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 subjects must be identified and selected30. A BSL-1 surrogate or non-infectious pathogen is necessary for this protocol, as the organism will be used to inoculate the bare hands of human volunteers. Second, approval from the local Institutional Review Board to conduct research with human subjects must be obtained before recruiting volunteers or beginning the experiment. Many aspects of this protocol can be adjusted to meet the specific needs of the research questions of interest.
1. Recruit Eligible Human Subjects
<ol>
	<li>Recruit volunteers by posting paper flyers on public notice boards and sending emails to groups with members who may be interested in participating. These announcements should include the study purpose, contact information, and eligibility criteria.</li>
	<li>Meet with the volunteers to evaluate eligibility. Confirm that the volunteers are healthy, between the ages of 18 and 65, and not currently pregnant or taking antibiotics and that they have no skin damage/disorders, known allergies to handwashing agents, or history of mental health issues related to hygiene.</li>
	<li>Have eligible volunteers read consent forms. Answer any questions they pose and have the volunteer and investigator sign two copies of the form. Retain one form and provide one to the volunteer.</li>
	<li>Administer a baseline survey, including questions on demographic information, personal history of skin conditions, and information on recent handwashing behavior. Examine hands for signs of dermatitis, skin injuries, or baseline skin abnormalities31.</li>
	<li>Schedule the volunteers for two testing sessions for each organism of interest (one for testing with soil load and one for testing without). Instruct the volunteers to avoid antimicrobial products for a seven-day washout period prior to testing to avoid confounding from personal product use.
	<ol>
		<li>Provide volunteers with antimicrobial products (shampoo, conditioner, and soap) to use in place of their usual products. Provide heavy-duty vinyl gloves and instruct the subjects to wear them when using products such as house cleaning products.</li>
	</ol>
	</li>
</ol>
2. Prepare Handwashing Solutions Commonly Used in Emergency Response (Soap, ABHS, 0.05% HTH, NaDCC, and NaOCl Solutions)
NOTE: Chlorine solutions can be prepared up to 12 h in advance of the experiment but will degrade if stored &#62;12 h.
<ol>
	<li>Choose and purchase a soap relevant to the context for which testing is being performed. 
	NOTE: In most cases, for infectious disease emergencies in the developing world, this will be a bar of soap.</li>
	<li>Choose and purchase an ABHS solution relevant to the context for which testing is being performed. 
	NOTE: The chosen ABHS should have an ethyl alcohol context greater than or equal to 70% to ensure efficacy.</li>
	<li>Prepare a 0.05% calcium hypochlorite (Ca(ClO)2) solution by adding granular Ca(ClO)2 powder to ultrapure water. Determine the amount of solution needed based on the number of subjects to be tested.
	<ol>
		<li>Using the following equation, determine the amount of powder needed to prepare the desired amount of solution in a given volume of water using a given percent of available chlorine: 
		<img alt="Equation" src="/files/ftp_upload/55604/55604eq1.jpg" /> 
		NOTE: Ca(ClO)2 powder typically has 60-80% available chlorine.</li>
	</ol>
	</li>
	<li>Prepare a 0.05% sodium dichloroisocyanurate (NaDCC) solution by adding a granular NaDCC powder to ultrapure water.
	<ol>
		<li>Using the following equation, determine the amount of powder needed to prepare the desired amount of solution in a given volume of water using a given percent of available chlorine: 
		<img alt="Equation" src="/files/ftp_upload/55604/55604eq2.jpg" /> 
		NOTE: NaDCC powder typically has about 50% available chlorine.</li>
	</ol>
	</li>
	<li>Prepare stabilized 0.05% NaOCl solution by adding stock sodium hypochlorite solution to ultrapure water.
	<ol>
		<li>Confirm the concentration of the NaOCl stock solution (likely to be 5-8%) using a titration test method according to the manufacturer&#39;s instructions (e.g., iodometric titration; see the Materials List for the suggested kit).</li>
		<li>Using the results of the test method, calculate the amount of solution to add to water using the following equation: 
		<img alt="Equation" src="/files/ftp_upload/55604/55604eq3.jpg" /></li>
	</ol>
	</li>
	<li>Prepare stabilized 0.05% NaOCl solution by adding sodium hypochlorite solution produced using an electrochlorinator, ultrapure water, and laboratory-grade sodium chloride (NaCl) to ultrapure water.
	<ol>
		<li>Prepare a 1% chlorine solution with ultrapure water and NaCl, using an electrochlorinator according to the manufacturer&#39;s instructions.</li>
		<li>Use a titration test method (e.g., iodometric titration) to confirm the concentration of the NaOCl stock solution32.</li>
		<li>Using the results of the test, calculate the amount of solution to add to water using the following equation: 
		<img alt="Equation" src="/files/ftp_upload/55604/55604eq3.jpg" /></li>
	</ol>
	</li>
	<li>Confirm the concentration of each of the chlorine handwashing solutions using a titration method (e.g., iodometric titration) and adjust the solutions by adding water or chlorine source powder/solution until they are within a 10% error of the target concentration (0.045-0.055%).</li>
</ol>
3. Prepare Organisms and Soil Load and Combine to Produce the Inoculate
NOTE: In the following sub-sections, E. coli and Phi6 are used as sample bacterial and viral organisms for the methods description.
<ol>
	<li>Prepare the organism to be used for testing at a concentration greater than 10 x 108 CFU/mL for bacteria and greater than 10 x 107 PFU/mL for viruses.
	<ol>
		<li>To prepare E. coli, streak a nonpathogenic strain of E. coli onto Luria-Bertani (LB) agar plates and incubate at 37 &#176;C for 24 h to obtain single colonies. Store at 4 &#176;C. 
		NOTE: This can be done several days before experimentation.
		<ol>
			<li>One day before the start of the experiment, pick a single colony from the plate and inoculate 10 mL of LB broth using a sterile loop. Incubate overnight at 37 &#176;C with shaking.</li>
			<li>On the morning of the experiment, start a fresh culture by adding 1 mL of the overnight culture to 20 mL of fresh LB broth. Incubate for approximately 2.5 h to achieve a cell density greater than 108 CFU/mL.</li>
			<li>Use a spectrophotometer to estimate the concentration of the culture. 
			NOTE: Use a previously established conversion factor from a standards curve for the E. coli strain used, ensuring a concentration greater than 108 CFU/mL33. If the cell density is not high enough, return the culture to the incubator and test again until ready.</li>
		</ol>
		</li>
		<li>Confirm the concentration using membrane filtration34. 
		NOTE: Perform serial dilutions of the culture in phosphate-buffered saline (PBS) so that the solution filtered will produce a countable number of colonies on the plate (the exact number will depend on the medium used).
		<ol>
			<li>Set up a flame and a filtration manifold with sterile filtration funnels and a vacuum connection. Sterilize forceps by flaming them with ethanol. Use them to place a 0.45-&#181;m filter on the filtration manifold, with the grid facing up. Wet the filter with a small amount of sterile PBS.</li>
			<li>Place the funnel on the base and add the sample solution to be processed by pipetting or pouring directly onto the filter. Engage the vacuum until the entire sample has passed through the membrane. Rinse the sides of the funnel with sterile PBS and engage the vacuum again. 
			NOTE: Samples should be at least 100 &#181;L and up to 100 mL. If a sample is less than 10 mL, add approximately 20 mL of PBS to the filtration funnel prior to filtering to ensure the uniform filtration of the sample.</li>
			<li>Remove the funnel, flame-sterilize the forceps, and lift the filter from the base. Place the filter gently on the LB agar in a Petri dish, with the grid facing up, ensuring that the filter lies flush against the surface. Invert the plates and incubate for 24 h at 37 &#176;C.</li>
			<li>After 24 h, remove the plates from the incubator and count the E. coli colonies. Use this data and the known dilution factor and volume of the solution to calculate the concentration of the filtered solution in CFU/mL.</li>
		</ol>
		</li>
		<li>Propagate Phi6 in Pseudomonas syringae host using the double agar overlay method35.
		<ol>
			<li>Add 100 &#181;L of Phi6 stock suspension and 100 &#181;L of P. syringae overnight culture directly to 6 mL of Nutrient Broth Yeast (NBY) soft agar (0.3%). Pour it onto plates with NBY hard agar (1.5%) and incubate overnight at 26 &#176;C. Prepare enough plates to produce enough inoculum for the experiment, estimating a yield of approximately 4 mL of viral suspension per plate.</li>
			<li>The next day, add 5 mL of PBS on top of the soft agar layer. Leave it at room temperature for 4 h, retrieve it with a pipette, and filter it using a 0.45-&#181;m filter. Store at 4 &#176;C. 
			NOTE: This solution will serve as the viral inoculate.</li>
		</ol>
		</li>
		<li>Use a plaque assay to confirm that the concentration is greater than 107 PFU/mL35. Perform serial dilutions of the viral suspension in PBS so that 100 &#181;L produces a countable number of plaques on the plate.
		<ol>
			<li>Pipette 100 &#181;L of an appropriate diluted sample and 100 &#181;L of overnight host culture directly into a tube containing 6 mL of NBY soft agar. Pour soft agar over NBY hard agar and incubate at 26 &#176;C for 24 h.</li>
			<li>The next day, remove the plates from the incubators and count the number of plaques per plate. Use this data and the known dilution factor and volume of the solution to calculate the concentration of the filtered solution in PFU/mL.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare the tripartite soil load, intended to mimic human serum.
	<ol>
		<li>Combine 7.80 mg/mL bovine serum albumin, 10.92 mg/mL tryptone, and 2.52 mg/mL bovine mucin to produce the required volume of soil load. After mixing the soil load, filter it through a 0.22-&#181;m filter to sterilize. Store it at 4 &#176;C until use. Do not heat sterilize, as the proteins will denature.</li>
	</ol>
	</li>
	<li>Prepare a 0.9% NaCl solution for mixing the inoculate for conditions without soil load.</li>
	<li>Immediately prior to testing, prepare an inoculum composed of 68% bacterial or viral suspension and 32% soil load. For example, use 1.02 mL of bacterial or viral suspension from steps 3.1.1.2 or 3.1.3.2 and 0.48 mL of either soil load (step 3.2.1) or 0.9% NaCl solution (step 3.3). Swirl or vortex gently to mix. 
	NOTE: 1.5 mL of this inoculum will be used for each volunteer under each condition, so ensure that the total volume of the inoculum prepared is sufficient for the intended number of tests.</li>
</ol>
4. Preparing Volunteers for the Experiment
NOTE: Determine the organism and soil load condition to be tested on that day. The same volunteers may be used for testing multiple conditions, but each volunteer should only be subjected to one round of testing within a 48 h period.
<ol>
	<li>Before beginning testing, confirm that the volunteers remain eligible by verbally verifying that they adhered to the 7 day antimicrobial washout period and by visually confirming that they have not developed any breaks or abnormalities on their skin.</li>
	<li>Using a random number generator, assign each volunteer to use either their right or left hand for sampling on this day of testing. Assign an order in which the handwashing conditions will be performed. 
	NOTE: For example, ABHS may be assigned #3 and will be performed third.</li>
	<li>Perform a &#34;cleansing wash&#34; once at the start of testing to strip the skin of dirt and oils so that each subsequent test is conducted under equivalent conditions.
	<ol>
		<li>To do a cleansing wash, run through each step of the experiment (section 5, below), using a blank inoculate (LB broth or PBS only) and taking a sample without handwashing.</li>
	</ol>
	</li>
</ol>
5. Experimental Procedure
<ol>
	<li>To test the pH of the skin of each volunteer (to control for variation), place a flat-tipped skin pH probe on the palmar surface skin and the web space between the pointer and middle fingers. Ensure that the electrode is flat against the skin. Record the pH reading.</li>
	<li>Spike the hands.
	<ol>
		<li>Have the volunteers cup both hands together. Spike the hands with 1.5 mL of the inoculate by carefully pipetting 750 &#181;L slowly into each palm.</li>
		<li>Have the volunteers gently rub their hands together until all surfaces of the hand are coated with the inoculate while subjecting the hands to as little friction as possible.</li>
		<li>Have the volunteers hold their hands still and away from their body for an additional 30 s to allow the inoculate to dry. The inoculate may not dry completely.</li>
	</ol>
	</li>
	<li>Wash the hands.
	<ol>
		<li>For all following wash steps, capture the rinse water from the hands in a large sample collection bag. Add 4.5 mL of a 12% sodium thiosulfate solution to the bag to neutralize the chlorine on contact and process within 2 h. 
		NOTE: Sodium thiosulfate should be added to all samples (even those without chlorine) to control for any impact that it may have on the organism.</li>
		<li>After inoculation (section 5.2), wash the hands with the next method in the designated order.
		<ol>
			<li>For Control A, do not perform a handwashing step and move directly to step 5.5.</li>
			<li>For Control B, wash the hands with only ultrapure water at room temperature (approximately 21 &#176;C) through a funnel with a known flow rate. 
			NOTE: Here, a flow rate of 1.5 L/m and 500 mL of water were used.</li>
			<li>For handwashing with soap, wet the hands with 10 mL of ultrapure water. Have the volunteers lather their hands with soap and then rub their hands together for an additional 20 s. Rinse their hands by pouring 500 mL of ultrapure water at room temperature through a funnel at a flow rate of 1.5 L/m.</li>
			<li>For all chlorine solutions (e.g., ABHS, HTH, NaDCC, and NaOCl), pour 200 mL of chlorine solution through a funnel at a flow rate of 1.5 L/m and have the volunteer rub their hands thoroughly.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Hand rinse using a modified glove juice procedure.
	<ol>
		<li>After handwashing, immediately place each volunteer&#39;s hand (i.e., the hand (right or left) selected for testing in step 4.2) into a sample bag containing 75 mL of eluent (e.g., PBS) up to the wrist. Hold the top of the bag tightly around the wrist. 
		NOTE: Use an eluent for sampling that contains sufficient sodium thiosulfate to neutralize any chlorine used for handwashing. PBS is a commonly used eluent that is appropriate for many organisms.</li>
		<li>Have the volunteers gently rub their hand in the solution for 30 s, taking care to reach in between the fingers and underneath the fingernails. Massage the hand from outside the bag gently for 30 s to ensure that the entire hand is rinsed thoroughly in the eluent, all the way up to the wrist.</li>
		<li>Seal the bag and process it according to the appropriate assay, described in section 6, within 2 h.</li>
	</ol>
	</li>
	<li>Decontamination.
	<ol>
		<li>Before repeating the process with each handwashing method, have the volunteers wash their hands thoroughly in a sink with soap and warm water. Spray the volunteers&#39; hands with 70% ethanol until they are coated on both sides. Allow them to dry.</li>
		<li>Repeat all steps in section 5 for each handwashing condition, only using the hand randomly selected in step 4.2 (Figure 1).</li>
	</ol>
	</li>
</ol>
6. Quantification
<ol>
	<li>Perform assays appropriate for the organism of choice (e.g., membrane filtration for bacteria or plaque assay for viruses, described above in sections 3.1.2 and 3.1.4, respectively).</li>
	<li>After counting the plates, record the estimated CFU/mL or PFU/mL for each test for the analyses (sections 3.1.2 and 3.1.4).</li>
</ol>
7. Analysis
<ol>
	<li>Using the results from step 6.2, calculate the log reduction value of organisms on the hands, for each organism and soil load status and for each subject and handwashing method.
	<ol>
		<li>For handwashing efficacy, compare the concentration of bacteria/virus in each handwashing sample to control A (no handwashing). For rinse water persistence, compare each rinse water sample to control B (washing with water only). Use the following standard formula: 
		Log Reduction (handwashing) = <img alt="Equation" src="/files/ftp_upload/55604/55604eq4.jpg" /> <img alt="Equation" src="/files/ftp_upload/55604/55604eq5.jpg" /> 
		Log Reduction (rinse water) = <img alt="Equation" src="/files/ftp_upload/55604/55604eq4.jpg" /><img alt="Equation" src="/files/ftp_upload/55604/55604eq6.jpg" /> 
		NOTE: Log reduction can also be expressed as log10(without handwashing) - log10(with handwashing)</li>
	</ol>
	</li>
	<li>Use a one-way repeated measures analysis of variance (ANOVA) to assess the significant differences in the calculated log reduction values between handwashing methods and a post-hoc Tukey&#39;s HSD test for significant models to pairwise assess significant differences (p &#60;0.05)36.
	<ol>
		<li>Prior to running the ANOVA, assess each dataset for sphericity (e.g., using Bartlett&#39;s test). Apply a correction (e.g., the Greenhouse-Geisser correction) when the test indicates that sphericity was violated37.</li>
	</ol>
	</li>
</ol>

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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. 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>

The authors declare that they have no competing financial interests.

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 to infectious disease.
During the use of the method, two key data quality recommendations were noted as important. First, the inoculate must be applied both as similarly as possible across the rounds of testing and in a manner, that minimizes loss. This is to ensure that sufficient inoculate is applied to the hands to allow for statistically significant results. Second, be sure to complete the "cleansing wash" step, in which the protocol is performed without handwashing prior to testing, as previous work has shown that there are likely to be significant differences between a first wash and subsequent washes, but not between subsequent washes performed after a cleansing round29. Additionally, this step clears residual hand contamination, which would interfere with results. 
The main limitation of this protocol is that it can be uncomfortable for volunteers. During each round of testing, which lasted about 2 h, volunteers' hands became cold. Some volunteers reported mild pain from their hands being constantly wet. Additionally, after a few rounds of testing, volunteers' hands became supersaturated, no longer fully drying between rounds. Although the randomization of the order of handwashing methods for each volunteer accounted for supersaturation, it is possible that the supersaturation could act as a confounding or modifying factor in this type of testing. To address this limitation, it is recommended that volunteers are appraised of this risk during consent disclosures and are reminded of their right to drop out of the study at any time. Volunteers should not undergo testing for more than 2 h per day to allow time for the hands to return to a baseline state and to minimize discomfort. A second limitation is the need to use a surrogate organism or non-infectious variant of a pathogenic organism to protect the health of volunteers. This might cause concern about the generalizability of results. However, for some pathogenic organisms (such as the Ebola virus), this limitation cannot be ethically overcome. Care must be taken during surrogate organism selection. Lastly, this is a laboratory study on efficacy. Results may only translate to effective disease prevention in real-word contexts where handwashing methods are made accessible to those in need and are used properly and consistently.
This protocol draws on previous work on handwashing efficacy but attempts to streamline methods and emphasizes the use of human hands (rather than surrogate surfaces) for testing. Additionally, rinse water is a transmission risk that had previously not been assessed. Existing studies on handwashing efficacy vary in methodology, leading to non-comparable data. We hope that standardized protocols for conducting handwashing method comparisons will encourage comparable and replicable results. Previous work has demonstrated that in vitro testing on surrogate surfaces such as pig skin, where, for example, the actual Ebola virus could be used, produced results that do not match those found after testing on human hands38. Therefore, a method using human hands and surrogate or non-infectious organisms is currently the best available approach to estimate handwashing efficacy and rinse water persistence for infectious microorganisms.
Handwashing is critical to prevent disease transmission. However, there is a lack of evidence on the comparative efficacy of handwashing methods that are commonly recommended. This protocol can be used to generate evidence about handwashing efficacy and rinse water persistence. This is especially important for infectious diseases with the potential to cause large outbreaks, such as the Ebola virus. We hope that other researchers will find this protocol useful to generate much-needed additional evidence on handwashing method efficacy and rinse water persistence that will assist in developing recommendations to reduce the transmission of infectious diseases.

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 significantly greater log reductions than handwashing with water only (F(6,102) = 2.72, p = 0.034). With soil load, HTH resulted in a significantly greater log reduction of E. coli than water only, HWWS, and ABHS (F(6,102) = 3.94, p &#60;0.001). There was no significant difference between methods for Phi6 without soil load (F(6,66) = 2.04, p = 0.073). However, for Phi6 with soil load (F(6,102) = 7.01, p &#60;0.001), water alone resulted in a greater log reduction than ABHS or stabilized NaOCl, and HWWS 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. While HTH performed most consistently well across conditions, we would caution against over-interpretation of significant results, as many confidence intervals were large, ranging from less than 0.5 log to more than 1.5 log reduction in many cases.
In rinse water, chlorine resulted in a significantly greater log reduction of E. coli persisting in the rinse water than HWWS (without soil load, F(4,68) = 331.7, p &#60;0.001; with soil load, F(4,68) = 162.44, p &#60;0.001) (Figure 4). This same pattern was found in Phi6 without soil load ((F(4,43) = 8.95, P &#60;0.001), with all chlorine solutions resulting in a significantly greater reduction of Phi6 in rinse water than HWWS. There were no significant differences in persistence in rinse water with Phi6 and soil load ((F(4,67) = 3.35, p = 0.071) (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55604/55604fig1.jpg" /> 
Figure 1: Experiment overview. The five steps undertaken for each round of handwashing include: 1) pH testing, 2) inoculating the hands, 3) handwashing, 4) rinsing the hands, and 5) decontaminating the hands for each of the eight conditions tested. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55604/55604fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55604/55604fig2.jpg" /> 
Figure 2: E. coli handwashing results. Compared to no handwashing, the handwashing methods tested resulted in an average log reduction in E. coli of 1.94-3.01 without soil load and 2.18-3.34 with soil load. Handwashing with water demonstrated the least reduction in E. coli in both conditions (1.94 and 2.18 log). Handwashing with NaDCC resulted in the greatest reduction without soil load (3.01), and HTH resulted in the greatest reduction with soil load (3.34). In the charts, the line represents the percent reduction in organisms, and the error bars represent the standard error of log reduction. Ctrl B, control B; HWWS, handwashing with soap; ABHS, alcohol-based hand sanitizer; HTH, high-test hypochlorite; NaDCC, sodium dichloroisocyanurate; st NaOCl, stabilized sodium hypochlorite; gen NaOCl, generated sodium hypochlorite. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55604/55604fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55604/55604fig3.jpg" /> 
Figure 3: Phi6 handwashing results. Compared to no handwashing, the handwashing methods tested resulted in an average log reduction in Phi6 of 2.44-3.06 without soil load and 2.71-3.69 with soil load. Handwashing with soap demonstrated the least reduction in Phi6 without soil load (2.44), and handwashing with stabilized NaOCl resulted in the smallest reduction with soil load (2.71). Handwashing with generated NaOCl resulted in the greatest reduction without soil load (3.06), and handwashing with soap resulted in the greatest reduction with soil load (3.69). In the charts, the line represents the percent reduction in organisms, and the error bars represent the standard error of log reduction. Ctrl B, control B; HWWS, handwashing with soap; ABHS, alcohol-based hand sanitizer; HTH, high-test hypochlorite; NaDCC, sodium dichloroisocyanurate; st NaOCl, stabilized sodium hypochlorite; gen NaOCl, generated sodium hypochlorite. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55604/55604fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55604/55604fig4.jpg" /> 
Figure 4: E. coli hand rinse results. Compared to hand washing with water only, the average log reduction of E. coli remaining in the rinse water was 0.28-4.77 without soil load and 0.21-4.49 with soil load. Both with and without soil load, the smallest reduction was found in handwashing with soap (0.28 and 0.21). The greatest reductions were observed with stabilized and generated NaOCl without soil load (both 4.77) and with HTH and generated NaOCl with soil load. In the charts, the line represents the percent reduction in organisms, and the error bars represent the standard error of log reduction. HWWS, handwashing with soap; ABHS, alcohol-based hand sanitizer; HTH, high-test hypochlorite; NaDCC, sodium dichloroisocyanurate; st NaOCl, stabilized sodium hypochlorite; gen NaOCl, generated sodium hypochlorite. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55604/55604fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55604/55604fig5.jpg" /> 
Figure 5: Phi6 hand rinse results. Compared to hand washing with water only, the average log reduction of Phi6 remaining in the rinse water was 1.26-2.02 without soil load and 1.30-2.20 with soil load. With soil load, the smallest reduction was found in handwashing with soap (1.26). Without soil load, HTH resulted in the smallest reduction (2.02). The greatest reductions were observed both with and without soil load with NaDCC (2.02 and 2.20). In the charts, the line represents the percent reduction in organisms, and the error bars represent the standard error of log reduction. HWWS, handwashing with soap; ABHS, alcohol-based hand sanitizer; HTH, high-test hypochlorite; NaDCC, sodium dichloroisocyanurate; st NaOCl, stabilized sodium hypochlorite; gen NaOCl, generated sodium hypochlorite. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55604/55604fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

18.8K Views.  Tufts University. 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. 

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.

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.

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 ...

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.

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