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UV disinfection processes have been identified as a potential alternative for high-level disinfection (HLD) of heat-sensitive non-lumened semi-critical medical devices, such as endocavitary probes.
UV-C disinfection process cannot be tested and approved as a sterilant or HLD using the method recommended by the Food and Drug Administration (FDA) for liquid chemicals.
UV-C devices are much more effective than usual FDA approved chemical HLD products to kill spores in real use conditions.
FDA Standards for HLD approval should include a test demonstrating the sporicidal activity in recommended use conditions.
Endocavitary probes are semi-critical devices and must undergo, at least, high level disinfection (HLD) between uses. Therefore, they should be high level disinfected between uses (i.e., with a product/process that kills all forms of microbial life; bacteria, fungi, mycobacteria, and virus, and in some countries, a demonstrated potential for sporicidal activity). In this study, the sporicidal activity of three common Food and Drug Administration cleared sterilants (CIDEX OPA Solution, SPOROX II Sterilizing and Disinfection Solution and CIDEX Activated Dialdehyde Solution) was compared with the sporicidal activity of an ultraviolet disinfection technology (Hypernova Chronos, Germitec) against Bacillus subtilis ATCC 19659 spores spread on silicone flat carriers in the presence of inorganic and organic soil.
The results indicate that the UV disinfection process presented within a 35 seconds exposure time a sporicidal efficacy substantially higher than the chemical sterilants used according to manufacturer instructions for HLD.
This study demonstrated that even if it cannot be tested/approved as a sterilant according to AOAC 966.04, the UV unit is much more effective than usual Food and Drug Administration approved chemical HLD products to kill spores in real use conditions. This finding questions the relevancy of evaluating product efficacy within extended conditions giving results that could mislead users to select the most effective HLD product/process for the reprocessing of their medical devices.
endocavitary probes or external probes scanning non-intact skin are considered semi-critical devices and must undergo, at least, high level disinfection between uses. Most disinfection guidelines and international recommendations follow the Spaulding classification principles and recommend to disinfect probes using a product or process with a demonstrated activity against bacteria, fungi, mycobacteria, and viruses, even if probes are covered with a single-use probe cover.
as a lethal process utilizing a sterilant under less than sterilizing conditions and killing all forms of microbial life except for large numbers of bacterial spores. Thus, according to FDA a high level disinfectant shall demonstrate broad spectrum efficacy and potential for sporicidal activity (even if this sporicidal activity is obtained for a longer contact time than the recommended use conditions) whereas in France, no sporicidal activity is required for intermediate level disinfectant.
As a consequence, in United States, products with HLD claims should first qualify as a sterilant by passing the Association of Official Analytical Chemists (AOAC) Sporicidal Test (Sporicidal Activity of Disinfectants, AOAC 6.3.05:1995, Official Method 966.04) as a sterilant.
This test is a carrier-based, qualitative test method used to evaluate water-soluble chemicals using 2 carrier types (porcelain penicylinders and suture loops) artificially inoculated with Bacillus subtilis and Clostridium sporogenes. To support a sterilant claim, 3 batches of the chemical disinfectant and 60 carriers/batch/strain/carrier type shall be tested and all test carriers must be negative for growth.
UV disinfection processes have been identified as a potential alternative for HLD of heat-sensitive non-lumened semi-critical medical devices, such as endocavitary probes (eg, rectal and vaginal).
In the absence of an international standard dedicated to the evaluation of a UV disinfection process, it seemed logical to follow the same evaluation scheme that for liquid chemical. Since Hypernova Chronos is intended to disinfect flat non-porous surfaces, penicylinders or silk suture loops used in the 966.04 sporicidal tests were not appropriate for the evaluation of its sporicidal activity, as they are adapted for liquid or gas processes. Therefore, we proposed a different approach taking into account that the ultimate purpose of requiring of a high level disinfectant to pass the AOAC Sporicidal Test 966.04 is to ensure that the tested chemical can kill at least a small number of bacterial spores in routine usage.
The purpose of this study was to evaluate if the Germitec UV process can be considered as a high level disinfectant by comparing the sporicidal activity of the UV disinfection unit with sterilants already cleared by the FDA according to a test method applicable to both products. The method proposed is a quantitative carrier test based upon French and European carrier tests
for chemical antiseptics and disinfectants and is intended to evaluate the ability of the tested product/process to inactivate, in the presence of organic and inorganic challenge, bacterial endospores spread on silicone carriers (predominant material of acoustic lens rubber for ultrasonic probes).
UV disinfection process: Hypernova Chronos (GERMITEC France) is a liquid-free HLD system that uses UV-C as a surface-acting germicide dedicated to non-lumened ultrasound probes. According to the manufacturer, a 35 second contact time providing a UV-C dose of at least 100 mJ/cm² is required for a high level disinfection.
Sterilant/High level disinfectant: the characteristics of the tested products are presented in Table 1.
Table 1Characteristics of the chemical disinfectants tested
CIDEX OPA ortho-Phthalaldehyde
Advanced Sterilization Products
Sterilant in 32 h at 20°C and 25°C. HLD: 12 min at 20°C
Interfering substance solution (mixture of calf serum and hard water): 5% (vol/vol) heat inactivated new-born calf serum (SIGMA, N4762), 12.7 mg MgCl2 anhydrous (SIGMA, M 82 66), 29.6 mg CaCl2 anhydrous (SIGMA C1016) and 29.8 mg NaHCO3 (FISHER-SCIENTIFIC DA04799) prepared in distilled water. This level of interfering substance is compliant with FDA requirement for the evaluation of High Level disinfectant and more restrictive than what is required in European standards for the evaluation of disinfectant intended to be used on cleaned medical devices.
Bacterial growth control: To validate that each neutralizing solution can support the growth of low numbers of B. subtilis ATCC 19659 spores, 0.1 mL of a validation suspension (Nv) containing from 5.0 × 102 to 5.0 × 103 spores/ml was transferred into 23 mL portions of recovery medium. After 10 minutes’ contact time, the number of viable spores in the test tube (Tv2) was determined using the pour-plate method (1 mL) and filtration (22 mL) on 0.45 µm membrane filters. After 48-72 hours of incubation at 37 ± 1°C, plates were counted and results were expressed as a number of colony-forming units (CFU) per millili ter. The ability of the recovery medium to support the growth of low number of viable spores was validated if Tv2 is equivalent to 0.1 x Nv.
Neutralization control: For the UV disinfection unit, 2 sterile silicone carriers were treated in the chamber with the maximum UV-C dose intended to be tested.
For the disinfectants, 2 sterile silicone carriers were immersed independently in 2 tubes containing 10 mL of the disinfectant solution intended to be tested. Carriers were maintained in the solution for the maximum contact time intended to be tested (1,310 seconds for CIDEX OPA Solution and CIDEX Activated dialdehyde Solution or 1,800 seconds for SPOROX II).
After the maximum contact time, carriers were transferred into cylindrical screw tubes filled with 23 mL of recovery solution. The surface of the test carriers was scraped with a cell scrapper. 0.1 mL of the validation suspension (Nv) containing between 5.0 × 102 to 5.0 × 103 CFU per milliliter is then introduced in the test tubes.
Test tubes, containing test carriers, were then mixed for 30 seconds and the number of viable spores in the test mixture was determined after tenfold serial dilution, using the pour plate technique (1 mL) and filtration (22 mL) on 0.45 µm membrane filters.
After 48-72 hours of incubation at 37 ± 1°C for B. subtilis, plates were counted and results were expressed as a mean number of CFU per test tube (TV1).
A correlation between the number of CFU per tube (Tv1) and the number of CFU per 0.1 mL of validation suspension (Nv) validate that the recovery/neutralization method neutralizes any residues coming from the disinfectant solution and supports the growth of low numbers of Bacillus subtilis ATCC 19659.
Interfering substance control: 5 mL of 2x concentrated interfering substance was added to 5 mL of a suspension (Nv2) containing 5.0 × 102 to 5.0 × 103 spores/mL. After 10 minutes’ contact time, the number of viable spores in the test tube (Nv”) is determined using the pour-plate method. After 48-72 hours of incubation at 37 ± 1°C, plates were counted and results were expressed as a number of CFU per milliliter.
At the same time, 5 mL of the diluent (sterile distilled water) was added to 5 mL of a suspension (Nv2) containing 5.0 × 102 to 5.0 × 103 spores/mL. After 10 minutes’ contact time, the number of viable spores in the test tube (Nv’) is determined using the pour-plate method. After 48-72 hours of incubation at 37 ± 1°C, plates were counted and results were expressed as a number of CFU per milliliter.
The absence of effect of the interfering substances on spore viability/germination was validated if Nv’ is equivalent to Nv” and if Nv’ and Nv” are equivalent to 0.5 x Nv2.
Inoculation of carriers: A contamination solution was prepared by mixing 5 ml of 2x concentrated interfering substance solution and 5 mL of a spore suspension containing 1.0 × 108 to 5.0 × 108 spores/mL. With this mixture the final concentration in the test will be 5% for the calf foetal serum and 400 ppm CaCO3 for the hardness.
One thousand and fifty-six drops of about 0.050 μL of the contamination solution were then equally distributed onto a predefined 5 cm² “inoculation square” of the tests carriers using a specific dispensing robot (Model DR-2203N, Nordson EFD). The carriers were maintained at room temperature until visible dryness (no more than 60 minutes) and used immediately after the end of the drying time.
Main test: For the UV disinfection unit: for each tested dose, 5 silicone carriers were loaded in the device's chamber using a specific hanger. Carriers are hung vertically on the hangers (see Fig 1). The hanger was positioned in the middle of the unit and remain in the same position during the cycle.
At the same time, 1 contaminated silicone carrier was maintained outside the UV disinfection unit during the exposure time to determine the initial contamination level of test carriers (Nw) and validate experimental conditions.
Five UV doses were tested (50, 100, 266, 556, 55,550 mJ/cm²) corresponding respectively to contact times with UV of 18, 35, 84, 145, and 1,310 seconds.
For the chemicals, for each tested contact time, 5 carriers were placed on 5 test tubes containing the disinfectant to be tested at the specified temperature (ie, 25°C for CIDEX Activated Dialdehyde Solution and 20°C for SPOROX II and CIDEX OPA). At the same time, 1 contaminated silicone carrier was maintained at room temperature during the exposure time to determine the initial contamination level of test carriers (Nw) and validate experimental conditions. Five contact times were tested: 18, 35, 84, 145, and 1,310 seconds (1,800 seconds for SPOROX II).
After treatment, carriers treated by UV technology or the chemicals (assays) or not treated (controls) were transferred into cylindrical screw tubes filled with the neutralizing solution. The inoculated surface was then scraped with a sterile cell scraper.
Test tubes containing test carriers were then mixed for 30 seconds and the number of viable microorganisms in the test mixture was determined after tenfold serial dilution, using the pour plate technique. A filtration on 0.45 µm membrane filters of the 22 mL of neutralizing solution remaining in the test tube was also performed to increase the sensitivity of the counting medium. After 48-72 hours of incubation at 37 ± 1°C, plates were counted and results were expressed as a mean number of CFU per carrier (Nw for control and E for assays).
Preliminary tests: Results presented in Table 2 demonstrate that when 100 µL of the validation suspension are added to 10 mL of recovery medium, the number of CFU per tube (Tv2) was comparable to the number of CFU per 100 µL of validation suspension. These results validated that the medium can support the growth of low numbers of B. subtilis ATCC 19659 spores (Bacterial growth control).
Table 2Preliminary tests:
CIDEX Activated Dialdehyde Solution
8.5 × 102
1.3 × 103
8.5 × 102
8.5 × 102
Bacterial growth control
Tv2 (CFU/tube) [Tv2/Nv]
9.4 × 101 (0.11)
1.2 × 102 (0.09)
7.8 × 101 (0.09)
7.4 × 101 (0.09)
Tv1 (CFU/tube) [Tv1/Nv]
7.5 × 101 (0.09)
1.2 × 102 (0.09)
1.1 × 102 (0.13)
8.8 × 101 (0.10)
Interfering substance control
1.4 × 103
Nv’ (CFU/ml) [Nv’/Nv2]
8.1 × 102 (0.60)
Nv” (CFU/ml) [Nv”/Nv2]
8.4 × 102 (0.62)
NOTE. Tv1: Number of viable spores recovered after addition of 50-500 colony-forming units (CFU) of Bacillus subtilis in a capped test tube filled with 23 mL of recovery medium and a sterile carrier previously treated with the test disinfectant or with by UVC. Tv2: Number of viable spores recovered after addition of 50-500 colony-forming units (CFU) of Bacillus subtilis in a capped test tube filled with 23 mL of recovery medium. Nv, Nv2: Number of viable spores in the validation suspension. Nv’’: Interfering substance control. Nv’: Number of viable spores in the validation suspension diluted in SDW.
The data presented confirm also that the recovery/neutralization method neutralizes any active residues remaining on the test support after treatment with the three tested disinfectants and with the UV disinfection unit (Neutralization validation) and that the interfering substances do not present any adverse effects on spore viability/germination (interfering substances control).
Main tests: Results presented in Tables 3 and in Figure 2 indicate that in the presence of organic (5% foetal calf serum) and inorganic substances (400 ppm CaCO3) the UV disinfection unit induces an important and rapid decrease of the number of viable spores (ie, 4.8-log10 reduction in 18 seconds) whereas for the chemicals the reduction obtained after a contact time of 18 seconds vary from 0.0-log10 for CIDEX OPA to 0.2-log10 for CIDEX Activated Dialdehyde Solution.
Table 3Main tests: Evolution of the number of CFU per carrier (E) according to the contact time with the tested process/chemicals
Contact time (sec)
CIDEX activated dialdehyde solution
1.5 × 108
1.7 × 108
1.3 × 108
1.5 × 108
Nw (CFU/carrier) [Log10]
5.3 × 106 (6.7 ± 0.1)
3.8 × 106 (6.6 ± 0.1)
2.9 × 106 (6.5 ± 0.2)
4.1 × 106 (6.6 ± 0.1)
E (CFU/carrier) [Log10 ± SD]
8.9 × 101 (1.9 ± 0.8)
3.0 × 106 (6.5 ± 0.2)
1.9 × 106 (6.3 ± 0.1)
5.3 × 106 (6.7 ± 0.3)
1.6 × 101 (1.2 ± 0.1)
2.8 × 106 (6.4 ± 0.1)
1.6 × 106 (6.2 ± 0.1)
4.1 × 106 (6.6 ± 0.1)
3.0 × 100 (0.4 ± 0.4)
1.4 × 106 (6.1 ± 0.2)
4.4 × 105 (5.6 ± 0.1)
2.8 × 106 (6.4 ± 0.1)
2.6 × 100 (0.4 ± 0.4)
1.3 × 106 (6.1±0.2)
3.0 × 105 (5.4 ± 0.3)
2.6 × 106 (6.4 ± 0.0)
0.0 × 100 (0.0)
1.8 × 104 (4.2 ± 0.3)
1.3 × 106 (6.1 ± 0.0)
3.1 × 105 (5.5 ± 0.1)
NOTE. Nw: number of viable spores per carrier after inoculation. Tc: number of viable spores in the test suspension.
For a contact time of 35 seconds (minimum contact time in the UV disinfection unit to reach the recommended dose for a disinfection of ultrasound probes, ie, at least 100 mJ/cm²), the cycle induces a 5.5-log10 reduction of the number of viable spores of Bacillus subtilis. For the chemicals the reductions obtained after the labelled contact time for a high disinfection level or estimated from the 2 values that flank this recommended use condition are much lower (ie, between 1.1 to 2.3-log10 for CIDEX Activated Dialdehyde Solution after a 20 minutes contact time at 25°C, 1.1-log10 for SPOROX II after a 30 minutes contact time at 20°C and between 0.2 and 0.5-log10 for CIDEX OPA after a 12 minutes contact time at 20°C).
The results obtained on CIDEX Activated Dialdehyde and CIDEX OPA are in line with the data published by Myner in 2017
who demonstrate that OPA (0.5% wt/vol) was not sporicidal against B. subtilis within 270 minutes of exposure whereas à 2% (vol/vol) alkaline glutaraldehyde solution induce a 5-log10 reduction in B. subtilis spore count within 180 minutes.
Several studies dealing with the antimicrobial efficacy of UV radiation have been published.
who summarized all peer-reviewed fluence response data for UV exposure of various microorganisms, a 5-log10 reduction of B. subtilis spores is obtained for a mean UV dose of 75 ± 11 mJ/cm². This efficacy level is consistent with the results obtained in this study but the test conditions of the two studies are different. The data cited by Masjoudi et al represents doses that were delivered in water (not on hard surfaces) against a different strain of B. subtilis (ATCC 6633 and not ATCC 19559) and as explained by Boyce et al,
these variables may affect the UV dose required to achieve a defined log10 reduction.
In the test conditions described, this study demonstrated that, under HLD use conditions, the UV based system is more efficient, at least on bacterial endospores, than the compared FDA approved chemical HLD products. Thus, for disinfection process that do not claim for liquid chemical sterilization, passing the AOAC Sporicidal Test (within conditions that are far away from real use conditions) should not be the only way to demonstrate the ability of a tested product to kill bacterial spores. As suggested by Miner et al
other sporicidal tests as the carrier test described in this study may be added to the standards for approval of a high-level disinfectant to measure the antimicrobial activity of disinfectants against spore-forming bacteria.
However, sporicidal activity is not the first requirement for a HLD and bacterial spores may not represent the most difficult microorganism to kill for UV based disinfection processes.
Therefore, the demonstration of a high potential for sporicidal activity shall not neglect the need, as required for HLD to perform potency tests against viruses, vegetative bacteria, fungi, and mycobacterium as well as simulated use tests to address more complex surfaces.
The results of the tests performed demonstrated that in the presence of organic (5% fetal calf serum) and inorganic substances (400 ppm CaCO3) the UV disinfection unit showed a rapid killing effect on B. subtilis ATCC 19659 spores. The comparison of the reduction measured for each process/chemical at the labelled contact time for a high disinfection level indicates that the UV disinfection unit, within 35 seconds exposure time, presents a much higher sporicidal efficacy than the 3 High Level Disinfectants already cleared by the FDA.
Considering the substantial discrepancy between results obtained according to FDA guidelines (ie, AOAC 966.04 in extended contact time) and the sporicidal activity demonstrated in real use conditions, there might be an interest to question the relevancy of evaluating sporicidal efficacy under extended conditions when the main objective is to ensure that in real use conditions, the HLD product/process present a potential for sporicidal activity.
Add to the standards for approval, a test demonstrating the sporicidal activity in recommended use conditions, would allow the user to select the most efficient HLD product/process for the real use contact time.
The role of chemical disinfection in the prevention of nosocomial infections.
in: Brachman PS Eickoff TC Proceedings of the International Conference on Nosocomial Infections. 1971. American Hospital Association,
Funding/support: Eurofins Biotech Germande received financial support from Germitec to performed the tests presented in this study.
Conflicts of interest: Eurofins Biotech Germande reports having been consulted and having received financial support from medical device manufacturers to performed studies on the efficacy of medical devices.