Assessment of Performance Qualification of UV-C LED Disinfection and Hydrogen Peroxide Mist-Based Systems Against Geobacillus stearothermophilus

Abstract

UV-C LED systems have emerged as a chemical-free alternative to hydrogen peroxide (H₂O₂) mist-based technologies. This study assesses the sporicidal efficacy of a UV-C LED HLD system under clinically relevant conditions using performance qualification (PQ) and compares its outcomes with two established H₂O₂ mist devices. The effectiveness of Lumicare ONE^® UV-C LED system and two H₂O₂ mist systems (trophon^® EPR and trophon^®2) in achieving a 6 log₁₀ reduction was evaluated using Geobacillus stearothermophilus spores as challenge test. Tests were conducted under three conditions: bare-metal non-flamed, bare-metal flamed, and packaged (glassine or Tyvek^®), with indicators positioned at both upper and lower chamber locations. AOAC 966.04 carrier tests using bacterial spores were also performed to confirm log-reduction performance. Under clinically representative bare-metal conditions, both non-flamed and flamed, Lumicare ONE achieved complete sporicidal efficacy at all chamber positions, matching the performance of the H₂O₂ mist systems. Under non-relevant (packaged) conditions, only the H₂O₂ systems passed the test, which is consistent with packaging materials that allowed H₂O₂ penetration, but blocked UV-C. AOAC carrier testing confirmed > 6 log₁₀ reductions following a 90 s UV-C cycle. Overall, when evaluated using appropriate PQ criteria, the UV-C LED system delivered efficacy equivalent to H₂O₂ systems while providing a fast and chemical-free HLD option for semi-critical probes.

1. Introduction

The increasing demand for high-level disinfection (HLD) of ultrasound probes has driven significant advancements in disinfection technologies. Among the emerging technology, Ultraviolet Light (UV-C) irradiation High-Level Disinfection (HLD) systems have attracted considerable attention because they offer a non-chemical, fast, and efficient method of disinfection [1,2]. Ensuring the effectiveness of these technologies is critical for preventing healthcare-associated infections and maintaining patient safety.

Currently, three automated devices are used in clinical settings across Australia and New Zealand (ANZ) to provide high-level disinfection (HLD) of ultrasound probes. These systems employ either ultraviolet-C (UV-C) light-emitting diodes (LEDs), UV-C lamps, or hydrogen peroxide (H₂O₂) mist within enclosed chambers to achieve effective disinfection. Compared with chemical disinfection systems, UV-C technology is cost-effective, environmentally friendly, and effectively controls microbial contamination (>99.9%) without producing harmful chemical residues [3]. UV-C effectively inactivates microorganisms including bacterial and fungal spores, and viruses [4,5,6,7,8]

All the disinfectant systems require regular in-field validation testing of disinfection processes by both Australian (AS 5369:2023) [9] and International (ISO 15883–1:2024) [10] standards. Performance qualification reflects one stage of in-field validation and requires testing the biological inactivation of microorganisms after installation, upon servicing and requalification annually under Australian standards (AS 5369:2023) [9]. Performance qualification is performed using a biological indicator which should be non-pathogenic, easy to culture and handle, shows high resistance to the disinfection processes, has a long shelf life and is easily commercially available [11].

Biological indicators (BIs) are used to demonstrate microbicidal efficacy in gaseous and vapor sterilization or disinfection processes such as ethylene oxide, steam, and vaporized hydrogen peroxide (VH2O2) processes. Usually, BIs are enclosed within process challenge devices (PCDs) during the validation of sterilization modalities to ensure adequate resistance to the applied process [12]. Such packaging is clinically relevant for hydrogen peroxide-based HLD, which is performed in controlled environments and requires packaging for safe storage and transport, and not for UV-C. UV-C LED HLD systems are designed for point-of-care use and require direct line-of-sight exposure; therefore, packaging, which blocks UV-C transmission is considered clinically non-relevant for UV-C. Therefore, BIs with packaging are not recommended to validate the sterilization of all types of devices. Using the right biological indicator, in right condition for evaluating the performance of disinfectant system is necessary as described in the AS 5369:2023 [9]. Currently no biological indicators have been specifically designated for validating the sporicidal efficacy of UV-C disinfection systems [13]. Previous studies, including [13], have utilized commercially available metal coupons impregnated with Geobacillus stearothermophilus.

To date, no HLD system designed for ultrasound probe reprocessing has been evaluated for its efficacy using spores suspension of G. stearothermophilus in comparison with commercially available spore coupons. In the present study, the performance of a UV-C LED disinfection system was assessed using both spores of G. stearothermophilus and commercially available metal coupons as challenge tests under clinically relevant and non-relevant conditions. G. stearothermophilus were used as the target organism because they represent a more robust biological challenge than other bacteria

2. Materials and Methods

The study was conducted in the PC2 microbiology facility at the School of Optometry and Vision Science, UNSW Sydney.

2.1. HLD Disinfectant Systems

Two different technologies including UV-C LED (Lumicare ONE, Lumicare, Drummoyne, New South Wales, Australia) [14] and a hydrogen peroxide mist device (H₂O₂ mist: trophon^®2, and trophonEPR, Nanosonics Ltd., Macquarie Park, New South Wales, Australia) [15] were used to evaluate their effectiveness against Geobacillus stearothermophilus ATCC 7953 spores.

2.2. Challenge Test

The challenge test was performed using biological indicators (BT93/6 Terragene^®, Santa Fe, Argentina) containing 10⁶ G. stearothermophilus ATCC 7953 spores inoculated onto stainless steel coupons (dimensions: 34.0 mm × 7.0 mm × 0.8 mm) enclosed in a glassine package/Tyvek^® (dimensions: 25 mm × 70 mm). They were used in this study to test the UV-C and H₂O₂ mist-based disinfectant system’s effectiveness. The following conditions were tested: (1) The biological indicator in sealed glassine packaging/Tyvek^®; (2) biological indicator with packaging removed and non-flamed (bare metal coupon), to directly expose the metal coupon to the UV-C LED disinfection system; (3) biological indicator with packaging removed and flame-sterilized metal coupon at the tip to simulate sublethal heat effects. This was performed by flame sterilizing 5 mm (0.5 cm) of the coupons’ ends for 2 s on each side of the same end (1 min cooling time in between) (Figure 1). This was performed to mimic potential clinical scenarios where surface changes might occur due to heat exposure. For each test condition, positive controls were treated in the same way, except treatments with a disinfectant system were used.

2.3. Test Protocol

Each biological indicator was positioned at both the top and bottom positions within the disinfection chamber to ensure no shadowing or occlusion of UV-C exposure would happen. To minimize the shadowing effect, an adhesive tape was used to hang the coupon inside the chamber following the suspension method (Figure 1).

Biological indicators were positioned, with the inoculation site facing outwards, in the top and bottom positions inside the disinfection chambers to represent locations corresponding to where a clinical probe would be placed with direct exposure condition (Figure 2). Three replicates were used for each test condition for both top and bottom positions in the chamber (six replicates per device). The holder was suspended in each disinfection device and removed from the chamber via the cavity located at the top where an ultrasound probe cable would typically emerge. A standard disinfection cycle was carried out according to the disinfection device manufacturer’s Instruction For Use (the UV-C LED disinfection cycle was 90 s, the hydrogen peroxide (H₂O₂) mist-based device cycles were 7 min).

Figure 1 and Figure 2 illustrate the suspension method used to provide UV-C exposure and the orientation of biological indicators within the disinfection chamber, respectively, highlighting clinically representative probe positioning and direct exposure conditions.

All three devices were tested simultaneously. The inclusion of positive controls which were processed similar to others but without disinfection treatment ensured that the incubation process did not contribute to a false-negative result. After the disinfection cycle, each coupon was aseptically collected into suitable growth media (Bionova MC1020-2, Terragene, Santa Fe, Argentina). All vials were then incubated at 56 °C for 48–72 h and assessed for growth/no growth according to the manufacturer’s Instructions for use (IFU). Two positive controls were included, one for the non-flamed test conditions and one for the flamed test conditions, and were prepared by aseptically transferring the biological indicator directly into the corresponding culture media vial.

2.4. Bacterial Culture and Spores Production

A bacterial culture carrier test was conducted to assess the efficacy of the Lumicare ONE system against vegetative cells and spores of Geobacillus stearothermophilus ATCC 7953 dried onto glass slides, following the AOAC 966.04 standard procedure. The bacteria were cultured overnight in Nutrient Broth (Oxoid, Basingstoke, UK) and subsequently washed with phosphate-buffered saline (PBS, NaCl 8 g/L, KCl 0.2 g/L, Na₂HPO₄ 1.4 g/L, KH₂PO₄ 0.24 g/L). The method for spores production of G. stearothermophilus was adapted from Park et al. [16]. G. stearothermophilus was grown in nutrient broth for 24 h at 55 °C. After incubation, bacterial suspension 400–500 mL was aliquoted on nutrient agar (Oxoid, UK) supplemented with 10 ppm MnSO₄ to induce sporulation. The palates were incubated for 10–14 days at 55 °C. After incubation, the bacterial spores were harvested by flooding with sterile chilled distilled water. The spore suspension was washed three times with sterile water by centrifugation at 8000× g for 10–20 min followed by sonication for 10 min. The spores were heated at 80 °C for 10 min in water bath to destroy any remaining vegetative cells.

An aliquot of 50 μL containing vegetative or spores of G. stearothermophilus (1–5 × 10⁸ CFU/mL) diluted in high organic soil (5% horse serum in hard water) was inoculated to autoclaved glass slides (30 mm × 20 mm) and air-dried for 30–45 min in the biosafety cabinet. The dried slides were then exposed to UV-C irradiation for 90 s in the Lumicare UV-C device and maintained at a controlled temperature of 20–25 °C. Control carriers were left untreated and exposed to ambient light for the same duration. Following treatment, viable bacteria from both test and control slides were recovered by placing the carriers into sterile tubes containing PBS and small glass beads. The samples were sonicated for 5 min and then vortexed for 4–5 min to dislodge bacterial cells. Aliquots (1 mL) of the resulting suspension were plated on Tryptic Soy Agar (Becton Dickinson, MD, USA) and incubated at 56 °C for 48–72 h to determine the number of surviving bacteria.

Statistical Analysis

The relationship between HLD devices and test conditions (flame sterilized, non-flamed) was assessed using chi square test (fisher’s exact test) using GraphPad prism software (version 8.0.2). Activity against spores and vegetative cells was analyzed using one sample T test. A p-value less than 0.05 indicated statistical significance.

3. Results

The Lumicare ONE (UV-C LED HLD) showed no sporicidal activity against G. stearothermophilus when the coupons were covered with glassine packaging or Tyvek^® as a clinically non-relevant condition (

Table 1. Inactivation of G. stearothermophilus coupons by UVC and H₂O₂ Mist disinfectant system across different test conditions.

Test Condition	BI Chamber Position	UV-C LED (Lumicare ONE)/Pass Rate (%)	H2O2 Mist (trophon2)/Pass Rate (%)	H2O2 Mist (trophonEPR)/Pass Rate (%)	p-Value
Glassine package/Tyvek®	Top	0 ± 0 (0)	3 ± 0 (100)	3 ± 0 (100)	p < 0.05
Glassine package/Tyvek®	Bottom	0 ± 0 (0)	3 ± 0 (100)	3 ± 0 (100)
BI bare metal coupon (non-flamed)	Top	3 ± 0 (100)	3 ± 0 (100)	3 ± 0 (100)	p > 0.05
BI bare metal coupon (non-flamed)	Bottom	3 ± 0 (100)	3 ± 0 (100)	3 ± 0 (100)
BI bare metal coupon (flamed)	Top	3 ± 0 (100)	3 ± 0 (100)	3 ± 0 (100)
BI bare metal coupon (flamed)	Bottom	3 ± 0 (100)	3 ± 0 (100)	3 ± 0 (100)

The results shown here in the table are the average of three replicates used in duplicates. ± represent the SD.

). There was 0% pass rate (0/6) for Lumicare ONE disinfectant system. The hydrogen peroxide mist-based systems, trophon2 and trophon EPR, successfully inactivated spores on all covered coupons (6/6) at both the top and bottom positions within the chamber, demonstrated 100% pass rate. Overall, the hydrogen peroxide mist-based systems demonstrated significantly greater efficacy against covered G. stearothermophilus spores than the UV-C LED-based system under clinically non-relevant conditions (Table 1; p < 0.05).

In clinically relevant condition, the Lumicare ONE (UV-C LED) system effectively disinfected uncovered metal coupons, demonstrating sporicidal performance comparable to hydrogen peroxide mist-based systems without glassine packaging or Tyvek^®. Complete inactivation of G. stearothermophilus was achieved on all metal coupons (6/6) with 100% pass rate at both the top and bottom positions within the chamber. Similarly, the H₂O₂ mist-based devices, trophon EPR and trophon2, also achieved complete spore inactivation on all metal coupons (6/6) at both chamber positions. There was no significant difference in the performance of both (UV-C LED) and hydrogen peroxide mist-based systems in inactivating G. stearothermophilus on non-heated uncovered metal coupons (p > 0.05; Table 1).

In another clinically relevant conditions after the coupons end had been exposed to heat, the Lumicare ONE (UV-C LED) disinfection and H₂O₂ mist devices (TrophonEPR and Trophon2) completely inactivated the G. stearothermophilus with a 100% pass rate (6/6) at both top and bottom positions in the chamber of the devices. The results of all the disinfectant systems at both the positions were statistically similar (p > 0.05).

Under clinically relevant conditions, positive controls of each test condition—flamed and non-flamed—showed consistent results, showing bacterial growth and further confirming that proper incubation and test conditions were met. There were no instances of false negatives results caused by the heat or other stress.

The Lumicare ONE UV-C disinfection system demonstrated high efficacy against vegetative cells and spores of G. stearothermophilus. Following the standard 90 s disinfection cycle, the Lumicare ONE device completely killed the vegetative cell and spores and achieved a 6.05 log₁₀ and 6.15 log₁₀ reduction in viable G. stearothermophilus counts compared to the untreated control, respectively (p < 0.05; Figure 3).

4. Discussion

This study aimed to evaluate the efficacy of UV-C LED disinfection in comparison to hydrogen peroxide (H₂O₂) mist-based systems under clinically relevant and non-relevant conditions. The results obtained showed that the UV-C system demonstrated comparable sporicidal efficacy to H₂O₂ mist-based devices under clinically relevant conditions by inactivating the G. stearothermophilus (6 log₁₀) impregnated on metal surface.

The present study focused on two testing conditions clinically relevant and non-relevant. Under the clinically relevant condition, G. stearothermophilus spores impregnated on metal coupons without the glassine or Tyvek^® packaging were evaluated after being flame-sterilized and positioned at two locations (top and bottom) within the disinfection chamber. This setup aimed to replicate real-world disinfection scenarios and address factors such as heat-induced surface changes. Localized heat exposure to metallic surfaces is known to alter surface characteristics, including micro-topography and surface continuity, which can contribute to the formation of uneven or mated surface interfaces [17]. Such interfaces may create regions of reduced UV-C irradiance due to partial occlusion or surface shadowing, particularly in optical disinfection systems. In the context of this study, the flame sterilization step was therefore used as a conservative control to ensure that any heat-induced or contact-associated surface effects did not confound interpretation of UV-C efficacy on the exposed test surface. In previous PQ studies that suspended BIs using reusable metal clamps [13], sterile adhesive tape was used in the present study in place of a clamp. This approach was adopted to eliminate potential sterility issues associated with reusable clamps. Adhesive tape allowed controlled attachment of the BI coupon without introducing additional sources of contamination. Additionally, provision of insufficient heat can induce sublethal stress, and may trigger G. stearothermophilus spore activation rather than inactivating them [18]. Despite these challenging conditions, the current study demonstrated that the UV-C LED disinfection system effectively inactivated G. stearothermophilus spores on metal surfaces.

To confirm further whether the UV-C LED system was effective against vegetative and spores of G. stearothermophilu, suspension of each G. stearothermophilus dried on glass carrier slides was tested separately. The UV-C LED system killed all the vegetative and spores of G. stearothermophilus on glass carrier by reducing more than 6 log₁₀ bacteria and spores as was demonstrated against coupons. In a previous study, the UV-C LED system reduced the numbers of Bacillus subtilis ATCC 19659 spores by >7 log₁₀ CFU within 90 s on probes after five repetitive inoculations [19]. This demonstrates that the UV-C LED system exceeds the mandatory requirements set by the TGA of Australia. In contrast, the H₂O₂-based Trophon EPR system achieved > 6 log₁₀ reductions using G. stearothermophilus spores in recent study but showed only 4.6–5.2 log₁₀ reductions against Mycobacterium terrae in an earlier study [20], suggesting BI results alone cannot be fully relied upon for H₂O₂ mist-based systems. These findings highlight the need for regulatory-grade microbiological studies beyond simple BI testing to accurately establish true high-level disinfection performance.

Under non-relevant conditions (glassine/Tyvek^® packaging), the UV-C LED system did not completely inactivate all spores. Glassine/Tyvek^® packaging is considered non-relevant for UV-C LED high-level disinfection because UV-C LED HLD systems are designed for point-of-care use, with probes disinfected immediately prior to clinical use and stored in protective covers with appropriate labeling. A recent study, similar to another earlier study [13], showed that UV-C did not inactivate the G. stearothermophilus on biological indicators when tested with glassine and Tyvek^®. UV-C light is known to have limited penetration depth, which can result in its reduced antimicrobial activity [21]. The biological indicators used in the current study were originally designed and validated for use only with plasma or vaporized H₂O₂ sterilization, which are able to penetrate packaging. Glassine/Tyvek^® packaging is clinically relevant only when ultrasound probes are high-level disinfected in central sterilization departments using high concentrations of H₂O₂ and subsequently transported to clinical areas. In this context, packaging is required to maintain the disinfected state during storage and transport, as probes cannot be reprocessed within clinical departments due to chemical safety and ventilation requirements because H₂O₂ is classified as a hazardous chemical and a dangerous good, and therefore, laws under the Transportation of Dangerous Goods, Rules and Regulations, Australia, must be adhered to, as well as other restrictions in many other countries. The manufacturer clearly states that the Bionova^® BT93 spore coupons should not be used with other disinfection modalities, including UV-C [22]. Therefore, their use in UV-C testing, particularly when sealed in their original packaging (clinically non-relevant condition) is inappropriate and not reflective of clinical practice, where reusable ultrasound probes are disinfected outside of peel-pouch packaging inside the disinfection system chamber.

Recent studies have indicated that UV-C LED technology offers a rapid, chemical-free alternative to hydrogen peroxide (H₂O₂) mist systems for high-level disinfection of ultrasound probes. All challenge tests were conducted in accordance with the manufacturer’s Instructions for Use (IFUs) by placing them at the top and bottom positions facing towards the front. The standard disinfection cycle was 90 s for the UVC device and 420 s for the Trophon hydrogen peroxide (H₂O₂) devices. The Lumicare device delivers UVC radiation generated by light-emitting diodes at wavelengths of 265–275 nm for 90 s. This radiation damages microbial DNA by blocking replication and transcription, resulting in rapid microbial death. The efficacy of UVC primarily depends on dose, distance from the light source, and shadowing effects, making its action most effective when microorganisms are directly exposed. The current study demonstrated that the Lumicare device delivered a sufficient UVC dose within 90 s to achieve complete inactivation of >6 log₁₀ spores. Previous studies have shown that UVC doses 220.1 ± 24.3 mJ/cm² and 123.8 ± 6.3 mJ/cm² could achieve 2 log₁₀ reduction in spores of Aspergillus niger and Penicillium sp. [23]. The differences in spore inactivation may be attributed to the use of fungal spores in previous studies, which are more resistant to UVC than bacterial spores, as well as differences in UVC dose. The dose of the Lumicare UVC device required to inactivate different types of spores, will be investigated in future studies. Shadowing remains a known limitation of UV-C disinfection, particularly for complex surfaces; however, such an effect is not observed using UVMESH technology, providing a 360° UV-C light coverage to ensure a comprehensive surface exposure in the present study and compared to hanging coupons after flame sterilizing in previous study [13]. In contrast, the Trophon EPR and Trophon2 systems operate by using ultrasonic vibration to generate a fine mist of hydrogen peroxide, which circulates within a closed chamber. Microbial inactivation occurs through the generation of reactive oxygen species (ROSs) [24]. The efficacy of H₂O₂ mist systems depend on factors such as hydrogen peroxide concentration and diffusion. The limited volume and concentration-dependent diffusion of H₂O₂ likely contribute to the longer disinfection cycle (420 s) required to achieve microbial killing with these systems compared to UVC LED device cycle (90 s). Hence, UVC LED technology is particularly advantageous for clinical departments aiming to reduce chemical exposure, prevent toxic residues, and minimize device downtime associated with H₂O₂-based systems [25]. The robustness and efficacy of UV-C LED disinfection make it suitable for high-throughput or time-sensitive settings, including emergency departments, gynecology and radiology units, outpatient clinics, infectious disease wards, and rural or remote healthcare facilities where quick turnover of medical devices is essential. To ensure consistent performance in these clinical settings, proper validation of UV-C LED devices is essential and should be conducted through rigorous performance qualification testing under real-world clinical conditions using an appropriate biological indicator to ensure reliable assessment of sporicidal efficacy and operational performance.

5. Conclusions

This study demonstrates that the UV-C LED system can achieve consistent sporicidal efficacy equivalent to hydrogen peroxide mist-based systems when evaluated under clinically relevant performance qualification conditions. Both flamed and non-flamed bare-metal biological indicators in challenge tests showed 100% inactivation of G. stearothermophilus spores at both upper and lower chamber positions for all devices tested. This study further demonstrates that conducting challenge tests using BIs packaged in glassine/Tyvek^® is not recommended for testing with UV-C LED systems as well as in-field validation testing. Complementary AOAC 966.04 carrier tests with G. stearothermophilus spores further confirmed that the UV-C LED system demonstrated > 6 log₁₀ reductions in high-organic-load conditions, aligning with TGA and international HLD efficacy expectations. This positions UV-C LED HLD as an effective and potentially advantageous alternative system to provide HLD with ultrasound probes.