עבודה זו נתמכה על ידי הסוכנות האמריקאית לפיתוח בינלאומי, משרד סיוע חוץ מאסון (AID-OFDA-A-15-00026). מרלין וולף נתמך על ידי הקרן הלאומית למדע (מענק 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+effect+of+hand+wash+duration,+soap+use,+ground+beef+debris,+and+drying+methods+on+the+removal+of+Enterobacter+aerogenes+on+hands.">Quantifying the effect of hand wash duration, soap use, ground beef debris, and drying methods on the removal of Enterobacter aerogenes on hands.</a> J Food Prot. 78 (4), 685-690 (2015).</li><li>Girou, E., Loyeau, S., Legrand, P., Oppein, F., Brun-Buisson, C. <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+handrubbing+with+alcohol+based+solution+versus+standard+handwashing+with+antiseptic+soap:+randomised+clinical+trial.">Efficacy of handrubbing with alcohol based solution versus standard handwashing with antiseptic soap: randomised clinical trial.</a> BMJ. 325 (7360), 362 (2002).</li><li>Kac, G., Podglajen, I., Gueneret, M., Vaupr&#233;, S., Bissery, A., Meyer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+evaluation+of+two+hand+hygiene+procedures+achieved+by+healthcare+workers+during+routine+patient+care:+a+randomized+study.">Microbiological evaluation of two hand hygiene procedures achieved by healthcare workers during routine patient care: a randomized study.</a> J Hosp Infect. 60 (1), 32-39 (2005).</li><li>Lages, S. L. S., Ramakrishnan, M. A., Goyal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vivo+efficacy+of+hand+sanitisers+against+feline+calicivirus:+a+surrogate+for+norovirus.">In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus.</a> J Hosp Infect. 68 (2), 159-163 (2008).</li><li>Holton, R. H., Huber, M. A., Terezhalmy, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+efficacy+of+soap+and+water+hand+washing+versus+an+alcohol-based+hand+cleanser.">Antimicrobial efficacy of soap and water hand washing versus an alcohol-based hand cleanser.</a> Tex Dent J. 126 (12), 1175-1180 (2009).</li><li>Salmon, S., Truong, A. T., Nguyen, V. H., Pittet, D., McLaws, 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=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. A. <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+selected+hand+hygiene+agents+used+to+remove+Bacillus+atrophaeus+(a+surrogate+of+Bacillus+anthracis)+from+contaminated+hands.">Efficacy of selected hand hygiene agents used to remove Bacillus atrophaeus (a surrogate of Bacillus anthracis) from contaminated hands.</a> JAMA. 289 (10), 1274-1277 (2003).</li><li>Grayson, M. L., Melvani, S., 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=Efficacy+of+Soap+and+Water+and+Alcohol-Based+Hand-Rub+Preparations+against+Live+H1N1+Influenza+Virus+on+the+Hands+of+Human+Volunteers.">Efficacy of Soap and Water and Alcohol-Based Hand-Rub Preparations against Live H1N1 Influenza Virus on the Hands of Human Volunteers.</a> Clin Infect Dis. 48 (3), 285-291 (2009).</li><li>Oughton, M. T., Loo, V. G., Dendukuri, N., Fenn, S., Libman, M. 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+hygiene+with+soap+and+water+is+superior+to+alcohol+rub+and+antiseptic+wipes+for+removal+of+Clostridium+difficile.">Hand hygiene with soap and water is superior to alcohol rub and antiseptic wipes for removal of Clostridium difficile.</a> Infect Control Hosp Epidemiol. 30 (10), 939-944 (2009).</li><li>Liu, P., Yuen, Y., Hsiao, H. -. M., Jaykus, L. -. A., Moe, C. <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+liquid+soap+and+hand+sanitizer+against+Norwalk+virus+on+contaminated+hands.">Effectiveness of liquid soap and hand sanitizer against Norwalk virus on contaminated hands.</a> Appl Environ Micro. 76 (2), 394-399 (2010).</li><li>Savolainen-Kopra, C., Korpela, T., 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=Single+treatment+with+ethanol+hand+rub+is+ineffective+against+human+rhinovirus--hand+washing+with+soap+and+water+removes+the+virus+efficiently.">Single treatment with ethanol hand rub is ineffective against human rhinovirus--hand washing with soap and water removes the virus efficiently.</a> J Med Virol. 84 (3), 543-547 (2012).</li><li>Tuladhar, E., Hazeleger, W. C., Koopmans, M., Zwietering, M. 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. M., Pittet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guideline+for+Hand+Hygiene+in+Health-Care+Settings+Recommendations+of+the+Healthcare+Infection+Control+Practices+Advisory+Committee+and+the+HICPAC/SHEA/APIC/IDSA+Hand+Hygiene+Task+Force.">Guideline for Hand Hygiene in Health-Care Settings Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force.</a> Infect Control Hosp Epidemiol. 23 (12 Suppl), S3-S40 (2002).</li><li>. 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. D., Agner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hand+eczema+severity+index+(HECSI):+A+scoring+system+for+clinical+assessment+of+hand+eczema.+A+study+of+inter-+and+intraobserver+reliability.">The hand eczema severity index (HECSI): A scoring system for clinical assessment of hand eczema. A study of inter- and intraobserver reliability.</a> Br J Dermatol. 152 (2), 302-307 (2005).</li><li>. Method 1604: Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a Simultaneous Detection Technique (MI Medium) Available from: <a target="_blank" href="https://www.epa.gov/sites/production/files/2015-08/documents/method_1604_2002.pdf">https://www.epa.gov/sites/production/files/2015-08/documents/method_1604_2002.pdf</a> (2002)</li><li>Adams, M. H., Anderson, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacteriophages. , (1959).</li><li>Kao, L. S., Green, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Variance:+Is+There+a+Difference+in+Means+and+What+Does+It+Mean?.">Analysis of Variance: Is There a Difference in Means and What Does It Mean?.</a> The Journal of surgical research. 144 (1), 158-170 (2008).</li><li>Schutz, R. W., Gessaroli, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Analysis+of+Repeated+Measures+Designs+Involving+Multiple+Dependent+Variables.">The Analysis of Repeated Measures Designs Involving Multiple Dependent Variables.</a> Research Quarterly for Exercise and Sport. 58 (2), 132-149 (1987).</li><li>Woolwine, J. D., Gerberding, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+testing+method+on+apparent+activities+of+antiviral+disinfectants+and+antiseptics.">Effect of testing method on apparent activities of antiviral disinfectants and antiseptics.</a> Antimicrob Agents Chemother. 39 (4), 921-923 (1995).</li></ol>

המחברים מצהירים כי אין להם אינטרסים פיננסיים מתחרים.

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...

שטיפת ידיים מומלצת באופן נרחב על מנת למנוע את התפשטות המחלה, במיוחד אלה המועברים על ידי הצוואר הפה או אוראלי, כולל מחלות שלשולים ונשימה. באופן מפתיע, יש עדויות רבות על יעילותן של שיטות שטיפת הידיים, כגון שטיפת ידיים במים וסבון (HWWS) ועם אלכוהול על בסיס אלכוהול (ABHS), על הסרת אורגניזמים מן הידיים. מחקר ראשוני מצא כי הפעולה המכנית של שטיפת ידיים, בניגוד לשיטת שטיפת הידיים, עשויה להסביר את רוב הסרת האורגניזם 2 , 3 . בנוסף, יש ראיות השוואתי קטן על אשר שיטת שטיפת ידיים היא היעילה ביותר. בסקירה ספרותית לא רשמית, 14 מחקרים אשר השוו את יעילות הסבון ותמצית היד על הסרת אורגניזמים זוהו. מתוך מחקרים אלה, חמישה מצאו ABHS להיות יעיל יותר 4 , </Sup&gt; 5 , 6 , 7 , 8 , 7 מצאו כי HWWS יעילים יותר 9 , 10 , 11 , 12 , 13 , 14 , 15 , ושניים לא מצאו הבדל משמעותי בין השיטות 16 , 17 . ממצאים אלה אינם עקביים ואינם מתייחסים לסיכון המתמשך למחלות עקב התמשכות האורגניזמים במי השטיפה לאחר שטיפת ידיים. בסך הכל, הראיות על היעילות היחסית של שיטות שטיפת ידיים להסרת פתוגנים הגורמים למחלות זיהומיות מוגבלות. עדות מוגבלת זו הובילה לחוסר ודאות לגבי השיטות המתאימות ביותר להגדרות התפרצות המחלה. לדוגמה, במהלך התפרצות מחלת הנגיף (EVD) במערב אפריקה בשנים 2013 עד 2016, מספר מגיבים בינלאומיים גדולים סיפקו המלצות סותרות לפתרונות כלור HWWS, ABHS או 0.05%. Mddecins Sans Frontières (MSF) ממליצה על שימוש בפתרון של 0.05% כלור לשטיפת ידיים, בעוד ארגון הבריאות העולמי (WHO) ממליץ על HWWS או ABHS (אם הידיים אינן מלוכלכות). ארגון הבריאות העולמי מרחיק לכת על מנת לקבוע כי אין להשתמש בכלור אלא אם כן אין אפשרויות אחרות, כי הוא פחות יעיל מאשר שיטות אחרות עקב הביקוש כלור המופעל על ידי העור 18 , 19 , 20 , 21 , 22 . בנוסף, הפתרונות כלור מופקים בדרך כלל מארבעה תרכובות כלור שונות, כולל hypochlorite הבדיקה גבוהה (HTH), מקומי שנוצר וייצוב hypochlorite נתרן (NaOCl), ואת sodDichloroisocyanurate ium (NaDCC). סקירה שיטתית שהוזמנה על ידי ארגון הבריאות העולמי בתגובה לפריצת ה- EVD במערב אפריקה מצאה לאחרונה רק ארבעה מחקרים שחקרו את היעילות היחסית של שטיפת ידיים עם כלור 23 . מחקרים אלה גם הניבו תוצאות סותרות, ואף אחד מהמחקרים הללו לא השתמש בריכוז הכלור המומלץ של 0.05% לשטיפת ידיים או למיקרואורגניזמים שנבדקו בדומה לנגיף האבולה 10 , 24 , 25 , 26 , 27 . לפיכך, ההמלצות לא נמצאו מבוססות על ראיות, ולא ברור אילו המלצות היו יעילות ביותר. יש צורך במחקר נוסף כדי להשוות בין גישות שטיפת ידיים למניעת התפשטות של פתוגנים זיהומיים, שכן התערבויות שטיפת כלים הן כלי חשוב למניעת העברת מחלות מגיפה. אלה חוהמלצות שוטפות חייבות להתבסס על ראיות. לפיכך, פותחה שיטה 2 , 28 , 29 לבדיקת יעילות שטיפת ידיים ושטיפת מים, המבוצעת באמצעות תחליפים או פתוגנים לא זיהומיים. תוצאות לדוגמה, באמצעות Phi6 כמו תחליף עבור וירוס אבולה ושימוש Escherichia coli כאורגניזם מחוון משותף, מוצגים כאן. בפרוטוקול זה, יעילות שטיפת ידיים ושטיפת מיצוי המים מוצגים.

<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

שטיפת ידיים מומלץ באופן נרחב כדי למנוע שידור מחלות מדבקות. עם זאת, קיימות עדויות רבות על יעילותן של שיטות שטיפת ידיים באופן כללי. בנוסף, קיימות עדויות קטנות להשוואת שיטות שטיפת ידיים כדי לקבוע אילו יעילים ביותר על הסרת פתוגנים זיהומיות. המחקר נדרש כדי לספק ראיות עבור גישות שונות לשטיפת ידיים, כי ניתן להשתמש במהלך התפרצויות מחלות זיהומיות. כאן, שיטה מעבדה כדי להעריך את היעילות של שיטות שטיפת ידיים על הסרת מיקרואורגניזמים מן הידיים ואת ההתמדה שלהם במי השטיפה מתואר. ידיים של מתנדבים הם הראשון spiked עם אורגניזם הבדיקה ולאחר מכן שטף עם כל שיטת שטיפת ידיים של עניין. בדרך כלל, מיקרואורגניזמים פונדקאים משמשים להגנה על בני אדם מפני מחלות. מספר האורגניזמים שנותרו על ידי מתנדבים לאחר הכביסה נבדק באמצעות שיטת &quot;מיץ כפפות&quot; שונה: הידיים ממוקמות כפפות עם eluEnt ו הם scrubbed להשעות את מיקרואורגניזמים ולהפוך אותם זמינים לניתוח על ידי סינון קרום (חיידקים) או assay פלאק (וירוסים / bacteriophages). לשטוף מים המיוצר מן שטיפת הידיים נאסף ישירות לניתוח. יעילות שטיפת ידיים היא לכמת על ידי השוואת ערך הפחתת יומן בין דגימות נלקח לאחר שטיפת ידיים לדגימות ללא שטיפת ידיים. לשטוף מים התמדה הוא לכמת על ידי השוואת דגימות מים לשטוף משיטות שונות שטיפת ידיים לדגימות שנאספו לאחר שטיפת ידיים עם מים פשוטים. בעוד ששיטה זו מוגבלת על ידי הצורך להשתמש באורגניזמים חלופיים כדי לשמור על שלומם של מתנדבים אנושיים, היא לוכדת היבטים של שטיפת ידיים שקשה לשכפל במחקר במבחנה וממלא פערים במחקר על יעילות שטיפת ידיים והתמדה של אורגניזמים זיהומיות בשטיפה מַיִם.

הנה, פרוטוקול ( איור 1 ) הושלמה עם 18 מתנדבים, אשר נבדקו כל אחד באמצעות E. קולי ו Phi6. נמצאו הבדלים משמעותיים בין תוצאות שטיפת ידיים עם E. coli הן עם וללא עומס קרקע ו ​​Phi6 עם עומס הקרקע ( איור 2 איור 3 ). ...

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

שטיפת ידיים מומלץ באופן נרחב כדי למנוע שידור מחלות מדבקות. עם זאת, יש ראיות מועטות בהן שיטות שטיפת ידיים יעילות ביותר בהסרת פתוגנים למחלות זיהומיות. פיתחנו שיטה כדי להעריך את היעילות של שיטות שטיפת ידיים בהסרת מיקרואורגניזמים.

שיטה לבדיקת יעילות שטיפת הידיים להסרת הפתוגנים המזהמים

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 Views.  Tufts University.  שטיפת ידיים מומלץ באופן נרחב כדי למנוע שידור מחלות מדבקות. עם זאת, יש ראיות מועטות בהן שיטות שטיפת ידיים יעילות ביותר בהסרת פתוגנים למחלות זיהומיות. פיתחנו שיטה כדי להעריך את היעילות של שיטות שטיפת ידיים בהסרת מיקרואורגניזמים. 

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.

96 microtiter ובכן פלייט מבוססי שיטה ליצירת ניטור ובדיקה של רגישות נגד פטריות קנדידה אלביקנס Biofilms

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

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.

בידוד של המוח ותאי חוט השדרה mononuclear שימוש Percoll Gradients

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.

באמצעות assay הפאן ויראלי microarray (Virochip) אל מסך דוגמאות קליניות עבור פתוגנים ויראליים

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;Bioluminescent&quot; כתב phage לצורך זיהוי של קטגוריה פתוגנים חיידקיים

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.

אבחון שחפת ריאתית עם מבחן אקספרט MTB / RIF

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.

שימוש בינוני כיח מלאכותית לבדיקת יעילות אנטיביוטי נגד<em&gt; Pseudomonas aeruginosa</em&gt; בתנאי רלוונטיים יותר ריאות סיסטיק פיברוזיס

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.

שימוש בכימיה לחץ כדי למדוד את ההשפעה של זיהום ויראלי בסינתזת RNA Host-Cell

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.

בניתוח חוץ גופייה של תיווך Myd88 הסלולרי תגובה החיסונית לוירוס הנילוס המערבי זיהום הזנים Mutant

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.

אסטרטגית ראפיד עבור הבידוד של ניו Faustoviruses ממדגמים סביבת שימוש<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.

Intratracheal חיסון של פישר 344 חולדות עם Francisella tularensis

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