Los estudios fueron patrocinados por AdvanDx.

1. Recogida y preparación de <ol><li> Recoger la sangre venosa de pacientes con sospecha de sepsis y poner en dos BACTEC (BD, Sparks, MD) botellas de hemocultivos, una aeróbica y anaeróbica uno, con un total un conjunto de hemocultivo. </li><li> Botellas lugar en el BACTEC 9240 instrumento a 35 ° C. </li><li> Incubar hasta que el instrumento se activa la alarma por el crecimiento de organismos en la botella de hemocultivo. </li><li> Retire la botella del instrumento hemocultivo. </li></ol> 2. Preparación de los reactivos antes de la tinción <ol><li> Prepare la solución de lavado mediante la adición de 4 ml de solución de lavado 60x seguido por 240 ml de agua dulce desionizada o destilada directamente al plato de tinción. </li><li> Coloque el plato en la tinción de la 55 ° C baño de agua a calentar. </li><li> Almacenar el concentrado restante a 2-8 º C. </li><li> Quite el soporte de montaje del refrigerador y la temperatura ambiente. </li></ol> 3. La tinción de Gram <ol><li> Agite suavemente el frasco de cultivo de sangre. </li><li> Usando una aguja y una jeringa o un aparato de subcultivo, remover la sangre (aproximadamente 5 ml) de la botella y colocar en un tubo estéril tornillo tope. </li><li> Con una pipeta estéril, coloque una gota grande de sangre sobre un portaobjetos de vidrio. </li><li> Aire seco de la diapositiva y fijar con metanol durante 10 segundos. </li><li> Permita que seque al aire libre de diapositivas. </li><li> Realizar una tinción de Gram del frotis de sangre para determinar la morfología del organismo en crecimiento en la botella de hemocultivo. </li><li> El uso de un lente de inmersión en aceite, la vista de diapositivas. Cocos gram positivos en pares y cadenas cortas observado en la tinción de Gram es más consistente con especies de Enterococcus. </li><li> Este resultado tinción de Gram se determina la selección del kit de la sonda adecuada PNA FISH para el uso de la identificación de bacterias. Esta cultura de la sangre se colorean con la E. faecalis / OE PNA FISH mancha. </li></ol> 4. PNA FISH manchas <ol><li> Mezclar el tubo que contiene la sangre suavemente por agitación antes de la preparación de frotis. </li><li> Coloque una gota de solución de fijación en el bienestar de un portaobjetos de microscopio PNA PESCADO. </li><li> Transferencia de 10 l, o una pequeña gota del tubo con el hemocultivo positivo a la solución de fijación y mezclar suavemente para emulsionar. </li><li> Fijar los frotis por calentamiento durante 20 minutos. a 55 ° C en una placa calefactora. </li></ol> 5. La calidad del material de control Uno positivo y otro negativo de diapositivas de control de calidad deben ser probados con cada lote de diapositivas para la tinción. <ol><li> Use E. faecalis / OE Control de diapositivas comprado AdvanDx para este fin o preparar los frotis de cultivos líquidos de cepas de referencia de E. faecalis y E. faecium como control positivo, ya sea en diapositivas independientes o mezclados en una diapositiva y Staphylococcus spp o Streptococcus spp como material de control negativo. </li></ol> Los resultados de control de calidad debe ser capaz de controlar las condiciones de las pruebas adecuadas, en particular los que afectan a la hibridación rigor y la penetración de la pared celular, ya que la metodología PNA está diseñado para optimizar la penetración de la pared celular 6. Hibridación <ol><li> Añadir una gota de E. faecalis / OE a la Autoridad Nacional Palestina y en el portaobjetos con la extensión. </li><li> Añadir cubreobjetos. Evite las burbujas de aire. </li><li> Incubar durante 30 ± 5 min. a 55 ± 1 ° C en la placa de calefacción </li><li> Después de la incubación a cabo las diapositivas en soporte de diapositivas </li></ol> 7. Lávese las estrictas <ol><li> Soporte de diapositivas sumergirse en la solución de lavado precalentado a 55 ° C y retirar con cuidado el cubreobjetos. A menudo, los cubreobjetos se desliza suavemente agitando el portaobjetos en la solución de lavado. En ocasiones, los cubreobjetos se empujan suavemente con unas pinzas. </li><li> Incubar en la solución de lavado durante 30 ± 5 min. a 55 ± 1 ° C. </li><li> Eliminar las diapositivas de la solución de lavado </li><li> Permita que la diapositiva se seque al aire </li></ol> 8. Montaje <ol><li> Añadir una gota de medio de montaje a cada diapositiva </li><li> Añadir cubreobjetos. Evite las burbujas de aire. </li><li> Examinar el portaobjetos en un microscopio de fluorescencia en 2 horas. </li><li> No exponga las transparencias a la luz solar directa u otras fuentes de luz intensa, ya que puede conducir a la extinción de fluorescencia. </li></ol> 9. Interpretación de los resultados El microscopio de fluorescencia para el examen de diapositivas debe estar equipado con el filtro de banda AdvanDx doble y un objetivo de aceite de 60x o 100x. Las diapositivas de control de calidad deben examinarse en primer lugar para confirmar que la hibridación se produjo. E. faecalis debe aparecer como de color verde brillante fluorescente cocos en múltiples campos de la vista y el E. faecium aparecerá como cocos brillantes de color rojo. No enterococos portaobjetos de control debe aparecer no fluorescentes. Después de confirmar que el sistema en los controles, las diapositivas de los pacientes pueden ser examinados. En un primer momento la sangre film se adopta tonos rojizos, pero las de color rojo brillante y los cocos verdes será muy evidente. <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="Figura 1" /> Figura 1. Ejemplos representativos de verde-positivas E. faecalis (izquierda), rojo positivo E. faecium (centro) y negativa (derecha) resultados de la prueba. 10. Resultados representante Tres instituciones se incluyeron en un ensayo clínico multicéntrico evaluando este PNA FISH mancha y la comparación de un protocolo de 2.5 horas para el protocolo de hora acortó 1.5 que acaba de ser revisado. Un total de 152 cocos Gram positivos rutina en pares y cadenas (GPC) frascos de hemocultivo positivo se incluyeron en los estudios. Hubo un 100% (152/152) concordancia entre los resultados de la modificación y el procedimiento de ensayo original de E. faecalis / OE PNA FISH: 41/41 E. faecalis, 33/33 otros enterococos y 78/78 GPC otros (Tabla 1). Este método de tinción había exquisita sensibilidad y especificidad en este estudio clínico (tabla 2). <table border="1"><tbody><tr><td></td><td> Los métodos de rutina </td><td> E. faecalis / OE PNA FISH procedimiento estándar </td><td> E. faecalis / OE PNA procedimiento FISH corto </td></tr><tr><td> E. faecalis </td><td> 41 </td><td> 41 (verde Positivo) </td><td> 41 (verde Positivo) </td></tr><tr><td> E. faecium </td><td> 27 </td><td> 27 (Rojo Positivo) </td><td> 27 (Rojo Positivo) </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2 (Rojo Positivo) </td><td> 2 (Rojo Positivo) </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2 (Rojo Positivo) </td><td> 2 (Rojo Positivo) </td></tr><tr><td> Otras especies de Enterococcus. </td><td> 2 </td><td> 2 (Rojo Positivo) </td><td> 2 (Rojo Positivo) </td></tr><tr><td> S. pneumoniae </td><td> 17 </td><td> 17 (Negativa) </td><td> 17 (Negativa) </td></tr><tr><td> S. viridans </td><td> 33 </td><td> 33 (Negativa) </td><td> 33 (Negativa) </td></tr><tr><td> S. mitis </td><td> 1 </td><td> 1 (negativo) </td><td> 1 (negativo) </td></tr><tr><td> S. pyogenes </td><td> 3 </td><td> 3 (negativo) </td><td> 3 (negativo) </td></tr><tr><td> S. bovis </td><td> 2 </td><td> 2 (negativo) </td><td> 2 (negativo) </td></tr><tr><td> S. salivarius </td><td> 1 </td><td> 1 (negativo) </td><td> 1 (negativo) </td></tr><tr><td> S. sanguinis </td><td> 2 </td><td> 2 (negativo) </td><td> 2 (negativo) </td></tr><tr><td> S. agalagtae </td><td> 7 </td><td> 7 (Negativo) </td><td> 7 (Negativo) </td></tr><tr><td> Otros Streptococcus spp. </td><td> 7 </td><td> 7 (Negativo) </td><td> 7 (Negativo) </td></tr><tr><td> Abiotrophia spp. </td><td> 3 </td><td> 3 (negativo) </td><td> 3 (negativo) </td></tr><tr><td> Peptostrepococcus spp. </td><td> 2 </td><td> 2 (negativo) </td><td> 2 (negativo) </td></tr><tr><td> Total </td><td> 152 </td><td> 152/152 100% de acuerdo </td><td> 152/152 100% de acuerdo </td></tr></tbody></table> Listado de la Tabla 1. Bacterias de prueba utilizando el estándar (2.5hr) y el protocolo rápido (1,5 horas). <table border="1"><tbody><tr><td> E.faecalis sensibilidad </td><td> Sensibilidad de otras especies de Enterococcus. </td><td> Especificidad </td></tr><tr><td> 100% (41/41) IC del 95% (93,0 a 100) </td><td> 100% (33/33) 33/33 IC del 95% (91,3 a 100) </td><td> 100% (78/78) IC del 95% (96,2 a 100) </td></tr></tbody></table> Tabla 2. Sensibilidad y especificidad del protocolo PESCADO 1,5 horas PNA.

<ol>
	<li>Oliveira, K., Chapin, K. C., Musgnug, M. C., Haase, G., Weber-Heynemann, J., DeGirolami, P. C., Dakos, J., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures+by+dual+color+fluorescence+in+situ+hybridization+using+PNA+probes.">Rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures by dual color fluorescence in situ hybridization using PNA probes.</a> , (2002).</li><li>Chapin, K. C., Musgnug, M. C., Oliveira, K., DeGirolami, P. C., Dakos, J., Johansen, J. T., Procop, G. W., Wilson, D., Padilla, E., Gonzalez, V., Stender, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicenter+evaluation+of+E.+faecalis+PNA+FISH+for+rapid+identification+and+differentiation+of+Enterococcus+faecalis+and+other+Enterococcus+species+directly+from+positive+blood+cultures.">Multicenter evaluation of E. faecalis PNA FISH for rapid identification and differentiation of Enterococcus faecalis and other Enterococcus species directly from positive blood cultures.</a> , (2003).</li><li>Rausei, V., Youssef, E., Morgan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+identification+of+Staphylococcus+aureus,+Enterococcus+faecalis,+and+Candida+albicans+from+blood+cultures+using+fluorescence+in+situ+hybridization+with+nucleic+acid+probes.">Direct identification of Staphylococcus aureus, Enterococcus faecalis, and Candida albicans from blood cultures using fluorescence in situ hybridization with nucleic acid probes.</a> , (2005).</li><li>Novak-Weekley, S. M., LaForga, M., Carey, L., Frazier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+S.+aureus+PNA+FISH,+E.+faecalis+PNA+FISH+&#38;+C.+albicans+PNA+FISH+for+Rapid+Identification+of+Positive+Blood+Culture+Bottles.">Validation of S. aureus PNA FISH, E. faecalis PNA FISH &#38; C. albicans PNA FISH for Rapid Identification of Positive Blood Culture Bottles.</a> , (2006).</li><li>Johnson, J. K., Roberts, A. A., Forrest, G. N., Lincalis, D. P., Venezia, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Identification+of+Enterococcus+Species+in+Positive+Blood+Cultures+with+Therapeutic+Implications.">Rapid Identification of Enterococcus Species in Positive Blood Cultures with Therapeutic Implications.</a> , (2006).</li></ol>

La producción de este video-artículo fue patrocinado por AdvanDx. Benjamin Cristal es un empleado de AdvanDx, que hace que las herramientas y los reactivos utilizados en este artículo.

La E. faecalis / OE ensayo PNA FISH de enterococo puede proporcionar la identificación de 2-3 días antes que los métodos estándar de cultivo. El procedimiento abreviado para el ensayo (1,5 hourr) proporciona una rápida identificación que pueden ser utilizados para un mejor enfoque a la terapia antimicrobiana y la evolución del paciente. Un estudio para apoyar este fue realizado por Forrest, et al. (6). Durante dos años consecutivos a partir de 2005 el laboratorio de microbiología identificado cocos Gram positivos en pares y cadenas que crecen en frascos de hemocultivo por métodos microbiológicos convencionales y en 2006 agregando el E. faecalis / OE PNA PESCADO. Además, un algoritmo de tratamiento desarrollado por el equipo de las instituciones a los antimicrobianos (AMT) para utilizar con eficacia los datos PNA FISH generados por el laboratorio en forma oportuna. Resultado primario evaluado fue el tiempo de la cultura de extracción de sangre a la aplicación de la terapia antimicrobiana efectiva antes y después de PNA FISH fue instituido en el flujo de trabajo de los laboratorios. Gravedad de la enfermedad, la ubicación del paciente y el tratamiento antimicrobiano empírico se midieron. Un total de 224 pacientes con bacteriemia por enterococo adquiridas en el hospital fueron evaluados con 129 en el período pre-intervención y 95 en el período PNA FISH. PNA FISH identificó E. faecalis 3 días antes de que los cultivos convencionales (1,1 frente a 4,1 días, p &lt;0,001). PNA FISH identificó E. faecium una mediana de 2,3 días anteriores (1,1 frente a 3,4 días, p &lt;0,001) y se asoció con una reducción estadísticamente significativa en el tiempo de iniciar el tratamiento eficaz (1,3 vs 3,1 días, p &lt;0,001) y la disminución de 30 días la mortalidad (26% versus 45%, p = 0,04). Este grupo llegó a la conclusión de que la E. faecalis / OE ensayo PNA FISH junto con un algoritmo de tratamiento AMT resultó en un inicio más temprano de la terapia adecuada antimicrobiano empírico para pacientes con E. faecium adquiridas en el hospital bacteriemia. <ol><li> Los reactivos no deben utilizarse después de la fecha de vencimiento impresa en las etiquetas y los reactivos no se deben modificar de ninguna manera. La persona debe permanecer en el refrigerador cuando no esté en uso o podrían perder su potencia. </li><li> Botellas para hemocultivo deben ser mezclados antes del muestreo. </li><li> Es importante no permitir que la punta del frasco gotero toque el frotis de sangre, ya que puede causar la contaminación cruzada de material entre las diapositivas, o causar la contaminación del reactivo. </li><li> Asegúrese de que el baño de agua y el bloque de calefacción se mantienen a una temperatura constante a 55 ° C. </li><li> No utilice filtros que no sea el filtro de doble banda o los colores no discernir con claridad. </li><li> No utilice portaobjetos de microscopio que no sea el portaobjetos, la hibridación no se producirá. </li><li> Es importante que el microscopio está funcionando correctamente. Asegúrese de que la bombilla microscopio en el número de horas aprobadas para su uso. </li></ol><ul><li> Algunas especies de Enterococcus raras que ocurren no son detectados por la sonda de PNA que hibrida con otras especies de Enterococcus, tales como E. asinii, E.dispar, haemoperoxidus E., cecorum E., E. columbae, saccharolyticus E., E. solitarius y E . sulfuroso. </li><li> Moraviensis E. es identificado como E. faecalis debido a las similitudes de secuencia. </li><li> Algunas cepas de Streptococcus anginosus producir una falsa fluorescencia verde positiva debido a las similitudes de secuencia. </li><li> VersaTREK frascos de hemocultivo no se han evaluado en los estudios clínicos. El rendimiento para detectar E. faecalis y otras Enterococcus spp. con botellas VersaTREK hemocultivo es desconocida. </li><li> Falsos positivos autofluorescencia verde puede ocurrir si un filtro estándar FITC se usa en lugar del filtro de doble banda. </li><li> Los resultados falsos negativos pueden ocurrir con poca frecuencia debido al crecimiento mixto o debido a un error en la técnica de ensayo. </li><li> El tipo y el estado de la instrumentación utilizada influirá en el aspecto visual de la imagen obtenida. La fluorescencia puede varydue con el tipo de microscopio empleado, la fuente de luz y el nivel de rRNA en las células </li><li> El aislamiento en medio sólido es necesario para diferenciar el crecimiento se mezcla con otros organismos e identificar cultivos positivos de sangre da un resultado negativo. </li><li> El producto no ha sido validado con muestras diferentes a los cultivos de sangre. </li></ul>

Reactivos E. faecalis / PESCADO OE PNA está formado por los componentes del equipo siguiente: <ul><li> Solución de fijación Fijación 3 ml de solución tampón fosfato salino con detergente. </li><li> E. faecalis / OE PNA E. faecalis / OE PNA 1,5 ml sondas de hibridación PNA. solución. Contiene 30% formamida. </li><li> Lave 60x Solución Solución de Lavado 60x 50 ml de Tris-salina tamponada con detergente </li><li> Medio de Montaje Montaje Medio 3 ml de inhibidor de photobleaching en glicerol </li><li> Diapositivas de control de calidad </li><li> <a href="http://www.advandx.com">AdvanDx</a> </li></ul>

a 1.5 hour procedure for identification of enterococcus species directly from blood cultures

Los enterococos son una causa común de bacteriemia por E. faecalis es la especie predominante seguido por E. faecium. Dado que la resistencia a la ampicilina y la vancomicina en E. faecalis es todavía poco común en comparación con la resistencia de E. faecium, el desarrollo de las pruebas rápidas que permitan diferenciar entre las especies de enterococos es importante para la terapia apropiada y vigilancia de la resistencia. La E. faecalis OE ensayo PNA FISH (AdvanDx, Woburn, MA) utiliza especie de ácido nucleico peptídico (ANP), sondas de fluorescencia en el formato de hibridación in situ y ofrece un tiempo de resultados de 1,5 horas y el potencial de proporcionar información importante para la especie el tratamiento. Estudios multicéntricos, para evaluar el desempeño de las 1,5 horas E. faecalis / OE procedimiento PNA FISH en comparación con el procedimiento del ensayo original de 2,5 horas y de métodos bacteriológicos estándar para la identificación de enterococos directamente de una botella de hemocultivo positivo.

Watch this Scientific Journal Video about A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures at JoVE.com

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Un protocolo rápido para la identificación directa de Enterococcus faecalis y otras especies de Enterococcus en un hemocultivo positivo con un ácido péptido nucleico ensayo de hibridación fluorescente in situ (FISH ANP).

Un procedimiento de 1,5 horas para la identificación de Enterococcus Especies directamente los cultivos de sangre

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

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Research

JoVE Journal

Immunology and Infection

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

20.7K vistas.  Cedars-Sinai Medical Cente. Un protocolo rápido para la identificación directa de Enterococcus faecalis y otras especies de Enterococcus en un hemocultivo positivo con un ácido péptido nucleico ensayo de hibridación fluorescente in situ (FISH ANP).

Video: A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Investigación

Inmunología e Infección

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Un día de flujo de trabajo Método de detección de patógenos bacterianos y pruebas de resistencia a los antimicrobianos de cultivos de sangre

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Aislamiento de los precursores de células B subconjuntos de Sangre del Cordón Umbilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extracción de proteínas totales y 2-D Métodos gel de electroforesis para<em&gt; Burkholderia</em&gt; Especies

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedimientos para la Identificación de priones infecciosos después de pasar por el sistema digestivo de una especie de aves

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

La identificación rápida de bacterias gram negativas de Cultura sangre caldo de cultivo utilizando MALDI-TOF espectrometría de masas

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparación de una Cultura de pellets de sangre para Rapid bacteriana Identificación y Prueba de Susceptibilidad a los antibióticos

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Generation and Identification of GM-CSF Derived Alveolar-like Macrophages and Dendritic Cells From Mouse Bone Marrow

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.

Bone marrow cells cultured with granulocyte macrophage colony stimulating factor (GM-CSF) generate a heterogeneous culture containing macrophages and dendritic cells (DCs). This method highlights using MHCII and hyaluronan (HA) binding to differentiate macrophages from the DCs in the GM-CSF culture. Macrophages in this culture have many similarities to alveolar macrophages.

Generación e identificación de GM-CSF Derivado alveolares-como los macrófagos y las células dendríticas A partir de médula ósea de ratones

Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

El uso de una monocapa de monocitos Ensayo para evaluar la fagocitosis mediada por el receptor Fc gamma

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...