Esta labor contó con el apoyo de la Agencia de los Estados Unidos para el Desarrollo Internacional, Oficina de Asistencia para Desastres en el Extranjero (AID-OFDA-A-15-00026). Marlene Wolfe recibió el apoyo de la National Science Foundation (subvención 0966093).

<ol>
	<li>Kampf, G., Kramer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiologic+Background+of+Hand+Hygiene+and+Evaluation+of+the+Most+Important+Agents+for+Scrubs+and+Rubs.">Epidemiologic Background of Hand Hygiene and Evaluation of the Most Important Agents for Scrubs and Rubs.</a> Clin Microbiol Rev. 17 (4), 863-893 (2004).</li><li>Miller, T., Patrick, D., Ormrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+decontamination:+influence+of+common+variables+on+hand-washing+efficiency.">Hand decontamination: influence of common variables on hand-washing efficiency.</a> Healthc Infect. 16 (1), 18 (2013).</li><li>Jensen, D. A., Danyluk, M. D., Harris, L. J., Schaffner, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+care+workers'+hand+contamination+levels+and+antibacterial+efficacy+of+different+hand+hygiene+methods+used+in+a+Vietnamese+hospital.">Health care workers' hand contamination levels and antibacterial efficacy of different hand hygiene methods used in a Vietnamese hospital.</a> Am J Infect Control. 42 (2), 178-181 (2014).</li><li>Steinmann, J., Nehrkorn, R., Meyer, A., Becker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+in-vivo+protocols+for+testing+virucidal+efficacy+of+handwashing+and+hand+disinfection.">Two in-vivo protocols for testing virucidal efficacy of handwashing and hand disinfection.</a> Int J Hyg Environ Health. 196 (5), 425-436 (1995).</li><li>Weber, D. J., Sickbert-Bennett, E., Gergen, M. F., Rutala, W. 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H., Duizer, E., Beumer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+viral+contamination+from+finger+pads:+handwashing+is+more+effective+than+alcohol-based+hand+disinfectants.">Reducing viral contamination from finger pads: handwashing is more effective than alcohol-based hand disinfectants.</a> J Hosp Infect. 90 (3), 226-234 (2015).</li><li>Steinmann, J., Paulmann, D., Becker, B., Bischoff, B., Steinmann, E., Steinmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+virucidal+activity+of+alcohol-based+hand+sanitizers+versus+antimicrobial+hand+soaps+in+vitro+and+in+vivo.">Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.</a> J Hosp Infect. 82 (4), 277-280 (2012).</li><li>de Aceituno, A. F., Bartz, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ability+of+Hand+Hygiene+Interventions+Using+Alcohol-Based+Hand+Sanitizers+and+Soap+To+Reduce+Microbial+Load+on+Farmworker+Hands+Soiled+during+Harvest.">Ability of Hand Hygiene Interventions Using Alcohol-Based Hand Sanitizers and Soap To Reduce Microbial Load on Farmworker Hands Soiled during Harvest.</a> J Food Protect. 78 (11), 2024-2032 (2015).</li><li>Boyce, J. 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UNDP Medical Waste Experts Assessment and Recommendations Regarding Management of Ebola-Contaminated Waste Available from: <a target="_blank" href="https://noharm-global.org/sites/default/files/documents-files/3127/Report%20to%20WHO%20WASH%20and%20Geneva%20on%20Ebola%20final.pdf">https://noharm-global.org/sites/default/files/documents-files/3127/Report%20to%20WHO%20WASH%20and%20Geneva%20on%20Ebola%20final.pdf</a> (2015)</li><li>Hopman, J., Kubilay, Z., Allen, T., Edrees, H., Pittet, D., Allegranzi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+chlorine+solutions+used+for+hand+hygiene+and+gloves+disinfection+in+Ebola+settings:+a+systematic+review.">Efficacy of chlorine solutions used for hand hygiene and gloves disinfection in Ebola settings: a systematic review.</a> Antimicrob Resist Infect Control. 4 (1), 1 (2015).</li><li>Lowbury, E. J. L., Lilly, H. A., Bull, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disinfection+of+hands:+removal+of+transient+organisms.">Disinfection of hands: removal of transient organisms.</a> BMJ. 2 (5403), 230-233 (1964).</li><li>Edmonds, S. L., Zapka, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+Hand+Hygiene+for+Removal+of+Clostridium+difficile+Spores+from+Hands.">Effectiveness of Hand Hygiene for Removal of Clostridium difficile Spores from Hands.</a> Infect Control Hosp Epidemiol. 34 (3), 302-305 (2013).</li><li>Rotter, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=150+years+of+hand+disinfection-Semmelweis'+heritage.">150 years of hand disinfection-Semmelweis' heritage.</a> Hyg Med. (22), 332-339 (1997).</li><li>Hitomi, S., Baba, S., Yano, H., Morisawa, Y., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+effects+of+electrolytic+products+of+sodium+chloride--comparative+evaluation+with+sodium+hypochlorite+solution+and+efficacy+in+handwashing.">Antimicrobial effects of electrolytic products of sodium chloride--comparative evaluation with sodium hypochlorite solution and efficacy in handwashing.</a> Kansensh&#333;gaku Zasshi. 72 (11), 1176-1181 (1998).</li><li>. Standard E1174-13. Standard Test Method for Evaluation of the Effectiveness of Health Care Personnel Handwash Formulations Available from: <a target="_blank" href="https://www.astm.org/">https://www.astm.org/</a> (2013)</li><li>Casanova, L. M., Weaver, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+eluents+for+the+recovery+of+an+enveloped+virus+from+hands+by+whole-hand+sampling.">Evaluation of eluents for the recovery of an enveloped virus from hands by whole-hand sampling.</a> J Appl Microbiol. 118 (5), 1210-1216 (2015).</li><li>Sinclair, R. G., Rose, J. B., Hashsham, S. A., Gerba, C. P., Haas, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criteria+for+Selection+of+Surrogates+Used+To+Study+the+Fate+and+Control+of+Pathogens+in+the+Environment.">Criteria for Selection of Surrogates Used To Study the Fate and Control of Pathogens in the Environment.</a> Appl Environ Microbiol. 78 (6), 1969-1977 (2012).</li><li>Held, E., Skoet, R., Johansen, J. 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Los autores declaran que no tienen intereses financieros en competencia.

The method described here provides an approach for testing handwashing efficacy in a controlled laboratory setting. This method highlights the use of human volunteers and surrogate, non-infectious organisms. Using the method, it was possible to demonstrate differences in: 1) the efficacy of handwashing methods and 2) organism persistence in rinse water. The purpose of presenting this protocol is to provide a general framework that can be adapted to test a wide range of surrogate organisms and handwashing methods relevant...

El lavado de manos es ampliamente recomendado para prevenir la propagación de enfermedades, particularmente las transmitidas por vía fecal-oral o aérea, incluyendo enfermedades diarreicas y respiratorias 1 . Sorprendentemente, hay poca evidencia comparable sobre la eficacia de los métodos de lavado de manos, como el lavado de manos con jabón y agua (HWWS) y con desinfectante de manos a base de alcohol (ABHS), en la eliminación de organismos de las manos. La investigación inicial ha encontrado que la acción mecánica del lavado de manos, en comparación con el método de lavado de manos, puede explicar la mayor parte de la eliminación del organismo 2 , 3 . Además, hay poca evidencia comparativa sobre qué método de lavado de manos es más eficaz. En una revisión informal de la literatura, se identificaron 14 estudios que compararon la eficacia del jabón y el desinfectante para las manos en la eliminación de organismos. De estos estudios, cinco encontraron ABHS para ser más eficaz 4 , </Sup&gt; 5 , 6 , 7 , 8 , siete encontró HWWS a ser más eficaces 9 , 10 , 11 , 12 , 13 , 14 , 15 , y dos no encontraron diferencias significativas entre los métodos 16 , 17 . Estos hallazgos son inconsistentes y no abordan el riesgo continuo de enfermedad por la persistencia de organismos en el agua de enjuague después del lavado de manos. En general, las pruebas sobre la eficacia comparativa de los métodos de lavado de manos para la eliminación de patógenos causantes de enfermedades infecciosas son limitadas. Esta evidencia limitada ha llevado a la incertidumbre sobre qué métodos son los más apropiados en los ajustes del brote. Por ejemplo, Durante el brote de Ebola Virus Disease (EVD) en África Occidental de 2013 a 2016, varios grandes respuestas internacionales proporcionaron recomendaciones contradictorias para HWWS, ABHS o soluciones al 0,05% de cloro. Médecins Sans Frontières (MSF) recomienda el uso de solución de cloro al 0,05% para el lavado de manos, mientras que la Organización Mundial de la Salud (OMS) recomienda HWWS o ABHS (si las manos no están visiblemente sucias). La OMS llega a afirmar que el cloro no debe utilizarse a menos que no haya otras opciones disponibles, ya que es menos eficaz que otros métodos debido a la demanda de cloro ejercida por la piel 18 , 19 , 20 , 21 , 22 . Además, las soluciones de cloro se producen comúnmente a partir de cuatro compuestos de cloro diferentes, incluyendo hipoclorito de alta prueba (HTH), hipoclorito de sodio generado localmente y estabilizado (NaOCl), y sodDicloroisocianurato de ium (NaDCC). Una revisión sistemática encargada por la OMS en respuesta al brote EVD en África Occidental recientemente encontró sólo cuatro estudios que investigan la eficacia comparativa del lavado de manos con cloro 23 . Estos estudios también produjeron resultados contradictorios, y ninguno de estos estudios utilizó la concentración de cloro recomendada del 0,05% para el lavado de manos o investigó microorganismos similares al virus Ebola 10 , 24 , 25 , 26 , 27 . Por lo tanto, no se encontró que las recomendaciones estuvieran basadas en pruebas científicas y no estaba claro cuáles de las recomendaciones eran más eficaces. Se necesitan más investigaciones para comparar los métodos de lavado de manos para prevenir la propagación de patógenos infecciosos, ya que las intervenciones de lavado de manos son una herramienta importante para prevenir la transmisión de enfermedades epidémicas. Estos hY las recomendaciones de lavado deben basarse en pruebas. Por lo tanto, se desarrolló un método para probar la eficacia del lavado de manos y la persistencia del agua de enjuague, realizada con sustitutos o patógenos no infecciosos2 , 28 , 29 . Se presentan aquí los resultados de la muestra, usando Phi6 como un sustituto del virus Ebola y usando Escherichia coli como un organismo indicador común. En este protocolo se presentan las pruebas de eficacia de lavado de manos y de persistencia del agua de enjuague.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Soap bar</td>
			<td>Dove</td>
			<td colspan="2">White Beauty Bar soap</td>
		</tr>
		<tr>
			<td>Alcohol-based hand sanitizer</td>
			<td>Purell</td>
			<td colspan="2">Advanced Instant Hand Sanitizer with 70% Ethyl Alcohol</td>
		</tr>
		<tr>
			<td>HTH Powder</td>
			<td>Acros Organics</td>
			<td>300340010</td>
			<td></td>
		</tr>
		<tr>
			<td>NaDCC Powder</td>
			<td>Medentech</td>
			<td>Klorsept granules</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOCl Solution</td>
			<td>Acros Organics</td>
			<td>419550010</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrochlorinator</td>
			<td>AquaChlor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodometric titrator</td>
			<td>Hach</td>
			<td>1690001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MP Biomedicals</td>
			<td>NC0117242</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>BP1421-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Mucin</td>
			<td>EMD Milipore</td>
			<td>49-964-3500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m Filter</td>
			<td>EMD Milipore</td>
			<td>GVWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin pH probe</td>
			<td>Hanna Instruments</td>
			<td>H199181</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Whirlpak Sample Bag</td>
			<td>Nasco</td>
			<td>B01447WA</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Whirlpak Sample Bag</td>
			<td>Nasco</td>
			<td>B01323WA</td>
			<td></td>
		</tr>
		<tr>
			<td>Funnel bottle</td>
			<td>Thermo Scientific</td>
			<td>3120850001</td>
			<td>You may drill an appropriately sized hole in the lid of a bottle to form a funnel that will dispense water at the appropriate flow rate</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>ThermoScientific</td>
			<td>615090010</td>
			<td>Mix with water to produce 70% ethanol</td>
		</tr>
		<tr>
			<td>Spray bottle</td>
			<td>Qorpak</td>
			<td>PLC06934</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli</td>
			<td>ATCC</td>
			<td>25922</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>Fisher BioReagents</td>
			<td>BP1426-2</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Fisher BioReagents</td>
			<td>BP1425-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile loop</td>
			<td>Globe Scientific</td>
			<td>22-170-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi6</td>
			<td>HER</td>
			<td>102</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient broth</td>
			<td>BD Difco</td>
			<td>BD 247110</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneQuant 100 Spectrophotometer</td>
			<td>General Electric</td>
			<td>28-9182-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>Fisher Chemical</td>
			<td>S445-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane filter (47mm, 0.45 &#181;m)</td>
			<td>EMD Millipore</td>
			<td>HAWP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>m-ColiBlue24 broth media</td>
			<td>EMD Millipore</td>
			<td>M00PMCB24</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish with pad (47mm)</td>
			<td>Fisherbrand</td>
			<td>09-720-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Manifold</td>
			<td>Thermo Scientific/Nalgene</td>
			<td>09-752-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter funnels</td>
			<td>Thermo Scientific/Nalgene</td>
			<td>09-747</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas syringae</td>
			<td>HER</td>
			<td>1102</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Thermo Scientific</td>
			<td>10010031</td>
			<td>Solution may also be mixed from source compounds according to any basic recipe</td>
		</tr>
	</tbody>
</table>

a method to test the efficacy of handwashing for the removal of emerging infectious pathogens

El lavado de manos es ampliamente recomendado para prevenir la transmisión de enfermedades infecciosas. Sin embargo, hay poca evidencia comparable sobre la eficacia de los métodos de lavado de manos en general. Adicionalmente, existe poca evidencia que compara los métodos de lavado de manos para determinar cuáles son los más eficaces para eliminar patógenos infecciosos. Se necesitan investigaciones para aportar pruebas de los diferentes enfoques del lavado de manos que pueden emplearse durante los brotes de enfermedades infecciosas. Aquí se describe un método de laboratorio para evaluar la eficacia de los métodos de lavado de manos en la eliminación de microorganismos de las manos y su persistencia en el agua de enjuague. Las manos de los voluntarios se puntean primero con el organismo de prueba y luego se lavan con cada método de lavado de manos de interés. En general, los microorganismos sustitutivos se utilizan para proteger a los seres humanos de la enfermedad. El número de organismos que permanecen en las manos de los voluntarios después del lavado se prueba usando un método modificado de &quot;jugo de guante&quot;: las manos se colocan en guantes con un eluY se lavan para suspender los microorganismos y hacerlos disponibles para su análisis por filtración en membrana (bacterias) o ensayo de placa (virus / bacteriófagos). El agua de enjuague producida por el lavado de manos se recoge directamente para su análisis. La eficacia del lavado de manos se cuantifica comparando el valor de reducción logarítmica entre las muestras tomadas después del lavado de manos y las muestras sin lavado de manos. La persistencia del agua de enjuague se cuantifica comparando muestras de agua de enjuague de varios métodos de lavado de manos con muestras recogidas después de lavarse las manos con agua justa. Aunque este método está limitado por la necesidad de usar organismos sustitutivos para preservar la seguridad de los voluntarios humanos, captura aspectos del lavado de manos que son difíciles de replicar en un estudio in vitro y llena vacíos de investigación sobre la eficacia del lavado de manos y la persistencia de organismos infecciosos en el enjuague agua.

Aquí, el protocolo ( Figura 1 ] se completó con 18 voluntarios, que fueron cada uno de los ensayos utilizando tanto E. coli y Phi6. Se encontraron diferencias significativas entre los resultados de lavado de manos con E. coli con y sin carga de suelo y Phi6 con carga de suelo ( Figura 2 y Figura 3 ). Para E. coli sin carga del suelo, el lavado de manos con HTH, NaDCC y N...

Watch this Scientific Journal Video about A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens at JoVE.com

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

El lavado de manos es ampliamente recomendado para prevenir la transmisión de enfermedades infecciosas. Sin embargo, hay poca evidencia de que los métodos de lavado de manos sean más eficaces para eliminar patógenos de enfermedades infecciosas. Se desarrolló un método para evaluar la eficacia de los métodos de lavado de manos en la eliminación de microorganismos.

Un método para probar la eficacia del lavado de manos para la eliminación de patógenos infecciosos emergentes

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

a-method-to-test-efficacy-handwashing-for-removal-emerging-infectious

Research

JoVE Journal

Immunology and Infection

18.8K vistas.  Tufts University.  El lavado de manos es ampliamente recomendado para prevenir la transmisión de enfermedades infecciosas. Sin embargo, hay poca evidencia de que los métodos de lavado de manos sean más eficaces para eliminar patógenos de enfermedades infecciosas. Se desarrolló un método para evaluar la eficacia de los métodos de lavado de manos en la eliminación de microorganismos. 

Video: A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

A 96 de microtitulación y placa basada en el método para la formación de Control y pruebas de sensibilidad antifúngica Candida albicans Las biopelículas

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Investigación

Inmunología e Infección

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Aislamiento de células del cerebro y la médula espinal mononucleares uso de gradientes de Percoll

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

El uso de un ensayo de microarrays Pan-viral (Virochip) a la pantalla de muestras clínicas de los patógenos virales

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminiscente &#39;reportero fagos para la detección de patógenos bacterianos de la categoría A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Diagnosing Pulmonary Tuberculosis with the Xpert MTB/RIF Test

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world population1, and almost two million people are killed by TB each year.2 Universal access to high-quality, patient-centered treatment for all TB patients is emphasized by WHO's Stop TB Strategy.3 The rapid detection of MTB in respiratory specimens and drug therapy based on reliable drug resistance testing results are a prerequisite for the successful implementation of this strategy. However, in many areas of the world, TB diagnosis still relies on insensitive, poorly standardized sputum microscopy methods. Ineffective TB detection and the emergence and transmission of drug-resistant MTB strains increasingly jeopardize global TB control activities.2
Effective diagnosis of pulmonary TB requires the availability - on a global scale - of standardized, easy-to-use, and robust diagnostic tools that would allow the direct detection of both the MTB complex and resistance to key antibiotics, such as rifampicin (RIF). The latter result can serve as marker for multidrug-resistant MTB (MDR TB) and has been reported in &gt; 95% of the MDR-TB isolates.4, 5 The rapid availability of reliable test results is likely to directly translate into sound patient management decisions that, ultimately, will cure the individual patient and break the chain of TB transmission in the community.2
Cepheid's (Sunnyvale, CA, U.S.A.) Xpert MTB/RIF assay6, 7 meets the demands outlined above in a remarkable manner. It is a nucleic-acids amplification test for 1) the detection of MTB complex DNA in sputum or concentrated sputum sediments; and 2) the detection of RIF resistance-associated mutations of the rpoB gene.8 It is designed for use with Cepheid's GeneXpert Dx System that integrates and automates sample processing, nucleic acid amplification, and detection of the target sequences using real-time PCR and reverse transcriptase PCR. The system consists of an instrument, personal computer, barcode scanner, and preloaded software for running tests and viewing the results.9 It employs single-use disposable Xpert MTB/RIF cartridges that hold PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is eliminated.6 Current nucleic acid amplification methods used to detect MTB are complex, labor-intensive, and technically demanding. The Xpert MTB/RIF assay has the potential to bring standardized, sensitive and very specific diagnostic testing for both TB and drug resistance to universal-access point-of-care settings3, provided that they will be able to afford it. In order to facilitate access, the Foundation for Innovative New Diagnostics (FIND) has negotiated significant price reductions. Current FIND-negotiated prices, along with the list of countries eligible for the discounts, are available on the web.10

The Xpert MTB/RIF test integrates sample decontamination, hands-free operation, on-board sample processing, and ultra-sensitive hemi-nested PCR for the simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance, either in expectorated sputum or concentrated sputum sediments, in approximately two hours. Testing is standardized and requires only moderate laboratory infrastructure and training.

El diagnóstico de tuberculosis pulmonar con el Xpert MTB / RIF prueba

Tuberculosis (TB) due to Mycobacterium tuberculosis (MTB) remains a major public health issue: the infection affects up to one third of the world ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Uso de un medio artificial de esputo para probar la eficacia de antibióticos contra la<em&gt; Pseudomonas aeruginosa</em&gt; En condiciones más pertinentes a la fibrosis quística pulmonar

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Usando Click Química para medir el efecto de la infección viral en el Host-Cell ARN Síntesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

El análisis in vitro de la respuesta inmune celular Myd88 mediada por el Virus del Nilo Occidental cepa mutante Infección

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Una estrategia rápida para el aislamiento de nuevos Faustoviruses de muestras ambientales Uso<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the current lack of an approved tularemia vaccine for humans, research is focused on vaccine development utilizing appropriate animal models. The Fischer 344 rat has emerged as a model that reflects human susceptibility to F. tularensis infection, and thus is an attractive model for tularemia vaccine development. Intratracheal inoculation of the Fischer 344 rat with F. tularensis mimics pulmonary exposure in humans. The successful delivery into the rat trachea is critical for pulmonary delivery. A laryngoscope with illumination is used to properly intubate the tracheae of anesthetized rats; the correct placement within the trachea is determined by a simple device to detect breathing. Following intubation, the F. tularensis culture is delivered in a measured dose via syringe. This technique standardizes pulmonary delivery of F. tularensis within the rat trachea to evaluate vaccine efficacy.

Intratracheal Inoculation of Fischer 344 Rats with Francisella tularensis

This protocol describes intratracheal inoculations of Fischer 344 rats with Francisella tularensis. This procedure mimics pulmonary exposure of humans to this potential biothreat agent and can be used to test vaccine and therapeutic efficacy against pulmonary tularemia.

La inoculación de Fischer 344 ratas con Francisella tularensis

Pulmonary infection with the bacterium Francisella tularensis can lead to the serious and potentially fatal disease, tularemia, in humans. Due to the ...