High level disinfection and possible sterilization of computer hardware material using the flowing afterglow of a reduced-pressure N2-O2 discharge

P.Levif, S. Larocque, M. Moisan and J. Barbeau Groupe de physique des plasmas, Université de Montréal, Montréal, Québec Faculté de médicine dentaire, Laboratoire de contrôle des infections, Université de Montréal, Montréal, Québec

Outline

• Introduction Brief description of our method Comparison with FDA cleared low-temperature techniques

• Generation of N and O atoms and their ability to diffuse before forming NO* excited molecules yielding UV photons

• Some specific features of the late N2-O2 flowing-afterglow Broadband spectrum of the UV emission of NO* molecules An electron-free medium Innocuity of the effluents coming out from the N2-O2 discharge flowing afterglow

• Packaging-pouch material adapted to the N2-O2 late flowing-afterglow • First results on computer keyboards and laptops • Conclusion

Introduction Brief description of the N2-O2 discharge flowing afterglow used for disinfection/sterilization

Microorganisms inactivating agents: UV photons resulting from the formation of NO* excited molecules through the interaction of O and N atoms provided initially by a (microwave) discharge dissociating the N2 and O2 molecules of a N2-O2 mixture. The UV photon irradiation causes in the end enough lesions to the microorganism DNA, preventing their repair.

"Artist’s view" representing the discharge outflow of N and O atoms, long-lived species, as they enter the chamber after travelling a distance xd from the microwave-plasma source. The figure illustrates the case where xd is relatively short (≈100 mm). At xd ≥ 820 mm, the whole 50 litre chamber is fully filled with species from the late afterglow of the N2-O2 discharge.

Maximum UV intensity, i.e. shortest exposure time required for inactivating a given number of endospores (used as bio-indicators), is obtained when: i. the O2 percentage in the N2-O2 mixture is set for maximum UV intensity

(≈ 0.2 % O2 in the present operating conditions) (US Patent 6 707 254 (2004)); ii. when the sterilizer chamber is filled only with the later part of the discharge flowing

afterglow (xd ≥ 820 mm) (also preventing the presence of electrons); iii. when total gas pressure is set at 3.5 torrs (under a 1.4 slm N2 flow rate in a

50 litre chamber); iv. when plasma is sustained at the highest microwave frequency currently available, namely

2450 MHz

Introduction Brief description of the N2-O2 discharge flowing afterglow used for disinfection/sterilization

Introduction Advantages/shortcomings of the late flowing-afterglow of the N2-O2 discharge compared to FDA approved low-temperature (< 60-650C) sterilization techniques

FDA cleared sterilization techniques Late flowing-afterglow of the N2-O2 discharge

EtO Toxic, carcinogenic, polluting gas with a high degree of impregnation in many materials

Non-toxic and environmental friendly gases: N2 and O2

Aeration time required (minimum 8 hrs) No aeration time: immediate release Porous packaging (few months on shelf only) Non-porous packaging: long term storage time (> 1 year)

Immersion in peracetic acid

Immersible instruments only No damage to computer keyboards and laptops No sterile storage Full diffusivity around/through complex-form objects Long term storage time (> 1 year) Hydrogen peroxide plasma

Presence of electrons: dislodgment and release of viable microorganisms Possible interaction with some polymers

No electrons in the late N2-O2 flowing-afterglow

Ozone exposition

Powerful oxidant: not all polymers welcomed, but can process metals

Not efficient with metals (too long or incomplete inactivation), but all polymers accepted

Generation of N and O atoms and their ability to diffuse before forming NO* excited molecules yielding UV photons Setting conditions for achieving the highest possible UV intensity in the processing chamber UV emission intensity increases with the concentration of N atoms in the chamber. The higher the microwave frequency at which N2-O2 discharge is sustained, the higher the dissociation degree of N2 molecules, thus, the higher the intensity of photon radiation.

Survival curves for 107 B. atrophaeus spores exposed to the late flowing-afterglow from a N2-O2 microwave discharge sustained either at 915 MHz or 2450 MHz in the 50 litre chamber. Operating conditions optimized for maximum UV intensity (N2-0.2 % O2 at 3.5 torrs (~ 470 Pa)) and axial uniformity (1.4 slm N2 flow). The safety assurance level (SAL) of 6 logs is reached after 60 min at 2450 MHz.

Concept of photon fluence (dosis)

The O2 % admixed to N2 determines the UV intensity, thus the characteristic time for spore inactivation: the higher the UV intensity, the shorter the decimal times, as shown in figure (a). Plotting the number of survivors as a function of fluence (dosis) shows a "universal" survival curve (b).

Survival curves of B. atrophaeus spores following their exposure to the N2-O2 flowing afterglow at three different UV intensity levels obtained by setting the O2 admixture percentages to N2 at 0.3%, 1% and 10%. The D1 time values for phase 1 are indicated; (b) same survivor data counts as in Figure (a), but plotted as functions of fluence instead of exposure time

Early vs. late flowing afterglow in terms of UV intensity

UV emission intensity increases with the concentration of N atoms present in the chamber. The higher the microwave frequency at which the N2-O2 discharge is sustained, the higher the dissociation degree of N2 molecules, thus, the higher the intensity of photon radiation. No electron in the late flowing afterglow

Relative emission intensity from a NOγ band head as a function of the O2 percentage in the N2-O2 mixture at two distances xd of the connecting tube. Operating conditions: 915 MHz with 100 W absorbed power, N2 flow rate of 1.4 slm at a 3.5 torr (~ 470 Pa) pressure.

Influence of microwave power on N atom yield

UV intensity increases with microwave power transferred to plasma up to approximately 300 W, then saturates. Concentration of N atoms can be increased by connecting a second plasma source to the sterilization chamber, for example on its opposite side.

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Intensity of UV emission (≈ 268 nm) measured in the chamber as a function of microwave power to plasma source.

Ability of long-lived N and O atoms to efficiently diffuse, ensuring UV irradiation of complex-form objects

Photons from a UV lamp travel in straight line, not reaching all surfaces of a complex object: parts of a device can remain contaminated

UV Lampfartest leftposition

UV Lampfartest rightposition

A UV lamp cannot illuminate all surfaces within an object: case of a crevice.

Ability of long-lived N and O atoms to efficiently diffuse, ensuring UV irradiation of complex-form objects Definite ability of the N and O atoms to move around objects through diffusion before forming NO molecules emitting UV photons : case of a hidden spore under a stack.

Schematized representation of how N and O atoms can infiltrate, through diffusion, in-between spores and afterwards form a NO* excited molecule, which then emits a UV photon that reaches the DNA of a spore concealed within the stack.

Ability of N and O atoms to diffuse efficiently through very small openings

Entry of N and O atoms into the pouch is macroscopically restricted, but these atoms actually penetrate into the pouch by diffusion and efficiently spread within it. The non-porous pouch (Baglight) used is only partially transparent to UV radiation (20, 50 and 60% transmission factor at 200, 250 and 300 nm, respectively). Diffusion of the N and O atoms is effective in providing NO molecules, i.e. UV photons, within the whole bag itself.

Photograph of a (cover-less polystyrene) Petri dish

enclosed in a (non-porous) pouch, unsealed at one extremity. Flaps of the open-ended side are simply resting one on the other.

No difference, in terms of inactivation rate, between a freely exposed Petri dish and one enclosed in the partially open pouch. A similar behavior is observed with a spore-contaminated forceps with a nickel-plated steel. Characteristic inactivation time of the B. atrophaeus spores is, however, much slower due to a higher recombination rate of N atoms (into N2 molecules), γN, on the forceps surface.

Petri dish inoculated with a deposit of B. atrophaeus spores in a Petri dish, either enclosed or not in a BagLight Polysilk non-porous pouch with one end unsealed, and subjected to the (early) flowing-afterglow of a N2-O2 discharge

A nickel-plated forceps inoculated, either bare or enclosed in a BagLight polysilk pouch with one end unsealed

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Probability of recombination of N atoms onto the surface of noteworthy materials

The recombination rate of N atoms, γN, is indicative of how many times (statistically) N atoms can collide on (interact with) a given type of surface and bounce back without being "lost" (by recombining into N2) for the formation of NO* excited molecules.

Material γN

Fused silica ~10-4

Pyrex ~10-4 *

Nylon 2x10-3 *

Alumina 10-3 *

Aluminum 4x10-3 *

Stainless steel 10-2

Copper 5x10-3 *

Brass 10-2 *

Sterilizer load coefficient: area of the exposed surface times γN. The higher this coefficient, the larger the number of N atoms required to achieve inactivation of microorganisms in a given exposure time.

* 3.5 torr values : Sarrette et al, Plasma Proc. Polym. (2006)

Some specific features of the late N2-O2 flowing afterglow Broadband spectrum of the UV emission of NO* molecules Broad UV wavelength domain of NO*molecules

Emission intensity from the flowing afterglow of the N2-O2 discharge sustained at 2450 MHz under optimum operating conditions

Endospores have different spectral windows as far as their inactivation rate is concerned upon exposure to UV radiation: a broad wavelength radiation range ensures their adequate inactivation in contrast to a "monochromatic" UV lamp, like the 254 nm Hg lamp.

An electron-free medium

In most plasma sterilization systems, the device to be disinfected/sterilized is exposed to electrons: charged microorganisms are dislodged from the surface of the exposed objects, some of these microorganisms still being viable. Late flowing-afterglow provides N and O atoms in an electron-free inactivation volume (not the early flowing-afterglow).

Exposure conditions Total number of

recovered spores

Non-exposed 1.3 ± 0.3 x 107

Exposure to the argon discharge

2 min 0.4 ± 0.1 x 107

5 min 0.3 ± 0.1 x 107

Exposure to the N2-O2 late afterglow

2 min 1.1 ± 0.1 x 107

5 min 1.1 ± 0.3 x 107

Total number (active and inactivated ones) of endospores recovered after exposure for a period of 2 and 5 min to the argon discharge or to the late N2-O2 flowing-afterglow, compared to the initial number of spores (non-exposed spores)

Innocuity of the effluents coming out from the N2-O2 discharge flowing afterglow

In contrast to the sterilization/disinfection processes using EtO, the gases used in our system (N2 and O2) are harmless to human health (non-toxic, non-carcinogenic), without greenhouse imprint and do not necessitate venting time: the processed device is available immediately after the required inactivation time is over. Most of the NO gas present in the N2-O2 flowing afterglow is eliminated as it collides with the pump exhaust walls.

Packaging-pouch material adapted to the N2-O2 late flowing afterglow Polyolefin polymers, a low-recombinant surface for N and O atoms

With conventional sterilization methods (steam, ozone, chemicals), the devices to be processed are wrapped/enclosed in packaging materials (after being "cleaned") and are then closed/sealed before initiating the sterilization process: these packaging materials need to be porous. The most frequently utilized packaging materials are highly recombinant for N and O atoms: i. recombination on their surface reduce the UV intensity around the bag from 30% (Tyvek)

and 40 % (Kimguard) up to 55% (SteriPouch), whereas it is less than 10% with BagLight polysilk.

ii. damage inherent to the afterglow exposure made the conventional materials lose their antimicrobial barrier

iii. because the BagLight Polysilk material is non-porous it requires to be (thermally) sealed after the sterilization process, the counter-part of this disadvantage being that it ensures a much longer on-shelf storage time (the manufacturer guarantees sterility for a full year).

Polyolefin polymers, a protective barrier ensuring less structural damage to spores When exposed freely to the flowing afterglow, the membrane of B. atrophaeus spore is damaged, which is not the case for exposure time less than an hour for those enclosed in the open-ended Baglight pouch: in this latter case, the spores are uniquely inactivated due to lesions, by the UV photons, to the DNA (single strand breaks). Damage to unpackaged spores, revealed by Dapi staining, is attributed to the presence of singlet oxygen O2(1∆g), a metastable-state molecule: when quenched on a surface, it transfers 0.97 eV (94.3 kJ/mole) to it.

Micrographs of Dapi stained B. atrophaeus endospores: (a) non-exposed (control); (b) autoclaved for 30 min: the membrane has ruptured since the staining has reached the spore cortex; (c) exposed for 30 min unpackaged in the (early) flowing afterglow of the N2-O2 discharge sustained with 200 W at 915 MHz: similar comments as in (b); d) same conditions as in c), but the spores are enclosed in the BagLight Polysilk (open ended) pouch: the membrane is intact.

First results on computer keyboards and laptops

Computer keyboards 1) Gram-negative vegetative bacteria, Serratia marcescens, yielding a reddish-orange pigment when growing, were inoculated. The computer keyboard was divided into three sections, each key receiving 105 bacteria with a total of 6 keys involved per Petri, with four Petris sharing the same protocol (24 keys under the same conditions: 2.4 M bacteria in total per given condition). Cleaned means that the contaminated keys were afterwards wiped with quaternary ammonium (efficient surfactant). Harvesting was achieved with Petris, filled with Agar, brought in contact with the keys (Rodac plate technique). Exposure time was 2 hrs in the late flowing-afterglow.

1) "cleaned" through wiping without exposure to the afterglow; 2) cleaned through wiping and exposed; 3) directly exposed

2) 500 k bacterial endospores B. atrophaeus deposited per key, letting the deposit dry for 24 h with 4 keys per harvesting Petris. Petris 1, 4 and 6 (orange coloured on keyboard) were wiped with quaternary ammonium and then exposed in the 50 litre chamber under optimized conditions in the late flowing-afterglow configuration for 6 hrs. Petris 2, 3 and 5 were directly exposed under the same operating conditions.

Photograph of the keyboard inserted into the 50 litre processing chamber

Photograph of the keyboard used with the location of the various Petris filled with Agar and used to harvest the viable B. atrophaeus endospores via the Rodac plate method followed by swabbing the remaining spores (Petri). Petri 1, 4 and 6 correspond to keys that were wiped with a surfactant once the spore deposit had dried (necessity of a stringent cleaning: a standard procedure).

Viable spore counts obtained through the Rodac plate technique followed by swabbing the keys for remaining spores (petri) The keyboard remained mechanically and electrically operating after having been subjected to the late flowing-afterglow for at least 6 hours.

Pre-cleaned and exposed Exposed only

1 4 6 3 5 2

Rodac (CFU) 0 8 1 TNC TNC TNC

Petri (CFU) 9 120 >200 TNC TNC TNC

TNC: Too Numerous to Count

The highest reduction in the number of spores is observed in the same group (1, 4, 6): on Petri 1, closest to the gas inflow, while the highest count is on the opposite side, on Petri 6, where the number of available N atoms has decreased continuously due to surface recombination.

Laptop decontamination Three groups of keys of an HP laptop were inoculated with 500 k B. atrophaeus endospores and, after drying 24h, were then wiped with quaternary ammonium before being subjected to the late flowing-afterglow of the N2-O2 discharge run at a power of 200 W and at a microwave frequency of 2450 MHz. The laptop was exposed first for 4 hrs in a given orientation with respect to the gas inflow and then turned at a 180 o for a further 4 hrs.

Schematized representation of the laptop used.

(a) (b)

Laptop seen from the top opening side of the sterilization chamber: (a) in a given orientation with the gas inflow and (b) in the 180o opposite direction. Full opening of the laptop is limited by the dimensions of the 50 litre chamber.

Summary of the viable spore counts: 4 keys per Petri Petris number 7 and 9 (at each extremity) have the lowest counts in contrast to the center one (number 8), suggesting a strong depletion of the N and O atoms as the flow moves away from the chamber entrance toward the right-hand side of the chamber and, possibly, because the screen opening is restricted, limiting access of the species flow to Petri number 8. The laptop was found to work as usual, as we plugged it back.

Sample Rodac Petri Total

7 11 4 15

8 TNC > 300 > 300

9 3 > 300 > 300

TNC: Too Numerous to Count

Summary and conclusion

The biocidal agent of the sterilization system based on the late flowing-afterglow system are the UV photons; they can, in the end, cause sufficient lesions to the DNA (single-strand break) of the microorganism such that it cannot repair. The efficient action of these UV photons, in contrast to those emitted by a UV lamp, stems from the fact that they are emitted by the excited NO* molecules formed through interaction of long-lived N and O atoms present in the flowing afterglow, atoms which beforehand were able to diffuse around complex-form objects and penetrate into holes and crevices.

The main limitation of our technique depends on the probability of recombination of N and O atoms (into N2 and O2 molecules as a rule) on a given surface material. When this probability is small enough to allow, for instance, 105 N atom surface collisions before it recombines, then the N atom can move freely around in the chamber through diffusion and reach locations on a given device otherwise not accessible from a fixed UV source. Metallic materials such as, for example, brass and copper are too much recombinant to enable the N atoms to bounce back many times on their surface, hindering their further diffusion. It seems that dielectric materials (fused silica, glasses in general, some polymers like PTFE)) are little recombinant compared to conductors. Hence sterilizing metallic surfaces with the present system, even if they present a very small surface, is not recommended. The total surface dimensions of the devices and the nature of their materials determine the total N atoms losses through surface recombination (load coefficient). The concentration of N atoms can be increased by implementing more than one plasma source.

A specific advantage of using the late flowing afterglow of the N2-O2 discharge is that, in contrast to electron-containing discharges or some afterglows, it does not dislodge microorganisms from their substrate. The present disinfection/sterilization system provides a low-temperature, non-harmful to human health and non-polluting process, with immediate availability of the treated devices once exposure time is over. Furthermore, it appears to be particularly suited to disinfect/sterilize computer-keyboards or tablets since it does not damage their (polymeric) material or their electrical/mechanical properties.

The original system described is far from being a universal sterilizer, but it offers exceptional niche opportunities among the low-temperature sterilization systems. The present 50 litre chamber should be extended to a larger one (e.g. 125 litres) with additional microwave-plasma sources for increased usefulness. Acknowledgments The work on keyboards and laptops was supported by Getinge (Infection Control, UK), thanks

going to Dr. Stephen Morley and Tim Swales.