Browsing by Subject "PCR"

Now showing 1 - 6 of 6
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    Characterization of natural occurring Pneumocystis carinii pneumonia in pigs by histopathology, electron microscopy, in situ hybridization and PCR amplification
    (F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 1998) Ramos Vara, J. A.; Lu, J. J.; da Silva, A. J.; Montone, K. T.; Pieniazek, N. J.; Lee, C. H.; Pérez, L.; Steficek, B. A.; Dunstan, R. W.; Craft, D.; Watson, G. L.
    Macroscopic. histolog ic . ultrastruc tural. microbiologic. ill sirll hybridi za ti on (ISH ) and PCR detection results in three 8-week-old pi gs naturall y infected with Pllel.llllonsris c{lrinii (PC) are described . All an imals had a nonsu ppurati ve interstitial pneumoni a and intra-alveolar PlleulIlucysris organisms with foamy eos in ophili c and PAS positi ve appearance . Ultrastructurally. PC trophozoites and cysts we re observed in pigs No.2 and NO .3. with the fo rmer being much more numerous. PC organisms we re located on the alveolar surface or within the alveolar septa . Trophozoites had numerous filopodia and were thin-wall ed. Cysts had no o r few filopodia. we re thick- wa ll ed and co nt a ined intracysti c bodies. Using non-isotopic ISH on formalinfixed. paraffin-embedded lung tissue sections. PC DNA from pigs No.2 and No . 3 hyb ridi zed with a probe specific for PC ribosomal RNA (rRNA). Using primers spec ific for mit oc ho ndrial rRNA ge ne (pA Z I0 2- E/pAZ I 02-H). and for the interna l transcriber space rs of ribosomal gene of PC. PCR methods amplified a product in the lung of pigs No.2 and No.3 using either frozen or formalin -fi xed and paraffin -embedded lung tissue. DNA from Pig No. I samples did not amplify with any primer. This is the first time that molecular biology techniques (ill siru hybridi zation and PCR) have been applied to the study of porc ine pneumocystosis.
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    Correlation between 3D microstructural and 2D histomorphometric properties of subchondral bone with healthy and degenerative cartilage of the knee joint
    (F. Hernández y Juan F. Madrid. Universidad de Murcia: Departamento de Biología Celular e Histología, 2014) Lahm, Andreas; Kasch, Richard; Spank, Heiko; Erggelet, Christoph; Esser, Jan; Merk, Harry; Mrosek, Eike
    Cartilage degeneration of the knee joint is considered to be a largely mechanically driven process. We conducted a microstructural and histomorphometric analysis of subchondral bone samples of intact cartilage and in samples with early and higher- grade arthritic degeneration to compare the different states and correlate the findings with the condition of hyaline cartilage. These findings will enable us to evaluate changes in biomechanical properties of subchondral bone during the evolution of arthritic degeneration, for which bone density alone is an insufficient parameter. From a continuous series of 80 patients undergoing implantation of total knee endoprosthesis 30 osteochondral samples with lesions macroscopically classified as ICRS grade 1b (group A) and 30 samples with ICRS grade 3a or 3b lesions (group B) were taken. The bone samples were assessed by 2D histomorphometry (semiautomatic image analysis system) and 3D microstructural analysis (high-resolution microCT system). The cartilage was examined using the semiquantitative real-time PCR gene expression of collagen type I and II and aggrecan. Both histomorphometry and microstructural and biomechanical analysis of subchondral bone in groups A and B consistently revealed progressive changes of both bone and cartilage compared with healthy controls. The severity of cartilage degeneration as assessed by RT PCR was significantly correlated with BV/TV (Bone Volume Fraction), Tb.Th (Trabecular Thickness) showed a slight increase. Tb.N (Trabecular Number), Tb.Sp (Trabecular separation) SMI (Structure Model Index), Conn.D (Connectivity Density) and DA (Degree of Anisotropy) were inversely correlated. We saw sclerotic transformation and phagocytic reticulum cells. Bone volume fraction decreased with an increasing distance from the cartilage with the differences compared with healthy controls becoming greater in more advanced cartilage damage. The density of subchondral bone alone is considered an unreliable parameter for classifying changes evolving over time. The progressive damage of subchondral bone seen in the present study correlates well with cartilage changes. Trabecular orientation is also impaired, which explains the changes in biomechanical parameters and the inadequate load transfer and excessive loading of cartilage. Besides subchondral bone density, which in turn correlates with cartilage thickness, other parameters such as structure model index and grade of anisotropy best reflect mechanical properties such as Young modulus, compressive strength, tensile stress, and failure energy. However, it remains unclear whether the mechanical interaction of the mineralized subchondral tissues with articular cartilage works vice versa. The possibility of a biochemical signalling from the degenerating cartilage via the synovial fluid and bone- cartilage crosstalks via subchondral pores may indeed explain a certain depth dependency of subchondral bone changes.
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    Descripción y análisis del impacto de las estrategias de control de legionelosis en un hospital de tercer nivel
    (Universidad de Murcia, 2024-07-29) Tomás Borja, Antonio; Torres Cantero, Alberto Manuel; Segovia Hernández, Manuel; Escuela Internacional de Doctorado
    Introducción: Aunque los aspectos relacionados con la Legionella han sido ampliamente estudiados sigue habiendo una tasa creciente de legionelosis en los hospitales. El análisis de temperatura y cloro en instalaciones de agua sanitaria (AS) y de resultados analíticos de muestras ambientales de AS son indicadores de nivel de riesgo de infección. Objetivos: Describir el grado de colonización de Legionella en el periodo 2016-2022 en el sistema de AS del Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA), valorando la efectividad de las medidas de control adoptadas en 2019 en el pabellón Hospital General (HG) y el comportamiento de la instalación, correlacionando los resultados de parámetros fisicoquímicos de AS. Material y método: Estudio longitudinal con los resultados analíticos disponibles de la base de datos de muestras ambientales del periodo 2016 a 2023, incluyendo los parámetros fisicoquímicos y microbiológicos realizados antes y después de las actuaciones de control en 2019 en la instalación de AS del HG. Resultados: Se obtienen valores del comportamiento de la instalación a nivel general, observando la inexistencia de patrón estacional exacto de la bacteria y un riesgo en el sistema de producción central. Los resultados de las actuaciones en hospitalización de HG proporcionan información para la confección del mapa de riesgos a partir de los parámetros fisicoquímicos y los resultados de análisis de Legionella en muestras ambientales por la técnica de PCR y qPCR. Discusión: Los resultados muestran la importancia que tiene el correcto diseño y mantenimiento de las instalaciones de agua sanitaria, evitar condiciones de estancamiento del agua y de mal funcionamiento de los circuitos de retorno de ACS como medidas para evitar la presencia de Legionella en las instalaciones de fontanería, así como alcanzar y mantener homogéneamente niveles de temperatura y cloro en dichas instalaciones. Indicadores como nivel de ATP en AS y técnicas de determinación de Legionella por PCR y qPCR son complementarias a la técnica validada de cultivo y sirven para diseñar el mapa de riesgos de la instalación de AS y priorizar actuaciones de control. Conclusiones: En instalaciones complejas y de muchos años de uso colonizadas no se consigue la erradicación de Legionella aunque se haga correctamente el mantenimiento técnico legal. Es por ello que se hace necesario realizar intervenciones de reforma y limpiezas severas en las instalaciones hídricas como estrategia para poder minimizar la legionelosis en este tipo de edificaciones. Es necesario el exhaustivo mantenimiento y medición de niveles de temperaturas, cloro y ATP en puntos terminales por ser éstas herramientas eficaces para evitar el crecimiento y proliferación de Legionella. Es necesario el control analítico de la bacteria por técnicas más sensibles y rápidas como PCR y PCRq, junto con el método validado de cultivo, herramientas de control para la gestión de la prevención de legionelosis.
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    Massive microfilaremia in a dog subclinically infected with Acanthocheilonema dracunculoides
    (Elsevier, 2020-06) Muñoz, Clara; Gonzálvez, Moisés; Rojas, Alicia; Martínez Carrasco, Carlos; Baneth, Gad; Berriatua, Eduardo; Ortiz Sánchez, Juana; Sanidad Animal
    Canine filarioids are worldwide distributed nematodes transmitted by arthropods with variable virulence depending on the species. Dirofilaria immitis is the most virulent and serological antigen tests are commonly employed to detect it. This study reports on the heaviest cavity filariasis recorded so far in a dog, which showed no apparent clinical signs of infection. The 6-year-old male was positive to a D. immitis antigen test. Blood samples collected and analyzed with the modified Knott's test for microfilariae revealed 264,367 microfilariae/ml. In a post-mortem examination 791 adult filarial nematodes were found in the dog's thoracic and peritoneal cavities. Morphological and molecular analysis identified the nematode as Acanthocheilonema dracunculoides and no other species were present. This is evidence that massive A. dracunculoides infections in dogs may not be clinically evident, they may cause serologic cross-reaction with D. immitis infection and become a life-threatening condition if dogs are treated with a microfilaricidal treatment without previously performing an adequate diagnosis.
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    Preventing legionellosis outbreaks by a quick detection of airborne Legionella pneumophila
    (Elsevier, 2019-01-15) Sánchez-Parra, Beatriz; Núñez, Andrés; Moreno, Diego A.; Genética y Microbiología
    Legionellosis is a severe pneumonic infection caused by inhaling bacteria of the genus Legionella. Most cases reported in the USA and Europe are associated with the species Legionella pneumophila. This Gram-negative bacterium can survive within a wide spectrum of temperatures, and be transmitted via aerosols from multiple aquatic sources: fountains, thermal spas and other water systems. Although the PCR is one of the most popular methods to verify its presence in environmental or clinical samples, the direct application of this technique to ambient air samples is unusual because of the scarce material in the specimens. Here, we have developed a two-PCR assay, carried out over the V3 and V5 hypervariable regions of the 16S rRNA gene, to detect specifically the pathogenic bacteria Legionella pneumophila in outdoor air samples with low concentration of DNA. The application of this protocol does not require culture and retrieves quick results to activate the corresponding public alerts to prevent legionellosis outbreaks.
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    Sensitivity of two methods to detect Mycoplasma agalactiae in goat milk
    (BioMed Central, 2015) Tatay-Dualde, Juan; Prats- Van der Ham, Miranda; Gómez-Martín, Angel; Paterna, Ana; Corrales, Juan Carlos; Sánchez, Antonio; Fe, Christian de la; Amores, Joaquín; Amores, Joaquín; Sanidad Animal
    Background: Laboratory diagnostic techniques able to detect Mycoplasma agalactiae are essential in contagious agalactia in dairy goats. This study was designed: 1) to determine the detection limits of PCR and culture in goat milk samples, 2) to examine the effects of experimental conditions including the DNA extraction method, PCR technique and storage conditions (fresh versus frozen stored milk samples) on these methods and 3), to establish agreement between PCR and culture techniques using milk samples from goats with mastitis in commercial dairy herds. The study was conducted both on artificially inoculated and field samples. Results: Our findings indicate that culture is able to detect M. agalactiae in goat milk at lower concentrations than PCR. Qualitative detection of M.agalactiae by culture and PCR was not affected by sample freezing, though the DNA extraction method used significantly affected the results of the different PCR protocols. When clinical samples were used, both techniques showed good agreement. Conclusions: The results from this study indicate that both culture and PCR are able to detect M. agalactiae in clinical goat mastitis samples. However, in bulk tank milk samples with presumably lower M. agalactiae concentrations, culture is recommended within the first 24 h of sample collection due to its lower limit of detection. To improve the diagnostic sensitivity of PCR in milk samples, there is a need to increase the efficiency of extracting DNA from milk samples using protocols including a previous step of enzymatic digestion.