Evaluation of an Ozone Cabinet for Disinfecting Medical Equipment

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Evaluation of an Ozone Cabinet for

Disinfecting Medical Equipment

Ida Ljungberg

Examinator, Thomas Ederth Tutor, Maria Lerm

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2018-11-05 Department of Physics, Chemistry and Biology

Linköping University

URL för elektronisk version

ISBN

ISRN: LITH-IFM-A-EX—18/3583—SE

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Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel Title

Evaluation of an Ozone Cabinet for Disinfecting Medical Equipment

Författare Author Ida Ljungberg Nyckelord Keyword Sammanfattning Abstract

The spreading of infection is a significant and well-known problem in all healthcare environments today. The most prevalent ways that infection spreads are either by direct contact between two individuals where one has an infection, or with an intermediate person or object as an infection carrier. This thesis aims to evaluate a method that could operate to disinfect the type of medical equipment which is not suited to be disinfected by the commercially existing methods.

In keeping with the long term goal of preventing the spread of infection, this project evaluates an ozone cabinet according to its antimicrobial properties and investigates if the cabinet is suited to work as a disinfectant for some chosen test objects. The objects were borrowed from different hospital institutions at Motala Lasarett and the antimicrobial effect was evaluated according to the reduction of colony forming units (CFUs) of samples taken from the object's surfaces after the treatment. The results show that the ozone cabinet is not able to kill bacterial spores (Geobacillus stearothermophilus), but could be very efficient at killing living bacteria. Concentration setting 4 (56 ppm) in combination with a treatment period of at least 40 minutes proves bacterial reductions varying between 83-98 %. Nevertheless, the sources of error are numerous and there is a great variation between identical runs which indicates that more studies need to be performed in order to obtain clearer results.

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Master of Science in Applied Physics

Evaluation of an Ozone Cabinet for Disinfecting Medical

Equipment

Ida Ljungberg

LITH-IFM-A-EX—18/3583—SE

Supervisors:

Maria Lerm

IKE, Linköping University

Magnus Stridsman

Clinicum Test and Innovation, Region Östergötland

Martin Berglund

Medicinsk teknik, Region Östergötland

Magnus Roberg

Vårdhygien, Region Östergötland

Examiner:

Thomas Ederth

IFM, Linköping University

Department of Physics, Chemistry and Biology Linköping University SE-581 83 Linköping, Sweden

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The spreading of infection is a significant and well-known problem in all healthcare en-vironments today. The most prevalent ways that infection spreads are either by direct contact between two individuals where one has an infection, or with an intermediate per-son or object as an infection carrier. This thesis aims to evaluate a method that could operate to disinfect the type of medical equipment which is not suited to be disinfected by the commercially existing methods.

In keeping with the long term goal of preventing the spread of infection, this project evaluates an ozone cabinet according to its antimicrobial properties and investigates if the cabinet is suited to work as a disinfectant for some chosen test objects. The objects were borrowed from different hospital institutions at Motala Lasarett and the antimicro-bial effect was evaluated according to the reduction of colony forming units (CFUs) of samples taken from the object’s surfaces after the treatment.

The results show that the ozone cabinet is not able to kill bacterial spores (Geobacillus

stearothermophilus), but could be very efficient at killing living bacteria. Concentration

setting 4 (56 ppm) in combination with a treatment period of at least 40 minutes proves bacterial reductions varying between 83-98 %. Nevertheless, the sources of error are nu-merous and there is a great variation between identical runs which indicates that more studies need to be performed in order to obtain clearer results.

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First, I would like to thank Clinicum Test and Innovation for giving me the opportu-nity to do this Master Thesis project. I would like to thank Magnus Stridsman for giving me a great introduction to the project and supporting me along the way with great en-couragement. Special thanks to Martin Berglund for helping me with all the problems that arose during this project and always making time for me and supporting me. This project would never have been possible without your help. Also, thank you to Magnus Roberg for being a source of guidance and your interest in my work.

I would also like to thank Maria Lerm for valuable opinions, feedback and providing me with a lab for my work. Also, thank you to Thomas Ederth for examining this work. Finally, thank you to Sepideh Kamrani for teaching me how to work in the lab and advising me from a biomedical analyst’s point of view.

Linköping, October 2018 Ida Ljungberg

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1.1 Motivation . . . 1 1.2 Background . . . 1 1.3 Problem statement . . . 2 1.4 Limitations . . . 3 2 Theory 4 2.1 Ozone . . . 4 2.1.1 Ozone as a disinfectant . . . 4 2.1.2 Ozone production . . . 5 2.1.3 Current research . . . 5

2.2 The ozone cabinet . . . 6

2.2.1 Settings . . . 7

2.2.2 Previous bacteria tests in the ozone cabinet . . . 9

2.3 Bacteria . . . 9

2.3.1 Spores . . . 10

2.4 Colony forming units . . . 10

2.5 Agar . . . 11

2.5.1 Hematin agar . . . 11

2.5.2 UTI agar . . . 11

2.5.3 Blood agar . . . 11

2.6 Spore specimens . . . 12

2.7 Dry disinfection methods . . . 12

2.7.1 Hydrogen peroxide . . . 13 2.7.2 UV light . . . 13 2.7.3 Ethylene oxide . . . 15 2.7.4 Gamma radiation . . . 16 2.7.5 Formalin . . . 16 2.8 Degrees of cleanliness . . . 17 2.9 Defining disinfection . . . 17

2.10 The test objects . . . 18

2.10.1 Blood pressure cuff . . . 18

2.10.2 Drug pump . . . 19

2.10.3 X-ray neck collar . . . 20

2.10.4 Transportation bag . . . 21

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4.2 Obtaining data . . . 24 4.3 Statistical analysis . . . 25 4.3.1 Antimicrobial efficiency . . . 25 4.3.2 ANOVA . . . 26 5 Results 27 5.1 Spore specimens . . . 27 5.2 ANOVA . . . 27

5.3 Mean and standard deviation . . . 31

5.4 Antimicrobial efficiency . . . 33

5.5 Disinfection with Meliseptol . . . 36

5.6 Variation . . . 37 5.7 Visual effects . . . 38 6 Discussion 39 6.1 Results . . . 39 6.1.1 Spore specimens . . . 39 6.1.2 ANOVA . . . 39

6.1.3 Mean and standard deviation . . . 40

6.1.4 Antimicrobial efficiency . . . 41

6.1.5 Disinfection with Meliseptol . . . 43

6.1.6 Variation . . . 44 6.1.7 Visual effects . . . 44 6.2 Method . . . 45 6.3 Future work . . . 48 7 Conclusion 49 Appendices 50

Appendix A Specimen collection and analysis

-a description 50

Appendix B ANOVA 53

Appendix C Data 57

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Abbreviations

Abbreviation Definition

ANOVA Analysis of Variance BI Biological carrier BPC Blood Pressure Cuff CFU Colony Forming Unit

CPAP Continuous Positive Airway Pressure

CxDy Concentration setting x in combination with duration setting y (x = 1, 2, 3, 4), (y = 1, 2, 3, 4, 8)

eSwab Copan Liquid Amies Elution Swabs NC X-ray Neck Collar

UTI Urinary Tract Infection UVR Ultraviolet Radiation

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1 Introduction

This thesis work has been carried out at Clinicum test and innovation, Region

Östergöt-land and examined at IFM, the Department of Physics, Chemistry and Biology at Linköping

University. The purpose of the project was to investigate if an ozone cabinet could be used to disinfect some different types of medical equipment. There is no previously published literature covering this, however, there is literature that describes the effect of ozone in general as a disinfectant.

1.1 Motivation

The most common way of infection spreading within Sweden’s healthcare institutions today is through intermediate contact spreading when the infection is spreading from one person to another through a third person or object. [1] Various pieces of medical equip-ment which are frequently used in hospitals today are not being cleaned or disinfected. This could be due to their material which might be complicated to clean or disinfect with the techniques that are available today. The equipment might also be of a complicated structure with many cavities and joints which could be challenging to clean properly. If it were possible to use an ozone cabinet to disinfect some of these objects, this might reduce the spread of infections in Sweden’s healthcare institutions.

1.2 Background

Elozo Oy is a company stationed in Finland which produces different solutions for safer

and healthier surroundings. Among their many products are different kinds of ozone cabinets. The ozone cabinets are sold commercially and are utilized mainly for removing odors from fire fighter’s clothes, theater costumes or smelly mattresses, rugs or other textiles. [2] One of the ozone cabinets produced by the company, Elozo D800 Cleaning System (see Figure 1), was donated to the Medical Technology department at Linköping

University Hospital, Region Östergötland. It is of interest for Elozo Oy to expand the

areas of use for the cabinet and it is of interest for the hospital to find new disinfection methods to reduce the spread of infections.

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Figure 1: The ozone cabinet (Elozo D800 Cleaning System) [3]

1.3 Problem statement

This thesis work investigated the possibility of ozone being used as a disinfectant for different objects that are hard to disinfect with other methods that are standard within hospitals in Sweden. The objects were chosen due to their different materials and shapes to better understand what objects are suitable for being disinfected in the cabinet. The questions that this thesis work aimed to answer are the following:

- Can a blood pressure cuff, a drug pump, an X-ray neck collar and a transportation bag for Continuous Positive Airway Pressure (CPAP) and similar devices be disinfected by being placed in the ozone cabinet?

- What concentrations of ozone in combination with what duration of treatment has the best effect on each of those different objects, respectively?

This project also investigated the effect that the ozone cabinet had on bacterial spores. Another question that was answered in this study was, therefore:

- Does the ozone cabinet have the ability to kill bacterial spores (Geobacillus

stearother-mophilus)?

If time allowed, the following question was aimed to be answered by this thesis work: - Can a CPAP, a mattress cover, an emergency room pillow cover, ambulance ECG electrodes or a sling lift be disinfected by being placed in the ozone cabinet?

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raised an additional question to extend the work if the cabinet was found not to be suit-able for disinfection purposes. In this case, the thesis would examine the efficiency of the existing cleaning procedure for each of the chosen objects by finding an answer to the following question:

- How well do the currently used cleaning methods work for a blood pressure cuff, a drug pump, an X-ray neck collar, a transportation bag for CPAP (and similar) devices, a CPAP, an emergency room pillow cover and ambulance ECG electrodes (if they are being cleaned by the hospital staff)?

1.4 Limitations

The project was limited to testing only the objects listed in Section 1.3 and no other. The project was also limited to testing the ozone cabinet. A comparison would be made to the existing disinfection methods for the individual objects only if applicable and if there was time or if the ozone cabinet did not exhibit satisfying levels of antimicrobial properties.

Another limitation of this project was the number of objects that could be tested. If the objects were hard to access that might affect the number of tests that could be per-formed and so that will have an influence on the statistical validity of the results. The project was also limited to the material that is available from Vårdhygien and the lab time at IKE, the Department of Clinical and Experimental Medicine at Linköping University.

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2 Theory

This chapter contains the theory related to this project along with a description of the objects that will be investigated and the current method used to disinfect them.

2.1 Ozone

Ozone is a chemical compound consisting of three oxygen atoms put together, forming O3. Ozone exists naturally in the atmosphere and is a colorless (or blue like) gas. [4] The

ozone in the atmosphere is generated by oxygen, organic compounds or nitrogen oxides that are catalyzed by UV radiation. [5] Ozone has many different areas of use and can, for example, be used to sterilize bottled water, disinfect wastewater and eliminate odors. Ozone is also used as an antimicrobial agent in food processing and can be utilized in fire restoration. [6] The ozone molecule can be visualized in Figure 2.

Figure 2: The ozone molecule [7]

Ozone has, among many other hazards, the ability to cause and intensify a fire, cause skin irritation, eye irritation, respiratory irritation and may be fatal if inhaled. [5]

2.1.1 Ozone as a disinfectant

Ozone is an unstable molecule that will give away an oxygen atom and form O2 at any

given opportunity. Ozone can work as a disinfectant since it will oxidize molecules that build up the bacteria and thereby destroy them in the process when the ozone molecule gets reduced and gives away an atom. This process, by which ozone eliminates bacteria, is called an oxidative burst. When the ozone comes in contact with the bacterial cell wall, this process will take place and generate a hole in the cell wall. [8] The burst orig-inates from a reaction between the ozone molecules and the double bonds of the lipids that constitute the cell membranes. [9] This will lead to the bacteria having difficulties maintaining their structure at the same time as more ozone molecules create more holes. This will eventually lead to the bacteria not being able to keep their content and hence it will die. [8] The ozone will also enter the cells after a hole is created and oxidize the nucleic and amino acids, which also leads to cell lysis. [9]

Since the ozone will give away an oxygen atom in this reaction, this means that the byproduct after the process is oxygen (O2), which is completely harmless. This indicates

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favorable. [10] When using this technique, the only instrument required is the cabinet in addition to a power outlet to provide the cabinet with electricity. There should also be an extraction pipe to pump out any indwelling ozone molecules that might still be present after the process.

2.1.2 Ozone production

Ozone can be produced in several ways, for example (as mentioned in section 2.1) through UV radiation in the atmosphere. The ozone cabinet used in this thesis work utilizes a method called the corona discharge to produce ozone. The corona discharge is the process when a high voltage is applied to an oxygen gas flow, which splits the O2 molecules into

individual oxygen atoms (see Figure 3). When these individual O atoms get in contact with naturally occurring O2, the two will collectively form O3 molecules. [11]

Figure 3: The corona discharge [11]

2.1.3 Current research

There is a great deal of research regarding the usage of ozone as a disinfectant but most of it is in relation to smaller laboratory experiments. By reading about the applica-tions for the ozone cabinet at the vendor’s website, the only thing that is mentioned for hospital use is for eliminating odor from mattresses and bed linen and nothing con-cerning disinfecting medical equipment. [2] However, regarding the smaller experiments, there is a large number of articles published regarding the usage of ozone as a disinfectant. There are studies showing that ozone can be used to reduce the number of bacteria in the tooth root canal. In a study from 2013, researchers found that 82 % of all the bacteria (Streptococcus mitis and Propionibacterium acnes) in the tooth root canal were eliminated as a result of being treated with ozone gas for 40 seconds. The ozone concen-tration used in this study was 2100 ppm (± 10 %). [12]

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Another study from 2015 shows that corrugated tubes (used for tracheostomized pa-tients) that have been in contact with non-intact skin and need high-level disinfection could be exposed to ozone gas and successfully be disinfected. The study used an ozone generator which produced 33 mg/L of ozone and treated the tube for 15 minutes. The result showed that 99.99 % of all bacteria (Pseudomonas spp., Alcaligenes spp.,

Acineto-bacter, Klebsiella spp., Citrobacter, and Enterobacter) died from the treatment. [13]

Most studies that are published on ozone as a disinfectant show that the research fo-cus lies in disinfecting wastewater and foods, even though there are a few that fofo-cus on disinfecting medical equipment (such as the one above). There are no scientific studies on ozone cabinets’ effect on medical equipment, which means that this project will include new research.

This project will also investigate the effect that the ozone cabinet has on bacterial spores. Several articles describe how ozone can successfully kill spores in liquid samples. For ex-ample, in a study from 2014, it was proved that a 40-minute treatment (when a bubbling stream of ozone of 5.3 mg/L was applied to a liquid) induced a 99 % reduction of A.

acidoterrestris spores. [14] Several other articles describe how ozone in combination with

some other disinfectant can prove to be sporicidal. One example is a study from 2011 where a combination of 80 ppm ozone together with a 3 % hydrogen peroxide vapor proved a 99.9999 % reduction of Bacillus subtilis spores. In this study, the spores were dried onto steel discs or cotton gauze pads and the treatment lasted for 30 to 90 minutes. [15] The number of published research papers that covers the sporicidal properties of gaseous ozone alone are very few but they exist. An article published in 2006 describes how ozone showed successful results in killing Bacillus subtilis spores. The treatment was made using a very high concentration of ozone (1500 ppm) which yielded a 99.9 % reduction of the spores after a 4-hour exposure. In this study, the spores were dried onto a glass surface prior to ozone treatment. [16]

2.2 The ozone cabinet

The ozone cabinet that will be used in this project is the Elozo D800 Ozone Cleaning System, see Figure 1. The cabinet has a volume of 758 L and is made from stainless steel components. It contains an ozone generator, three fans (that circulate the ozone air inside the cabinet) and supportive electronics. [17] Its specifications can be read in the table below.

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Table 1: Table of specifications for the ozone cabinet [17]

Specifications

Approvals Electricity safety IEC-60335-1 EMC-tests EN 55022

EN 610004-2,-3,-4,-5-6 ja -11 CE-certified

Name Elozo D800 Ozone Cleaning System

Voltage Uin 220-240 VAc 1

Imax 5.3 A

Power consumption Pmax 0.35 kW/h

Ozone production 100 % power 1500 mg/h

Safety Door safety / auto stop mechanism Electromagnetic lock mechanism

Connection to a ventilation system required

Elozo Oy recommend that the cabinet is used in a clean and dry environment where

the temerature is 15-25 °C and the relative humidity is lower than 50 %. [18] Both temperature and humidity might have an effect on the final result since they both can affect bacterial growth. [19] The temperature and humidity inside the cabinet cannot be changed from the outside, but they will both be measured and used as a potential error source if the values are not within the limits set by the manufacturer.

2.2.1 Settings

The cabinet offers different settings for the treatment duration in four steps; 20, 40, 60 and 120 minutes. The cabinet also admits different settings for ozone concentration, named step 1, 2, 3 and 4. All settings can be set at the cabinet’s instrument panel which can be seen in Figure 4. The power settings correspond to maximum concentrations according to Table 2. The concentrations were measured by Elozo Oy during the 60 minute program with an industrial ozone analyzer (Teledyne model 465L). The values are approximate and are affected by the external suction and how many items are being placed in the cabinet. It is also affected by how many microorganisms exist on the surfaces of the objects. Upon request, Elozo Oy made further measurements of the shorter programs to investigate whether the ozone concentration would reach the same levels as the one hour program. The results show that the 20-minute program yields maximum ozone levels 18-35 % lower than the one hour program. The maximum ozone concentration from the 20-minute program can be visualized in the middle column in Table 2.

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Table 2: Maximum concentrations of the 20-minute and one hour program, respectively

Power setting Conc at 20-minute program Conc at 1-hour program

1 24 ppm 37 ppm

2 32 ppm 48 ppm

3 42 ppm 54 ppm

4 46 ppm 56 ppm

Figure 4: The instrument panel

The Elozo ozone cabinet produces ozone without considering the current ozone concentra-tion inside the cabinet. The cabinet takes in surrounding air and produces ozone pulses every 10 minutes. This can be seen in Figure 5.

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Figure 5: Measurement of the ozone concentration in the cabinet during the one hour program

2.2.2 Previous bacteria tests in the ozone cabinet

The Finnish company SeiLab Oy have done some testing on one type of bacterium and mold in the ozone cabinet to investigate how well they survive in the cabinet. The tests were done in collaboration with Elozo Oy, and was made with the D-series ozone cabinets on hospital textiles. The organisms tested were Listeria monocytogenes (causes stomach flu, infections, and meningitis) which can be discovered in foods and animals and the second was mold (causes asthma and respiratory infections) which can be found in moist interiors and old structures. The results from the experiments were that a treatment period of 58 minutes for Listeria monocytogenes and 2 hours for mold resulted in a 100 % elimination of the bacteria or fungi (mold). [20] The concentration settings were not mentioned in the report.

2.3 Bacteria

Bacteria exist everywhere and their main function is to break down organic substances. The bacterial cell contains a single-stranded DNA molecule which flows around in the bacterial cytoplasm. The cytoplasm consists of water, protein, fat, and carbohydrates. Along with these substances, ribosomes are also present in the cytoplasm to control the replication of the cell. The bacterial cell membrane consists of phospholipids and pro-teins but their appearance differ depending on if it is a gram-positive or gram-negative bacterium. Gram-positive cells have one cell membrane while gram-negative cells have a double membrane. The bacteria also have a cell wall to help it stay together and improve the strength of the cell. The appearance of bacteria can be very different as they can, for example, take the shape of a globe, a rod or be spring shaped. [21]

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When an infection has occurred, the infectious subject has broken through the protective tissue (our skin) somewhere on our body. This could happen through a wound, by eating or drinking something, by inhalation, or through mucous membranes. [21]

2.3.1 Spores

When bacteria live under environmental conditions that are not beneficial (too warm surroundings, in the presence of chemicals or lack of nutrition), some bacteria species might transform into spores as a way to survive. The DNA in the cell encapsulates itself and becomes much more resilient to environmental stresses. When this happens, the cytoplasm, cell membrane, and cell wall dry up together and create a waterproof and heat resistant shell around the DNA strand. The bacterial cell can survive as a spore for a long time (up to several years) until the spore is exposed to a more beneficial environment. Then it has the ability to go back to its normal cell structure. The spore structure makes bacteria much more resistant to disinfection. To obtain a trustworthy disinfection it is therefore very beneficial to use a method that is able to kill spores as well as vital bacteria. [21]

2.4 Colony forming units

Bacteria are present everywhere and are measured in colony forming units (CFUs) since they form colonies when inoculated onto different agar plates. Everybody has CFUs all over their bodies but a person with an infection or some kind of skin disease has a much higher CFU quota. These persons are therefore more likely to transmit bacteria to others, which might cause a spread of infection. [22]

The colony forming unit is a measure of bacterial colonies that are present in a sam-ple. CFUs are measured in CFU/mL (or CFU/g for solid samples or CFU/area for surface samples) and they reveal the amount of viable bacterial cells in a sample. [23] After taking a sample, the diluted specimen is placed evenly on the surface of an agar plate before being incubated for a certain time. After this, the bacterial colonies can be counted with the naked eye. By doing this before and after the objects are placed in the ozone cabinet, it is possible to measure a change in CFUs to see if the method is effective. Different bacteria have different life spans and according to Sepideh Kamrani, biomedical analyst at Region Östergötland, the bacteria used in this project have life spans varying between one day to several months. It could be of great value to determine the bacterial species that will be grown in this project to see how long they would survive without the ozone treatment. However, this is something that will not be done in this project due to limited time and resources.

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2.5 Agar

To grow bacteria, the sample taken from the test subject is transferred to a growth medium which is left for incubation in a heated cabinet for one to two days. This will provide the medium with optimal conditions for maximal bacterial growth. Different bacteria prefer different mediums (that provide specific nutrients) which means that it is possible to control which species are grown. [21] In this project, three different agar plates will be used as a growth medium for the bacteria. The reason for these specific ones is that they provide growth platforms for the most common bacteria that are being spread through indirect contact within healthcare environments. The three agar types will be presented below along with the bacteria which can be characterized using them. These three different types of agar plates provide growth mediums for both gram positive and gram negative bacteria, which is beneficial for this study since it is possible that they react differently to ozone.

2.5.1 Hematin agar

Hematin agar is a non-specific growth medium which is used to isolate fastidious bacteria. The hematin agar is a brown agar with a heated blood additive. [21] The bacteria that can be cultivated on the hematin agar are Neisseria gonorrhoeae, Neisseria meningitidis,

Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae. [24] They

can cause diseases such as gonorrhea, meningitis, pneumonia, and tonsillitis. [21] Each of the bacteria can be characterized by studying the shape, color, and size of the colonies.

2.5.2 UTI agar

Urinary Tract Infection (UTI) agar is used to identify the main microorganisms that cause urinary tract infections. The prepared UTI agar is a white opaque gel placed in Petri dishes. The bacteria which can be observed and characterized using this agar are Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas

aerugi-nosa, Proteus mirabilis, and Staphylococcus aureus. [25] The different bacteria can all be

individually identified according to their appearance (color, size and shape of colonies).

2.5.3 Blood agar

Blood agar is an agar with blood additive which most bacteria can grow on. [21] The bacteria species that grow on blood agar are Streptococcus pneumonia, Streptococcus

pyogenes, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. [26] These

bacteria species can, for example, cause pneumonia, tonsillitis, scarlet fever, impetigo, sepsis, severe wound infections and muscle infections.

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2.6 Spore specimens

Spore specimens are often used as a control or reference for sterilization processes. A biological carrier (BI) is used and loaded with a known concentration of spores. In this project and in many other, Geobacillus stearothermophilus is used. The reason behind this choice is that this bacterium is highly resistant to environmental stresses and provides a good test that does not depend on what material it grows on. The BIs are loaded with a large number of microbial populations compared to the amounts which are present on items in hospital environments, which means that if the disinfection method is capable of killing the spores, it is a good disinfection method in general. [27]

In this project, Geobacillus stearothermophilus spores are inoculated onto stainless steel discs (BIs) which are placed in permeable Tyvek envelopes. The spores are inoculated on the inside curves of the discs prior to placement in the Tyvek envelopes. The material of the envelopes allows a flow of gas to pass through its walls, which will make the ozone reach the disc when it is placed in the ozone cabinet. [28] The stainless steel capsules can be seen from two angles in Figure 6, and the Tyvek envelope (which contains a disc) can be seen in Figure 7.

Figure 6: Stainless steel disc

Figure 7: A spore specimen in the Tyvek envelope [28]

2.7 Dry disinfection methods

The most common way of sterilizing hospital equipment is by steam sterilization with an autoclave. The technique rapidly kills 100 % of all bacteria and spores and is

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inexpen-subjected to this form of sterilization must be able to withstand heat and moisture. [29] One of the advantages of working with ozone is that the objects do not have to be resistant to moisture and heat. The medical equipment that is sensitive to heat and moisture can be hard to disinfect by these procedures and because of this, some of the most commonly used dry disinfection methods will be presented in this section.

2.7.1 Hydrogen peroxide

Hydrogen peroxide, H2O2, is a compound of great interest since its byproducts are

com-pletely harmless (water and oxygen). Hydrogen peroxide has the ability to produce hydroxyl radicals, OH, which react with lipids, DNA, and proteins. [30] Studies have also shown that the hydroxyl radicals carry strong oxidative properties which can degrade nucleic acids, enzymes, and cell membrane components. [31] This means that there are several ways that the gas can cause injuries to bacterial cells which might kill them, and thereby clean or disinfect a contaminated object.

Hydrogen peroxide has advantages including its byproducts being non-hazardous, a rel-atively short disinfection period (items can be sterilized in 80 minutes or less) and is not temperature dependent. Another benefit of using the gas is that the treated objects do not have to be ventilated after the process. [32] Hydrogen peroxide is a gas of great inter-est due to its favorable properties and there are constantly new studies being published in this field. For example, there is research carried out by Vårdhygien, Region Östegötland that examines how an entire room can be disinfected by a single device that exudes the gas. However, there are also disadvantages of employing the gas, including that it has shown to not possess the same antimicrobial effects when it comes to textile materials which are recommended to be disinfected in other ways. [33] The gas is also flammable, can cause eye damage and should not be inhaled due to its toxicity, which could make hydrogen peroxide a gas difficult to handle. [34]

2.7.2 UV light

Ultraviolet radiation (UVR) mainly induces two different photoproducts (cyclobutane pyrimidine dimers and 6–4 photoproducts) when it comes in contact with a DNA helix. The cyclobutane pyrimidine dimer is a result of UVR induced bindings between two adjacent thymine or cytosine nucleobases. In the case of thymine, the thymine molecule includes an aromatic heterocyclic compound that double binds to its neighboring thymine through bonds between the two carbon atoms at the fourth and fifth position, respectively (see Figure 8). The same process applies to two adjacent cytosine molecules. [35]

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Figure 8: Cyclobutane pyrimidine dimer [35]

The 6-4 photoproducts (Figure 9) are a result of UV radiation that induces a binding between the sixth carbon atom in the thymine aromatic heterocyclic compound and the fourth carbon atom in an adjacent cytosine molecule. The same process can take place between two adjacent cytosine molecules. [35]

Figure 9: 6-4 photoproduct [35]

The formation of these products will break the bindings between the base pairs in the helix and instead form bonds to other nucleotides on the same strand, see Figure 10. If the damage is not repaired, the new bindings might disrupt DNA transcription and replication processes. This can lead to errors in the reading of genetic code and cause mutations and death. [36, 37]

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Figure 10: UVR causing a bond between same stranded base pairs [38]

UV light sources have been used for decontamination purposes within healthcare envi-ronments for the last 60 years but have become of great interest especially during the present time due to the excessive use of mobile phones and tablets in today’s hospital care. [39] Since these devices are not originally made for use in this environment, they are not suited for being disinfected with the liquid disinfectants that are most commonly used. The usage of UVR has been shown to be a good alternative for disinfecting plane surfaces (glass or PPE), such as for smartphones and tablets. [40] New research in this field is published continuously and it is of great interest to hospitals to prevent infection spreading by broadening the spectrum of materials that can be disinfected with UV light in a convenient manner. [41]

Since UV light can split O2 molecules to create single O atoms and thereby create ozone

(for example in the atmosphere), this should be something that also happens when UVR is used as a disinfectant if there is normal air surrounding the test environment. However, no scientific articles have been published to cover this matter. Nonetheless, there have been many articles describing the effect of UVR in combination with ozone when the methods are used simultaneously to increase the antimicrobial effect of the treatment. [42, 43]

2.7.3 Ethylene oxide

Ethylene oxide has a strong alkylation reaction (reaction of giving away an alkyl group) with nucleic acids and functional proteins existing in cells. By exposing contaminated equipment to this gas, the alkylation reaction disrupts the normal cell activity of the bac-terial cells in the equipment and can thereby disinfect the object since the denaturation could lead to cell death. [44]

Ethylene oxide is a gas with bactericidal and fungicidal properties and is effective against most materials. [45] The main advantage of using ethylene oxide as a disinfectant is

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that it can be used on materials that are sensitive to both heat and moisture without destroying the material. [46] However, when exposed to humans, the gas causes irritation in the eyes, throat, and skin and also leads to feelings of nausea and vomiting. A chronic exposure can lead to the development of leukemia, cancer of the pancreas, stomach can-cer, and non-Hodgkin lymphoma. [45] In addition to the health hazards connected to this method, the process of disinfecting materials with ethylene oxide is expensive and time-consuming (one treatment can take up to six hours). Ethylene oxide is also a gas that is known to be flammable, a serious risk to be considered when using this gas. Due to the fact that many materials absorb the gas, there is a need for the products to be left in air for some time after the process to remove superfluous ethylene oxide molecules. [32, 46]

2.7.4 Gamma radiation

Gamma rays are high energy photons which bombard the object that is going to be dis-infected. This causes a displacement of electrons within the material of the object which creates free radicals that help in breaking chemical bonds. These radicals can break the bonds between the base pairs in the DNA helix of bacterial cells which will hinder reproduction and cause apoptosis. In this manner, gamma radiation can be used as a disinfectant since the rays break down DNA in bacterial cells that contaminate the object. Medical devices that are made of polymers are however not suited for this method since the crosslinking of polymers change when they are being exposed to gamma radiation. This can change the tensile strength, impact strength and elongation at break. Metals are normally not affected negatively by gamma radiation, but metals in contact with polymers have shown to corrode when being exposed to the process. Because of this, the composition of materials in the object needs to be considered before being irradiated. [47] The advantages of using gamma radiation is that the process does not leave any residues which need to be removed afterward. [47] It is also independent of external temperature and pressure and has a deep penetration depth. [32]

2.7.5 Formalin

Formaldehyde (CH2O) is a frequently used compound in disinfection procedures. The

chemical reacts with free amino groups in the nucleosides and forms different derivatives which breaks down the DNA strands. [48] The water-based solution of formaldehyde is called formalin and has been proven to be an efficient killer of bacteria, viruses, spores and tuberculosis bacteria. It is used in vaccine production, sterilization procedures of surgical instruments and disinfection of entire patient rooms. [49]

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ir-ritation. Ingestion of formaldehyde might be fatal, which means that the substance must be handled carefully and that human exposure should be limited in as great extent as possible. [49]

2.8 Degrees of cleanliness

There are different degrees of cleanliness that are defined by Vårdhandboken, which is a service provided by Sweden’s county councils and regions. There are three degrees of cleanliness for medical equipment; sterile medical equipment, highly clean medical equip-ment and clean medical equipequip-ment. The sterile medical equipequip-ment refers to the objects that penetrate the skin or are in contact with different body fluids. These objects have to be completely free from microorganisms to be classified as sterile, which they are if the probability of finding a microorganism on the object is less than one in a million. The highly clean medical equipment are the objects that come in contact with mucous membranes or damaged skin but do not penetrate it. Highly clean medical equipment is defined as in the case when the probability of finding a microorganism on the object is less than one in a thousand. The final degree of cleanliness, clean medical equipment, are objects that normally do not come in contact with damaged skin or mucous membranes. [50] There is no defined limit of the probability of finding a microorganism on objects in this category (clean medical equipment).

The objects that are going to be investigated in this project all belong to the last category, clean medical equipment. These objects are not supposed to be used on open wounds but might be used on patients that have infections. The main reason behind infections being spread in hospital environments is through physical contact. This can be both in direct contact with an infected patient or with an intermediate person or object. This could be through a third person, clothes or medical equipment. [1] Because of this, it is important to clean or disinfect the equipment when needed and maybe even make it a routine to clean/disinfect them periodically to prevent diseases from spreading between patients or from a patient to someone in the hospital staff.

2.9 Defining disinfection

There are no set values available that describe what classifies an object as disinfected. However, according to SS-EN ISO 15883-1:2009, which is a standard set for washer-disinfectors, disinfection is defined as "reduction of the number of viable microorganisms on a product to a level previously specified as appropriate for its intended further handling or use". There are no standards published that covers this for non-washer disinfectors, but by applying the same standard to the ozone cabinet it is possible to state that the level of disinfection depends on the type of equipment and its areas of use. Because of this, it is not possible to prove that the ozone cabinet can work as a disinfectant when

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it reaches a certain level of CFU reduction since the object and its areas of use have to be taken into consideration. This means, for example, that an object which belongs to the category of highly clean medical equipment (see Section 2.8) will be classified as disinfected when the probability of finding a microorganism on the object is less than one in a thousand.

Since the objects that are investigated in this project all belong to the category of clean medical equipment, there are no specific levels that classify the objects as disinfected. However, it is still of interest to examine how a high degree of disinfection can be reached for both current and future applications. According to Thomas Wilhelmsson, hygiene nurse at Landstinget Värmland, a disinfection method does not have to be sporicidal to be declared a disinfection method. [51] This would mean that even though the ozone cabinet might not be able to kill bacterial spores, it could still be a good disinfection method if it would present a great reduction of living bacteria.

2.10 The test objects

This Section will contain a description of all of the individual test objects, their areas of use and how they are currently being cleaned or disinfected.

2.10.1 Blood pressure cuff

A blood pressure cuff (BPC) is a device that is used to measure the blood pressure of patients for many various reasons. It is done frequently in both emergency rooms, health centers and routine visits to the doctor. The cuff is used to check the blood pressure of a patient by strapping the inflatable cuff around the upper arm while pumping in the air into the cuff to make it shut off the blood flow in the arm. When this happens, the pressure should be lowered slowly in order to measure the blood pressure. By using a stethoscope, the systolic (during a heartbeat) and diastolic (between heartbeats) pressure can be measured this way. A blood pressure measurement with either too high or too low values can be a first indication that a person is ill or that something in the body is incorrect. The blood pressure cuff used in this study can be seen in Figure 11.

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Figure 11: The blood pressure cuff

The BPC which is used in this project is produced by the company Boso and is of the model CA01. The blood pressure cuff is hard to clean today due to its arm wrapping component which is of a textile-like material. The strap is often made out of velcro which also is a material which is hard to clean with the conventional cleaning methods. The BPC is a device that only comes in contact with intact skin and is not normally cleaned or disinfected at all if there are no stains of blood or other body liquids visible on it. However, to prevent infection from spreading these kinds of devices should be cleaned or disinfected periodically even though the contamination is not noticeable with the naked eye.

2.10.2 Drug pump

The drug pumps are constructed to pump all kinds of drugs, suspensions, blood or nutri-tion to patients. In the cavity to the right (see Figure 12), a syringe with the requested drug is placed. In the settings of the pump’s software system, the doctor or nurse can specify the pump’s flow rate. The device then pumps the drug through a tube into the patient’s bloodstream. The pumps are used by one patient at a time but are transferred from patient to patient frequently (often daily). [52]

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Figure 12: The drug pump

The pumps used in this project are produced by Fresenius Kabi and the model is Agilia SP MC. According to Martin Berglund, medical technician at Motala Lasarett, the drug pumps are currently cleaned with Meliseptol Foam pure but due to their many joints, they could be hard to clean properly.

2.10.3 X-ray neck collar

The thyroid is a very sensitive organ to X-ray exposure and the need for X-ray collars (see Figure 13) is therefore high in clinics which uses radiation as a treatment method. [53]

Figure 13: The X-ray collar

The X-ray collar used in this project is produced by MAVIG, a company that produces X-ray Protection and Medical Suspension Systems. The collar is made out of a material called ComforTex HPMF (High Performance Medical Fabric), which according to Carola Larsson (Area Manager at MAVIG, Nordic and Baltic Countries) should withstand being disinfected with ozone. Larsson describes the material as a woven microfiber fabric with

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incorporated carbon fiber to make it non-electrostatic. The material is coated to prevent fluids and bacteria to penetrate the fabric and reach the inner material, which is a lead rubber and has an unknown reaction to ozone. The X-ray neck collar is only cleaned by the hospital staff in the case of it having an unpleasant smell. If this is the case, the neck collar is cleaned with a liquid disinfectant and a moist cloth.

2.10.4 Transportation bag

The transportation bag that is investigated in this project is not being disinfected or cleaned at all by the hospital staff. This bag, which can be seen in Figure 14, is used to transport CPAP devices that are used by patients in their home environment. There are many similar bags that are utilized to transport many other kinds of medical equipment for the same purpose. The bags are used over and over again and can be used by many different patients.

Figure 14: The transportation bag

The bag is made of a textile fabric and has zippers made for closing and opening. The bag is not suitable to be cleaned by the currently used methods and is thereby transferred from patient to patient without any cleaning procedure at all.

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3 Materials

Of the instruments used in this project, the primary one was the ozone cabinet (Elozo D800 Cleaning System). To measure the temperature and the humidity in the cabinet, a combined thermometer and hygrometer (Extech Instruments RHT10) was used. To measure the ozone concentration outside the cabinet, an ozone meter (Aeroqual 500) was employed. The device was equipped with a sensor head suited to measure ozone con-centrations between 0 and 0.15 ppm. All of these were provided by Motala Lasarett and

Clinicum Test and Innovation, Region Östergötland.

To take samples of the bacteria on the objects, Copan Liquid Amies Elution Swabs (eSwabs) were used along with spore specimens (Apex Biological Indicators), originally manufactured for testing with gaseous hydrogen peroxide. The eSwabs and the spore specimens were provided by Vårdhygien and Clinicum Test and Innovation. The inocu-lation was done at a hygiene lab located at Linköping University Hospital. The bacteria were inoculated onto three different agar plates; hematin, UTI, and blood agar.

The test objects, which all are presented in Section 2.10 were provided by Motala Lasarett and were temporarily borrowed from the hospital environment for testing.

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4 Method

This Section will describe how the specimen collection was performed, how the data was obtained and analyzed, and finally how the statistical analysis was performed.

4.1 Specimen collection

The specimen collection was performed by swabbing the objects with eSwabs both before and after the object had been treated with ozone. The eSwabs were transported to a lab for inoculation and bacterial growth to examine the amount of CFUs that were present before and after the cleaning process. The specimen collection process can be seen in Figure 15.

Figure 15: Specimen collection with eSwab [54]

When the first test was performed, all of the objects were marked with a permanent marker to create six equally sized spots. All the marked parts covered the same area and included equal amounts of complicated structures (zippers, seams, joints or cavities). The objects were touched by the author of this project and by some of the staff at the Medical Technology department to provide some extra contamination to the objects. The contamination was done carefully and all of the marked pieces on the objects were con-taminated in the same way. The swabbing was done in a standardized (to the greatest extent possible) way by applying the same (experienced) force to the swabs and moving them in the same way during the same time period. The first marked spot was swabbed with an eSwab and transported to a hygiene lab for inoculation, incubation and bacte-rial growth. This sample worked as a reference to know how much bacteria was present before the treatment. This was done for all objects before they were placed in the ozone cabinet. After 20 minutes (duration 1) the cabinet was opened and the second spot was swabbed and the sample was sent to the lab. This was repeated when the objects had been treated for 20, 40, 60, 120 and 240 minutes. There were also one or two spore spec-imens placed in the cabinet for each of the setting combinations. The spore specspec-imens were also transported to a lab for analysis.

Due to the results from the spore specimens (presented in Section 5.1), there was a need for another control test in this project; nose control samples. These samples were taken with eSwabs directly from a nostril and then placed in the cabinet together with the other objects. This supplied results that were not dependent on the different

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mate-rials and also gave significantly higher numbers of CFUs and thereby a more apparent reduction. Additionally, the nose control samples were shown to be advantageous when examining the standard deviation from these tests since these numbers were much smaller than for the tests done on the different objects.

Out of all the test objects it is only the drug pump that is currently cleaned period-ically when used in hospitals. The currently used cleaning procedure (spraying with Meliseptol Foam pure and wiped with paper) was tested and evaluated in the same way as the ozone treatment.

4.2 Obtaining data

The eSwabs and spore specimens were transported to a hygiene lab at Linköping

Uni-versity Hospital and were inoculated onto three different types of agar plates (hematin,

UTI, and blood agar). The agar plates were then incubated for 48 hours in 35-37 °C. After this time, the CFUs could be counted in all of the agar plates. By studying the number of CFUs both before and after the ozone treatment it was possible to draw some conclusions regarding the effect of the treatment. Figure 16 shows an example of the bacterial growth from two different samples (one taken before ozone treatment and one taken after ozone treatment).

Figure 16: Example of CFUs on an UTI agar plate where the left has been inoculated with a sample that has not been treated with ozone, and the right has been inoculated with an equivalent sample that has been treated with ozone

To obtain data from the spore specimens, they were placed in a control liquid and stored in a heating cabinet for 40-48 hours at a temperature of 56 °C. The control liquid (pro-prietary formulation of soybean casein digest medium) is a pH indicator (bromocresol purple) which shifts into a yellow color if the sample is positive. [55] A positive sample means that the spores have not been completely eliminated and that there still are living spores in the sample. A positive result does not say anything about how many spores were killed, only that it was not 100 %. If the sample is negative (100 % elimination of spores), the liquid will retain its purple color.

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The whole process of transporting the specimens to reading out the results is further described in Appendix A. This approach was used at all times during this project.

4.3 Statistical analysis

There were several tests under the same conditions (same kind of object, same duration, and concentration of ozone). For these tests, there was an average value calculated for the number of CFUs before and after the treatment. The mean value, x, was calculated according to the following formula:

x= 1 n n X j=1 xj= x1+ x2+ . . . + xn n . (4.1)

Where n is the number of tests. The standard deviation could then be calculated for each of the measurements that contributed to the mean value calculations. The stan-dard deviation, s, was calculated before and after treatment according to the following formula: s= v u u t 1 n −1 n X j=1 (xj− x)2. (4.2)

These values were calculated using Python.

4.3.1 Antimicrobial efficiency

Since there is no official documentation regarding the definition of a disinfected object, as many of the settings as possible will be tested and each evaluated according to their antimicrobial efficiency. There is no percentage number that is considered to be a limit for successful disinfection which means that it will be hard to state whether the method is working or not. However, the antimicrobial efficiency will give a percentage number of the reduction of CFUs which the treatment produces. The bacteria specimens from the different objects will be inoculated onto three different types of agar plates (hematin agar, UTI agar, and blood agar) and the number of CFUs will be counted on each of them (both before and after the ozone treatment). To investigate if the cabinet can work as a disinfectant or not, the following equation will be used:

Antimicrobial efficiency=(x0− x1) + (y0− y1) + (z0− z1)

x0+ y0+ z0

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Where x0 and x1 are the number of CFUs on the hematin agar before and after the

ozone treatment, y0 and y1are the number of CFUs on the UTI agar before and after the

treatment and z0 and z1 are the number of CFUs on the blood agar before and after the

treatment. This equation gives the antimicrobial efficiency of the cabinet (the reduction of CFUs) as a percentage value. This equation was used for all different objects and all different duration and ozone concentration settings to find the most optimal settings for all objects.

The tests were run as many times as possible to gain statistical validity to the results. For tests that were done at least three times, equation 4.1 and 4.2 could be used to calculate the mean reduction of CFUs along with the standard deviation between the different runs.

4.3.2 ANOVA

There were two different two-way ANOVA (analysis of variance) tests performed. The first, ANOVA for different objects, explains if the ozone cabinet works differently on different objects or not. The second, ANOVA for different bacteria, explains if the cabinet works differently on different bacteria. The theory behind the ANOVA tests can be read in Appendix B. The conditions for the tests are also mentioned there. The software used to analyze this was SPSS and the level of significance was set to α = 0.05.

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5 Results

The results from the spore specimens, the statistical analysis, the CFU quantification and the antimicrobial efficiency will be presented in this Section.

5.1 Spore specimens

A total of 19 spore specimens that were placed in the ozone cabinet at the highest concentration all showed positive results (not eliminating 100 % of the spores). The specimens were left in the cabinet for 20 to 240 minutes.

5.2 ANOVA

This Section provides the results from the ANOVA tests, done for both different agar and different objects. The effects of interest are "Agar" and "Object" and the results show that there is no significant difference in how the ozone cabinet works for the different objects or the different agar. The ANOVA tests were done for all concentration and duration settings with data from Appendix C.

Table 3: Results from the ANOVA test for different agar (concentration 4)

Feature Settings Effect F(2,66) p

CFUs C4D8 Time 5.278 0.024

Agar 0.321 0.726

Time*Agar 0.320 0.727

CFUs C4D4 Time 5.035 0.028 Agar 0.326 0.722 Time*Agar 0.313 0.733 CFUs C4D3 Time 7.570 0.007 Agar 0.336 0.716 Time*Agar 0.329 0.720 CFUs C4D2 Time 7.748 0.007 Agar 0.343 0.710 Time*Agar 0.323 0.725

CFUs C4D1 Time 4.658 0.034

Agar 0.315 0.731

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Table 4: Results from the ANOVA test from different objects (concentration 4)

CFUs C4D8 Time 5.202 0.037 Object 1.390 0.282 Time*Object 1.384 0.248 CFUs C4D4 Time 5.665 0.029 Object 1.616 0.221 Time*Object 1.580 0.229 CFUs C4D3 Time 5.245 0.034 Object 2.081 0.139 Time*Object 2.183 0.125 CFUs C4D2 Time 5.417 0.032 Object 2.111 0.134 Time*Object 2.165 0.128 CFUs C4D1 Time 0.742 0.399 Object 3.071 0.051 Time*Object 0.049 0.985

CFUs C3D8 Time 5.876 0.018 Agar 0.042 0.959 Time*Agar 0.039 0.962 CFUs C3D4 Time 5.580 0.021 Agar 0.041 0.960 Time*Agar 0.040 0.961 CFUs C3D3 Time 4.193 0.045 Agar 0.033 0.967 Time*Agar 0.046 0.955 CFUs C3D2 Time 2.901 0.093 Agar 0.068 0.934 Time*Agar 0.015 0.985 CFUs C3D1 Time 2.784 0.100 Agar 0.027 0.973 Time*Agar 0.053 0.949

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5.3 Mean and standard deviation

Since there is no significant difference in the ozone cabinet’s effect on the different objects or different bacteria, the mean and standard deviation of CFUs were calculated with data from all objects and all agar together. The results (plotted in Figure 17-20) are based on data from Appendix D. The mean and standard deviation are presented in four separate bar plots that describe each of the four concentration settings. The plot to the left shows the average number of CFUs before and after ozone treatment for all objects. The plot to the right shows the average number of CFUs before and after ozone treatment on the nose control samples. The error bars show the standard deviation. The x-axis describes the five different durations (20, 40, 60, 120 and 240 minutes) and the y-axis shows the number of CFUs. Note the different scales on the y-axes. The plots describing all objects are based on data from at least 12 runs and the plot describing the nose control samples are based on data from 3 runs.

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Figure 17: Average number of CFUs before and after ozone (concentration 4)

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5.4 Antimicrobial efficiency

The antimicrobial efficacy of the ozone cabinet was calculated according to equation 4.3. The average reduction of CFUs for each of the objects will first be presented separately in Figure 21-24. Further down in this section is a presentation of the average reduction of CFUs on all objects (not separated) in Figure 25. Below this, the result from the nose control samples can be visualized in Figure 26. The y-axis describes the reduction of CFUs as a percentage value and the x-axis shows the treatment duration in minutes. The error bars show the standard deviation. The plots describing all objects are based on data from at least 12 runs and the plot describing the individual objects and the nose control samples are based on data from 3 or 4 runs.

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Figure 22: Mean reduction of CFUs on the drug pump

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Figure 24: Mean reduction of CFUs on the transportation bag

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Figure 26: Mean reduction of CFUs on the nose control samples

5.5 Disinfection with Meliseptol

The drug pump was contaminated and disinfected with Meliseptol pure foam spray three times. The mean and standard deviation of the number of CFUs before and after treat-ment can be seen in Figure 27.

Figure 27: Average number of CFUs before and after Meliseptol spray

The mean average reduction of CFUs was 97.8 ± 3.0 %. The pump was swabbed im-mediately after being disinfected with the Meliseptol spray, and the treatment time was therefore set to one minute (which is the approximate time it takes to wipe the pump). The average reduction of CFUs with this method was incorporated in Figure 22 in or-der to be visually compared to the ozone disinfection. The result can be seen in Figure

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28, where the reduction of CFUs due to the Meliseptol spray treatment is represented through the black line.

Figure 28: Mean reduction of CFUs on the drug pump when using ozone and Meliseptol pure foam separately

5.6 Variation

The contamination was done manually and presented large variations in the number of CFUs that were counted between the different runs. To prove this difference, the number of CFUs on the blood agar plates for all C4D8 (concentration 4 and duration 8) runs will be presented in Table 11 below.

Table 11: CFUs on the blood agar, data from C4D8 runs

Object Run Before ozone After ozone BPC 1 6 0 2 41 0 3 88 0 Pump 1 19 0 2 58 2 3 17 1 NC 1 11 0 2 384 4 3 24 0 Bag 1 1 0 2 248 4 3 344 5

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5.7 Visual effects

The effect that the ozone had on the different objects has not been evaluated in this study, however, there were clear visual effects on two of the objects. The ozone had a negative effect (which could easily be seen) on the blood pressure cuff and the X-ray neck collar. Figure 29 shows the blood pressure cuff after all the ozone treatments. The metal ring that works to tighten the cuff has become rusty from the ozone.

Figure 29: The blood pressure cuff after all ozone treatments

The X-ray neck collar was also affected by the ozone. The lead rubber inside the collar has crumbled apart as a result from being treated with ozone. This can be seen in Figure 30, where the outer material was cut open to visualize eventual effects.

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6 Discussion

This Section includes a discussion regarding the results, the methods and sources of error and ends with suggestions for future work.

6.1 Results

The discussion about the results has been divided into seven parts covering the same headlines that were included in the Results Section.

6.1.1 Spore specimens

The ozone cabinet was proven not to have sporicidal properties due to its inability to kill the spore specimens even with the toughest treatment. According to previous stud-ies (presented in Section 2.1.3), ozone can be sporicidal at higher concentrations which indicates that the ozone cabinet does not provide high enough concentrations of ozone to kill spores. The study mentioned in Section 2.1.3, which proved a 99.9 % reduction of spores, used an ozone concentration of 1500 ppm for 4 hours. This is much higher than the maximum ozone concentration in the Elozo cabinet (56 ppm). When comparing these two, it is reasonable to conclude that the cabinet does not provide sufficient ozone levels to be sporicidal. However, the spore specimens used in this study only gave a negative result (proving a treatment to be sporicidal) if all spores in the specimen were killed. This means that it is not possible to know if the cabinet killed many spores but not all, or if the cabinet did not kill a single one.

However, according to Section 2.9, a disinfection method does not have to be sporici-dal in order to be accepted. Hence the ozone cabinet could still be used as a disinfector if it is able to reduce the number of living bacteria to a satisfying level.

6.1.2 ANOVA

The results from the ANOVA tests (Table 3-10) show that there is no significant differ-ence in how the ozone cabinet works for the different objects or the different agar. This was tested for all concentration and duration settings and is thereby valid for all possible settings of the cabinet. However, this does not prove if the materials are suited for ozone treatment since the effect on the materials themselves have not been examined. Also, this does not state anything regarding how the cabinet affects other materials that were not tested in this project. Since there was no significant difference in how the cabinet works for different agar (i.e. different bacteria), this means that the cabinet is efficient against both gram positive and gram negative bacteria, which is very advantageously for this method.