Situations Leading to Reduced Effectiveness of Current Hand Hygiene against Infectious Mucus from Influenza Virus-Infected Patients

Viruses and cells.Madin-Darby canine kidney (MDCK) cells were purchased from the Riken BioResource Center Cell Bank (Ibaragi, Japan) and cultured in minimal essential medium (MEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and standard antibiotics (6, 41). IAV (PR8; A/Puerto Rico/8/1934; H1N1) was cultured in MDCK cells and stored as a working stock at −80°C. Viral titers were measured by focus-forming assays in MDCK cells and are expressed as the number of focus-forming units (FFU)/ml (21).

Collection of sputum samples and preparation of mucus.Figure 7 shows the overall workflow of this study. Mucus samples (sputum samples [≥2 g]) were obtained from 52 individuals who were diagnosed with acute upper respiratory tract infection at the Kyoto Prefectural University of Medicine Hospital and its affiliated hospitals between September 2016 and March 2018. Individuals <20 years old, with chronic respiratory illness, or who were taking expectorants were excluded.

• Open in new tab
• Download powerpoint

FIG 7

Outline of the study.

The 52 mucus samples were evaluated by quantitative reverse transcription-PCR (qRT-PCR) to distinguish between IAV-infected and IAV-uninfected samples. Nucleic acids were extracted from the mucus samples with a PureLink RNA minikit (Thermo Fisher, Waltham, MA, USA), and cDNA was prepared using a ReverTra Ace qPCR RT master mix (Toyobo, Osaka, Japan) (6, 21). IAV was detected and quantified by qRT-PCR with a Thunderbird SYBR qPCR mix (Toyobo). IAV-specific PCR was performed with an IAV matrix protein gene-specific primer set (M30F, 5′-TTCTAACCGAGGTCGAAACG-3′; M264R2, 5′-ACAAAGCGTCTACGCTGCAG-3′) designed by the National Institute of Infectious Diseases (Tokyo, Japan). Results of the qRT-PCR analysis showed that 19 of the 52 samples were positive for IAV, and these 19 samples were used in this study.

To completely inactivate infectious IAV derived from the individuals, the samples were irradiated with UV light (10 mJ/cm²). Viral titration with MDCK cells was performed to confirm that no infectious IAV was present. Bubbles were removed from irradiated samples by centrifugation at low speed to avoid destroying the mucous structure.

Rheological analysis.The viscosity of the mucus samples was measured with a DHR-1 controlled-stress rheometer (TA Instruments, New Castle, DE, USA) (42). A cone plate geometry (20 mm, 2°) was used at 25°C. A solvent trap containing distilled water prevented sample dehydration during measurement (43). Mucus (400 mg) was loaded onto the rheometer and left for 5 min to enable relaxation to the original gel structure. Steady-flow viscosities were measured with the flow-sweep mode (steady-flow measurement). Steady-flow viscosity (in Pa s) and shear stress (in Pa) were determined with the Rheology Advantage data-analysis software (TRIOS Software, TA Instruments) for a range of shear rates (0.01, 0.016, 0.025, 0.04, 0.063, 0.1, 0.16, 0.25, 0.4, 0.63, 1.0, 1.6, 2.5, 4.0, 6.3, 10, 16, 25, 40, 63, and 100 s⁻¹). Steady-flow viscosity was calculated as shear stress divided by shear rate.

Analysis of other physical properties.The contact angles of pure water, saline, and sputum samples on a glass surface were measured with a contact angle meter (Asumigiken, Tokyo, Japan), and surface tensions were measured with a surface tension meter (Asumigiken).

Analysis of complete drying time of saline and mucus.The time required for 5 μl of saline or mucus samples to completely dry and solidify on a glass plate was measured. In an indoor environment (temperature, 25°C; humidity, 40%), 5 μl of each sample was placed on a glass dish and weighed over time. The weight decreased gradually due to the drying of the sample, and the time when the weight ceased to decrease was defined as the “complete drying time.” An analytical balance (XFR-135; Shinko Denshi, Tokyo, Japan) was used to measure the weight of the samples.

Measurement of diffusion coefficients by PFG-NMR.Three representative mucus samples (no. 1, 10, and 19) were selected, and the ethanol diffusion coefficients in these samples and those in saline were measured by pulsed-field gradient nuclear magnetic resonance (PFG-NMR). Samples were prepared by mixing deuterium oxide at 99.9 atom% D (D₂O; Isotech Laboratories, Inc., Champaign, IL, USA) and 99.5% ethanol (Kanto Chemical, Tokyo, Japan) into the mucus samples. Here, D₂O minimized the signal intensity from the OH group in ¹H NMR measurement. ¹H NMR spectra of the mucus-alcohol-D₂O mixtures were acquired at 298 ± 1 K. All NMR measurements were conducted using a Diff-30 diffusion probe on a Bruker Avance 600 spectrometer (Hitachi, Tokyo, Japan) operating at a ¹H resonance frequency of 600.13 MHz. The chemical shifts were calibrated using the ¹H resonance of tetramethylsilane as an external reference. PFG-NMR self-diffusion measurements were carried out using the pulsed-gradient spin-echo sequence, starting from the minimum value of 0.05 midpoint temperature (T_m⁻¹). The maximum gradient strength was adjusted experimentally to 2.0 to 3.0 T_m⁻¹ to maximize the accuracy of fitting from signal attenuation. The diffusion time (Δ) was fixed to 20 ms, and the gradient-pulse duration (δ) was set to 1 ms. For all samples, 5-mm NMR glass tubes were used; the tubes were sealed to prevent evaporation of the solvent. In total, eight signal averages were acquired for each gradient increment (g) for a total of 16 increments. The recycle time was 5 s, resulting in a total acquisition time for an individual PFG-NMR data set of <12 min.

The area (Ψ) of the respective resonance peaks was measured as a function of the gradient strength (g), and the self-diffusion coefficient (D) was determined using equation 1: Ψ=exp(−γ2δ2g2[Δ−δ/3]) (1)where γ is the gyromagnetic ratio of the ¹H nucleus.

After measurement of the self-diffusion coefficients of the water and ethanol molecules, the interdiffusion coefficient (D) of the water-ethanol system was calculated from the self-diffusion coefficients of water (D_H2O) and ethanol (D_EtOH). D was determined using Darken’s equation, as follows (2, 44, 45):D=NEtOH×DH2O+NH2O×DEtOH (2)where N is the mole fraction.

Measurement of changes in ethanol concentration with a modified reflectometer.Changes in ethanol concentration in saline or mucus were measured after addition of 80% (wt/wt) ethanol-based disinfectant (EBD) (see Fig. S5A in the supplemental material). Ethanol concentrations were measured with a digital ethanol densitometer (ATAGO, Tokyo, Japan), which was modified for this measurement. The digital ethanol densitometer is a refractometer that measures the ethanol concentration at the center of a sample placed on a prism in a hemispherical region with a diameter of ∼1 mm; this enables measurement of changes in ethanol concentration over time in the region of the sample with the lowest ethanol concentration. For this manual procedure, 95 μl of EBD was added as slowly as possible to a 5-μl sample placed on a prism. The densitometer was then used for measurement of the ethanol concentration over time. A fitting curve was prepared from the ethanol concentration at each time point, and the time for the ethanol concentration to reach 5, 10, 15, 20, 25, 30, 31, 35, and 40% was calculated from the fitting curve. Three independent experiments were performed, and the results are expressed as the mean ± standard deviation (SD).

In vitro inactivation tests for evaluation of EBD efficacy against IAV.Tests for inactivation of IAV were carried out according to the following protocols (Fig. S5B), with EBD containing 80% (wt/wt) ethanol; established guidelines were followed for these tests (EN14476:2013/FprA1:2015) (19, 38, 46–48). IAV was mixed with saline or mucus prior to EBD exposure. Briefly, 95 μl of EBD or PBS was added to 5 μl of a mixture of IAV and mucus or saline (final viral titer, 1.0 × 10⁶ FFU/ml) in 24-well plates and incubated at 25°C for 15, 30, 60, 120, 180, 210, 240, or 300 s prior to neutralization by dilution with 900 μl MEM; titration of IAV was then performed. The survival ratio was calculated as the ratio of the titer after incubation with disinfectant to that measured after incubation with PBS; this ratio indicated the proportion of IAV that was not inactivated by the disinfectant. A fitting curve was prepared using the survival ratio at each time point, and complete inactivation time (time required for IAV to be completely inactivated) was calculated from the fitting curve. Complete inactivation was defined as a survival ratio below 10⁻⁵. In addition, the mixture of IAV and saline or mucus was dried completely by letting the mixture stand for 40 min before EBD exposure, and the same inactivation test described above was carried out. For each measurement, three independent experiments were performed, and the results are expressed as the mean ± standard deviation.

Clinical research (in situ inactivation tests for evaluation of EBD efficacy against IAV).Clinical research was performed with 10 test subjects who provided informed consent. Three representative mucus samples were selected (no. 1, 10, and 19), and the effectiveness of EBD against IAV in mucus or saline was examined using in situ inactivation tests conducted under established guidelines (finger pad protocol, ASTM E1838; hand wash protocol, EN14476) (32, 33, 47).

First, clinical research concerning the efficacy evaluation of AHR was performed (Fig. S5C). IAV was mixed with saline or mucus (final viral titer, 1.0 × 10⁶ FFU/ml) and applied in 5-μl aliquots to washed and disinfected fingertips of test subjects in a marked area. A tube containing 95 μl of EBD was placed on the contaminated area and incubated for 30, 60, 120, or 240 s prior to neutralization and elution with 500 μl MEM; IAV was then titrated. The mixture of IAV and saline or mucus on fingertips was completely dried by standing for 40 min before exposure to EBD, and the same efficacy evaluation described above was carried out.

Next, clinical research concerning the efficacy evaluation of AHW was performed (Fig. S5D). IAV was mixed with saline or mucus (final viral titer, 1.0 × 10⁶ FFU/ml) and applied in 5-μl aliquots to washed and disinfected fingertips of test subjects in a marked area. The contaminated fingers were hand washed with running water for 30, 60, 120, or 240 s prior to neutralization and elution with 500 μl MEM; IAV was then titrated. The mixture of IAV and saline or mucus on fingertips was completely dried by standing for 40 min before hand washing, and the same efficacy evaluation described above was carried out.

OpenFOAM simulation of changes in ethanol concentrations.OpenFOAM is an open-source software for continuum physics simulations and computational fluid dynamics. The InterMixingFoam solver of OpenFOAM 2.2.2 was used for this analysis. InterMixingFoam solved the incompressible Navier-Stokes equation and solved the conservation law of the volume fraction of each fluid (gas and two types of liquid). The volume fractions (α₁, α₂, and α₃) satisfied equation 3:α1+α2+α3=1 (3)

In this analysis, the volume fraction, α₁, was air, α₂ was an 80% ethanol aqueous solution, and α₃ was saline or mucus. The density (ρ) and viscosity (μ) of the whole fluid could be obtained from equation 4 and equation 5 using the density, viscosity, and volume fraction of each fluid.ρ=ρ1α1+ρ2α2+ρ3α3 (4)μ=μ1α1+μ2α2+μ3α3 (5)

Additionally, the ethanol concentration (χ) could be obtained from equation 6:χ=(80·ρ2α2)/(ρ1α1+ρ2α2+ρ3α3) (6)

The entire fluid domain was analyzed using the Navier-Stokes equations, which include the law of conservation of mass (equation 7) and the law of conservation of momentum (equation 8).∂ρ∂t+∂∂xi(ρUi)=0 (7)∂ρUi∂t+∂∂xj(ρUiUj)=−∂ρ∂xi+μ∂∂xi∂Ui∂xj+ρgi+(f12)i+(f13)i (8)where g_i is the gravitational acceleration and f₁₂ and f₁₃ are the surface tensions.

The surface tension (f₁₂) between the gas α₁ and the liquid α₂ and the surface tension (f₁₃) between the gas α₁ and the liquid α₃ can be obtained by the following equations, respectively (equation 9 and equation 10):f12=σ12(∇⋅n→12)n→12 (9)f13=σ13(∇⋅n→13)n→13 (10)where n₁₂ and n₁₃ are normal vectors of the interface between the α₁ and α₂ and α₁ and α₃, respectively, and σ₁₂ and σ₁₃ are the surface tension coefficients of the interface between α₁ and α₂ and gas α₁ and α₃, respectively.

Finally, α₁ and α₂ were obtained by equation 11 and equation 12:α2d1+Ui∂α1∂xi=0 (11)α2dt+Ui∂α2∂xi+D∂2α2∂xi2=0 (12)where D is an interdiffusion coefficient. α₃ was obtained from equation 3.

In the OpenFOAM code, the value of D was set to zero in places where α₂ and α₃ did not exist.

First, the initial simulation reproducing a mixing of 20 μl of saline or mucus and 480 μl of EBD in a 96-well plate was conducted (Fig. 3A). Fluid simulation was carried out for a total of four conditions using the physical property values of saline or those of the three mucus samples (no. 1, 10, and 19).

Next, the conditions of the additional simulation were set to reproduce the reaction conditions of EBD and saline or mucus in the inactivation tests as much as possible. Briefly, cubic meshes with sides of 0.2 mm were used for the simulation, and 95 μl of 80% ethanol was added at a flow rate of 250 μl/s from a height of 6.0 mm above 5 μl of saline or mucus (Fig. S5E). The physical properties of the sample (no. 10) with a median viscosity among all 19 mucus samples were adopted as the physical properties of the mucus used in the simulation. In the initial simple simulation of mucus conditions using the measured viscosity of mucus, the time required for regions containing <31% ethanol to disappear exceeded 120 s. Times exceeding 120 s are not realistic because the calculation time and cost are dramatically increased. Therefore, fluid simulation was performed with the mucus (sample no. 10) viscosity reduced to 16.7 or 12.5% of the measured value.

The physical properties and ethanol diffusion coefficient of the saline and mucus used in all simulations are shown in Fig. S6 in the supplemental material.

Ethical considerations.Informed consent was obtained from all participants. The study protocol was reviewed and approved by the Institutional Review Board of the Kyoto Prefectural University of Medicine (ERB-C-716-2, UMIN000030152).

Statistical analysis.Data were analyzed using GraphPad Prism 7 software (GraphPad, La Jolla, CA, USA). Differences between the means of continuous variables were evaluated by Student's t test. Pearson’s correlation coefficient was used to assess the correlation between viscosity and 10, 20, and 31% ethanol concentration achievement time or between complete inactivation time and 31% ethanol concentration achievement time. For clinical data, the Wilcoxon signed rank test was performed. In this clinical study, under the condition that the Wilcoxon signed-rank test was performed under a normal distribution with an expected difference between log reductions of 2 and a standard deviation of 1, the required number of subjects was calculated to be 10 by the Monte Carlo simulation. All reported P values are two sided; P values of <0.05 were considered significant.