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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.
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Humanos , Masculino , Femenino , Adulto , Persona de Mediana EdadRESUMEN
Introduction: Photobiomodulation with low-level laser treatment can enhance bone formation by stimulating the cell division of osteoblasts and increasing the amount of protein deposition, thus encouraging the formation of new bone. The aim of this study was to evaluate the effects of photobiomodulation with a low-level laser on proliferation and gene expression related to calcium signaling in human osteoblasts. Methods: Osteoblastic cell lines of the hFOB1.19 lineage, human osteoblasts, were grown and assigned into two groups, control (C; n=78 cultured wells) and photobiomodulation (L; n=78 cultured wells) with n=6 per day of the experimental period. Cells were cultured (immature at 34 ºC), and after maturation at 37 ºC, group L cells were exposed to laser irradiation with a low-level laser device (gallium and aluminum arsenide), at a wavelength of 808 nm, a power output of 200 mW, and a power density of 200 mW/cm2. The energy delivered to the cells was 37 J/cm2, with a beam area of 0.02 mm2 and an exposure time of 5 seconds. This treatment was applied daily for a period of 13 days. Following this, the number of cells was counted, and RNA was isolated, measured, and then converted into cDNA for further quantification using a comparative Ct method with real-time polymerase chain reaction. The results were then subjected to statistical analysis through a Mann-Whitney test, with a significance level of P<0.05. Results: The cell count in the L group (37.25x10±4±22.02) was statistically higher compared to the control group (22.75x10±4±7.660) with a P value of 0.0259. The values of 2-ΔΔCt for S100A6, plasma membrane calcium ATPase (PMCA), and calmodulin genes indicated hyper-expression on the thirteenth day, while the osteocalcin gene showed hypo-expression. Conclusion: The study suggests that the photobiomodulation mechanism with a low-level laser may regulate gene expression in human osteoblasts in a dose-dependent and cumulative manner.
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This study aimed to investigate the presence of murine astrovirus (MuAstV) in Brazil. Fecal samples from mice belonging to four Brazilian animal facilities were collected and tested for MuAstV using real-time polymerase chain reaction. Of the 162 samples tested, 38 (23.5%) were positive for MuAstV, 33 (91.7%) of which came from specific-pathogen free colonies. Although most of the samples were obtained from asymptomatic animals, three mice presented diarrheal symptoms, and MuAstV was the only agent detected by molecular assay. Phylogenetic analysis revealed similarities between the MuAstV strains from this study and prototypes from the USA. MuAstV's high prevalence, environmental stability, genetic diversity and potential for persistent infections must be considered when evaluating health monitoring programs for laboratory rodents.
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Advanced diagnostic technologies have made accurate and precise diagnosis of pathogens easy. Herein, we present a new diagnostic method, droplet digital PCR (ddPCR), to detect and quantify Acinetobacter baumannii in mini bronchoalveolar lavage (mini-BAL) samples. A. baumannii causes ventilator-associated pneumonia (VAP), a severe healthcare infection affecting patients' lungs. VAP carries a high risk of morbidity and mortality, making its timely diagnosis crucial for prompt and effective management. Methodology. The assay performance was evaluated by comparing colonization data, quantitative culture results, and different generations of PCR (traditional PCR and Real-Time PCR-qPCR Taqman® and SYBR® Green). The ddPCR and qPCR Taqman® prove to be more sensitive than other molecular techniques. Reasonable analytical specificity was obtained with ddPCR, qPCR TaqMan®, and conventional PCR. However, qPCR SYBR® Green technology presented a low specificity, making the results questionable in clinical samples. DdPCR detected/quantified A. baumanni in more clinical samples than other methods (38.64% of the total samples). This emerging ddPCR technology offers promising advantages such as detection by more patients and direct quantification of pathogens without calibration curves.
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The collective involvement of virulence markers of Escherichia coli as an emerging pathogen associated with periodontitis remains unexplained. This study aimed to implement an in vitro model of infection using a human epithelial cell line to determine the virulome expression related to the antibiotic and disinfectant resistance genotype and pulse field gel electrophoresis (PFGE) type in E. coli strains isolated from patients with periodontal diseases. We studied 100 strains of E. coli isolated from patients with gingivitis (n = 12), moderate periodontitis (n = 59), and chronic periodontitis (n = 29). The identification of E. coli and antibiotic and disinfectant resistance genes was performed through PCR. To promote the expression of virulence genes in the strains, an in vitro infection model was used in the human epithelial cell line A549. RNA was extracted using the QIAcube robotic equipment and reverse transcription to cDNA was performed using the QuantiTect reverse transcription kit (Qiagen). The determination of virulence gene expression was performed through real-time PCR. Overall, the most frequently expressed adhesion genes among the isolated strains of gingivitis, moderate periodontitis, and chronic periodontitis were fimH (48%), iha (37%), and papA (18%); those for toxins were usp (33%); those for iron acquisition were feoB (84%), fyuA (62%), irp-2 (61%), and iroN (35%); those for protectins were traT (50%), KpsMT (35%), and ompT (28%); and those for pathogenicity islands were malX (45%). The most common antibiotic and disinfectant resistance genes among gingivitis, moderate periodontitis, and chronic periodontitis strains were sul-2 (43%), blaSHV (47%), blaTEM (45%), tet(A) (41%), dfrA1 (32%), marR-marO (57%), and qacEA1 (79%). The findings revealed the existence of a wide distribution of virulome expression profiles related to the antibiotic and disinfectant resistance genotype and PFGE type in periodontal strains of E. coli. These findings may contribute toward improving the prevention and treatment measures for periodontal diseases associated with E. coli.
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Antibacterianos , Desinfectantes , Farmacorresistencia Bacteriana , Infecciones por Escherichia coli , Escherichia coli , Factores de Virulencia , Humanos , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/patogenicidad , Factores de Virulencia/genética , Antibacterianos/farmacología , Infecciones por Escherichia coli/microbiología , Farmacorresistencia Bacteriana/genética , Desinfectantes/farmacología , Periodontitis/microbiología , Virulencia/genética , Células A549 , Células Epiteliales/microbiología , Genotipo , Adulto , Femenino , Masculino , Persona de Mediana Edad , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Electroforesis en Gel de Campo PulsadoRESUMEN
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.
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Campylobacter coli , Campylobacter jejuni , Pollos , Carne , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Pollos/microbiología , Ovinos/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Campylobacter coli/genética , Campylobacter coli/aislamiento & purificación , Campylobacter coli/clasificación , Campylobacter jejuni/genética , Campylobacter jejuni/aislamiento & purificación , Campylobacter jejuni/clasificación , Carne/microbiología , Campylobacter fetus/genética , Campylobacter fetus/aislamiento & purificación , Campylobacter fetus/clasificación , Campylobacter/genética , Campylobacter/aislamiento & purificación , Campylobacter/clasificación , Microbiología de Alimentos , ADN Bacteriano/genéticaRESUMEN
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.
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Enfermedades de los Gatos , Leishmaniasis , Animales , Gatos , Brasil/epidemiología , Enfermedades de los Gatos/epidemiología , Enfermedades de los Gatos/parasitología , Prevalencia , Femenino , Masculino , Leishmaniasis/veterinaria , Leishmaniasis/epidemiología , Leishmaniasis/parasitología , Leishmania/aislamiento & purificación , Leishmaniasis Visceral/veterinaria , Leishmaniasis Visceral/epidemiología , Leishmaniasis Visceral/parasitología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Hospitales Veterinarios , Leishmania infantum/aislamiento & purificaciónRESUMEN
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.
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Carcinoma de Células Escamosas , Enfermedades de los Perros , Receptores ErbB , Inmunohistoquímica , Receptor ErbB-2 , Neoplasias Cutáneas , Animales , Perros , Enfermedades de los Perros/genética , Enfermedades de los Perros/metabolismo , Carcinoma de Células Escamosas/veterinaria , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Neoplasias Cutáneas/veterinaria , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inmunohistoquímica/veterinaria , Femenino , Regulación Neoplásica de la Expresión Génica , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinariaRESUMEN
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.
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Antibacterianos , Proteínas Bacterianas , Helicobacter pylori , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Corteza de la Planta , Extractos Vegetales , Helicobacter pylori/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Extractos Vegetales/farmacología , Extractos Vegetales/química , Corteza de la Planta/química , Brasil , Factores de Virulencia , Meliaceae/química , Claritromicina/farmacología , Ureasa , Sinergismo Farmacológico , Antígenos BacterianosRESUMEN
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.
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Virus de la Rabia , Rabia , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Rabia/diagnóstico , Rabia/veterinaria , Rabia/virología , Brasil , Virus de la Rabia/genética , Virus de la Rabia/aislamiento & purificación , Virus de la Rabia/clasificación , Humanos , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Lyssavirus/genética , Lyssavirus/aislamiento & purificación , Lyssavirus/clasificación , ARN Viral/genética , Carga ViralRESUMEN
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.
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Basidiomycota , Coffea , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Coffea/microbiología , Coffea/genética , Basidiomycota/genética , Basidiomycota/patogenicidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Perfilación de la Expresión Génica/métodos , Mutación , Hojas de la Planta/microbiología , Hojas de la Planta/genética , Interacciones Huésped-Patógeno/genéticaRESUMEN
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.
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Queso , Microbiología de Alimentos , Listeria monocytogenes , Reacción en Cadena de la Polimerasa , Reacción en Cadena en Tiempo Real de la Polimerasa , Queso/microbiología , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/métodos , Microbiología de Alimentos/métodos , Proteínas Hemolisinas/genética , Toxinas Bacterianas/genética , ADN Bacteriano/genética , Proteínas de Choque TérmicoRESUMEN
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.
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Especies en Peligro de Extinción , Reacción en Cadena en Tiempo Real de la Polimerasa , Animales , Masculino , Femenino , Contaminantes Químicos del Agua/toxicidad , Fundulidae/genética , Monitoreo del Ambiente/métodos , Glifosato , Factores Sexuales , Herbicidas/toxicidad , Peces KilliRESUMEN
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.
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Antígenos de Superficie , Infertilidad Masculina , Varicocele , Adulto , Humanos , Masculino , Expresión Génica , Infertilidad Masculina/genética , Infertilidad Masculina/etiología , Análisis de Semen , Varicocele/genética , Varicocele/complicaciones , Varicocele/metabolismo , Antígenos de Superficie/genéticaRESUMEN
Bacterial meningitis is still a significant public health concern, with high morbidity and mortality rates. Despite this, it is still a rare event that requires the bacterial invasion of the meninges. However, some predisposing factors can trigger recurrent episodes of meningitis. This study is aimed at determining the clinical characteristics and the molecular epidemiology of episodes of recurrent community-acquired meningitis with and without predisposing factors. For this purpose, we performed a retrospective study of our laboratory database during the period of 2010 to 2020. Additionally, using molecular tools developed in our previous works, the epidemiology of the pathogens causing these episodes was analyzed using cerebrospinal fluid samples, especially in the absence of isolated strains. We observed a total of 1,779 meningitis cases and 230 were caused by Streptococcus pneumoniae. Of those, 16 were recurrent meningitis episodes (16/1,779; 0.9%) from seven patients. Pneumococcus was the main agent responsible in these recurrent episodes and only two episodes were caused by Haemophilus influenzae. The mean age of these patients was 20 years old and three had predisposing factors which could have led to contracting meningitis. The samples presented different pneumococcal serotypes. Most of them were non-vaccine-covered serotypes and antibiotic susceptible strains. Therefore, it was demonstrated how the practical employment of molecular tools, developed for research, when applied in the routine of diagnosis, can provide important information for epidemiological surveillance. Furthermore, it was shown how pneumococcus was the leading cause of recurrent community-acquired meningitis without predisposing factors, suggesting that pneumococcal vaccination may be necessary, even in those groups of individuals considered to be less susceptible.
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Infecciones Comunitarias Adquiridas , Meningitis Neumocócica , Recurrencia , Streptococcus pneumoniae , Humanos , Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/epidemiología , Meningitis Neumocócica/epidemiología , Meningitis Neumocócica/microbiología , Adulto , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pneumoniae/clasificación , Estudios Retrospectivos , Femenino , Masculino , Adulto Joven , Persona de Mediana Edad , Adolescente , Factores de Riesgo , Serogrupo , Antibacterianos , Haemophilus influenzae/genética , Haemophilus influenzae/aislamiento & purificación , Haemophilus influenzae/clasificaciónRESUMEN
The objective of this study was to compare, by qPCR, the circulating blood parasite load of Trypanosoma cruzi in the buffy coat, and in whole blood mixed with boiled and unboiled guanidine hydrochloride-EDTA buffer, of individuals with chronic ChD. The concentration and purity of DNA were evaluated in a Nanodrop Denovix DS-11FX Series Spectrophotometer (DeNovix Inc., Wilmington, NC, USA). The parasite load was determined with the Taqman® qPCR system using a Stratagene Mx3000P thermocycler (Agilent Technologies, Santa Clara, CA, USA) with Cruzi 1 and Cruzi 2 satellite primers. Student's t-test with Bonferroni correction, Chi-squared (χ2) tests and Spearman's correlation coefficient were applied. The concentration and purity of DNA were higher in the buffy coat. Parasite DNA was detected and quantifiable in the three types of samples in seven patients, without statistically significant differences in the parasite load obtained. Higher correlations were found between the total DNA concentrations and the parasite loads obtained in the samples of the buffy coat.
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Since human papillomavirus (HPV) is recognized as the causative agent of cervical cancer and associated with anogenital non-cervical and oropharyngeal cancers, the characterization of the HPV types circulating in different geographic regions is an important tool in screening and prevention. In this context, this study compared four methodologies for HPV detection and genotyping: real-time PCR (Cobas® HPV test), nested PCR followed by conventional Sanger sequencing, reverse hybridization (High + Low PapillomaStrip® kit) and next-generation sequencing (NGS) at an Illumina HiSeq2500 platform. Cervical samples from patients followed at the Family Health Strategy from Juiz de Fora, Minas Gerais, Brazil, were collected and subjected to the real-time PCR. Of those, 114 were included in this study according to the results obtained with the real-time PCR, considered herein as the gold standard method. For the 110 samples tested by at least one methodology in addition to real-time PCR, NGS showed the lowest concordance rates of HPV and high-risk HPV identification compared to the other three methods (67-75 %). Real-time PCR and Sanger sequencing showed the highest rates of concordance (97-100 %). All methods differed in their sensitivity and specificity. HPV genotyping contributes to individual risk stratification, therapeutic decisions, epidemiological studies and vaccine development, supporting approaches in prevention, healthcare and management of HPV infection.
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The present study investigates molecular-based PCR techniques to estimate the prevalence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia cultures has been observed. Sample of internal organs and lesions of Nile tilapia were taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using PCR. No samples were taken in 2021 due to the COVID-19 pandemic. The real-time PCR conditions were optimized to allow a qualitative reliable detection of the bacteria from fixed fish tissue. A total of 599 pond- and cage-cultured tilapia from the southwestern Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures were generally with the optimal range to grow Nile tilapia, although extreme values were recorded on some farms. Most of the tilapia sampled were apparently healthy. No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019 at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first detection of these pathogenic bacteria in rural farms using real-time PCR and constitutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of these pathogens to other aquaculture areas.
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Cíclidos , Enfermedades de los Peces , Tilapia , Animales , Cíclidos/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , México/epidemiología , Prevalencia , Pandemias , Enfermedades de los Peces/microbiología , AcuiculturaRESUMEN
SUMMARY OBJECTIVE: The aim of this study was to assess the results and efficiency of two real-time polymerase chain reaction procedures for detecting human papillomavirus utilizing urine samples. METHODS: This study comprised 151 patients who had previously tested positive for human papillomavirus in their cervical samples. Two different commercial real-time polymerase chain reaction techniques were used for identification and genotyping human papillomavirus in urine specimens. The urine samples of 151 patients were evaluated via the Roche Cobas test, and the urine samples of 91 patients were also evaluated via the Qiagen test. RESULTS: The overall consistency of urine and cervical swab specimens for the identification of human papillomavirus in Roche Cobas and Qiagen tests were 44.8 and 44%, respectively. The rates of positive human papillomavirus results from urine samples were 57 and 70.3%, respectively. The overall concordance among Roche Cobas and Qiagen tests utilizing urine samples for human papillomavirus type 16/18 was 84.3% with a kappa value of 0.675, and for other high-risk-human papillomavirus, it was 75.60% with a kappa value of 0.535. Roche Cobas showed high concordance with Qiagen test. CONCLUSION: human papillomavirus positivity was not detected in all urine samples. It is still inappropriate to recommend the use of urine liquid biopsy for the accurate and reliable detection of human papillomavirus. Due to the lack of a standardized tool, the utilization of urine samples as a screening human papillomavirus test remains a challenge.
RESUMEN
Hepatitis C virus infection (HCV) is the foremost reason of progressive hepatic fibrosis and cirrhosis, with an elevated risk of hepatocellular carcinoma (HCC) development. Medicinal plants have been used for human health benefits for several years, but their therapeutic potential needs to be explored. The main objective of this study was to figure out the in vitro antiviral and anticancer characteristics of total crude protein of Iberis gibraltarica against HCV and HCC. Total crude protein of Iberis gibraltarica was isolated and quantified. The level of cytotoxicity was measured against the HepG2 cell line and it shows no significant cytotoxicity at the concentration of 504µg/ml. The anti-HCV effect was determined by absolute quantification via real time RT-PCR method and viral titer was reduced up to 66% in a dose dependent manner against the total protein of Iberis gibraltarica. The anticancer potential of Iberis gibraltarica was also examined through mRNA expression studies of AFP and GPC3 genes against the total protein of Iberis gibraltarica-treated HepG2 cells. The results show up to 90% of the down-regulation expression of AFP and GPC3. The obtained results indicate the therapeutic potential of total protein of Iberis gibraltarica against HCV and hepatocellular carcinoma in vitro.
A infecção pelo vírus da hepatite C (HCV) é a principal causa de fibrose hepática progressiva e cirrose, com risco elevado de desenvolvimento de carcinoma hepatocelular (HCC). As plantas medicinais vêm sendo utilizadas para benefícios à saúde humana há vários anos, mas seu potencial terapêutico precisa ser explorado. O principal objetivo deste estudo foi descobrir as características antivirais e anticancerígenas in vitro da proteína bruta total de Iberis gibraltarica contra HCV e HCC. A proteína bruta total de Iberis gibraltarica foi isolada e quantificada. O nível de citotoxicidade foi medido contra a linha celular HepG2 e não apresenta citotoxicidade significativa na concentração de 504µg/ml. O efeito anti-HCV foi determinado por quantificação absoluta através do método RT-PCR em tempo real e o título viral foi reduzido em até 66% de forma dose-dependente contra a proteína total de Iberis gibraltarica. O potencial anticancerígeno de Iberis gibraltarica também foi examinado através de estudos de expressão de mRNA dos genes AFP e GPC3 contra a proteína total de células HepG2 tratadas com Iberis gibraltarica. Os resultados mostram até 90% da expressão de regulação negativa de AFP e GPC3. Os resultados obtidos indicam o potencial terapêutico da proteína total de Iberis gibraltarica contra HCV e carcinoma hepatocelular in vitro.