Name: determining viral disinfection efficacy of hot water laundering
Uploaded: 2022-06-21T15:00:00.000+00:00
Duration: 6 min 57 s
Description: Watch this Scientific Journal Video about Determining Viral Disinfection Efficacy of Hot Water Laundering at JoVE.com

The U.S. Environmental Protection Agency (EPA) through its Office of Research and Development directed the research described herein under EP-C-15-008 with Jacobs Technology Inc. It has been reviewed by the Agency but does not necessarily reflect the Agency&#39;s views. No official endorsement should be inferred. EPA does not endorse the purchase or sale of any commercial products or services. The authors would like to thank EPA contractors Denise Aslett for the oversight of the EPA RTP microbiology, Brian Ford, Rachael Baartmans, and Lesley Mendez Sandoval for their work on this project in the EPA RTP microbiology lab, Ramona Sherman for providing the EPA quality assurance review, and Worth Calfee and Shannon Serre for providing EPA technical reviews.

Phi6 was received from a collaborator laboratory as a ~1 mL frozen aliquot and was stored at -80 &#176;C until use. It was initially used to propagate more virus stocks which were subsequently stored at -80 &#176;C until use. Phi6 was selected as the demonstration virus because it is commonly used as a model enveloped virus, can be propagated to high titers, and requires a low biosafety level laboratory to perform the testing22,23,24.
1. Prepare virus stock solution
<ol>
	<li>Propagate bacteriophage Phi6 in bacterial host Pseudomonas syringae using a modified Tryptic Soy Agar media preparation and soft agar overlay method as described below.
	<ol>
		<li>Prepare modified Tryptic Soy Agar by weighing out and mixing the ingredients from Table 1 in deionized water.</li>
		<li>Prepare an overnight culture of P. syringae by adding a 1 mL aliquot of P. syringae with an optical density (OD600) between 0.9 to 1.5,&#160;to 100 mL of modified Tryptic Soy Broth (Table 1) and incubating in a shaking incubator at ~260 rpm at room temperature (20-26 &#176;C).</li>
		<li>Prepare soft agar tubes by placing modified Tryptic Soy Agar (~6 mL) in test tubes and covering with an autoclavable cap. Store at 4 &#176;C until use. Autoclave soft agar tubes at 121 &#176;C for 15 min to melt agar. Hold at 48 &#176;C until plating. Equilibration at 48 &#176;C is important otherwise the virus can be inactivated in the assay.</li>
		<li>Add 1 mL of undiluted concentrated virus aliquot to soft agar and 100 &#181;L of a log phase (OD600 between 0.9 to 1.5) P. syringae culture. Pour soft agar onto the surface of a solidified 100 mm diameter modified Tryptic Soy agar plate. To prevent bubbles and/or spilling, swirl plates gently to evenly distribute the soft agar over the solid agar surface and incubate overnight at room temperature.</li>
		<li>Gently scrape25 the contents of the three plates with a sterile cell spreader into a sterile 50 mL conical tube containing 15 mL of SM buffer. Vortex tubes at maximum setting for 1-2 min to break up agar and then centrifuge at 7,000 x g for 15 min.</li>
		<li>Remove supernatant and filter through a 0.2 &#181;m syringe filter. Store 1 mL aliquots in cryovials at -80 &#176;C until use.</li>
	</ol>
	</li>
</ol>
2. Perform pre-test visual assessment of PPE item
<ol>
	<li>Place each PPE item on a clean, smooth surface (e.g., a laboratory bench covered with a disposable paper liner). Assess each PPE item in triplicate.</li>
	<li>Ensure proper lighting during the PPE examination. Measure and record the length and width of the unwashed items at various locations (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64164/64164fig1v2.jpg" /> 
Figure 1. PPE pre-test assessment measurement locations. Denim, scrub, and face-covering locations where the length was recorded for tracking material changes from the laundering process. <a href="https://www.jove.com/files/ftp_upload/64164/64164fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Prepare coupons
<ol>
	<li>Make 2 cm x 4 cm test coupons by cutting PPE, prepare two coupons per face covering (three face coverings were tested), five coupons per denim pant (three denim pants were tested), and three coupons per scrub shirt (three scrubs were tested).</li>
	<li>Prepare a set of 2 cm x 4 cm procedural blank coupons (a set for one full-sized PPE item) for each material type that will not be inoculated but will be laundered. Prepare two coupons for every day of face-covering experiments, five coupons for every day of denim experiments, and three coupons for every day of scrub experiments.</li>
	<li>Prepare a set of 2 cm x 4 cm positive control coupons that will be inoculated, but not laundered. Prepare two coupons for every day of face-covering experiments (three face coverings were tested), five coupons for every day of denim experiments (three denim pants were tested), and three coupons for every day of scrub experiments (three scrubs were tested), and three stainless steel coupons. 
	&#8203;NOTE: Different numbers of replicates were selected based on the size of the item. For example, it is physically difficult to adhere five coupons on the face covering, and two coupons would represent a limited area of the denim pants. The locations were selected to maximize coverage and in zones that might get folded during laundering and be more difficult to clean.</li>
</ol>
4. Perform inoculation
<ol>
	<li>Prepare a 10% beef extract solution by dissolving 1 g of beef extract in a total volume of 10 mL of 1x phosphate buffered saline. Filter sterilize the entire volume using a 0.2 &#181;m syringe filter.</li>
	<li>Thaw the virus stock solution prepared in section 1 at room temperature. On the day of use, add 100 &#181;L of the thawed Phi6 stock to 900 &#181;L of the 10% beef extract solution.</li>
	<li>Inoculate test coupons and positive control coupons with ~107 PFU/sample by pipetting a 10 &#181;L droplet of solution onto the PPE item and spreading the droplet using the tip of the pipette. Depending on the PPE material, the droplets will separate and reaggregate differently.</li>
	<li>Allow inoculated coupons to dry in a biosafety cabinet. Determined dry time(s) via observation for your specific materials. For the results presented here, the following times were used: scrubs = 30 min dry time; face-covering = 60 min dry time; denim = 30 min dry time; stainless steel = 120 min dry time.</li>
	<li>Attach inoculated coupons to full-sized PPE items according to Figure 2 using safety pins and aseptic techniques.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64164/64164fig2v2.jpg" /> 
Figure 2. Test coupon locations on denim, scrubs, and face coverings. Letters A-D corresponds to the unique coupon identifiers for all laundering experiments. <a href="https://www.jove.com/files/ftp_upload/64164/64164fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Perform laundering
<ol>
	<li>Prepare laundering detergent as follows.
	<ol>
		<li>Sterilize tap water that will be used in the washing machine and collect 10 mL of autoclaved water for a sterility check. For this protocol, autoclave 7 L of tap water on a 60 min liquid cycle.</li>
		<li>Prepare laundering solution according to manufacturer&#39;s dilution directions. For this protocol, dissolve 1.54 mL of detergent in 3.5 L of sterile tap water. Warm the laundering solution to 50 &#176;C using a hot plate and stir bar. Measure and record the pH and temperature of the laundering solution. Collect 10 mL of solution for sterility check.</li>
	</ol>
	</li>
	<li>Pour laundering solution into a sterilized washer (3.25 L). Pre-sterilize washers using a 250 ppm-4 h cycle of vaporous hydrogen peroxide between tests.</li>
	<li>Place PPE item(s) in a sterilized washer. Add one denim pant and one scrub shirt per washer. Add one inoculated face covering and four non-contaminated fill masks per washer; fill masks did not have coupons attached.</li>
	<li>Wash PPE item for 18 min (two 9 min wash cycles with normal agitation). Drain the washer and triple rinse with room temperature tap water (5 L each time) to remove foam. Add room temperature sterilized tap water into the washer (3.25 L) and perform a 9 min long rinse cycle.</li>
	<li>Move the PPE item(s) into the washer&#39;s spin side and spin for 5 min. Move the PPE item(s) to the dryer and dry for 80 min on the high heat setting (93 &#176;C).</li>
	<li>Move PPE from dryer to sterile workspace and aseptically remove the coupons from each item and place them in conical tubes. Pre-fill the tubes with 10 mL of 10% Dey-Engley broth extraction buffer and cover with aluminum foil.</li>
</ol>
6. Extract and enumerate viral loads on coupons
<ol>
	<li>Extract coupons in 10 mL of 10% Dey-Engley neutralizing broth by vortexing for 2 min at the maximum setting of your equipment.</li>
	<li>Plate extracts using a conventional soft top agar overlay method26.
	<ol>
		<li>Prepare tubes of soft modified Tryptic Soy Agar and a P. syringae culture as described in section 1. On the day of testing, autoclave the soft agar tubes at 121 &#176;C for 15 min to melt the agar. Hold the soft agar at 48 &#176;C until plating. Equilibration at 48 &#176;C is important otherwise the virus can be thermally inactivated in the assay.</li>
		<li>Prepare a tenfold dilution series in 1x phosphate-buffered saline for each test sample used in the laundering study. Use both serially diluted (100 &#181;L) and undiluted (1 mL and 100 &#181;L) aliquots for plating.</li>
		<li>Add test sample aliquots to the soft agar tube containing 6 mL of soft agar and 100 &#181;L of the log phase P. syringae culture (OD600 between 0.9-1.5). Pour the soft agar onto the surface of a solidified modified Tryptic Soy agar plate. Evenly distribute the soft agar over the solid agar surface by swirling the plate.</li>
		<li>Incubate plates overnight at room temperature and manually enumerate the following day by counting the plaque-forming units on each plate.</li>
	</ol>
	</li>
</ol>
7. Perform post-test visual assessment of PPE item(s)
<ol>
	<li>Document the following in the various PPE items used for testing: signs of fading, discoloration, and/or damage (e.g., tearing, stretching); odors; small holes, cuts, or tears (use a small flashlight to look for damage); separation of layers, missing threads, areas where binding is damaged; damage to seams or zippers; measure and record the stretching of elastic.</li>
</ol>

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There are no known conflicts of interest to disclose.

This protocol was developed to execute systematic laboratory testing to assess the laundering effectiveness of viral disinfection from full-sized PPE/clothing items. The procedures outline the critical steps for preparing the virus, inoculating the test material, assessing the changes to the items due to the laundering process, and quantifying the reduction in viral load as a result of the laundering (machine washing and drying) process. Additionally, the protocol outlines the necessary quality control samples for ensuring the experiments are not biased by contamination and measurements/observations that should be recorded to track the material integrity of the PPE items after multiple laundering cycles. The results using Phi6 indicate that the hot water laundering process used in this protocol achieved a greater than 3-log reduction in viral load for all samples (face covering, scrubs, and denim pants). The viral load reduction was also uniform across different locations on the PPE/clothing items. To demonstrate 3-log reduction, this protocol requires the use of a high viral load and a stabilizing agent (beef extract) that may not be representative of the soil load for all situations.
Mini washers and compact dryers were selected to optimize the number of replicate experiments that could be conducted in a space-constrained setting and to keep the sterilization of equipment and water volume used during the experiments manageable for laboratory staff. As a result of using the mini washer, the rinsing steps were manual as compared to most home laundering applications that are fully automated. It is also important to remember that machine washing predominates in developed countries, but handwashing is still practiced all over the world15. Additionally, some may not have access to hot water for washing, and others manually air-dry clothes rather than machine drying. These differences in laundering practices were not addressed in this current protocol but could easily be investigated with minor modifications such as substituting the washing and drying steps with using a bucket and a close line
There has been minimal focus on the cleaning/disinfecting of virally contaminated face coverings and street clothing in the scientific literature at the full scale. More commonly, studies assess the filtration performance of face coverings after repeated washing and drying but do not evaluate viral disinfection efficacy27,28. For example, Clapp et al. evaluated fitted filtration efficiency of cloth masks and modified procedure masks and found wide variation in performance, with simple modifications providing increased fit and filtration efficiency29. Another study looked at the filtration efficiency of four cloth masks of different materials30, again focusing on source control or personal protection. This may be due to a lack of specialization for both the microbial portion and mechanical testing in the same laboratory. The protocol presented here provides an evaluation of disinfection efficacy as well as material degradation.
There have been a number of decontamination/disinfection methods for disposable respiratory protection (primarily N95s) recently published in the scientific literature31,32,33. The primary focus on FFRs (e.g., N95s) is due to the critical respiratory protection they provide for healthcare workers and other front-line occupations. Primary technologies for respirator decontamination involved vaporized hydrogen peroxide (VHP), ultraviolet germicidal radiation (UVGI), and moist heat (steam) for virus inactivation. Viscusi et al. evaluated five decontamination methods for FFRs and UVGI; ethylene oxide and VHP were found to be the most promising decontamination methods31. Fischer et al. evaluated four different decontamination methods-UV light, dry heat, 70% ethanol, and VHP-for their ability to reduce contamination with SARS-CoV-2 and their effect on N95 respirator function32. There are many additional studies on effective decontamination technologies for FFRs which were summarized and published in 202033. However, these specialized methods aren&#39;t accessible or designed to be used safely by the average home or small business owner.
This protocol was developed using Phi6, an enveloped bacteriophage that is similar to SARS-CoV-2, has spike proteins, and is of similar size (80-100 nm)34, for all testing. Since Phi6 is not a known pathogen, it can be manipulated in a general microbiological Biosafety Level 1 (BSL-1) laboratory. Efficacy against Phi6 may indicate the efficacy of other enveloped viruses, however, empirical verification for each virus of interest is necessary35. By using a similar, nonpathogenic viral agent, it is hoped that this protocol can be repeated elsewhere and used for studying future viral epidemics/pandemics. Future research may include the use of disinfectants (e.g., bleach) in addition to detergents and a standardized protocol for hand washing and line drying.

The coronavirus disease 2019 (COVID-19) pandemic caused unprecedented global supply chain disruption and led to a critical shortage of many items, including essential personal protective equipment (PPE)1,2,3. Those in high-risk occupations had to adapt using recommended crisis capacity strategies and the public adopted the use of non-specialized items such as cloth material face coverings primarily for source control, but also to provide some respiratory protection for wearers. In the United States, specialized respiratory protection (i.e., filtering facepiece respirators (FFRs) such as N95s) was reserved for some of these high-risk occupations (e.g., healthcare) during supply shortages4. When little was known about severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) transmission, a variety of other types of clothing materials were also considered as barrier protection early in the pandemic5. With the diversity of fabrics being used for wearer protection, questions arose about the use, reuse, and disinfection/decontamination of these items. While in the United States it was generally accepted that routine home machine laundering of face coverings and other clothing items rendered viruses on those surfaces non-infectious, little data existed to validate this claim, and there was a lack of published laboratory protocols for testing. The purpose of the research protocol presented here is to provide an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol was developed for research during the COVID-19 pandemic, it is intended to be a framework adaptable to other virus disinfection studies.
The role of clothing in disease transmission is a difficult concept to quantify. The International Scientific Forum on Home Hygiene attempted this challenging task by conducting a review of the role of clothing in the spread of infectious disease coupled with a risk assessment of home hygiene practices6. Included in this work was the review of several scientific studies that examined the survival of different viral strains on different types of fabrics such as wool and cotton7,8,9,10,11. Each study focused on a different type of virus including vaccinia, poliovirus, respiratory syncytial virus, herpesvirus, and influenza virus. The survival times of the different viruses on the fabrics ranged from 30 min to 5 months depending on the virus-material combination. Several of the studies also demonstrated the transfer of viral contamination from the material onto hands. As part of the publication, effective laundering was discussed as an important management technique to reduce transmission but recognized that the magnitude of the impact of laundering on reducing disease burden was dependent on the specific viral contaminant and difficult to quantify7,8,9,10,11.
The laundering process destroys microorganisms using chemical, physical, and thermal treatment processes. For example, soaps and detergents can separate soils and may impart some chemically mediated antimicrobial action. Physically, dilution and agitation may aid in the reduction in viral loads. A study examining the persistence of human coronavirus HCoV-OC43 on cotton swatches using industrial and domestic wash cycles with and without temperature and detergent found no detectable virus when washing in unheated water without detergent, but that in the presence of a soil load (artificial saliva), domestic wash cycles required detergent for samples to have non-detect virus loadings12. Hot water itself can also provide an effective means of destroying some microorganisms13,14.
In a recent publication summarizing the state of current laundry practices, many factors such as fabric composition, storage conditions, dirt load, wash temperature and time, and drying temperature was identified as varying in global practices of laundering15. While laundering is a common cleaning method for a large percentage of the population, this large variation in existing practices makes issuing detailed guidance for how to do this safely and effectively, when an item may be contaminated by a virus, challenging and sparse. During the COVID-19 pandemic, the United States Centers for Disease Control and Prevention (CDC) issued guidance on how to launder items for homeowners16,17. Much of this laundering guidance was based on several older studies on bacterial disinfection18,19 and supported by several benchtop studies that have found enveloped viruses inactivated in water with detergents20,21. The guidance can be summarized as 1) follow the manufacturer&#39;s instructions for the detergent, 2) use the warmest appropriate water setting, and 3) dry items completely. The rationale of these recommendations was that washing on the warmest possible cycle with detergent combined with drying completely (with heat if possible) will kill the SARS-CoV-2 virus.
The sheer number of possible variations in the laundering process necessitates a uniform protocol, such as presented here, to be able to isolate variables and test the viral disinfection efficacy of specific processes. The intent of this protocol coupled with an instructional video is to demonstrate a laboratory-based hot water laundering process for replication in other research studies. Additionally, the results of this viral disinfection efficacy testing should build consumer confidence in the effectiveness of home laundering during viral-based pandemics.

<table><tbody><tr><td>&amp;nbsp;Freezer (- 80 &amp;deg;C)</td><td>ThermoFisher Scientific</td><td>FDE30086FA</td><td></td></tr><tr><td>&amp;nbsp;Hot Plate</td><td>VWR</td><td>97042-714</td><td></td></tr><tr><td>&amp;nbsp;Safety Pins (steel)</td><td>Singer</td><td>319921</td><td></td></tr><tr><td>&amp;nbsp;Shaker</td><td>Lab-Line Instruments, Inc.</td><td>3525</td><td></td></tr><tr><td>&amp;nbsp;SM buffer</td><td>Teknova,&amp;nbsp; Hollister, CA</td><td>S0249</td><td></td></tr><tr><td>&amp;nbsp;Syringe filter (0.2 &amp;mu;m)</td><td>Corning, Corning, NY</td><td>PES syringe filters, 431229</td><td></td></tr><tr><td>1X Phosphate Buffered Saline</td><td>Teknova, Hollister, CA</td><td>P0196, 10X PBS solution</td><td></td></tr><tr><td>Agar</td><td>Becton Dickinson</td><td>214010</td><td></td></tr><tr><td>Autoclavable caps</td><td>DWK Life Sciences, Millville, NJ</td><td>KIM-KAP Caps, 73663-18</td><td></td></tr><tr><td>Autoclave</td><td>Steris</td><td>AMSCO 250LS Steam Sterilizer Model 20VS</td><td></td></tr><tr><td>Beef Extract</td><td>Sigma-Aldrich, Millipore Sigma, St. Louis, MO, USA</td><td>P/N B4888-100g</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>793639</td><td></td></tr><tr><td>Cell spreaders</td><td>Busse Hospital Disposables</td><td>23600894</td><td></td></tr><tr><td>Centrifuge</td><td>ThermoFisher Scientific</td><td>75004271</td><td>Heraeus MegaFuge 16R Centrifuge</td></tr><tr><td>Certified Timer</td><td>https://nist.time.gov/</td><td>Not Applicable</td><td></td></tr><tr><td>Conical tubes (50 mL)</td><td>Corning Life Sciences</td><td>352098</td><td>Falcon 50-mL high-clarity polypropylene conical centrifuge tubes</td></tr><tr><td>Cryovials</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>AY509X33</td><td></td></tr><tr><td>Denim</td><td>Wrangler</td><td>Rustler Regular Fit Straight Leg Jean Four Pocket Jean with Scoop Front Pockets, PN:87619PW</td><td></td></tr><tr><td>Detergent</td><td>Proctor and Gamble</td><td>Tide Original Scent Liquid Laundry Detergent Product Number (PN): 003700023068</td><td></td></tr><tr><td>Dextrose</td><td>Fisher</td><td>BP350</td><td></td></tr><tr><td>Dey-Engley neutralizing broth</td><td>Becton Dickinson</td><td>DF0819172</td><td></td></tr><tr><td>Dryer</td><td>Magic Chef</td><td>MCSDRY15W</td><td></td></tr><tr><td>Face Coverings</td><td>Felina</td><td>Reusable Organic Cotton Face Masks, PN: 990121P4</td><td></td></tr><tr><td>Incubator (top agar)</td><td>Symphony</td><td>414004-596</td><td></td></tr><tr><td>Laboratory Notebook</td><td>Scientific Notebook Company</td><td>2001</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>Media sterilization and dispensing system</td><td>Integra</td><td>Media Clave/Media Jet</td><td></td></tr><tr><td>Petri Dishes (100 mm)</td><td>VWR</td><td>25384-342</td><td></td></tr><tr><td>pH Meter</td><td>Orion/Oakton</td><td>STARA1110/EW-35634-35</td><td></td></tr><tr><td>pH Probe</td><td>Orion</td><td>8157BNUMD</td><td></td></tr><tr><td>pH Standards</td><td>Oakton</td><td>00654-(00/04/08)</td><td></td></tr><tr><td>Phi 6 and Pseudomonas syringae</td><td>Battelle Memorial Institute, Columbus, OH</td><td>Not Applicable</td><td></td></tr><tr><td>Pipette &amp;amp; Tips</td><td>Rainin</td><td>(Pipettes) 17014391, 17002921; (Pipette Tips) 30389239, 17014382</td><td></td></tr><tr><td>Refrigerator</td><td>True Manufacturing Co., Inc.</td><td>GDM-33</td><td></td></tr><tr><td>Scrubs</td><td>Gogreen cool</td><td>PN: WS19100PT</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>57656</td><td></td></tr><tr><td>Stir Bar</td><td>Fisherbrand</td><td>16-800-512</td><td></td></tr><tr><td>Tape Measure</td><td>Lufkin</td><td>PS3425</td><td></td></tr><tr><td>Test Tubes for Soft agar (14 mL)</td><td>Corning, Corning, NY</td><td>352059</td><td></td></tr><tr><td>Thermometer</td><td>Fisherbrand</td><td>14-983-19B</td><td></td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T9410</td><td></td></tr><tr><td>Vaporous hydrogen peroxide sterilization bags</td><td>STERIS</td><td>62020TW</td><td></td></tr><tr><td>Vortex (during the plating process)</td><td>Daigger Scientific, Inc</td><td>3030A</td><td>Vortex Genie 2</td></tr><tr><td>Vortex (for sample extraction)</td><td>Branson Ultrasonics</td><td>58816-115</td><td>Multi-Tube vortexer</td></tr><tr><td>Washer</td><td>Kuppet</td><td>KP1040600A</td><td></td></tr><tr><td>Washer Sterilization</td><td>Steris</td><td>STERIS VHP ED1000 generator</td><td></td></tr><tr><td>Yeast extract</td><td>Gibco</td><td>212750</td><td></td></tr></tbody></table>

determining viral disinfection efficacy of hot water laundering

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol was developed for research during the coronavirus disease 2019 (COVID-19) pandemic, it is intended to be a framework, adaptable to other virus disinfection studies; it demonstrates the steps for preparing the test virus, inoculating the test material, assessing visual and integrity changes to the washed items due to the laundering process, and quantifying the reduction in viral load. Additionally, the protocol outlines the necessary quality control samples for ensuring the experiments are not biased by contamination and measurements/observations that should be recorded to track the material integrity of the personal protective equipment (PPE) items after multiple laundering cycles. The representative results presented with the protocol use the Phi6 bacteriophage inoculated onto cotton scrub, denim, and cotton face-covering materials and indicate that the hot water laundering and drying process achieved over a 3-log (99.9%) reduction in viral load for all samples (a 3-log reduction is the disinfectant performance metric in U.S. Environmental Protection Agency's Product Performance Test Guideline 810.2200). The reduction in viral load was uniform across different locations on the PPE items. The results of this viral disinfection efficacy testing protocol should help the scientific community explore the effectiveness of home laundering for other types of test viruses and laundering procedures.

Several different types of quality control data and results are generated after completion of this protocol. Plaque forming unit (PFU) plate counts along with the extracted sample volume enable the calculation of the number of PFU per test coupon. Table 2 is an example of a data recording sheet for serial dilution/plating results. Using the dilution factor, sample volume, and plate count from Table 1, Table 3 shows representative viral recovery results for a face covering test. Note that these data include the test coupons and the quality control samples for the inoculum, coupons, and wash water (with and without detergent). The procedural blank and sterility quality control samples are important for confirming that the water solutions and PPE materials were not contaminated with Phi6. Indication of contamination would cause erroneous calculations of disinfection efficacy and require the test to be repeated. The positive control samples are intended to verify that the virus stock solution did not environmentally/naturally degrade during the experiments, thereby inflating the effect of the laundering process in viral load reduction. These samples should remain within 1 log PFU of the inoculum controls to accept the test coupon results. A large reduction in PFU of the positive control samples also indicates that all steps of the coupon inoculation should be carefully reviewed to ensure that the analyst is executing the protocol with proper pipetting and spreading techniques.
This protocol also provides information for assessing changes to the material properties of clothing items due to laundering and quality control information pertaining to the protocol (Table 4 and Figure 3). These data are useful for several reasons. Recording the trends in measurements of the PPE items allows for the identification of an item with a manufacturing defect. This identification may help to explain outlier microbial data and contextualize the variation in product behavior. Taking notes of odors or damage may also provide an indication if the washer or dryer was operating sub-optimally during an experiment and if the tests should be repeated or the equipment serviced. Additionally, if the test plan calls for multiple laundering cycles of the same PPE item, the data may help determine how long the PPE items maintain their integrity for use when laundering. Keeping a record of the pH of the detergent solution provides an alert to changes in the water source or the detergent product. Maintaining a time log of the laundering steps ensures that the timer on the washer and dryer does not cause variations to the experimental protocol.
Ultimately, these data are used to report the disinfection efficacy of the hot water laundering procedure against a surrogate for viral pathogens. Laundering disinfection efficacy (Eqn. 1) is calculated by subtraction of the average log10 virus recoveries from PPE test coupon from the average log10 virus recoveries from PPE positive control results (Figure 4). For test coupon results that are non-detect, the log10 of the detection limit is used in the disinfection efficacy calculation. It is common to report disinfection efficacy as log values for comparison with other viral disinfection techniques and standards.
Disinfection Efficacy = Average log10 (Positive Controls) - Average log10 (Test Coupons) (Eqn. 1)
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64164/64164fig3v2.jpg" /> 
Figure 3. Change in PPE dimensions by location. &#916; = Pre-test measurement - post-test measurement. A negative &#916; value corresponds to the stretching of the item at the specified location and a positive value corresponds to shrinkage. <a href="https://www.jove.com/files/ftp_upload/64164/64164fig3v2large.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/64164/64164fig04.jpg" /> 
Figure 4. Efficacy of hot water laundering at disinfecting face coverings, denim, and scrub PPE materials from Phi6. Stars denote full disinfection occurred (non-detects on the test coupons). Error bars indicate standard deviation (n = 3). Location letters correspond to the placement depicted in Figure 2. <a href="https://www.jove.com/files/ftp_upload/64164/64164fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Solution recipes. Ingredients and amounts necessary to prepare tryptic soy agar, tryptic soy broth, and beef extract solutions. <a href="https://www.jove.com/files/ftp_upload/64164/Table1_Solutions.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2. An example portion of a serial dilution/plating results sheet. Template for reporting raw microbial data. <a href="https://www.jove.com/files/ftp_upload/64164/Table2_PlatingResultsSheet_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3. Face covering microbial results. Example summary sheet for processed plaque-forming unit (PFU) data. <a href="https://www.jove.com/files/ftp_upload/64164/Table 3 Viral Recovery Results_Re.xlsx" target="_blank">Please click here to download this Table.</a>
Table 4. Scrubs quality control and material assessment log. Template for reporting calibration of pH probe, pH of detergent solution, pre-and post-wash measurements, and laundering cycle times. <a href="https://www.jove.com/files/ftp_upload/64164/Table 4_scrubs log.xlsx" target="_blank">Please click here to download this Table.</a>

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Determining Viral Disinfection Efficacy of Hot Water Laundering

In response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a laboratory protocol was developed to test the viral disinfection efficacy of hot water laundering of cloth face coverings, cotton scrubs, and denim pants. The Phi6 virus (bacteriophage) was used as the organism to test disinfection efficacy.

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol ...

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2.5K Views.  U.S. Environmental Protection Agency. In response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a laboratory protocol was developed to test the viral disinfection efficacy of hot water laundering of cloth face coverings, cotton scrubs, and denim pants. The Phi6 virus (bacteriophage) was used as the organism to test disinfection efficacy.

Video: Determining Viral Disinfection Efficacy of Hot Water Laundering

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

Environment

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Immunology and Infection

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.

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 ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a ...

Medicine

Antimicrobial Efficacy of Nano-Herbal-Coated Fabrics

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and burns. These cotton-based protective coats provide ideal conditions for microbial growth and attachment sites due to their porous nature, moisture-holding capacity, and retention of warmth from the user&#39;s body. Several studies have demonstrated the survival of pathogenic bacteria on hospital garments and lab coats, acting as vectors of microbial transmission.
A common approach to fix these problems is the application of antimicrobial agents in textile finishing, but concerns have been raised due to the toxicity and environmental effects of many synthetic chemicals. The ongoing pandemic has also opened a window for the investigation of effective antimicrobials and eco-friendly and toxic-free formulations. This study uses two natural bioactive compounds, carvacrol and thymol, encapsulated in chitosan nanoparticles, which guarantee effective protection against four human pathogens with up to a 4-log reduction (99.99%). These pathogens are frequently detected in lab coats used in biohazard laboratories.
The treated fabrics also resisted up to 10 wash cycles with 90% microbial reduction, which is sufficient for the intended use. We made modifications to the existing standard fabric tests to better represent the typical scenarios of lab coat usage. These refinements allow for a more accurate evaluation of the effectiveness of antimicrobial lab coats and for the simulation of the fate of any accidental microbial spills that must be neutralized within a short time. Further studies are recommended to investigate the accumulation of pathogens over time on antimicrobial lab coats compared to regular protective coats.

Author Spotlight: An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils  

Antimicrobial lab coats prevent the cross-contamination of pathogen accumulation and accidental bio-spills. Here, we describe the protocol for developing a skin-friendly antimicrobial fabric using nano-herbal encapsulation and modified standard tests to precisely evaluate the efficacy and suitability for typical usage of the lab coat.

Lab coats are widely used in biohazard laboratories and healthcare facilities as protective garments to prevent direct exposure to pathogens, spills, and ...

Biology

Decontamination for Laboratory Biosafety

Robert M. Rioux and Zhifeng Chen, Pennsylvania State University, University Park, PA
Decontamination is essential for laboratory biosafety, as the accumulation of microbial contamination in the laboratory can lead to the transmission of disease. The degree of decontamination can be classified as either disinfection or sterilization. Disinfection aims to eliminate all pathogenic microorganisms, with the exception of bacterial spores on lab surfaces or equipment. Sterilization, on the other hand, aims to eliminate all microbial life. Different methods are available which include chemicals, heat, and radiation, and once again depend on the degree of decontamination, as well as the concentration of the contaminating microorganisms, presence of organic matter, and type of equipment or surface to be cleaned. Each method has its advantages and cautionary measures that need to be taken to avoid hazards.

Decontamination Methods for Laboratory Biosafety

Robert M. Rioux and Zhifeng Chen, Pennsylvania State University, University Park, PA
Decontamination is essential for laboratory biosafety, as the ...

Education

JoVE Science Education

Basic Biology

Lab Safety

Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay

Source: Tai, C. et al., <a href="https://app.jove.com/v/53124">Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds.</a> J. Vis. Exp. (2015)
This video demonstrates the viral inactivation assay, showcasing the evaluation of the impact of antiviral agents on viral particles.

1. Viral Inactivation Assay
Note: Examples of incubation period and viral dose for various viruses are listed in Figure 1A. Higher concentrations of the ...

Determining Viral Disinfection Efficacy of Hot Water Laundering

Summary

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A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Remote Laboratory Management: Respiratory Virus Diagnostics

An Antimicrobial Fabric Using Nano-Herbal Encapsulation of Essential Oils

Decontamination for Laboratory Biosafety

Assessing the Effectiveness of an Antiviral Test Compound through a Viral Inactivation Assay