitologia , Prevalência , Feminino , Masculino , Leishmaniose/veterinária , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Leishmania/isolamento & purificação , Leishmaniose Visceral/veterinária , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Hospitais Veterinários , Leishmania infantum/isolamento & purificaçãoRESUMO
Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.
Assuntos
Campylobacter coli , Campylobacter jejuni , Galinhas , Carne , Reação em Cadeia da Polimerase em Tempo Real , Animais , Galinhas/microbiologia , Ovinos/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Campylobacter coli/genética , Campylobacter coli/isolamento & purificação , Campylobacter coli/classificação , Campylobacter jejuni/genética , Campylobacter jejuni/isolamento & purificação , Campylobacter jejuni/classificação , Carne/microbiologia , Campylobacter fetus/genética , Campylobacter fetus/isolamento & purificação , Campylobacter fetus/classificação , Campylobacter/genética , Campylobacter/isolamento & purificação , Campylobacter/classificação , Microbiologia de Alimentos , DNA Bacteriano/genéticaRESUMO
Helicobacter pylori is a bacterium that is present in the stomach of about 50% of the global population and is associated with several gastric disorders, including cancer. Natural products with antimicrobial activity have been tested against H. pylori, among them Trichilia catigua (catuaba), which is widely distributed in Brazil. This study aimed to evaluate extracts of T. catigua bark against H. pylori via determination of the minimum inhibitory and bactericidal concentrations (MIC and MBC); evaluation of virulence factors by real-time PCR, synergism with standard antimicrobials and morphology by scanning electron microscopy and simulations of the mechanism of action by molecular docking. The ethyl acetate fraction provided the best results, with an MIC50 of 250 µg/mL and a 42.34% reduction in urease activity, along with reduced expression of the CagA and VacA genes, which encode for the main virulence factors. This fraction presented synergistic activity with clarithromycin, reducing the MIC of the drug by four-fold. Docking simulations suggested that the extracts inhibit fatty acid synthesis by the FAS-II system, causing damage to the cell membrane. Therefore, T. catigua extracts have potential as an adjuvant to treatment and are promising for the development of new anti-H. pylori drugs.
Assuntos
Antibacterianos , Proteínas de Bactérias , Helicobacter pylori , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Casca de Planta , Extratos Vegetais , Helicobacter pylori/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Casca de Planta/química , Brasil , Fatores de Virulência , Meliaceae/química , Claritromicina/farmacologia , Urease , Sinergismo Farmacológico , Antígenos de BactériasRESUMO
Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions.
Assuntos
Vírus da Raiva , Raiva , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Raiva/diagnóstico , Raiva/veterinária , Raiva/virologia , Brasil , Vírus da Raiva/genética , Vírus da Raiva/isolamento & purificação , Vírus da Raiva/classificação , Humanos , Animais , Reação em Cadeia da Polimerase em Tempo Real/métodos , Lyssavirus/genética , Lyssavirus/isolamento & purificação , Lyssavirus/classificação , RNA Viral/genética , Carga ViralRESUMO
The annual killifish Austrolebias charrua is an endangered species, endemic to the southern region of South America, which inhabits temporary ponds that emerges in the rainy season. The main anthropogenic threat driving the extinction of A. charrua stems from extensive agriculture, primarily due to the widrespread use of glyphosate-based herbicides near their habitats. Annual killifishes have been used as models for ecotoxicological studies but, up to now, there are no studies about reference genes in any Austrolebias species. This represents an obstacle to the use of qPCR-based technologies, the standard method for gene expression quantification. The present study aimed to select and validate potential reference genes for qPCR normalization in the annual killifish Austrolebias charrua considering different tissues, gender and environmental conditions. The candidate reference genes 18 s, actb, gapdh, ef1a, shox, eif3g, and the control gene atp1a1 were evaluated in male and female individuals in three different tissues (brain, liver, and gills) under two experimental conditions (control and acute exposition to Roundup Transorb®). The collected tissues were submitted to RNA extraction, followed by cDNA synthesis, cloning, sequencing, and qPCR. Overall, 18 s was the most stable reference gene, and 18 s and ef1a were the most stable combination. Otherwise, considering all variables, gapdh and shox were the least stable candidate genes. Foremost, suitable reference genes were validated in A. charrua, facilitating accurate mRNA quantification in this species, which might be useful for developing molecular tools of ecotoxicological assessment based on gene expression analysis for environmental monitoring of annual killifish.
Assuntos
Espécies em Perigo de Extinção , Reação em Cadeia da Polimerase em Tempo Real , Animais , Masculino , Feminino , Poluentes Químicos da Água/toxicidade , Fundulidae/genética , Monitoramento Ambiental/métodos , Glifosato , Fatores Sexuais , Herbicidas/toxicidade , Peixes ListradosRESUMO
The aim of the present study was to compare the performance of a nested polymerase chain reaction (nPCR) and a real-time PCR based on the amplification of the HlyA gene from Listeria monocytogenes using a plasmid DNA standard. Nested PCR was developed with an internal amplification control (IAC). Both techniques were validated in soft cheese samples by comparing their results with the results of the microbiological reference method ISO 11290-1:2017. Cheese samples artificially contaminated with 3.5 to 3,500 UFC/25 g were processed by ISO 11290-1:2017 and, at several times of culture, DNA samples were extracted. All cheeses contaminated with L. monocytogenes were positive for the microbiological method 96 h post contamination and for nPCR and real-time PCR 48 h post contamination. At this time, the HlyA gene was amplified in all contaminated samples. Both molecular techniques showed the same sensitivity, 30 copies/reaction or 3.5 UFC/25 g, when plasmid DNA standard or artificially contaminated cheese samples were used. Finally, eighty soft cheese samples obtained from local retail stores and tested by three methods were negative, indicating a 100% concordance in results. The development of an nPCR with IAC reinforces the reliability of the negative results without increasing the costs of the reaction. Besides, nPCR showed less sensitivity to the presence of inhibitory substances in the reaction. The use of one of these molecular techniques could be easily coupled to the microbiological method, serving as a screening method in the food industry for hygiene monitoring and early identification of contaminated foods.
Assuntos
Queijo , Microbiologia de Alimentos , Listeria monocytogenes , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Queijo/microbiologia , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase/métodos , Microbiologia de Alimentos/métodos , Proteínas Hemolisinas/genética , Toxinas Bacterianas/genética , DNA Bacteriano/genética , Proteínas de Choque TérmicoRESUMO
Coffea spp. is the source of one of the most widely consumed beverages in the world. However, the cultivation of this crop is threatened by Hemileia vastatrix Berk & Broome, a fungal disease, which reduces the productivity and can cause significant economic losses. In this protocol, coffee leaf segment derived from a chemical mutagenesis process are inoculated with uredospores of the pathogen. Subsequently, the gene expression changes are analyzed over the time (0, 5, 24, 48, and 120 h) using quantitative real-time polymerase chain reaction (RT-qPCR). The procedures and example data are presented for expression analysis in the CaWRKY1 gene. This procedure can be applied for quantitative analysis of other genes of interest to coffee breeders and scientists for elucidating the molecular mechanisms involved in the interaction between the plant and pathogen, potentially leading to the development of more efficient approaches for managing this disease.
Assuntos
Basidiomycota , Coffea , Regulação da Expressão Gênica de Plantas , Doenças das Plantas , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Coffea/microbiologia , Coffea/genética , Basidiomycota/genética , Basidiomycota/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Perfilação da Expressão Gênica/métodos , Mutação , Folhas de Planta/microbiologia , Folhas de Planta/genética , Interações Hospedeiro-Patógeno/genéticaRESUMO
OBJECTIVE: Sperm Associated Antigen 11A (SPAG11A) protein is a family of the epididymis-specific secretory proteins implicated in sperm maturation and function. Varicocele might cause pathophysiological difficulties in the testis and epididymis, with a harmful effect on the environment for spermatogenesis and sperm maturation. The aim of this study was to evaluate the expression level of the SPAG11A gene and sperm parameters in infertile men with grade 1 and 2 varicocele before and after treatment. METHODS: Semen specimens were collected from 20 infertile men with varicocele pre-and post-treatment and 10 healthy volunteers. Semen analysis was conducted according to world health organization guidelines. Real time PCR (qRT-PCR) reaction was applied for determination of SPAG11A mRNA expression. RESULTS: The results showed that there was a significant difference between the concentration and normal morphology between pre- and post-treatment groups and the controls. There were significant differences between pre-treatment and control groups in terms of progressive and non-progressive mobility. SPAG11A mRNA levels were significantly lower in the pre-treatment group than in healthy control subjects (p=0.007). There was no statistically significant difference in the expression of SPAG11A as well as semen parameters in the post-treatment group compared to the pre-treatment group. CONCLUSIONS: SPAG11A gene expression and semen parameters may be affected by varicocele. Whether varicocele treatment is an effective approach to reduce the adverse effect of this disease on SPAG11A expression and semen parameters needs further investigation.