Human noroviruses (HuNoV) are the leading cause of acute nonbacterial gastroenteritis (11, 21). The U.S. Centers for Disease Control and Prevention estimate that approximately 23 million people suffer from HuNoV gastroenteritis each year. Indeed, 81% of outbreaks of nonbacterial gastroenteritis are caused by this agent (7, 8). These viruses may contaminate food, water, hands, and inanimate surfaces and are readily transmitted by contact with contaminated objects, by consumption of fecally contaminated food or water, or between people. In particular, hands are thought to be a principal vehicle for HuNoV transmission. Although many different hand hygiene agents are available, these generally do not have specific virucidal claims against the HuNoV. Two widely used types of hand hygiene products are antibacterial liquid soaps and alcohol-based hand sanitizers. Little is known about the effectiveness of hand hygiene agents in reducing HuNoV on contaminated hands. One reason for this is that the HuNoV cannot be routinely cultured in vitro, a factor that complicates evaluation of the efficacy of disinfection strategies. As an alternative, investigators have used cultivable surrogates such as feline calicivirus (FCV) and murine norovirus (MNV) (10, 15, 22), but questions continue regarding their relevance. Quantitative real-time nucleic acid amplification technologies allow us to quantify RNA and can be used to estimate HuNoV titer, although the relationship to virus infectivity is not clear (5, 18, 26, 30). In this study, we used real-time reverse transcription-quantitative PCR (RT-qPCR) to test the effectiveness of sodium hypochlorite and ethanol against Norwalk virus (NV) in a suspension assay. This was followed by an evaluation of the efficacy of an antibacterial liquid soap, alcohol-based hand sanitizer, and water rinsing for the removal and/or inactivation of NV on the finger pads of human volunteers. To examine the likelihood that detection of residual HuNoV RNA was associated with intact and, hence, infectious virus, we also tested finger pad eluates by RT-qPCR both with and without prior RNase treatment.

Norwalk virus inoculum. NV was obtained from the stool of an experimentally infected human volunteer from a previous study (20). The stool was diluted 20% (wt/vol) in RNase-free water prior to inoculation on the finger pads. Study subjects. The study protocol was reviewed and approved by the Institutional Review Board of Emory University. A total of 10 adult volunteers were enrolled in this study after informed consent was obtained. Prior to each experiment, both hands of each volunteer were carefully inspected to ensure that they were free of any cuts, abrasions, or rashes. Suspension assays. Suspension tests for virucidal activity were performed in accordance with a modification of the method reported by Macinga et al. (22). Different concentrations of ethanol (3%, 17%, 31%, 47%, 62%, and 95%) were prepared using deionized water and were analyzed by gas chromatography. Similarly, various sodium hypochlorite concentrations (3 ppm, 22 ppm, 51 ppm, 160 ppm, and 1,600 ppm) were prepared using potassium phosphate buffer (pH 7.4), and total chlorine concentrations were determined by a digital titrator (Hach, Loveland, CO). Ten microliters of the 20% NV stool suspension was added to 990 #l of each test disinfectant solution or water (as a baseline control), quickly vortexed, and held for 30 s. Immediately following the exposure period, a 100-#l aliquot was removed from the tube and added to a tube containing 900 #l of 10% fetal bovine serum to neutralize the reaction. Then, a 100-#l aliquot was further diluted in 900 #l Hanks balanced salt solution (HBSS). Finally, 1 ml of the diluted virus-disinfectant mixture was concentrated to 50 #l by precipitation with 12% polyethylene glycol (Sigma, St. Louis, MO), followed by processing for RT-qPCR. Hand sanitizer and liquid soap. A hand sanitizer, containing 62% ethyl alcohol as the active ingredient, was purchased from a local retail source. The antibacterial liquid soap was obtained from Fisher Scientific International (Hampton, NH). The latter contained 0.5% triclosan as the active ingredient, with inactive ingredients including sodium lauryl ether sulfate, cocamidopropyl betaine, solulan, propylene glycol, sodium chloride, dyes, and fragrances. To screen for PCR inhibition, 10 #l of the test product was mixed with 980 #l of HBSS and 10 #l of 20% NV stool suspension, 10- and 100-fold serially diluted, and then tested by RT-qPCR. Inoculation of NV onto human finger pads. The volunteers first washed their hands with a nonmedicated soap for at least 10 s, rinsed with tap water, and dried them with a single-use paper towel. Approximately 4 ml of 70% ethanol was placed in the palm of one washed hand, and the volunteers were instructed to rub it over the entire surface of both hands until the alcohol and water had evaporated completely. The volunteers pressed each finger pad over the mouth of an empty 2.0-ml plastic vial (Sarstedt, Newton, NC) to demarcate the area (about 1 cm2 ) to receive the NV inoculum. Ten microliters of the 20% NV suspension was slowly placed at the center of each demarcated area using a pipettor. Recovery of NV from contaminated finger pads. Two controls consisted of (I) a single finger pad where the NV inoculum was recovered immediately after inoculation (input control) and (ii) a baseline (dried) control where the inoculum was recovered from another finger pad immediately after drying for 20 min. For collection of the input control, the inoculum on the thumb pad was eluted using a 10-#l aliquot of a 1-ml volume of HBSS by pipetting up and down three to four times. This eluate was returned to the collection tube, and the process was repeated three times. To recover the baseline control, the finger pad with the dried inoculum was placed over the mouth of a 2.0-ml plastic vial containing 1 ml of the HBSS, inverted so that the eluent was in contact with the contaminatedarea for 10 s, and then inverted rapidly in succession for a total of 20 inversions with the finger pad still in place. This soak-and-inversion process was repeated once. The vial was then turned upright, and any eluate remaining in contact with the finger pad was scraped against the inside rim of the vial to recover as much of the fluid as possible. After sample collection, the volunteers were instructed todecontaminate all their finger pads by pressing them onto a paper towel soaked with 10% bleach and then washing to remove the germs on hands with antibacterial hand sanitizer.  Thank you to RTS Clinics who specialize in What is Jock Itch and Tinea Cruris Treatment for their sponsorship.

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