Estudos foram patrocinados pela AdvanDx.

1. Coleta e Preparação <ol><li> Coleta de sangue venoso de pacientes com suspeita de sepse e colocar em duas BACTEC (BD, Sparks, MD) garrafas de cultura de sangue, uma aeróbica e anaeróbica um, juntos, constituído por um conjunto de hemocultura. </li><li> Coloque as garrafas no instrumento BACTEC 9240 a 35 ° C. </li><li> Incubar até que o instrumento é acionado em alarme devido ao crescimento de organismos no frasco de cultura de sangue. </li><li> Remover garrafa do instrumento hemocultura. </li></ol> 2. Preparação de reagentes antes da coloração <ol><li> Prepare a solução de lavagem adicionando 4 mL de solução de lavagem 60x seguido por 240 mL de água deionizada ou destilada fresca diretamente para o prato de coloração. </li><li> Coloque o prato coloração nos 55 ° C banho-maria para aquecer. </li><li> Armazenar os restantes concentrar a 2-8 ° C. </li><li> Remover o meio de montagem do frigorífico e à temperatura ambiente. </li></ol> 3. Coloração Gram <ol><li> Agite suavemente o frasco de hemocultura. </li><li> Usando uma agulha e seringa ou um aparelho de subcultura, remoção de sangue (cerca de 5 mL) da garrafa e coloque em um tubo de rosca estéril tampado. </li><li> Usando uma pipeta estéril, coloque uma gota grande de sangue em uma lâmina de vidro. </li><li> O ar seco do slide e fixar em metanol por 10 segundos. </li><li> Permitir que deslize para o ar seco. </li><li> Fazer a coloração Gram no filme de sangue para determinar a morfologia do organismo em crescimento no frasco de cultura de sangue. </li><li> Usando uma lente de imersão em óleo, slides. Cocos Gram positivos em pares e cadeias curtas observada na coloração de Gram é mais consistente com espécies de Enterococcus. </li><li> Este resultado Gram, em seguida, dirige a seleção do próprio kit sonda FISH PNA a ser usado para identificação bacteriana. Esta cultura de sangue serão coradas pela E. faecalis / OE PNA FISH mancha. </li></ol> 4. FISH PNA Stain <ol><li> Misture o tubo contendo o sangue gentilmente agitando antes da preparação de esfregaço. </li><li> Coloque uma gota de solução de fixação sobre o bem de uma lâmina de microscópio FISH PNA. </li><li> Transfira 10 mL ou uma pequena gota do tubo com a hemocultura positiva para a solução de fixação e misture delicadamente para emulsionar. </li><li> Fixar os esfregaços pelo aquecimento por 20 min. a 55 ° C em uma placa de aquecimento. </li></ol> 5. Material de controlo de qualidade Um positivo e outro negativo lâmina de controlo de qualidade devem ser testados com cada lote de slides para a coloração. <ol><li> Use E. faecalis / OE Controle Slides comprado de AdvanDx para esse fim ou preparar esfregaços de culturas líquidas de cepas de referência de E. faecalis e E. faecium como controle positivo quer em slides separados ou misturados em um slide e Staphylococcus spp ou Streptococcus spp como material de Controlo Negativo. </li></ol> Os resultados QC deve ser capaz de monitorar as condições de ensaio adequadas, particularmente aquelas que afetam rigor hibridação e penetração da parede celular, uma vez que a metodologia PNA é projetado para otimizar a penetração da parede celular 6. Hibridização <ol><li> Adicione uma gota de E. faecalis / OE PNA para o bem na lâmina de microscópio com o esfregaço. </li><li> Adicionar lamela. Evite bolhas de ar. </li><li> Incubar por 30 ± 5 min. a 55 ± 1 ° C na placa de aquecimento </li><li> Após lugar de incubação, as lâminas em porta corrediça </li></ol> 7. Lavar rigorosos <ol><li> Transportadora deslize mergulhar na solução de lavagem pré-aquecido a 55 ° C e remova cuidadosamente a lamínula. Muitas vezes, a lamela desliza suavemente agitando o slide na solução de lavagem. Ocasionalmente, a lamela deve ser delicadamente empurrado para fora com uma pinça. </li><li> Incubar na solução de lavagem para 30 ± 5 min. a 55 ± 1 ° C. </li><li> Remover as lâminas da solução de lavagem </li><li> Permitir que o slide para o ar seco </li></ol> 8. Montagem <ol><li> Adicione uma gota de meio de montagem a cada slide </li><li> Adicionar lamela. Evite bolhas de ar. </li><li> Examine a lâmina em um microscópio de fluorescência dentro de 2 horas. </li><li> Não exponha os slides para direcionar a luz do sol ou outras fontes de luz forte, pois isso pode levar a extinção de fluorescência. </li></ol> 9. Interpretação dos Resultados O microscópio de fluorescência usado para exame de slides deve ser equipada com o Filtro de Banda AdvanDx dupla e um objetivo de petróleo 60x ou 100x. Os slides QC deve ser examinado primeiro a confirmar que a hibridização ocorreu. E. faecalis deve aparecer como verde brilhante cocos fluorescentes em vários campos de vista e os E. faecium aparecerá como vermelho brilhante cocos. Não enterococos lâmina de controlo deve aparecer nonfluorescent. Após a confirmação do sistema nos controles, as lâminas paciente pode ser examinado. No início, o sangue film aparecerá avermelhada, mas o vermelho e verde cocos será bastante aparente. <img src="/files/ftp_upload/2616/2616fig1.jpg" alt="Figura 1" /> Figura 1. Exemplos representativos de verde-positivo E. faecalis (à esquerda), o vermelho positivo E. faecium (meio) e negativos (direita) de teste. 10. Resultados representante Três instituições foram incluídos em um estudo clínico multicêntrico avaliando este PNA FISH mancha e comparando um protocolo de 2,5 horas com o protocolo encurtado 1,5 hora que acaba de ser revistos. Um total de 152 cocos Gram positivos de rotina em pares e correntes (GPC) garrafas de cultura positiva de sangue foram incluídos nos estudos. Houve 100% (152/152) concordância entre os resultados da modificação e do procedimento do ensaio original de E. faecalis / OE PNA FISH: 41/41 E. faecalis; 33/33 enterococos e outros 78/78 GPC outros (Tabela 1). Este método de coloração tinha extraordinária sensibilidade e especificidade durante este ensaio clínico (Tabela 2). <table border="1"><tbody><tr><td></td><td> Métodos de rotina </td><td> E. faecalis / OE PNA Procedimento Padrão FISH </td><td> E. faecalis / OE Procedimento PNA FISH curto </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 (Red Positivo) </td><td> 27 (Red Positivo) </td></tr><tr><td> E. casseliflavus </td><td> 2 </td><td> 2 (Red Positivo) </td><td> 2 (Red Positivo) </td></tr><tr><td> E. gallinarum </td><td> 2 </td><td> 2 (Red Positivo) </td><td> 2 (Red Positivo) </td></tr><tr><td> Outros Enterococcus spp. </td><td> 2 </td><td> 2 (Red Positivo) </td><td> 2 (Red Positivo) </td></tr><tr><td> S. pneumoniae </td><td> 17 </td><td> 17 (negativo) </td><td> 17 (negativo) </td></tr><tr><td> S. viridans </td><td> 33 </td><td> 33 (negativo) </td><td> 33 (negativo) </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> Outros 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 concordância </td><td> 152/152 100% de concordância </td></tr></tbody></table> Tabela 1. Listagem de bactérias testados usando tanto o padrão (2.5hr) e rápida protocolo (1,5 h). <table border="1"><tbody><tr><td> E.faecalis sensibilidade </td><td> Outros sensibilidade Enterococcus spp. </td><td> Especificidade </td></tr><tr><td> 100% (41/41) IC 95% (93,0-100) </td><td> 100% (33/33) 33/33 IC 95% (91,3-100) </td><td> 100% (78/78) IC 95% (96,2-100) </td></tr></tbody></table> Tabela 2. Sensibilidade e Especificidade do protocolo de 1,5 horas FISH 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>

A produção deste vídeo-artigo foi patrocinado pela AdvanDx. Benjamin Cristal é um empregado de AdvanDx, que faz com que as ferramentas e os reagentes utilizados neste artigo.

O E. faecalis / OE teste FISH PNA para Enterococcus pode fornecer identificação de 2-3 dias mais cedo do que métodos de cultura padrão. O procedimento abreviado para o ensaio (1,5 hourr) fornece a identificação rápida que pode ser usado para uma melhor abordagem terapêutica antimicrobiana e evolução de pacientes. Um estudo para apoiar esta foi realizada por Forrest, et al. (6). Mais de dois anos consecutivos a partir de 2005, o laboratório de microbiologia cocos Gram positivos identificados em pares e cadeias em crescimento em garrafas de cultura de sangue convencionais por métodos microbiológicos e, em 2006, adicionando a E. faecalis / OE FISH PNA. Além disso, um algoritmo de tratamento desenvolvido pela equipe instituições antimicrobianos (AMT) para efetivamente utilizar os dados PNA FISH gerado pelo laboratório em tempo hábil. Desfecho primário avaliado foi o tempo de cultura de sangue sorteio para a implementação da terapêutica antimicrobiana eficaz antes e depois de FISH PNA foi instituído no fluxo de trabalho laboratórios. Gravidade da doença local, paciente e terapia antimicrobiana empírica foram medidos. Um total de 224 pacientes com bacteremia hospitalar adquirida enterococos foram avaliados com 129 no período pré-intervenção e 95 no período FISH PNA. FISH PNA identificados E. faecalis 3 dias mais cedo do que as culturas convencionais (1,1 vs 4,1 dias, p &lt;0,001). FISH PNA identificados E. faecium em média 2,3 dias mais cedo (1,1 vs 3,4 dias, p &lt;0,001) e foi associado à redução estatisticamente significativa no tempo de se iniciar a terapêutica eficaz (1,3 vs 3,1 dias, p &lt;0,001) e diminuição 30 dias mortalidade (26% vs 45%, p = 0,04). Este grupo concluiu que o E. faecalis / OE teste FISH PNA em conjunto com um algoritmo de tratamento AMT resultou em início precoce da terapia empírica antimicrobiano apropriado para pacientes com adquiridas no hospital E. faecium bacteremia. <ol><li> Reagentes não deve ser utilizado após a data de validade impressa nos rótulos e reagentes não devem ser modificados de qualquer forma. Eles devem permanecer na geladeira quando não em uso ou podem perder a potência. </li><li> Garrafas de cultura de sangue precisam ser homogeneizado antes da amostragem. </li><li> É importante para não permitir que a ponta frasco conta-gotas para tocar um esfregaço de sangue, porque isso pode causar a contaminação cruzada de material entre os slides, ou causar contaminação do reagente. </li><li> Verifique se o banho de água eo bloco de aquecimento são mantidos em constante a 55 ° C. </li><li> Não use outros filtros que o filtro Dual Band ou cores não serão claramente discernida. </li><li> Não use lâminas de microscópio diferente do Lâminas de microscopia ou hibridização não irá ocorrer. </li><li> É importante que o microscópio está funcionando corretamente. Verifique se a lâmpada do microscópio é aprovado dentro do número de horas de uso. </li></ol><ul><li> Algumas espécies raras de ocorrência Enterococcus não são detectados pela sonda PNA hibridação de espécies de Enterococcus, tais como E. asinii, E.dispar, haemoperoxidus E., cecorum E., columbae E., saccharolyticus E., E. e E solitarius . sulfurosos. </li><li> Moraviensis E. é identificado como E. faecalis devido a semelhanças seqüência. </li><li> Algumas cepas de Streptococcus anginosus produzir uma fluorescência verde falso positivo devido a semelhanças seqüência. </li><li> Garrafas de cultura VersaTREK sangue não foram avaliados durante os estudos clínicos. O desempenho para detectar E. faecalis e Enterococcus spp outros. com garrafas de cultura VersaTREK sangue é desconhecido. </li><li> Autofluorescência falso positivo verde pode ocorrer se um padrão FITC filtro é usado em vez do filtro Dual Band. </li><li> Resultados falso-negativos podem ocorrer com pouca freqüência devido ao crescimento misto ou por erro na técnica de ensaio. </li><li> O tipo e condição da instrumentação utilizada vai influenciar a aparência visual da imagem obtida. A fluorescência pode varydue com o tipo de microscópio de empregados, a fonte de luz eo nível de rRNA nas células </li><li> Isolamento em meios sólidos é necessária para diferenciar crescimento misturado com outros organismos e identificar hemocultura positiva gerando um resultado negativo. </li><li> O produto não foi validado com outras amostras de hemoculturas. </li></ul>

Reagentes E. faecalis / OE PNA FISH é composto dos componentes do kit seguintes: <ul><li> Solução de fixação Fixação Solução 3 mL tampão fosfato com detergente. </li><li> E. faecalis / OE PNA E. faecalis / OE PNA 1,5 mL PNA sondas de hibridização. solução. Contém formamida 30%. </li><li> Lavar 60x Solution Solução de Lavagem 60x 50 mL Tris-salina tamponada com detergente </li><li> Meio de Montagem Montagem Médio inibidor de 3 mL fotobranqueamento em Glicerol </li><li> Controle de qualidade de slides </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

Enterococos são uma causa comum de bacteremia com E. faecalis sendo a espécie predominante seguida pela E. faecium. Porque a resistência à ampicilina e vancomicina em E. faecalis ainda é incomum em relação à resistência em E. faecium, o desenvolvimento de testes rápidos permitindo a diferenciação entre as espécies de enterococos é importante para a terapêutica adequada e vigilância da resistência. O E. faecalis OE teste FISH PNA (AdvanDx, Woburn, MA) usa espécie-específica de ácidos nucleicos peptídeo (PNA) sondas de fluorescência em formato de hibridização in situ e oferece um tempo para resultados de 1,5 horas e o potencial de fornecer informações importantes para a espécie-específicos o tratamento. Estudos multicêntricos foram realizados para avaliar o desempenho do E. 1,5 horas faecalis / OE procedimento FISH PNA em comparação com o procedimento de ensaio originais 2,5 horas e aos métodos de bacteriologia padrão para a identificação de enterococos diretamente de uma garrafa de sangue positivo cultura.

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

Um protocolo rápido para a identificação directa de Enterococcus faecalis e Enterococcus outras espécies a partir de uma hemocultura positiva usando um ácido nucléico Peptide fluorescentes em ensaio de hibridização in situ (FISH PNA).

Um Procedimento de 1,5 horas para a Identificação de Enterococcus Espécies Diretamente de hemoculturas

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

a-15-hour-procedure-for-identification-enterococcus-species-directly

Research

JoVE Journal

Immunology and Infection

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

20.7K visualizações.  Cedars-Sinai Medical Cente. Um protocolo rápido para a identificação directa de Enterococcus faecalis e Enterococcus outras espécies a partir de uma hemocultura positiva usando um ácido nucléico Peptide fluorescentes em ensaio de hibridização in situ (FISH PNA).

Vídeo: 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.

Apresentando tensão de cisalhamento em o Estudo da adesão 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 ...

Imunologia e Infecção

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.

Um dia-Esquema de fluxo de trabalho para detecção de patógenos de bactérias e de resistência antimicrobiana de hemoculturas

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.

Isolamento de precursor de células B subconjuntos de Sangue do Cordão 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.

Extração de proteínas e 2-D métodos Eletroforese em Gel de<em&gt; Burkholderia</em&gt; Espécies

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.

Procedimentos para identificar Infecciosas príons após a passagem através do sistema digestivo de uma espécie 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.

Rápida identificação de bactérias gram-negativas de Sangue Cultura Caldo Usando MALDI-TOF Espectrometria de Massa

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.

Isolamento, identificação e purificação de células epiteliais do 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.

Preparação de uma Cultura Pellet sangue para rápida identificação bacteriana e Antibiótico Teste à Susceptibilidade

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.

Geração e identificação de GM-CSF macrófagos derivados e dendríticas células alveolares do tipo de ratinho de Medula Óssea

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.

Uso de um monócito monocamada Ensaio para Avaliar a fagocitose mediada pelo receptor Fcy

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