In vitro method for evaluation on virus-eliminating effectiveness of hand wash agents: an experimental study

  Objective  To develop a simple, safe, and low cost in vitro method for the evaluation on virus-eliminating effectiveness of hand-washing and hand wash agents.

  Methods  We used artificial skin as human skin surrogate and bacteriophage as virus surrogate to construct an in vitro model. We analyzed effects of various recovery diluent, initial concentration of bacteriophage inoculation, and recovery method on detection reproducibility of the method to be established. Three bacteriophage strains were used in evaluations of bar and liquid soap. Statistical analyses on experimental data were performed to assess the applicability of the method.

  Results  The recovery (logarithmic value) of φX174 bacteriophage was 5.57 and 5.54 for washing procedure with bar soap and liquid soap when using physiological saline with 0.1% peptone as the diluent and the values were obviously higher than those when using modified Letheen broth plus Tween (MLBT), polyvalent universal neutralizer (PVUN) or phosphate buffer saline (PBS) as the diluent. The results of one-way analysis of variance demonstrated that there was no significant difference in the recovery of bacteriophage among the three patterns of simulated 2-minute hand-washing (shaking the bottle manually, shaking violently with a shaker at 200 revolutions per minute or with a homogenizer) (P_0.05 = 0.72); however, the variation in the recovery of bacteriophage was the lowest (coefficient of variation CV = 0.74%) when using homogenizer in the simulated test. The bacteriophage plate count consistent with the experimental design (baseline recovery > 4 Log10, P_{student-t test} = 0.003) was achieved under two (10⁸ and 10⁷ plaque forming unit pfu/ml) of the four initial concentrations of bacteriophage inoculation; while, the simulated virus-eliminating rate was significantly higher with the initial inoculation concentration of 10⁸ pfu/ml than with that of 10⁷ pfu/ml, indicating that 10⁸ pfu/ml was the most optimal initial inoculation concentration. All the CV of variation in bacteriophage recovery were less than 15% for the simulated virus-eliminating tests of bar soap, liquid soap and only water using φX174, MS2 and φ6 bacteriophage as virus surrogate and optimized experimental conditions.

  Conclusion  A simple, safe, feasible, and highly applicable in vitro method was established for the evaluation on virus-eliminating effectiveness of hand wash agents using 10⁸ pfu/ml bacteriophage suspension contaminated artificial skin, 2-minute shaking of a homogenizer, and physiological saline with 0.1% peptone as recovery diluent.