Differential expression of sigma factor of Mycobacterium tuberculosis under in vivo infection and in vitro culture conditions / 公共卫生与预防医学

Objective To investigate the gene expression of sigma factors in vivo, and to explore the sigma factors that may be closely related to the virulence of pathogenic Mycobacterium tuberculosis Methods Tuberculosis (TB) patients diagnosed in the outpatient department of Tianjin Tuberculosis Control Center from January to December 2018 were selected, and 20 sputum-positive specimens were randomly selected from TB patients confirmed with Xpert-positive for the present study. Two immediate sputum specimens were collected from each case of pulmonary tuberculosis before treatment, one for RNA extraction and one for in vitro culture. In vitro cultured strains in the logarithmic phase of growth were harvested for RNA extraction. The specific primers for 13 sigma factors were designed. The differential expression of the 13 sigma factors between sputum isolates and in vitro cultured strains was analyzed by fluorescence quantitative PCR. Taking ribosomal 16s as the reference gene, the transcription level of sigma factors was analyzed by 2ΔCt. Using the stably expressed sigA as the control reference, the expression differences of other sigma factors were analyzed by one-way ANOVA. Results Within 0 days, stress-associated sigma factors have a different expression profile in clinical isolate strains vs H37Rv or in vitro. All the sigma factors induced up regulation in sputum ,while no difference transcription between clinical isolate strains vs H37Rv（P＞0.05）. When compared to in vitro culture ,only sigM transcript highest in sputum(P＜0.05). Conclusion SigM plays an important role in the initial stages of bacterial infection, but its exact role is unclear.We assumed it could have a role in the interplay between the host immune defenses and the bacterial escape mechanisms.

Efectividad de la reacción en cadena de la polimerasa en tiempo real (PCR-RT) e Inmunodot en el diagnóstico temprano de leptospirosis: análisis comparativo con la prueba de Microaglutinación (MAT) / Effectiveness of the real-time polymerase chain reaction (RT-PCR) and Immunodot in the early diagnosis of Leptospirosis: comparative analysis with the Microagglutination test (MAT)

Introducción: la leptospirosis es una enfermedad infecciosa producida por espiroquetas del género Leptospira. Se disemina a través de la orina de animales domésticos, con mayor frecuencia por roedores. En República Dominicana se necesitan pruebas confiables para el diagnóstico en etapas tempranas de la enfermedad. En el presente trabajo se aborda la validez diagnóstica del PCR en Tiempo Real y del IGM (INMUNODOT) en comparación con la prueba de Microaglutinación (MAT). Materiales y métodos: se realizó un estudio observacional, descriptivo, de corte transversal con 69 pacientes admitidos en el Hospital Regional "José María Cabral y Báez" con diagnóstico presuntivo de leptospirosis desde el 2010 hasta el 2012. Resultados: del total de los casos (69), la mayoría fueron del sexo masculino (94.2 %), menores de 49 años (79.4 %) y provenían de la provincia de Santiago (58 %). La mortalidad fue de 52.1 % de los cuales 52.3 % fueron reportados positivos para Leptospira y 47.6 % resultaron negativos según el MAT. Al comparar los resultados de PCR en relación al MAT se obtuvo una sensibilidad de 27.3 % y una especificidad de 80 %. Los resultados del PCR y del Immunodot fueron equivalentes. Conclusión: en el presente trabajo la realización del PCR en sangre, después del 5to día de inicio de la enfermedad, no demostró ser mejor que la Inmunodot para la detección temprana de la enfermedad, al contrastarlos con el resultado del MAT. En República Dominicana, los casos hospitalizados con diagnóstico presuntivo de leptospirosis permanecen con muy alta mortalidad. Por tanto, es prioritario optimizar el diagnóstico y el tratamiento de estos casos. En este estudio, los casos confirmados con Leptospirosis que fallecieron indican que debe actualizarse el protocolo de tratamiento y asegurarse que pueda implementarse. Por otra parte, los casos no confirmados fallecidos exigen investigar otras causas de enfermedad como la Infección por Hanta Virus.

Introduction: Leptospirosis is an infectious disease caused by spirochaetes of the genus Leptospira. It spread through the urine of domestic animals most frequently in rodents. Need reliable tests to diagnose in early stages of the disease and it has been proposed the use of the PCR in real-time as an option. The present work deals with the diagnostic real-time PCR and IGM (INMUNODOT) in comparison with the Microagglutination (MAT) test. Materials and methods: An observational, descriptive, cross-sectional study with 69 patients admitted as possible Leptospirosis in the Hospital "Jose Maria Cabral y Báez" of Santiago from 2010 to 2012. Results: Of the total cases (69), most were male (94.2%), under 49 years of age (79.4%) coming from the province of Santiago (58%). Mortality was 52.1% of which 52.3% were reported positive for leptospirosis and 47.6% were negative according to the MAT. To compare the results of PCR in relation to the MAT was obtained a sensitivity of 27.3% and a specificity of 80%. The results of the PCR and immunodot were equivalent. Conclusion: In this study, the realization of the PCR in blood after the 5th day of the disease not proved to be better than the Inmunodot for the early detection of the disease. Is necessary to evaluate cases less than 5th day. In the Dominican Republic, hospitalized with a presumptive diagnosis of Leptospirosis cases remain with very high mortality. Therefore, it is important to optimize the diagnosis and treatment of these cases. In this study, confirmed cases with Leptospirosis who died, indicate that you must upgrade the treatment protocol and ensure that it can be implemented. On the other hand, the deceased not confirmed cases require to investigate other causes of disease as the Hanta Virus infection.

Características epidemiológicas de los pacientes ingresados por COVID-19 en el Hospital Pediátrico Cerro (2021)

Introducción: La infección por SARS-CoV-2 en la población pediátrica mostró en el año 2021 un aumento en la morbilidad de la pandemia. Se elevó el número de pacientes por lo que fue necesario la apertura de hospitales idóneos. Objetivo: Caracterizar desde el aspecto epidemiológico a los pacientes atendidos en el Hospital Pediátrico Cerro con diagnóstico de COVID-19. Métodos: Estudio observacional, descriptivo transversal sobre las características epidemiológicas de los pacientes ingresados, entre el 3 de abril y 7 de octubre de 2021. El universo fue de 2699 pacientes menores de 18 años con diagnóstico confirmado por el test de reacción en cadena de polimerasa. Las variables analizadas fueron: grupos de edad, sexo, procedencia según municipio y territorio, mes e ingreso en sala de terapia, características sintomáticas y evolución. Los resultados se expresaron en valores absolutos y relativos y se estimó el odd ratio con su intervalo de confianza de 95 %, (p<0,05). Resultados: Predominó el grupo de edades entre 5-10 años (31,83 %), el sexo masculino (50,46 %) y los pacientes procedentes del municipio Cerro (12,41 %). Desde abril hasta junio hubo un rango promedio de 300 casos mensuales con aumento desde julio hasta septiembre y disminución en octubre. Los pacientes sintomáticos alcanzaron 76,0 % y en terapia intensiva se internaron 1,66 %. Conclusiones: El grupo de edad escolar sintomático alcanzó la mayor frecuencia, con un número superior de casos desde julio hasta septiembre sobre todo en los municipios Cerro y Arroyo Naranjo. Se registraron pocos ingresos en terapia intensiva por presentar la mayoría una evolución favorable.

Introduction: SARS-CoV-2 infection in the pediatric population showed an increase in the morbidity of the pandemic in 2021. The number of patients also increased, so it was necessary to open suitable hospitals. Objective: To characterize from the epidemiological aspect the patients treated at Cerro Pediatric Hospital with a diagnosis of COVID-19. Methods: Observational, descriptive cross-sectional study on the epidemiological characteristics of admitted patients, between April 3 and October 7, 2021. The universe consisted of 2699 patients under 18 years of age with a diagnosis confirmed by the polymerase chain reaction test. The variables analyzed were: age groups, sex, origin according to municipality and territory, month and admission to the therapy room, symptomatic characteristics and evolution. The results were expressed in absolute and relative values and the odd ratio was estimated with its 95% confidence interval (p<0.05). Results: The age group between 5-10 years (31.83 %), the male sex (50.46 %) and patients from Cerro municipality (12.41 %) predominated. From April to June there was an average range of 300 monthly cases with an increase from July to September and a decrease in October. Symptomatic patients reached 76.0 % and 1.66 % were hospitalized in intensive care. Conclusions: The symptomatic school age group reached the highest frequency, with a higher number of cases from July to September, especially in the municipalities of Cerro and Arroyo Naranjo. There were few admissions to intensive care because most of them showed a favorable evolution.

Value of Detecting Peripheral Blood Cell Morphology Combined with EBV-DNA Quantity in the Initial Diagnosis for Infants Patients with Infectious Mononucleosis / 现代检验医学杂志

Objective To evaluate the value of detecting abnormal lymphocyte morphology in peripheral blood and Epstein Barr Virus(EBV)DNA quantity through real-time quantitative PCR (RT-qPCR)in the initial diagnosis for infants patients with infectious mononucleosis (IM).Methods From Jan.2013 to Dec.2014 212 infants patients with IM were analysed ret-rospectively,which were all in-patients in the hospital.The abnormal lymphocyte morphology in peripheral blood and Epstein Barr Virus (EBV)DNA quantity were both detected in the initial fever period and a week later.The latter was detected by real-time quantitative PCR (RT-qPCR),combined with all the symptoms were all analysed comprehensively.The percentage of abnormal lymphocyte more than 10% was positive,and the EBV-DNA quantity more than 1.0×103 copy per ml was posi-tive,too.Results Of all the infants patients,in the initial fever period,82 patients had more than 10% positive abnormal lymphocyte and 100 patients had positive EBV-DNA quantity.But a week later,156 patients had more than 10% positive ab-normal lymphocyte,the maximum abnormal lymphocyte was 56%.And 180 patients had positive EBV-DNA quantity.When both abnormal lymphocyte morphology in peripheral blood and Epstein Barr Virus (EBV)DNA quantity were detected,in the initial fever period,125 patients were positive,it rose significantly more than that of abnormal lymphocyte morphology in peripheral blood (χ2=17.45,P0.05).Conclusion The detecting of peripheral blood cell morphology combined with EBV-DNA quantity are very important in the initial diagnosis for infants patients with infectious mononucleosis.Including all the symp-toms,they could improve the diagnosis timely and accurately.

Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H6N1 AIV / 中国病毒学·英文版

Chicken embryo fibroblasts(CEFs)are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus(AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR(QPCR)analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4(RPL4)and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ)are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene(ACTB)and the ribosomal protein L4(RPL4)gene are the best references.

Quantification of Simian Immunodeficiency Virus by SYBR Green RT-PCR Technique / 中国病毒学·英文版

Plasma viral RNA load is widely accepted as the most relevant parameter to assess the status and progression of Simian immunodeficiency virus (SIV) infections. To accurately measure RNA levels of the virus, a one-step fluorescent quantitative assay was established based on the SYBR green Real-time reverse transcription-polymerase chain reaction (RT-PCR). The lower detection limit of the assay was 10 copies per reaction for the virus. This method was successfully applied to quantify SIVmac251 and SIVmac239 viruses produced in CEM×174 cells. Additionally, the performance of the SYBR green RT-PCR was assessed in a SIVmac251 infected rhesus macaque. The result demonstrated that the method could detect as little as 215 copies per milliliter of plasma and the dynamic pattern of viral load was highly consistent with previous results. With regard to convenience, sensitivity and accuracy our assay represents a realistic alternative to both branched-chain DNA (b-DNA) assays or real-time PCR assays based on TaqMan probes.

Application of real-time fluorescent RT-PCR for quantitative detection of SARS-Coronavirus / 临床检验杂志

Objective To establish a real-time fluorescent quantitative RT-PCR for detecting SARS-Coronavirus (CoV) mRNA in gargling liquid and serum of SARS patients.Methods The assay is based on simplified nested fluorescent RT-PCR. Total RNA was extracted from gargling liquid and serum by using high performance system and reverse-transcription by antisense primer.The specific TaqMan probe was designed according to the published DNA sequence of SARS-CoV polymerase and labeled with FAM and TAMRA. Standard curves for SARS-CoV quantification were prepared with serial dilutions of the recombinant plasmid.Results The method is highly sensitive and specific. The sensitivity of assay was 1?105 copies/L. The positive rate of SARS-CoV mRNA in the gargling liquid of patients was 65.0% (26/40) and 11.5% (6/52) in suspected patients, respectively. SARS-CoV mRNA was not detectable in the gargling liquid from 40 healthy individuals. The positive rate of SARS-CoV mRNA in the serum of SARS patients was 25.6% (10/39). PCR amplification products of 6 suspected patients with SARS-CoV mRNA were confirmed by sequencing and the sequence data were consistent with that of BJ01 AY278488.Conclusions Real-time fluorescent quantitative RT-PCR should be a rapid, specific tool for early diagnosis of SARS-CoV infection.

Study on survivin mRNA and protein of BEP-2D cells during its malignant transformation by cigarette smoke condensate / 重庆医科大学学报

Objective:To study the change of the survivin mRNA and protein of BEP-2D cells during its malignant transformation.Methods:Normal BEP-2D cell and BEP-2D cells treated by cigarette smoke condensate(CSC)for 15 weeks(P-15),25 weeks(P-25)and 38 weeks(P-38)were respectively chosen to study the survivin gene and protein by RT-PCR(retro-translation PCR,RT-PCR)and immunohistochemical method.Results:The survivin mRNA was found in BEP-2D cells of P-15,P-25 and P-38,respectively with 0.56,0.80,and 0.81,but not in the normal BEP-2D cell.The survivin protein was found in the normal BEP-2D cell,but not in BEP-2D cells of P-15,P-25 and P-38.The levels of survivin protein expressian were different between BEP-2D cells of P-15,P-25 and P-38 and normal BEP-2D cell,with significant difference between P-15 BEP-2D cell and normal BEP-2D cell(P