Name: a method to test the efficacy of handwashing for the removal of emerging infectious pathogens
Uploaded: 2017-06-07T14:00:00
Description: Watch this Scientific Journal Video about A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens at JoVE.com

This work was supported by the United States Agency for International Development, Office of Foreign Disaster Assistance (AID-OFDA-A-15-00026). Marlene Wolfe was supported by the National Science Foundation (grant 0966093).

Ethics Statement: The study described here (on Phi6 and E. coli as surrogates for Ebola) was approved by the Institutional Review Board at Tufts Medical Center and Tufts University Health Sciences Campus (#12018); Harvard University ceded review to the Tufts Institutional Review Board.
NOTE: Prior to beginning this protocol, two steps must be completed. First, a Biosafety Level 1 (BSL-1) surrogate or non-infectious version of the pathogen to be studied that is safe to use on human sub...

<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. -. 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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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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...

Handwashing is widely recommended to prevent the spread of disease, particularly those transmitted by the fecal-oral or airborne route, including diarrheal and respiratory diseases1. Surprisingly, there is little comparable evidence on the efficacy of handwashing methods, such as handwashing with soap and water (HWWS) and with alcohol-based hand sanitizer (ABHS), on the removal of organisms from the hands. Initial research has found that the mechanical action of handwashing, as opposed to the handwashing method, may account for most organism removal2,3. Additionally, there is little comparative evidence on which handwashing method is most efficacious. In an informal literature review, 14 studies that compared the efficacy of soap and hand sanitizer on the removal of organisms were identified. Of these studies, five found ABHS to be more efficacious4,5,6,7,8, seven found HWWS to be more efficacious9,10,11,12,13,14,15, and two found no significant difference between the methods16,17. These findings are inconsistent and do not address the ongoing risk of disease from the persistence of organisms in the rinse water after handwashing. Overall, the evidence on the comparative efficacy of handwashing methods for the removal of infectious disease-causing pathogens is limited.
This limited evidence has led to uncertainty about which methods are most appropriate in outbreak settings. For example, during the Ebola Virus Disease (EVD) outbreak in West Africa from 2013 to 2016, several large international responders provided contradictory recommendations for HWWS, ABHS, or 0.05% chlorine solutions. M&#233;decins Sans Fronti&#232;res (MSF) recommends the use of 0.05% chlorine solution for handwashing, while the World Health Organization (WHO) recommends HWWS or ABHS (if the hands are not visibly soiled). The WHO goes so far as to state that chlorine should not be used unless no other options are available, because it is less effective than other methods due to the chlorine demand exerted by the skin18,19,20,21,22. Additionally, the chlorine solutions are commonly produced from four different chlorine compounds, including high-test hypochlorite (HTH), locally-generated and stabilized sodium hypochlorite (NaOCl), and sodium dichloroisocyanurate (NaDCC). A systematic review commissioned by the WHO in response to the EVD outbreak in West Africa recently found only four studies investigating the comparative efficacy of handwashing with chlorine23. These studies also produced conflicting results, and none of these studies used the recommended chlorine concentration of 0.05% for handwashing or investigated microorganisms similar to the Ebola virus10,24,25,26,27. Thus, the recommendations were not found to be evidence-based, and it was unclear which recommendations were most efficacious.
Additional research is needed to compare handwashing approaches to prevent the spread of infectious pathogens, as handwashing interventions are an important tool to prevent epidemic disease transmission. These handwashing recommendations must be based on evidence. Thus, a method for testing handwashing efficacy and rinse water persistence, performed with surrogates or non-infectious pathogens, was developed2,28,29. Sample results, using Phi6 as a surrogate for the Ebola virus and using Escherichia coli as a common indicator organism, are presented here. In this protocol, handwashing efficacy and rinse water persistence tests are presented.

<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

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Here, the protocol (Figure 1) was completed with 18 volunteers, who were each tested using both E. coli and Phi6. Significant differences were found between handwashing results with E. coli both with and without soil load and Phi6 with soil load (Figure 2 and Figure 3). For E. coli without soil load, handwashing with HTH, NaDCC, and stabilized NaOCl all resulted in signific...

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

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

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

The overall goal of this experiment is to test the comparative efficacy of handwashing options on hands that have been experimentally inoculated with an organism of interest. This method can provide information on key questions to help prevent of infectious diseases. By allowing researchers to investigate the efficacy of handwashing for particular organisms of interest.The main advantage of this technique is that it is flexible and can be adapted to allow for investigation of many different organisms. We first had the idea for this method when we realized chlorine solutions were being used for handwashing in Ebola contexts and there was no evidence for the efficacy of this at the time. After recruiting eligible subjects and preparing handwashing solutions according to the text protocol, prepare bacteria by streaking a non-pathogenic strand of E.Coli onto LB agar plates and incubate the cultures at 37 degrees celsius for 24 hours to obtain single colonies.One day before the start of the experiment, use a sterile lube to grab a single colony from the plate and inoculate 10 milliliters of LB broth. Incubate the cultures at 37 degree celsius, with shaking, overnight. On the morning of the experiment, add one milliliter of the overnight culture to 20 milliliters of fresh LB broth.Incubate the cultures for approximately two and half hours to achieve a salt density greater than 10 to the eighth CFU per milliliter. Then, use a spectrophotometer to estimate the concentration of the culture. Immediately prior to testing, prepare an inoculum composed of 68%bacteria or viral suspension and 32%sodium chloride or soil load.Swirl or gently vortex to mix. After preparing the volunteers according to the text protocol, test the pH of each volunteer's skin by placing a flat tip skin pH probe in the webbed space between the pointer and the middle fingers. Ensure that the electrode is flat against the skin and record the pH reading.Next, have the volunteer cup both hands together. Then, carefully pipette 750 microliters of the inoculate into each palm to spike the hands. When spiking volunteers'hands, it is important none of the inoculum is spilled.Remind volunteers this is important and give them the opportunity to practice with water before beginning the procedure. Have the volunteers gently rub their hands together until all surfaces of the hand are coded with the inoculate, while subjecting the hands to as little friction as possible. Instruct the volunteers to hold their hands still and away from their body for an additional 30 seconds to allow the inoculate to dry.Then wash the hands using one of the methods described in the text protocol. For hand washing with soap, use 10 milliliters of Ultrapure water to wet the hands. Have the volunteers lather their hands with soap and then rub their hands together for an additional 20 seconds.Rinse the volunteer's hands by pouring 500 milliliters of Ultrapure water at room temperature through a funnel at a flow rate of 1.5 liters per minute into a large sample collection bag and process the sample within two hours. For all chlorine solutions, pour 200 milliliters of chlorine solution over the volunteer's hands through a funnel at a flow rate of 1.5 liters per minute and have the volunteers rub their hands thoroughly. As another hand washing method, have the volunteers use hand sanitizer to wash their hands.It's important all surfaces of the hand are gently rubbed, instruct participants to focus on each part, palms, fingernails, in between fingers as they rub and then ensure the entire hand has been rinsed. For control A, after inoculation, do not perform a handwashing step. For control B, use only Ultrapure water at room temperature to perform handwashing through a funnel with a known flow rate.After handwashing, immediately place each volunteer's hand selected for testing, into a sample bag containing 75 milliliters of Leeuwin up to the wrist. Hold the top of the bag tightly around the wrist. Have the volunteers gently rub their hand in the solution for 30 seconds, taking care to reach in between the fingers and underneath the fingernails.Gently massage the hand from outside the bag for 30 seconds to ensure that the entire hand is thoroughly rinsed in the Leeuwin, all the way up to the wrist. Seal the bag and process it according to the appropriate essay. Before repeating the process with each handwashing method, have the volunteers use soap in warm water to thoroughly wash their hands in the sink.Finally, use 70%ethanol to spray the volunteer's hands until they are coated on both sides, and allow them to dry. To analyze the samples, set up a flame and a filtration manifold with sterile filtration funnels and a vacuum connection. Sterilize forceps by dipping them in ethanol and flaming them.Use the forceps to place a 0.45 micron filter onto the filtration manifold with the grid facing up. Then use a small amount of sterile PBS to wet the filter. Place the funnel on the base and pipette or pour the sample directly onto the filter.Engage the vacuum until the entire sample has passed through the membrane. Then, use sterile PBS to rinse the sides of the funnel and engage the vacuum again. Next, remove the funnel, flame sterilize the forceps and lift the filter from the base.Then place the filter gently on the medium in a petri dish with the grid facing up, ensuring that the filter lies flush against the surface. Invert the plates and incubate at 37 degrees celsius for 24 hours. Following the incubation, count the E.Coli colonies and record the data.Carry out data analysis according to the text protocol. The protocol described in this video was completed with 18 volunteers tested with E.Coli and Phi6, for E.Coli without soil load, handwashing with HTH, NaDCC and stabilized NaOCl, all resulted in significantly greater log reductions than handwashing with water only. With soil load, HTH resulted in a significantly greater log reduction of E.Coli than water only handwashing with soap and ABHS.There was no significant difference between methods for Phi6 without soil load. However, for Phi6 with soil load, water alone resulted in a greater log reduction than ABHS or stabilized NaOCl, and handwashing with soap in a greater log reduction than ABHS, stabilized NaOCl and generated NaOCl. HTH also had a greater log reduction than ABHS and stabilized NaOCl and NaDCC resulted in a greater log reduction than stabilized NaOCl and ABHS.As demonstrated in these graphs, chlorine resulted in a significantly greater log reduction of E.Coli persisting in the rinse water than handwashing with soap. Finally, the same pattern was found in Phi6 without soil load with all chlorine solutions resulting in a significantly greater reduction of Phi6 in rinse water than handwashing with soap. There were no significant decreases in rinse water with Phi6 and soil load.While attempting this procedure, it's important to remember efficacy results for each condition are calculated by comparing the results for the two controls. This means it is very important to perform the steps consistently for each handwashing option.

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

Research

JoVE Journal

Immunology and Infection

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.

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.

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.

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.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

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.

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.

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.

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.

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.

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.

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 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...