Moura et al.
10.3389/fpubh.2025.1664322
droplets over 1 mm in diameter in the sectioned areas. For each assay, droplets were counted manually by 2 people using a digitally counter pen, and the average value was recorded.
ventilation of the room between assays. This aimed to replicate the potential contamination occurring following repeated use of public washrooms. All assays were performed in presence of a standby user stationed at 1 m and at a 45-degree angle from the hand drying station. During hand drying, each participant wore a N95 respirator (FFP2NR, Omnitex) and a disposable plastic apron (Medisave UK Ltd., United Kingdom), for measurement of body/clothing contamination. Once hand drying was completed, one of the hands of the volunteer performing the hand drying was immediately sampled (palm and fingertips) to measure contamination remaining on hands after drying. With the other gloved hand, the volunteers touched a door handle as they would for standard use. All surfaces (hands, apron, gloves) were swabbed with a 3 M sponge-stick moistened with neutralizing buffer (SLS, United Kingdom). Surfaces were disinfected with chlorine wipes pre- and post-sampling, as previously described (1). Facemasks worn during hand drying were collected to measure mask contamination occurring as result of splattering. All volunteers then wore a new respirator and remained at their positions for 5 min, allowing any potential aerosols to settle on the clean masks. This process was performed twice at 10–15 min post-hand drying, and at 25–30 min post-hand drying, to assess if aerosolized particles could potentially deposit on the masks and be detected by qPCR analysis, as previously reported (22).
Power calculation for bacteriophage assays
The study was powered to detect a difference between two methods, based on the contamination remaining on hands following hand drying and then transferred to a surface (door handle). The calculation was based on the risk of microbial dissemination to surfaces, associated with poorly cleaned hands, as previously reported (1). Experimental data was used to inform the calculation (1). Assuming non-normally distributed data with a population standard deviation of 22,347 between the two arms and analyzed using a Mann– Whitney test, it was determined that at least 6 experiments per arm would be required to find a difference of 10 3 copies/ μ L (or 10-fold), with 90% power, and alpha error rate of 0.05%. 1 As different hand dryer models were used in this experiment, we have performed 10 assays per arm instead of the 6 deemed essential. This allowed us to also investigate standby user contamination during hand drying, and mask contamination via droplets and aerosolized particles, in addition to hand and surface contamination.
Preparation of bacteriophage filtrate
Bacteriophage recovery from masks
Bacteriophage PR772 (BAA-769-B1) was propagated using its recommended host strain Escherichia coli K12 (BAA-769), as previously described (1). The obtained bacteriophage filtrate was diluted to 10 8 pfu/ml and kept at 4 ° C until use.
All N95 respirators were individually bagged upon collection and transferred to the lab for immediate testing. The outer layer of each mask was removed using sterile scissors and saturated with 2 mL of DNA/RNA shield solution (Zymo Research, Germany), as described before (24). The elute was recovered via centrifugation for 1 min at 3,300 g and stored at 4 ° C until DNA extraction.
Bacteriophage dispersion and aerosol formation during hand drying
DNA extraction and quantitative PCR (qPCR)
All hand drying tests were carried out in a room measuring 48 m 3 without air-conditioning at the Leeds General Infirmary (United Kingdom) (20). Room air was renewed through standard ventilation, without applied active positive or negative pressure (i.e., no fans or air conditioning). Before each experiment, all surfaces were sanitized with chlorine wipes (Medipal, Pal, United Kingdom). Before each test, volunteers sanitized their gloved hands with 70% alcohol hand gel disinfectant (Sterillium, Germany), followed by immersion in 10 mL of bacteriophage solution. Hands were shaken thrice to remove excess liquid and dried using either the electric hand dryers A9KJ (Dyson, United Kingdom) for 10s, the Dyson AW+D wall hand dryer (Dyson, United Kingdom) for 14 s, or using three PT (Hand Towels H3, Tork, United Kingdom). Overall, each method was tested in 10 separate hand drying assays performed on 2 different days (5 assays per day). Surface and mask samples were collected after each individual assay; however, the 5 daily experiments were performed consecutively, without additional
DNA extraction was performed using the QIAamp 96 Virus QIAcube HT Kit and 400 μ L of elutes from masks or surfaces (sampled with 3 M sponge-stick). Bacteriophage quantification was performed as previously described (1) via qPCR and using primers specific for gene P3 of bacteriophage PR772 (Table 1). Standard curves were used to convert threshold cycle values to copies per μ L of template. Limit of detection was established at 500 copies.
Statistical analysis
SPSS version 29 was used for data analysis. Statistical significance was assessed using a two-sided Wilcoxon Signed Rank test for related samples, i.e., to assess surface and mask contamination occurring during hand drying; and using a two-sided Mann–Whitney U test for independent samples, i.e., to compare samples between hand drying methods. Both tests were assessed using a 95% confidence interval; p ≤ 0.05 was considered statistically significant.
1 https://www.benchmarksixsigma.com/calculators/ sample-size-calculator-for-mann-whitney-test/
Frontiers in Public Health
03
frontiersin.org
Made with FlippingBook interactive PDF creator