EP2919821A1

EP2919821A1 - Method and device for disinfection of volume

Info

Publication number: EP2919821A1
Application number: EP13792628.3A
Authority: EP
Inventors: Olivier Somville; Frédéric PRADELLE
Original assignee: Vigitechnics
Current assignee: Vigitechnics
Priority date: 2012-11-13
Filing date: 2013-11-08
Publication date: 2015-09-23
Also published as: WO2014076013A1

Abstract

The present invention relates to a method for disinfecting a volume or a volume of space by means of a biocide containing hydrogen peroxide or peroxone and a disinfection device with a biocide containing hydrogen peroxide or peroxone. The present invention also relates to a device for disinfecting a volume with a biocide based on hydrogen peroxide or peroxone. The present invention can be applied, particularly in the medical field, in the field of scientific research and more generally in all spaces requiring disinfection / decontamination.

Description

METHOD AND DEVICE FOR DISINFECTION OF VOLUME

Technical Field

The present invention relates to a method for disinfecting a volume or a volume of space by means of a biocide containing hydrogen peroxide or peroxone and a disinfection device with a biocide containing hydrogen peroxide or peroxone.

The present invention can be applied, particularly in the medical field, in the field of scientific research and more generally in all spaces requiring disinfection / decontamination.

State of the art

There are many processes and or techniques known for disinfecting a surface or a place. For example, one of the most common disinfectants is bleach which is currently used for disinfecting surfaces. A significant amount of research is still being carried out on biocide solutions in order to improve disinfection efficiency.

In the technical field of medicine, in particular in hospitals, it is also another necessary to completely disinfect rooms, such as surgical theatres. In particular, many infections are contracted whilst in hospital: studies indicate that 7 to 10% of hospitalized patients develop an infection contracted after arrival at the hospital.

Hundreds of millions of patients are affected by health care- associated infections worldwide each year, leading to significant mortality and financial losses for health systems. Of every 100 hospitalized patients at any given time, 7 in developed and 10 in developing countries will acquire at least one health care-associated infection (sources: World Health Organisation - Health care-associated infections - FACT SHEET).

Accordingly, there is a real need for finding a process allowing the complete disinfection of rooms in order to avoid or diminish the risk of contracting infections. Another technical domain wherein there is a need for disinfecting space is the food industry wherein there is a need to control every step of preparation to ensure that working surfaces and equipment in contact with food will not be contaminated with any pathogenic microorganisms which may be harmful and even deadly to consumers, not to mention their adverse impact on economy, management and production. Although food safety has dramatically improved in the last decades, outbreaks of nosocomial gastroenteritis continue to occur worldwide.

Another technical domain wherein there is a need for thoroughly disinfecting space is the pharmaceutical industry, for example for clean rooms, research labs and production sites, wherein prevention and infection control has long been a priority. The demand for reliable, safe and effective bi-decontamination equipment service has increased significantly in recent years. Due to, substantial growth in today's research animal facilities, the need for rapid and consistent methods of bio-decontamination of a wide variety of equipment, e.g., biological safety cabinets, incubators, and animal transport units, has become apparent. Since its inception in 1976, NSF standard 49, has contained formaldehyde gas bio- decontamination procedural guidelines for biological safety cabinets. However formaldehyde decontamination has the following challenges:

1 - Recognized as carcinogenic by the IARC (Ref: Agents Classified by the IARC Monographs, Volumes 1-100)

2- Requires a neutralization phase;

3- Leaves an unhealthy odor;

4- Deposits a white powder residue (paraformaldehyde) that requires pots clean-up;

5- Requires a long cycle time.

At present there are also other well-known sanitizing solutions available for disinfecting surfaces or spaces such as: Phenolic derivatives, Ethanol, Chlorine Dioxide, Sodium Hypochlorite, quaternary ammonium, glutaraldehyde based solutions, UV (ultraviolet) etc.. However most of these products have limited efficiency and do not allow achieve a total kill or a significant kill on multiresistant micro-organisms, more especially on micro-organisms that can rest on surfaces which are inaccessible and/or hidden.

Accordingly, there is a need in the art to find a novel process allowing an efficient and complete disinfection, for example, of a hospital room. Moreover, there is a real need in the art to find a fast, efficient and secure disinfection process in order to reduce or eliminate the risk of, for example patient infection. Disclosure of the Invention

The present invention solves the technical problems with the prior art by furnishing a method of disinfecting a volume by means of a biocide containing hydrogen peroxide (H2O2) or peroxone (03+Η₂Ο2), comprising step of:

a) diffusion of hydrogen peroxide (H2O2) into the volume to be disinfected, or

b) injection of ozone (O3) into the volume to be disinfected, followed by a diffusion of hydrogen peroxide in said volume. In particular, the inventors of the present invention have surprisingly dicovered that the process of the present invention allows the simultaneous disinfection of air and surfaces. In particular, the inventors have discovered unexpectedly that the process of the present invention which uses a diffusion principle allows the disinfectant to be in contact, by displacement, with all surfaces to be treated within the space, including those which appear inaccessible.

In the present application "disinfecting" means a total kill and/or a significant kill of micro-organisms and/or pathogenic viruses for example, by altering their structure and/or inhibiting their metabolism and/or some of their vital functions.

In the present application, "volume to be disinfected" means any room, place, box or any space that could require disinfection / decontamination. For example patient wards, emergency rooms, treatment rooms, operating theatres, Intensive Care Unit (ICUs), infectious disease units, oncology units and/or other units receiving severely immunocompromised patients, isolation rooms, air plane cabin, cloakroom, cool room, refrigerated room, cold storage room, changing room, ambulances/emergency vehicles, a laboratory, including a biological laboratory, incubators room and other enclosed volume of any nature requiring disinfection / decontamination.

According to the invention, steps a) and b) can be carried out separately as needed for decontamination

According to the invention, the hydrogen peroxide can be used in the process at a concentration of 2 to 35% by volume, and preferably of 4.5 to 7.5% by volume.

According to the invention, the hydrogen peroxide can be in the form of a solution, a powder, a gas and/or any adapted form known to one skilled in the art. It may be for example a solution, for example a commercially available peroxide solution, for example Amity HP 75 (registered trademark) commercialized by amity international, X-CID (registered trademark) commercialized by Airinspace SAS, or any similar chemical disinfectants.

According to the invention, diffusion of hydrogen peroxide can be carried out by any means known by one skilled in the art. For example, when the hydrogen peroxide is in a form of a solution, by spraying, atomizing, vaporizing, misting, and/or a nebulizing into the volume. For example, the hydrogen peroxide solution can be atomized with any adapted means known to from one skilled in the art, for example using a venturi nozzle, or a venturi nozzle coupled with a compressor. The Hydrogen peroxide can be aspired according to the air suction principle. It can be delivered through the nozzle into the room in the form of a dry mist by means of Venturi effect. The hydrogen peroxide can be delivered until the desired amount is achieved (ml/m³ - milliliter per cubic meter room to be treated) . According to the invention, the diffusion of hydrogen peroxide can be carried out by misting with micro droplets of diameter ranging between 3 to 9 μιτι, preferably 5 to 8 μιτι. Advantageously, when the hydrogen peroxide is diffused by misting with micro droplets of a diameter ranging between 3 μιτι to 9 μιτι, commonly named dry mist of hydrogen peroxide (DMHP), this allows a perfect diffusion in the whole treated volume as well as a homogeneous and/or uniform distribution over the surfaces. Advantageously, when the hydrogen peroxide is diffused by misting with micro droplets of a diameter ranging between 5 to 8 μιτι, its advantageously allows to preserve Hydrogen peroxide entire disinfecting properties while reaching all surfaces of the volume.

According to the invention, the hydrogen peroxide can be diffused into the volume in an amount of 6 to 20 g/m³, and preferably 8 to 12 g/m³ of volume to be disinfected.

According to the invention, the ozone used in the process can be in the form of a solution, a gas or any other form known by the skilled person. It can be for example in gas and/or form. Advantageously, ozone is in the from of a gas.

According to the invention, ozone can be generated during the process, for example using a ozone generator, for example a corona tube, preferably a corona tube provided with an air dryer mean.

According to the invention the injection of ozone in the volume can be carried out using any means known to one skilled in the art. For example when ozone is diffused in a gas form, by a fan. It can be for example a colloidal fan, a blower fan, an axial compact fan, preferably an axial compact fan.

According to the invention, ozone can be injected into the volume in an amount ranging from 6 x10³ g/m³ to 108 x10³ g/m³, preferably from 9.81 x10³ g/m³ to 17.66 x10³ g/m³ of the volume to be disinfected.

Advantageously, when the process involves injection of ozone (O3) into the volume to be disinfected, followed by a diffusion of hydrogen peroxide into said volume, the ozone and hydrogen peroxide can be allowed to contact one another.

Advantageously, the process of the invention can comprise two steps: ozone dissolution and hydrogen peroxide addition in the volume. In other word, hydrogen peroxide can be added after ozone in the volume, and thus advantageously can allow ozone oxidation and disinfection to occur first.

According to the invention the contacting time between ozone and hydrogen peroxide in step b) may be carried out for 4 to 420 minutes, preferably 30 to 120 minutes.

Advantageously, when the process involves injection of ozone (O3) into the volume to be disinfected, followed by diffusion of hydrogen peroxide into said volume, the inventors have demonstrated that it allows the ozone oxidation and the transformation of ozone molecules into hydroxyl radicals and ozone transfer from the gas phase to the liquid is improved due to an increase in ozone reaction rates. In particular, the process of the invention allows advantageously to produce highly reactive hydroxyl radicals that attack DNA, membrane lipids and/or other essential cell components allowing therefore a complete disinfection.

Advantageously, the process of the present invention when the process involves injection of ozone (O3) into the volume to be disinfected, followed by diffusion of hydrogen peroxide into said volume, the added hydrogen peroxide advantageously increases the hydroxyl radical concentration and then accelerates the decomposition of ozone. Moreover the inventors have demonstrated that the added hydrogen peroxide advantageously increases the hydroxyl radical concentration and accelerates the decomposition of ozone and thus Increase the hydroxyl radical concentration allowing advantageously increasing the oxidation rate.

Advantageously, with the addition of hydrogen peroxide, the process of the present invention can allow a net production of hydroxyl free radicals about 1 .0 mole of hydroxyl radical per mole ozone. In other words the inventors have surprisingly demonstrated that the process of the present invention due to the combination of ozone and hydrogen peroxyde have a better efficiency in disinfection than the use of ozone alone or hydrogen peroxide alone. They also demonstrated that the process of the present invention due to the combination of ozone and hydrogen peroxyde have a better efficiency in disinfection than the use of Hydrogen peroxide alone or ozone alone.

According to the invention, the method may be implemented at a temperature of 15-30 °C, preferably 18 to 22 °C.

Moreover and advantageously, the inventors have demontrated that the process of the invention allows better oxydation of the lipidic structure of germ membranes, production of hydroxyl radicals, detoriation of ribosomes and DNA by cytoplasmic penetration, accelerated lyse of microorganisms.

The present invention also relates to a device for disinfecting a volume using a biocide based on hydrogen peroxide or peroxone (ozone + hydrogen peroxide) said device comprising:

i . An injection system of ozone, also defined as ozone generation system, in said volume,

ii . A diffusion system of hydrogen peroxide; also defined as hydrogen peroxide feed system, in said volume, for example an atomizing system, for example with an air compressor and a venturi nozzle.

According to the invention, the device may comprise containers (6) for hydrogen peroxide, for example plastic containers, preferably High

Density Polyethylene (HDPE) plastic containers, for example metal containers, preferably stainless steal containers which possess high corrosion resistance to Hydrogen peroxide and ozone and a means for producing ozone, for example a mean selected from the group comprising a corona tube, a corona tube with a air dryer.

Advantageously, when the device itself comprises a mean for producing ozone it can allow the automatic regeneration of ozone. According to the invention, the injection system of ozone can be any means known to one skilled in the art. It can be, for example when ozone is injected in gas form, a fan, for example colloidal fan, for example an axial compact fan.

According to the invention, the hydrogen peroxide diffusion system can be any means known to one skilled in the art. It can be, for example a means comprising a compressor, for example an oil- free compressor, for example a 7 bar pressure compressor, for example a HPE 40C (registered trademark), for example a Thomas 668 series (registered trademark), for example a PLANET-R OF-S60 (registered trademark) a venturi nozzle, and/or a venturi nozzle coupled with a compressor

Advantageously, when the hydrogen peroxide diffusion system is an oil-free compressor, the diffusion noise is reduced, for example the diffusion noise can be less than 58 db.

Advantageously, when the hydrogen peroxide diffusion system is a venturi nozzle coupled with a compressor, it can advantageously allow atomization of hydrogen peroxide in the form of droplets of small diameters, in particular 5 to 8 μιτι.

According to the invention, when the diffusion system of hydrogen peroxide is a venturi nozzle coupled with a compressor, the air intake of the compressor can further comprise air filters .

The device according to the invention can comprise internal compartments for hydrogen peroxide containers (6). For example the compartments can comprise a lockable back door (4).

According to the invention, the hydrogen peroxide containers can be ready-to-use and sealed containers (6). For example for hydrogen peroxide it can be Amity HP 75 (registered trademark), X-CID (registered trademark) containers and/or any ready-to-use and sealed hydrogen peroxide containers.

According to the invention, the container (6) for hydrogen peroxide can be 2.5 liters containers. According to the invention, the hydrogen peroxide container can comprise an identification zone on its exterior surface. For example the identification zone can be covered with a readable code, for example a barcode, a flashcode, a detectable chip, for example a chip card, for example a RFID chip. Advantageously, the sealed containers are ready- to-use containers removing the need of tanks within the device.

According to the invention, the compartment for the container can comprise a mobile support (7) wherein the container may be fitted.

According to the invention, the compartment for the container can comprise an aspiration means (8), for example a tube, a needle to be introduced into the container.

Advantageously, the mobile support (7) can allows, for example, the movement of the container fitted into the support and direct connection with the aspiration means.

Advantageously, when the injection mean is a needle, it can directly perforate the hydrogen peroxide containers avoiding therefore any risk of contact between the user and hydrogen peroxide.

According to the invention, the compartment for the container can comprise at least 2 spaces for at least two containers. Advantageously, when the compartment for container comprises at least two spaces for at least two containers it advantageously allows avoiding any disruption in the process and can advantageously avoiding any unexpected stop of the process from a lack of hydrogen peroxide.

According to the invention, the compartment for the hydrogen peroxide containers can comprise at least one or two reader(s) mean(s), for example a barecode reader, a flashcode reader, a chip card reader, a

RFID chip reader.

Advantageously, identification of the container with the identification zone and the reader mean can allow to control and/or identify and/or confirm that the container is adapted to the device.

According to the invention, the ozone can be generated by a built-in system. The ozone can be generated by a mean selected from the group comprising a corona tube, a corona tube with a air dryer. Advantageously, when the ozone is generated by a built-in system the device does not need to require any space or compartment for any ozone container.

According to the invention, the device may further comprise a computerized system.

Advantageously the computerized system can automatically manages the containers; means can automatically switch from one container to another when one of them is empty and also can avoid unexpected stop due to a lack of hydrogen peroxide.

According to the invention, the device may further comprise a liquid sensor, for example an optical sensor, for example a flow sensor, for example a flow meter to monitor the hydrogen peroxide flow from the containers to the nozzle.

According to the invention, the device may further comprise an embedded computer system which, for example, can comprise a programming system.

According to the invention, the device may further comprise a programming system and/or a system for an automatic control of the ozone injection and / or hydrogen peroxide release.

According to the invention, the device may further comprise a programming system and/or a system for automatic management of ozone injection and / or hydrogen peroxide diffusion to coordinate the injection of ozone and peroxide diffusion.

The device according to the invention may further comprise at least one of the following: a temperature and/or humidity sensor, for example a sensor with a humidity working range from 0 to 100% RH, with a temperature working range from about -20 to 80 degree Celsius, for example the MQ3010 (registered trademark) and/or a E+E electronic EE06 and/or a ATEN EA1240 (registered trademark), allowing advantageously measuring both temperature and relative humidity of the volume to be disinfected, a sensor for detecting the presence of hydrogen peroxide in the volume, for example a Drager Pac III device (registered trademark) or a C16-PortaSens II Portable Gas Detector device with ATI C16 Hydrogen Peroxide Sensor Module (registered trademark), or a The Very High Level Peroxide strips - PERVHL box (registered trademark), a sensor for detecting ozone in the volume to be disinfected, for example a ATI C16 PortaSens II Portable Gas Detector device with ATI C16 ozone sensor module (registered trademark) or a Conspec's new Smart Head Gas Monitoring system (registered trademark) or a MN ozone test strips box (registered trademark), a motion detection system (9) for example a PIR 416 (registered trademark) and/or a PANASONIC AMN 141 1 1 (registered trademark), allowing advantageously stopping the running device if a movement is detected within the volume to be disinfected. Advantageously, the motion detection can be either turned-on or turned-off from the control panel if necessary, for example in glazed room, an alarm, that may interact, for example, with the temperature sensor or any other means and/or system and/or sensors described .

According to the invention, the device may further comprise a Touchscreen (5) selected from the group comprising a Programmable Logic Controller (PLC), for example Human-machine interface, for example Programmable Logic Controller, for example Programmable Logic Controller plus Human-machine interface, for example pocket computer, For example a UNITRONICS PLC V350 (registered trademark) and/or a OMRON NSJ8 (registered trademark).

According to the invention, the device may further comprise a embedded PLC for operating and controlling the device.

Advantageously, when the device comprises a touch screen, it can allow user(s) to set the device, control the embedded computer, and/or to program / specify, for example the volume of the area to be disinfected, the designation of the area, the name and surname of the user, the disinfection program, for example simple diffusion of hydrogen peroxide or injection of ozone followed with diffusion of hydrogen peroxide, the hour and date, to set a differed start, the language, to unlock the containers back door, to directly or remotely abort a cycle, to setup VPN connections and VPN ports, to transfer traceability data via Ethernet and/or Wifi to computer and/or remote appliance, to connect the device via Ethernet and/or Wifi to computer and/or remote appliances for example a tablet, a smartphone, a PLC, an HMI, a pocket computer, to enable or disable the Wifi function, to enable or disable the Motion detection system, to monitor the ambient conditions (temperature and relative humidity) of the area to be disinfected, the start and stop times, the duration of the disinfection process, the amount of hydrogen peroxide stored in containers, the amount of hydrogen peroxide being delivered, to access manufacturer settings menu for monitoring and/or changing parameters if necessary .

The device of the invention may further comprise a recording system, for example for recording the number of disinfection process or cycles completed, and/or the disinfected volume. It can be, for example, a hard disk on which may be recorded, for example the date, start and stop times, type of cycle, for example simple diffusion of hydrogen peroxide or injection of ozone followed with diffusion of hydrogen peroxide, user name and surname, area designation, volume of the area, amount of hydrogen peroxide delivered, hydrogen peroxide flow in milliliter, temperature and relative humidity of the area, chemical name, batch number, warning message for a disinfection process carried out.

Advantageously, when the device comprises a recording system, it can make accessible the full audit history of hydrogen peroxide, ozone used and process of disinfecting events.

According to the invention, the device may further comprise a wireless system to connect the device and/or to control the device from, for example, a remote. For example, the device can be connected via Wifi and/or Bluetooth to a smart phone, a computer or the likes. Advantageously, when the device is connected in such manner, for example via Wifi and/or Bluetooth to a smart phone, a computer and/or the likes, it allows, for example, control, launch, stop, analysis of and/or monitor the disinfection from, for example a remote localisation. The device according to the invention may be a mobile unit. For example it can comprise wheels allowing advantageously displacement of the device. For example, the device can comprise at least two wheels (2) located on each opposite side of the device The device can also comprise a handle (1 ) located on the top of the device.

The device of the invention may further comprise an energy source. For example, the energy source can be an electrical generator, a chargeable battery, for example a lithium-ion battery, and/or any energy source known to one skilled in the art.

The device of the invention can comprise an exterior surface or a body (3) in any adapted shape and any known adapted material known to one skilled in the art. It can be, for example made with plastic, for example plastic resin prefrom, aluminum, and/or carbon resin Advantageously, the exterior surface and/or body can protect the device and its components from shock.

The invention will be better understood and other features and advantages of the invention will be apparent from the following examples according to the invention. These examples are non-limiting. Brief description of the figures

- Figure 1 represents the diffusion and injection of ozone and hydrogen peroxide from a device.

- Figure 2 represents lateral and front views of the device according to the present invention comprising a handle (1 ), wheels (2) and the body (3)

- Figure 3 represents a frontal view of the device according to the present invention comprising a handle (1 ), wheels (2), the body (3) and the motion detection system (9)

- Figure 4 represents a top view of the device according to the present invention comprising a handle (1 ), wheels (2), a back door (4) and a touch screen (5). Figure 5 is a diagram representing the disposal of inoculated plates comprising E.coli, Staphylococcus aureus and Candida albicans on a counter in a room.

Figure 6 is a diagram representing a device implementing the process of the invention in a room with regards to inoculated plates with E.coli, Staphylococcus aureus and Candida albicans on a counter.

Figure 7 is a top view of a room comprising a device implementing the process of the invention in a room with regards to inoculated plates with E.coli, Staphylococcus aureus and Candida albicans on a counter.

Figure 8 is a top view of a room comprising a device after implementation of the process of the invention in a room with regards to inoculated plates with E.coli, Staphylococcus aureus and Candida albicans on a counter.

Figure 9 is a top view and pictures of a room specially equipped for conducting microbial tests and evaluating the efficacy of a device against bacteria, fungi, yeasts, spores and mycobacteria in accordance with the NF T 72-281 requirements comprising a device after implementation of the process of the invention in the room and the stainless steel disks 1 .4301 (NF EN 10088-1 ) from 2 cm to 4 cm in diameter, inoculated with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus hirae, Candida albicans, Aspergillus niger, Bacillus subtilis, Clostridium difficile and Mycobacterium terrae on a trolley.

Figure 10 represents a rear view of a device capable of implementing the process of the invention comprising a lockable back door (4), a handle (1 ), wheels (2) and an external surface (3); and a lateral view of a device implementing the process of the invention before introducting a hydrogen peroxide container (6) into said device.

Figure 1 1 represents a rear view of a device comprising a mobile support (7) into which a hydrogen peroxide container (6) has been fitted. Figure 12 represents a lateral and three-quater view of a mobile support (7) into which a hydrogen peroxide container (6) has been fitted and wherein a tube (8) is inserted into the hydrogen peroxide container. Example 1 : device for disinfecting a volume

Figures 2 to 4 and 10 to 12 shows an example of disinfection device according to the invention. The disinfection device comprises a handle (1 ), a plastic thermoformed exterior surface (3), wheels (2), a lockable back door (4) which allows entry into the inner part of the device, a touch screen (5) for controlling and/or programing the device. The device comprises two mobile supports (7) wherein two containers of 2.5 L each (6) of hydrogen peroxide solution are fitted, said mobile supports respectively allowing insertion of a tube (8) into the container, said tubes being connected to an oil free compressor with air intake filter to avoid particulate contamination of air supply, connected to an advanced air assisted venturi nozzle. The approaches allows hydrogen peroxide solution atomization with liquid droplets of 5 to 8μηη in diameter. The device also comprises a built-in ozone generator, i.e. a corona ceramic tube and an electric power supply. The device also comprises an embedded computer connected to the touch screen (5) and control means for regulating the diffusion of hydrogen peroxide and ozone into the volume.

The touch screen also allows one to control the opening of the lockable back door (5).

Finally, the device comprises gauges for controlling the levels of hydrogen peroxide solution.

Example 2: process for disinfection a volume and performances

In the present example, the efficiency of the disinfection of the process was studied.

In the present example, the process was tested using the device represented in figure 1 and described in example 1 . In particular, the hydrogen peroxide solution used was the Amity HP 75 commercialized by amity international (registered trademark) which is a colorless and odourless liquid that comprises a concentration of 7.5% hydrogen peroxide. The ozone was produced by a built-in ozone generator, in particular a corona ceramic tube.

1 . Materials and methods:

1 .1 General data:

Test room volume: 50 m³ (cub. Meter)

The example was carried out on microorganisms linked to pathologies.

Those microorganims were not the sole species responsible for these pathologies, since other microorganisms have also been identified as co-responsibles.

Microorganisms were collected from fresh carcasses in a breeding centre in France (Department of Maine-et-Loire) and were:

- Candida albicans collected from cows (abnormal milk) / Sabouraudis agar method. Diagnostics Pasteur

- Staphylococcus aureus collected from rabbits (womb) / Subculture onto blood agar. Diagnostics Pasteur

- E.coli collected from 5-day-old chickens (heart) / Rambach™ agar method. CHROMagar Microbiology

The materials used in the example were the following:

Sartorius Stedim Biotech (cellulose nitrate filter); Petri dishes : Diam 55;

Trypticase soy agar (tcs); sterile distilled water; SABOURAUD + CHLORAMPHENICOL (agar) - DIAGNOSTICS PASTEUR; TRYPTO-

CASEINE-SOJA (agar) - DIAGNOSTICS PASTEUR; RAMBACHTM (agar) - CHROMagar Microbiology; MUELLER-HINTON (agar) - DIAGNOSTICS PASTEUR The biocides used were: - Hydrogen peroxide [H2O2]: Amity HP75 (registered trademark) disinfectant contained in two 2.5 liter containers. (Hydrogen peroxyde based solution at 7.5% cone).

Atomizing time: 32mn

Contact time: 60 mn and 120 mn.

- Peroxone [O3+H2O2]: Ozone (<18.68ppm) + Amity HP75.

1 ppm of 0₃ = 2.14 X 10^"3g.m^"3

Hydrogen peroxide was added after ozone (thus allowing ozone oxidation and disinfection to occur first).

Atomizing time: 49 mn (See details in section 3.2).

Contact time: 60 mn & 120 mn.

Monitoring procedure: In order to verify the Hydrogen Peroxide and ozone uniformly dispersed throughout the room; the Operator has placed test strips: Peroxide test strips (Quantofix® - Peroxide 100 - 913 12/ Macherey-Nagel). Ozone test strips (Ozono Test - 907 36/ Macherey-Nagel).

1 .2 The trials:

A total of three (3) trials were conducted, in the same conditions at several days' interval, in order to monitor and ensure the repeatability and practicability of the test.

For Each trial, 3 inoculated plates were placed vertically on a counter at 75 cm height, at a distance of ± 5 cm from each other, the inoculated media facing the wall so they weren't directly exposed to the disinfectant chemicals delivered by the TwinCruiser's nozzle as represented in figures 5 and 6.

Preparation of the testing room:

The testing room was neither pre-cleaned nor ventilated before running the disinfection cycles (incl. between cycles). This aimed to evaluate the effectiveness of the disinfectant chemicals when poor cleaning routines or negligence that may rarely occur in healthcare environment. The trials were not conducted in the presence of blood, organic waste, greasy substances etc.

No items potentially labile to steam cleaning were removed. The testing room was preserved in its normal state.

The testing room was not sealed so the technicians could:

- Empirically evaluate the impact of chemicals leakage on respiratory tracts when no sealing decontamination targeted area (irritation).

- Subjectively identify the smell of disinfectants.

- Monitor the level of noise.

Temperature and relative humidity were constantly monitored.

Preparation of the system:

One TwinCruiser® unit corresponding to the device of example 1 was placed in the room, at a distance of 3.00 meters from the media.

The system was systematically placed in a standard position (in the left bottom corner of the room) as represented in figures 7 and 8.

The angle of the nozzle of the TwinCruiser was adjusted for better dispersion (from 30 to 45°C) for all the chemicals used. The machine's dispensing system was calibrated so that the flow was constant (12-15 ml/mn).

The 2.5L reservoirs of the TwinCruiser were filled with Amity HP75 biocide

The TwinCruiser® was programmed to disperse 12 ml/mn.

- Dispersing time: 32mn for 50m³ when using Amity HP75.

- Dispersing time: 49mn for 50m³ when using Peroxone.

2. Results of trials:

2.1 Repeatability and trials control:

After dispersion, a contact time of two (2) hours was allowed to reduce the residual peroxide concentration and residual ozone concentration. The operator has verified that both hydrogen peroxide (H2O2) and ozone (O3) levels are safe to re-enter the room:

• Hydrogen peroxide - PEL: 1 part per million (ppm) parts of air (1 .4 milligrams per cubic meter (mg/m³) as an 8-hour time-weighted average (TWA) concentration [29 CFR 1910.1000, Table Z-1 ].

• Ozone - PELThe following limits are widly accepted (USA, Sweden, UK and other parts of Europe): 0.1 ppm (0.2mg/m³) calculated as an 8 hour time-weighted average concentration.

There was also a short-term exposure limit for ozone of 0.3ppm

(0.6mg/m³) calculated as a 15 minute time-weighted average concentration.

After 2 hours contact time the Operative also checked the H2O2 strip detectors to ensure the chemicals dispersal uniformly all over the surfaces in the testing room.

After each trial, all plates were removed and brought to the incubation room of the laboratory for incubation.

After incubation for 24 hours, 48 hours, 72 hours, 96 hours the cfu on each plate was numerated and counted for further computation. 2.2 Results:

Table 1 belows describes the trial conditions, i.e temperature, duration spraying and atomizing time, and the contact time between peroxide and ozone.

Table 1 : tests conditions

TEST CONDITIONS TRIAL 1 TRIAL 2 TRIAL 3

Name of disinfectant delivered Peroxone AMITY HP75 Peroxone

Disinfectant active substances O3+H2O2 H₂0₂ O3+H2O2

Ambient Temp (C°)

21.0°C 19.3°C 17.6°C at the start of the cycle

R.H. (%)

44% 44% 37% at the start of the cycle Ambient Temp (C°)

2I .OC⁰ NA 17.2°C

After Ozone was fully sprayed

R.H. (%)

53% NA 45%

After Ozone was fully sprayed

Ambient Temp (C°)

at the end of cycle

19.8°C 17.97°C 16.4°C

After Ozone and/or Hydrogen

Peroxide were fully sprayed

R.H. (%)

After Ozone and/or Hydrogen 60% 56% 52% Peroxide were fully sprayed

Start Time 17H45 17H41 19H27

Ozone atomizing cycle starts at 17H45 N/A 19H27

Ozone atomizing cycle ends at 18H04 N/A 19H46

0₃ atomizing time 19mn N/A 19mn

Hydrogen peroxide atomizing

18H04 17h41 19H46 cycle starts at

Hydrogen peroxide atomizing

18H34 18H13 20H16 cycle ends at

H₂0₂ atomizing time 30mn 32mn 30 mn

Total atomizing time 49mn 32mn 49mn

Contact time 120mn 120mn 60mn

In table 1 above "N/A" means not tested or non-applicable

The table 2 below summarizes the disinfection results obtained:

Table 2: disinfection results

Strains TRIAL 1 TRIAL 2 TRIAL 3

Inocula CFU: 1 .9 Inocula CFU: Inocula CFU: log 8 4.4 log 7 2 log 8

Escherichia coli

CFU counted: CFU counted: CFU counted: After 96 hours

1 .4log2 3 log 2 1 log 2 incubation at 37°C

Log 5 Log 6

Log 6 reduction

reduction reduction Inocula CFU: Inocula CFU:2.5 Inocula CFU:

2.7 log 7 log 7 3 log 7

Staphylococcus aureus

CFU counted: 3.3 CFU counted: CFU counted: After 96 hours

log2 3 log 2 1 .5 log 2 incubation at 37°C

Log 5 Log 5

Log 5 reduction

reduction reduction

Inocula CFU: Inocula CFU: Inocula CFU: 1 log 6 1 log 6 1 log 6

Candida albicans

CFU counted: CFU counted: CFU counted: After 96 hours

5 logl 1 log 2 4 log 1 incubation at 30°C

Log 4 Log 5

Log 5 reduction

reduction reduction

3. Conclusions:

As demonstrated in above table 2 results clearly demontrate the high efficacy of the process of the present invention in effectively reducing the level of infectious agents, i.e. viable Escherichia coli, Staphylococcus aureus and Candida albicans which are commonly used to evaluate the bactericidal, fungicidal and yeasticidal activity of aerial disinfection processes. It is important to note that the device achieved very good results in only 60 mn contact time (trial 3) vs. 120 mn in trial 1 and trial 2.

As demonstrated in this example, the process of the present invention allows disinfecting a volume with a great efficiency. In particular, the results demonstrate that the process of the present invention allows one to improve the disinfection with the combination of ozone and hydrogen peroxide. Example 3: process for disinfection a volume and performances

Further study of the efficiency of the disinfection of the process was carried out in accordance with the NF T 72-281 :2009 method by an independent laboratory, member of the AFNOR standardization committee. The hydrogen peroxide solution used was the Amity HP 75 commercialized by amity international (registered trademark). The material and method used in this example were different to those of Example 2. In particular, the test room volume was 90 m³ (cub. Meter). 1 . Materials and methods:

The method of implementation and evaluation as described within the NF T 72-281 :2009 document (ISSN 0330-3931 ) provided by the AFNOR was fully respected.

The example was carried out on test microorganisms clearly specified wihin the norm and procured from collection centers : P.aeruginosa NCIMB 1042, S.aureus NCTC 10788, E.coli NCTC 10418, E.hirae NCIMB 8192, A.baumanii NCIMB 14421 , MRSA NCTC 12493, B.subtilis (spores) NCIMB 8054, C.albicans NCPF 3179, A.niger NCPF 2275, M.terrae NCTC 10856.

The biocide used was Hydrogen peroxide [H2O2]: Amity HP75

(registered trademark) disinfectant contained in two 2.5 liter containers (Hydrogen peroxyde based solution at 7.5% cone).

Misting rate : 10ml per minute - 6ml per cubic meter

Atomizing time: 53mn

Contact time: 60 mn.

2. Results:

The table 3 below summarizes the disinfection results obtained:

B.subtilis

P. aeruginosa 2.36 x 10⁹ 0.87 0.86 2.69 X 10⁷ >6.43

S. aureus 2.99 x 10⁹ 0.99 0.96 1 .94 X 10⁷ >6.29

E.coli 2.06 x 10⁹ 0.95 0.95 1 .46 X 10⁷ >6.16

E.hirae

2.62 x 10⁹ 0.90 0.85 1 .45 X 10⁷ >6.16

A.baumanii 2.51 x 10⁹ 0.93 0.99 2.82 X 10⁷ >6.45

MRSA 3.21 x 10⁹ 0.91 0.87 2.99 X 10⁷ >6.08

B.subtilis

2.54 x 10⁷ 0.95 0.95 1 .60 X 10⁶ 4.23 (spores)

C. albicans 1 .79 x 10⁸ 0.97 1 .1 1 1 .40 X 10⁷ 5.60

A.niger 2.51 x 10⁷ 0.92 0.93 1 .35 X 10⁶ 4.43

M.terrae 2.15 x 10⁹ 0.78 0.78 2.1 1 X 10⁷ 5.93

3. Conclusions:

As demonstrated in above table 3 and table 4 below results clearly demontrate the high efficacy of the process of the present invention in effectively reducing the level of infectious agents in 60 minutes, i.e. bacteria, fungi, spores, yeasts and Mycobaterium terrae which are commonly used to evaluate the bactericidal, fungicidal, sporicidal, yeasticidal and mycobatericidal activity of aerial disinfection processes. It is important to note that the process achieved a total kill i.e. greater than log 6 for all the bacteria and Candida, a significant kill for the bacillus spores and also Aspergillus. It is also important to note that the process achieved a significant kill (log 5.93) for the Mycobacteria while requirement is log 4.

The table 4 below compares the disinfection results obtained to those required by the NF T 72-281 :2009 and NF EN 14348

After 60 mn contact

time.

Rgom volume : 90 NF T 72-281

m requirements

Activity Strains (contact time required:

Tests: N/A)

reductions, d in in 30 to 150 m

log

base 10 Distance= 6.50 m

Pseudomon

as

aeruginosa

Escherichia

>6.43

coli

>6.16

Staphylococ

BACTERICID >6.29

cus aureus > 5 log

AL >6.16

Enterococcu

>6.08

s hirae

>6.45

SARM

Acinetobact

er

baumannii

FUNGICIDAL Candida

& albicans 5,60

> 4 log

YEASTICIDA Aspergillus 4,43

L niger

Bacillus

subtilis 4,23

SPORICIDAL > 3 log

Clostridium

difficile

After 60 mn contact

time.

Rgom volume : 90

m NF EN 14348

requirements

Activity Strains Tests: 60 mn contact time in

reductions, d in 30 to 150 m3

log

base 10

Distance= 6.50 m

MYCOBACTE Mycobacteri

5,93 ≥4 log

RICIDAL um terrae

Example 4: process for disinfection a volume and performances

Further study of the efficiency of the disinfection of the process was also carried out in accordance with the NF T 72-281 :2009 method by another independent laboratory, COFRAC accredited and also member of the AFNOR standardization committee.

The hydrogen peroxide solution used was the X-CID commercialized by Airinspace SAS (registered trademark) which is a colorless liquid which is a colorless and odourless liquid that comprises a concentration of 7% hydrogen peroxide and 0.4% PAA.

The dual device/ X-CID was tested twice (30 mn and 60 mn contact times).

The method used in this example was similar to one used in example 3. It is important to note that the test room, and the misting rates were different to those mentioned in example 3.

In particular, the test room volume was 50 m³ (cub. Meter), misting rate was 10ml per minute - 9ml per cubic meter 1 . Materials and methods:

The method of implementation and evaluation as described within the NF T 72-281 :2009 document (ISSN 0330-3931 ) provided by the

AFNOR was fully respected .

The example was carried out on test microorganisms clearly specified wihin the norm and procured from collection centers : Pseudomonas aeruginosa ATCC 15442 = CIP 103-467, Staphylococcus aureus ATCC 6538 = CIP 4 .83, Enterococcus hirae ATCC 10541 = CIP 5855, Escherichia coli ATCC 10536 = CIP 54127, Bacillus subtilis spores ATCC 6633 = CIP 52 62, Candida albicans ATCC 10231 = IP 4872, Aspergillus niger ATCC 16404 = IP 1431 -83, Candida albicans ATCC 10231 = IP 4872, Mycobacterium terrae ATCC 5755

The biocide used was Hydrogen peroxide [H2O2+PAA]: X-CID (registered trademark) disinfectant contained in two 2.5 liter containers (Hydrogen peroxyde based solution at 7% concentration + PAA at 0.4% concentration).

Test 1 - Misting rate : 10ml per minute - 9ml per cubic meter

Test 1 - Atomizing time: 56mn

Test 1 - Contact time: 60 mn.

Test 2 - Misting rate : 10 ml per minute - 9ml per cubic meter Test 2 - Atomizing time: 56 mn

Test 2- Contact time: 30 mn.

2. Results:

The table 5 below summarizes the disinfection results obtained:

After 60 mn

After 30 mn

contact time

Room

Room volyme :

volum^ : 50 NF T 72-281

50 m

m requirements

(contact time

ACTIVITY STRAIN Tests: Tests: required: N/A) reductions, d reductions, d in Room volume^ in log log 30 to 150 m base 10 base 10

Distance= Distance= 4.20

4.20 m m

Pseudomonas

aeruginosa

Escherichia 7,28 7,29

BACTERICID coli 7,29 7,31

≥ 5 log AL Staphylococcu 7,25 7,18

s aureus 7,13 6,98

Enterococcus

hirae

FUNGICIDAL Candida

& albicans 6,95 4,31

≥4 log

YEASTICIDA Aspergillus 6,07 6,40

L niger

Bacillus

subtilis 6,22 6,51

SPORICIDAL ≥3 log

Clostridium N/A 4,61

difficile

After 60 mn

After 30 mn

contact time

Room

Room volyme : NF EN 14348 volum^ : 50

50 m requirements m contact time

ACTIVITY STRAIN Tests: Tests: required: 60 reductions, d reductions, d in mn) in log log Room volum^ : base 10 base 10 30 to 150 m Distance= Distance= 4.20

4.20 m m MYCOBACTE Mycobacteriu

7,86 N/A ≥4 log RICIDAL m terrae

In tab e 5 above "N/A" means not tested or non-applicable

3. Conclusions:

As demonstrated in above table 5 results clearly demontrate the high efficacy of the process of the present invention in effectively reducing the level of infectious agents in 30 minutes and 60 minutes, i.e. bacteria, fungi, spores, yeasts and Mycobaterium terrae which are commonly used to evaluate the bactericidal, fungicidal, sporicidal, yeasticidal and mycobatericidal activity of aerial disinfection processes. It is important to note that the process achieved a total kill i.e. greater than log 7 for all the bacteria, log 6 for fungi, yeasts and spores in 60 minutes contact time. It is also important that the process achieved a significant kill in 30 minutes contact time, and most importantly achieved log 7.86 for the mycobacteria while requirement is log 4.

4. General conclusions:

As demonstrated in all those examples, the process of the present invention allows the powerful disinfection of a volume, for example when used with hydrogen peroxide based solutions at 7 to 8% concentration and/or when used with peroxone, i.e. ozone plus above mentioned hydrogen peroxide based solutions. In particular, the results clearly demonstrate that the process of the present invention achieved total kills and significant kills, for example by altering the growth and accelerating lyse of very different microorganisms such as bacteria, fungi, yeasts, spores and mycobacteria. Accordingly, the process of the present invention allows advantageously simultaneous disinfection of a surface and of a volume.

Claims

1 . A method of disinfecting a volume by means of a biocide containing hydrogen peroxide (H2O2) or peroxone (03+Η₂Ο2) comprising step of:

a) diffusion of hydrogen peroxide into the volume to be disinfected, or b) injection of ozone (O3) gas into the volume to be disinfected , followed by a diffusion of hydrogen peroxide in said volume.

2. The method according to claim 1 , wherein steps a) and b) are carried out individually as needed for decontamination

3. The method according to claim 1 or 2, wherein in step b) ozone and hydrogen peroxide are in contact for a period of 4 to 420 minutes, preferably 30 to 120 minutes.

4. The method according to any preceding claims , wherein in step b) is diffused into the volume to be disinfected from 6x10³ g/m³ to 108x10³ g/m³ of ozone, preferably from 9.81 x10³ g/m³ to 17.66 x10³ g/m³

5. The method according to any preceding claims, wherein in step b) ozone is obtained by means of a corona tube preferably a corona tube provided with an air dryer means.

6. The method according to any preceding claims, wherein in step b) the ozone is injected into the volume by means of a fan.

7. The method according to any preceding claims, wherein in step a) or b), hydrogen peroxide is diffused into said volume in an amount of 6 to-20 g/m³, preferably 8 to 12 g/m³ of volume to be disinfected.

8. The method according to any preceding claims, wherein the hydrogen peroxide is used at a concentration of 2 to 35% by volume, preferably 4.5 to 7.5% by volume.

9. The method according to any preceding claims, wherein the hydrogen peroxide used is diffused by spraying, misting, atomizing and/or nebulizing.

10. A method according to any one of claims 1 to 9, wherein the method is implemented at a temperature from 15 to 30 °C, preferably from 18 to 22 °C.

1 1 . The method of any preceding claim , wherein the volume to be disinfected is selected from the group comprising patient wards, emergency rooms, treatment rooms, operating theatres, Intensive Care Units (ICUs), infectious disease units, oncology units and other units receiving severely immunocompromised patients, Isolation rooms, a laboratory, including a biological laboratory, incubators room and other enclosed volume of any nature requiring disinfection / decontamination.

12. A device for disinfecting a volume with a biocide based on hydrogen peroxide or peroxone, said device comprising:

i. An injection system of ozone in said volume,

ii. A diffusion system of hydrogen peroxide in said volume,

13. A Device according to claim 12, further comprising at least one of the following: a material selected from a measuring probe and / or monitoring the ozone injection probe, a probe for measuring and / or monitoring of the distribution of hydrogen peroxide, a temperature sensor, a humidity sensor, a liquid hydrogen peroxide sensor, an ozone sensor, a motion detection sensor, an alarm.

14. A Device according to any one of claims 12 to 13, further comprising a wireless system for controlling the remote device.

15. A Device according to any one of claims 12 to 14, further comprising an embedded PLC for operating and controlling the device.

16. A Device according to any one of claims 12 to 15, wherein the diffusion system of hydrogen peroxide comprises a compressor.