High-level disinfection
New South Wales. The swabs were collected from an IVF clinic which participated in the study. The sterile swabs were removed from their packaging and then used on the ultrasound device by passing the swab twice over a 5-10 cm area from one end to the other of the probe, on one side (randomly selected). The probe was then subjected to treatment (for 90 seconds) with the high-level disinfection UVC system first – followed by swabbing of the opposite side of the probe. The swabs were then transported to the microbiology laboratory of the School of Optometry and Vision Science University of New South Wales (UNSW) and processed within in four hours of collection.
Sample processing All the swabs were processed in a microbiology laboratory at the School of Optometry and Vision Science (UNSW) under aseptic conditions. Microorganisms were recovered in Phosphate Buffer Saline (PBS) by vortexing the swabs in yellow cap vials in the presence of glass beads for two minutes. 200 mL of the resulting slurry was spotted
onto chocolate blood agar and incubated in three different atmospheric conditions (aerobic, microaerophilic and anaerobic) for 24-72 hours at 37o
C. A similar process was followed for
isolation of fungus and yeast except using Sabouraud’s agar and incubating for seven days at 25o
colonies of each bacterium and fungus were counted and purified. Pure cultures of microbes
C. Following incubation, numbers of similar were stored in 25% glycerol at -80o C. To confirm whether microbes that were
isolated from UVC-treated probes were susceptible to UVC treatment, aliquots 100 μl (10 CFU/ml) were added on probes and on glass slides and dried for 30-45 minutes. These were then treated in the Lumicare disinfection system with a standard cycle (90 seconds). Probes
Collected the probes from the Patients Removed sheath if still present; Swab the probes
Left Side or Right Side Control (random selection) / Left side or Right side after UVC treated (opposite from random selection)
Transported to Micro Lab (UNSW) in cold chain in Amies Transport media Removed swabs, mixed in 2ml PBS by vortexing with glass beads Inoculated 200 μL on each of the following agar plates
Incubate in O2 24-48h Incubate in ANO2 48h Incubate in CO2 24-48h Enumerated and Purified of Bacteria and Yeast
Stored at -80 degrees in 30% Glycerol until all samples collected Identified single colony using MALDI-TOF
Figure 1: Flow diagram of methods employed to isolate and identify bacteria and fungi in the study.
Incubate of SDA 48h-7days
UVC kills cells through the accumulation of DNA damage. The UVC light penetrates cell walls of living organisms, such as bacteria, spores, viruses, fungi and mycobacteria. It damages the DNA structures, rendering the cell non-functional and unable to replicate.
and slides were then processed to determine numbers of viable microbes. (The techniques used to isolate microbes from swabs are outlined in Figure 1.) Fresh overnight bacterial culture was used for analysis using MALDI-TOF. Bacteria were grown on chocolate blood agar plate and incubated overnight. Following incubation, a loopful bacterial growth was diluted in 300 μL of HPLC- grade water into an Eppendorf tube and mixed thoroughly until bacteria completely suspended in water. This was followed by addition of 900 μL of absolute ethanol with thorough mixing. Then the tubes were centrifuged at 13,000 rpm for two minutes. The supernatant was removed using a pipette and pellets were air-dried at room temperature. Pellets were then redissolved in 25 μL of 70% aqueous formic acid followed by addition of 25 μL of acetonitrile (100%) and centrifugation at 13,000 rpm for two minutes. Following centrifugation, one μL of the supernatant was plated onto a MALDI target plate. Samples were overlaid with one μL of HCCA matrix solution and air dried at room temperature. Samples were analysed for the identification of microbes using MALDI-TOF. The numbers of microbes and type of microbes
isolated from the transvaginal ultrasound probes after use and then after a disinfection cycle in the HLD device were compared using a nonparametric (Mann Whitney) test.
Results The rate of contamination was 50% (15/30) on untreated probes (p < 0.001; Table 1 & Figure 2). On other hand, the rate of contamination on UVC treated probes was 3.3% (1/30) (Table 1 & Figure 2). The rate of contamination on untreated control probes was significantly higher than the UVC treated probes (p < 0.001; Table 2). The following microbes were identified on the probes (see Table 2 & Figure 3). A single UVC treated probe had
Staphylococcus capitis (4.3%) (Table 2). No fungal species was detected on control and/or UVC treated probes. The numbers of bacteria on control (untreated) probes ranged from 2.4 log to 3.8 log colony forming units (CFU). Bacillus
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