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 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 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. Médecins Sans Frontières (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.
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
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 >12 h.
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.
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.
5. Experimental Procedure
6. Quantification
7. Analysis
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 <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 <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 <0.001; with soil load, F(4,68) = 162.44, p <0.001) (Figure 4). This same pattern was found in Phi6 without soil load ((F(4,43) = 8.95, P <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).
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
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).
Soap bar | Dove | White Beauty Bar soap | |
Alcohol-based hand sanitizer | Purell | Advanced Instant Hand Sanitizer with 70% Ethyl Alcohol | |
HTH Powder | Acros Organics | 300340010 | |
NaDCC Powder | Medentech | Klorsept granules | |
NaOCl Solution | Acros Organics | 419550010 | |
Electrochlorinator | AquaChlor | ||
Iodometric titrator | Hach | 1690001 | |
Bovine serum albumin | MP Biomedicals | NC0117242 | |
Tryptone | Fisher | BP1421-100 | |
Bovine Mucin | EMD Milipore | 49-964-3500MG | |
0.22 µm Filter | EMD Milipore | GVWP04700 | |
NaCl | Fisher | BP358-1 | |
Skin pH probe | Hanna Instruments | H199181 | |
Large Whirlpak Sample Bag | Nasco | B01447WA | |
Small Whirlpak Sample Bag | Nasco | B01323WA | |
Funnel bottle | Thermo Scientific | 3120850001 | 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 |
Ethanol | ThermoScientific | 615090010 | Mix with water to produce 70% ethanol |
Spray bottle | Qorpak | PLC06934 | |
E. coli | ATCC | 25922 | |
LB Broth | Fisher BioReagents | BP1426-2 | |
LB Agar | Fisher BioReagents | BP1425-500 | |
Sterile loop | Globe Scientific | 22-170-204 | |
Phi6 | HER | 102 | |
Nutrient broth | BD Difco | BD 247110 | |
GeneQuant 100 Spectrophotometer | General Electric | 28-9182-04 | |
Sodium thiosulfate | Fisher Chemical | S445-3 | |
Membrane filter (47mm, 0.45 µm) | EMD Millipore | HAWP04700 | |
m-ColiBlue24 broth media | EMD Millipore | M00PMCB24 | |
Petri dish with pad (47mm) | Fisherbrand | 09-720-500 | |
Vacuum Manifold | Thermo Scientific/Nalgene | 09-752-5 | |
Filter funnels | Thermo Scientific/Nalgene | 09-747 | |
Pseudomonas syringae | HER | 1102 | |
Phosphate Buffered Saline | Thermo Scientific | 10010031 | Solution may also be mixed from source compounds according to any basic recipe |