A double-blind, double cross-over study was conducted at Haven of Hope (HOHCS) Woo Ping Care and Attention Home in Sai Kung, Hong Kong. Formal ethical approval was not required for this environmental sampling of residents’ bedsheets; however, the study protocol and sampling plan were approved by HOHCS management. Sampling took place in the absence of residents, who were informed of the procedures. Bedsheets were collected from rooms on the third and fourth floors over an eight-week period from January to February 2019. A total of 288 samples were gathered from 96 bedsheets across four observation periods. To maintain blinding, matched-pair randomization was used, with two beds in each room serving as controls and the other two treated with MAP-1.

MAP-1 coating was developed in the laboratory at HKUST, comprising a combination of three polymeric materials: polyhexamethylene biguanide (PHMB), polyethyleneimine (PEI), and polyvinyl alcohol (PVA). All active ingredients were approved for use by the US Food and Drug Administration (USFDA) and the US Environmental Protection Agency (USEPA). The liquid MAP-1 formulation met safety requirements for human use. A 180-liter 1:16 dilution MAP-1 coating was prepared to treat 100 bedsheets. Clean bedding materials were immersed in the MAP-1 solution and gently agitated for 30 min before drying in the HOHCS laundry facility, coded, and placed on the designated beds.

The sample size for the main study was determined based on a laboratory-controlled test analyzing 80 samples over three weeks to compare viable microbial loads on control and treated bedsheets. The analysis revealed a significant reduction in total bacterial counts, with log 4.68 ± 0.05 CFU·m− 2 for the control group (n = 40) and log 4.41 ± 0.05 CFU·m− 2 for the treated group, confirming a 0.258 log reduction as per t-test results. To achieve 95% power with an alpha error probability of 0.05, a minimum of 185 specimens was required. Considering ward capacities and sampling logistics, a sample size of 288 was chosen for the interventional double crossover study.

In the 8-week study at LTCF, an initial phase involved collecting 108 samples from 36 bedsheets over four weeks to establish a baseline environmental bacterial load. Subsequently, a total of 288 samples were systematically collected from 96 bedsheets across four observation periods. Half of these samples (n = 144) were from control bedsheets subject to regular washing. Each week, the infection control team collected 72 samples from 24 bedsheets.

Environmental samples were taken from the upper, middle, and lower sections of the bedsheets, representing the head, body, and leg areas, respectively, after 7 days of use in the LTCF resident’s bed. Each sampling area, measuring 50 × 50 square cm, was swabbed using a sterilized sponge (POLYWIPE™, Medical Wire & Equipment, Corsham, UK), as illustrated in Fig. 1–(1). To ensure accuracy and minimize contamination risks, each sample was collected using a new sterile glove. The swabbed sponge was then promptly placed in a sterile, labeled bag (Fig. 1–(2)) containing 10 ml of a neutralizing solution composed of 30 g/L Polysorbate 80 (Tween 80), 30 g/L Saponin, and 3 g/L Lecithin. To maintain sample integrity during transportation, the bagged sponges were stored in a cold box before being transported to the laboratory.

Schematic drawing of the environmental sampling procedure (1), sample preservation (2), microbial extraction (3), bacterial plating (4), incubation (5), and enumeration (6)

The environmental samples were processed within 2 h of collection. Microorganisms were extracted by resuspending the sponge samples in the elution solution using a Vortex mixer (Thermolyne, Type 37600 Mixer, Maxi Mix II) set at maximum speed for 30 s (Fig. 1–(3)). Subsequently, as illustrated in Fig. 1–(4), 100 µl of the suspension were transferred onto 90 mm tryptone soya agar (TSA) culture plates to quantify total viable bacterial counts, with each sample plated in duplicate. Additionally, 100 µl of the suspension were plated on 90 mm CHROMagar™ MRSA agar plates, also in duplicate, to identify and confirm the presence of MRSA.

Following a 48-hour incubation at 35 °C to assess the bacterial load of the samples (Fig. 1–(5)), colony-forming units (CFUs), as shown in Fig. 1–(6), were enumerated. Colonies were counted based on distinct margins and colors, treating spreading colonies as single units. Optimal counts for accurate calculations ranged from 30 to 300 CFUs per plate. MRSA colonies, appearing pink mauve on CHROMagar™ MRSA agar, were differentiated from yellow, round-shaped colonies observed on TSA plates for total viable counts. Confirmatory testing for MRSA, including Gram staining, tube coagulase, and Staphaurex tests, all yielded positive results for Gram-positive cocci, confirming the presence of MRSA.

The use of duplicate plating was crucial for ensuring the validity of sample data, allowing for the identification and elimination of outliers. An average CFU count from the duplicate plates was calculated and reported. The total bacterial count provided an overall assessment of cleanliness, while the presence of MRSA indicates contamination by multidrug-resistant organism. Microbial loads were expressed in log10 CFU·m− 2, and plates with no growth were assigned a value of 0.5 CFU, based on the minimum detection limit determined in the dilution study.

Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 21.0 (SPSS, Inc., Chicago, IL, USA). The total bacterial counts from the bedsheets of the control and treatment groups were compared using a t-test. Categorical variables, such as the percentage of MRSA presence, were assessed using the Chi-square test. Parametric tests were employed to evaluate the baseline cleanliness of each bed before and after treatment. Furthermore, ANOVA was employed to compare microbial loads across different periods of the study, as well as variations across different sections of a bedsheet and among bedsheets within the same resident area. Statistical significance was established at a p-value of less than 0.05.