In this study, we examined the efficacy of several disinfectants against Norwalk virus with virus reduction evaluated by RT-qPCR both with and without sample treatment with RNase. In initial screening studies using the suspension assay, we confirmed a concentration-dependent reduction in virus genome copy number after a 30-s exposure to sodium hypochlorite, with virus elimination at concentrations of >160 ppm. In contrast, NV appeared to be quite resistant to ethanol. Previous studies indicate that sodium hypochlorite affects the integrity of both capsid protein and viral RNA, while ethanol affects the viral capsid protein but not the nucleic acid (12
). The fact that virus suspensions treated with higher concentrations of sodium hypochlorite were nondetectable by RT-qPCR is consistent with the proposed mode of action of this disinfectant. However, because the viral RNA does not appear to be degraded by ethanol, the true impact of this disinfectant on the infectivity of the virus cannot be definitively confirmed.
The relationship between detection of residual viral RNA and virus infectivity remains an important consideration in these studies. Some investigators have sought to address this issue by pretreating samples with RNase H with or without proteinase K, hypothesizing that naked (noninfectious) RNA will be degraded by RNase prior to the application of RT-qPCR. Hence, detectable amplicons will be more indicative of infectious virus. In our study, finger pad eluates were screened by RT-qPCR both with and without RNase H treatment prior to nucleic acid amplification; for samples pretreated with RNase H, the detection of viral RNA should be more indicative of the presence of infectious virus. Although overall residual virus titers after exposure to antibacterial soap or hand sanitizer were lower for samples that received the RNase pretreatment, the differences between RNase-treated and untreated samples were not statistically significant. We conclude that it is likely that at least some of the residual RNA after treatment was associated with intact virions (resistant to RNase treatment) and hence more indicative of infectious virus particles that were not affected by hand wash product treatment.
The efficacy of sanitizers against target microbes is impacted by a variety of factors, including disinfectant type and concentration, test method (in vitro
and in vivo
), target organism, and matrix. This complicates comparisons between studies. Studies of other nonenveloped enteric viruses and HuNoV surrogates probably provide the most relevant insights. For example, Sattar et al. (27
) assessed the activity of a 60% ethanol-based hand gel against human adenoviruses, rhinoviruses, and rotaviruses on finger pads, using an infectivity assay, and reported that the product reduced the titers of all three viruses by 3 to >4 log10
compared to a hard-water rinse. However, others have observed that 70% ethanol was able to reduce the infectivity titer of hepatitis A virus and poliovirus type 1 inoculated onto human skin by only about 1 log10
). Kampf et al. (15
) reported that a higher concentration of ethanol (>70%) was associated with better virucidal efficacy against FCV, although these results have been disputed by others (13
). Two other studies examined the in vivo
efficacy of 62% alcohol-based hand sanitizer against FCV and observed a <0.5 log10
). Other studies confirm the relatively poor efficacy of 60 to 70% ethanol for the inactivation of HuNoV surrogates (6
). Taken together, ethanol appears to have relatively poor efficacy against the nonenveloped enteric viruses.
Comparative studies have shown that some disinfectants intended for direct skin contact may be more effective than ethanol. While some investigators have shown that ethanol has comparatively better efficacy than isopropanol (15
), Gehrke et al. (13
) found that 1-propanol was more effective than ethanol for FCV inactivation. Recently, two studies (16
) reported separately that ethanol-based formulas in combination with other ingredients not only significantly improved the efficacy of hand sanitizers but also exhibited a broad spectrum of virucidal activity. Specifically, Macinga et al. (22
) reported on a hand sanitizer containing 70% ethanol with a synergistic blend of polyquaternium-37 and citric acid. In an in vitro
study, each ingredient alone had limited efficacy against bacteriophage MS2, but the combination of the three agents produced >3 log10
virus reductions when applied to a variety of human enteric viruses and surrogates. Similarly, the formulation of Kramer et al. (16
) consisting of 55% ethanol in combination with 10% propan-1-ol, 5.9% propan-1,2-diol, 5.7% butan-1,3-diol, and 0.7% phosphoric acid showed synergistic, broad-spectrum virucidal activity in both in vitro
and in vivo
studies. Agents such as these are promising alternatives to simple ethanol sanitizers and merit further evaluation and commercial development.
Consistent with other reports that have tested hand hygiene products against a variety of microorganisms (1
), we found that a simple water rinse or use of an antibacterial liquid soap was more effective than alcohol-based agents for reducing NV contamination on human hands. We also observed that rubbing fingers provided better reduction of residual virus titer, suggesting that physical removal plays a role in the efficacy of these hand hygiene methods—especially for a water rinse where there is no disinfection effect.
Clearly, ethanol-based hand sanitizers are an effective deterrent to the transmission of enveloped viruses such as influenza virus, hepatitis B virus, and herpes simplex viruses 1 and 2 (14
). However, they appear to be less effective for controlling the transmission of human enteric viruses, particularly the epidemiologically important HuNoV group. The relatively poor efficacy of ethanol-based hand sanitizers against NV, and perhaps other HuNoV, has important implications for infection control in health care settings and food service establishments where these products are commonly used and where HuNoV outbreaks are most frequent (11
). Even though water washing, with or without the addition of an antibacterial soap, can remove some NV from fingers, it should not be relied upon to eliminate this virus that can be shed in titers of up to 1012
genome copies per gram of stool (3
). Clearly, there remains a need to develop commercial hand hygiene agents with specific, high-level activity (≥4 log10
reduction) against human gastrointestinal viruses. The search for effective products continues.