WO2011104567A1

WO2011104567A1 - Apparatus and method for detection of ammonia in exhaled air

Info

Publication number: WO2011104567A1
Application number: PCT/GB2011/050385
Authority: WO
Inventors: Trevor Smith; Ron Logan-Sinclair; Deborah Norris
Original assignee: Bedfont Scientific Limited
Priority date: 2010-02-25
Filing date: 2011-02-25
Publication date: 2011-09-01
Also published as: GB201003190D0

Abstract

Apparatus for measuring the concentration of ammonia in exhaled air comprising a sampling device and a sensor device, the sensor device comprising a nitric oxide sensor, characterised in that the apparatus further comprises a heating device configured to heat the exhaled air to affect conversion of ammonia in the exhaled air to nitric oxide prior to reaching the sensor device. Methods of using the apparatus in diagnosis are also provided.

Description

APPARATUS AND METHOD FOR DETECTION OF AMMONIA IN EXHALED AIR

The present invention relates to an apparatus and method for measuring the concentration of ammonia in exhaled air. It also relates to the use of such apparatus in the detection of gastrointestinal diseases.

Ammonia given off in exhaled air can be used as an indicator of gastrointestinal diseases, in particular Helicobacter pylori (H. pylori) infection. H. pylori is one of the most common bacterial pathogens in humans and is now recognised as a worldwide problem. It causes chronic gastritis, peptic ulcer disease, and lymphoproliferative disorders and is a major risk factor for gastric cancer.

Traditionally, once a patient begins to show the symptoms of H. pylori, they are routinely given an endoscopy to rule out certain diseases and diagnose certain conditions. The typical cost of endoscopy varies from as low as £120 to as high as £400. Recent figures suggest that the number of endoscopies carried out in the UK is about 280,000 per year; therefore the total cost of endoscopy to the NHS is possibly in the region of £40-70 million a year. Recent evidence has suggested that screening patients for H. pylori can result in a reduction of 20-25% in the number of endoscopies performed. The systematic use of H. pylori testing and doing fewer endoscopies as a result would be likely to save £10-15 million each year, even including the cost of the testing. These savings are equivalent to £600 per practice or £200 per GP per year.

The ability to be able to diagnose gastrointestinal diseases during triage, prior to an endoscopy, can lead to a significant reduction in the number of endoscopies performed, leading to significant savings for health authorities. However, no previous apparatus exists which provide real- time results and so none of the prior art apparatus are suitable for use in triage prior to an endoscopy.

At the present time there are a number of different tests that are available to test for the presence of the H. pylori infection, although they do not provide instant results and can be invasive. These tests are also expensive to perform and involve sending samples to a laboratory to be tested which is time consuming and expensive.

One such test is based on providing four simple breath test samples, two baseline samples followed by two indicator samples, where the patient ingests a tablet containing ¹³C urea then blows into a test-tube. The tubes are then sent off to a laboratory where they are analysed and the results sent to a doctor. This is time-consuming for the patient and also expensive to the hospital to purchase the testing kit.

The present invention is concerned with providing apparatus for measuring the concentration of ammonia in exhaled air which will provide rapid results and is more economical per unit test than those currently available. The provision of such apparatus would make testing more accessible to the population and further, would aid earlier detection of gastrointestinal diseases such as H. pylori which will of course lead to a reduction in patients going on to have peptic ulcers and/or gastric cancer.

According to one aspect of the present invention there is provided apparatus for measuring the concentration of ammonia in exhaled air comprising a sampling device and a sensor device, the sensor device comprising a nitric oxide sensor, characterised in that the apparatus further comprises a heating device configured to heat the exhaled air to affect conversion of ammonia in the exhaled air to nitric oxide prior to reaching the sensor device.

Preferably, the heating device comprises a heating element configured to heat the exhaled air sample to a temperature of 500 to 1000°C, preferably 500 to 800°C, especially to a temperature not exceeding 650°C, in the presence of oxygen. The oxygen present in the heating device may originate from the exhaled air, in which case the oxygen is at a level of greater than 15wt% of the exhaled air, preferably from 15 to 17wt%.

Ammonia present in the exhaled air preferably undergoes thermal oxidation upon heating, as follows:

4 NH₃ + 5 O₂ → 4 NO + 6 H₂O

A catalyst, such as platinum, platinum oxide, ruthenium oxide, rhodium oxide, palladium oxide, iridium oxide, cobalt oxide, vitreous carbon and copper oxide, may be present in the heating device which can assist to maintain high efficiency of the conversion of ammonia to nitric oxide, e.g. 90% or higher.

The heating element may be provided with in association with an oxidation chamber, e.g. a chamber defined by a length of tubing, through which the exhaled air sample passes. Other suitable forms of chambers and relationships with the associated heating element can readily be envisaged (e.g. a honeycomb structure associated with the heating element).

In a preferred embodiment, the heating element provides a core around which the tubing is wound. The tubing may be formed of any material which conducts heat from the heating element to the exhaled air sample. In a preferred embodiment, the tubing is formed of stainless steel.

Suitably it may have a length of between 0.5 and 2.5 metres, more preferably between 1 and 2 metres long, and have a diameter of from 1 to 10 mm, more preferably from 2 to 4 mm. Tubing having a length of 1 .5 m and a diameter of 3.2 mm has been found to be highly suitable.

In general the heating device must be adapted to allow thermal oxidation of ammonia to a sufficient degree to allow analysis of the resultant NO levels. 100% oxidation of ammonia is not required, but oxidation of 90% or greater is generally desirable. It is typically within the ability of the skilled person to provide a suitable heating device, and the efficacy of such a device can be tested in accordance with the procedures described below. The device should be adapted to allow the sample of gas to be oxidised to reach a high enough temperature for oxidation to occur, and retain the sample at or above this temperature for a suitable time. The presence of catalysts or the like may reduce the temperature required or the amount of time required.

By providing a suitable oxidation chamber which is heated to the required temperature it is straightforward for the person skilled in the art to provide suitable conditions. In general an oxidation chamber which has a relatively high surface area to volume ratio allows for effective heating of the sample. This can be achieved within many configurations of chamber, but a simple narrow tube wound around a heating element has been found to be both simple and surprisingly effective.

The temperature of the heating element (and in turn the oxidation chamber) may be controlled by a suitable thermostat, e.g. temperature control circuit with a thermocouple, which serves to monitor the temperature. Preferably, the temperature of the heating element is displayed at a user interface. It is preferred that the heating device comprises a high temperature cut-out device as a safety feature to reduce the risk of damage to other components of the device or to the

patient/user. Furthermore, the heating device may be provided with insulation to protect the user and the other components of the apparatus from exposure to excessive heat. Suitable forms of insulation are well- known in the art.

The heating device may be disposed between the sampling device and the nitric oxide sensor device to effect conversion of ammonia in the exhaled air sample to nitric oxide prior to the exhaled air entering the sensor device. Preferably the heating device further comprises one or more heat absorbers or cooling devices positioned at the inlet and/or outlet of the heating element to ensure that excess heat does not affect (for example, damage) other parts of the apparatus or becomes a safety hazard. In one embodiment, the heating device comprises one or more heat absorbers in the form of stainless steel tubing connected to one or more peltier coolers. In an alternative embodiment, the heating device comprises one or more cooling devices in the form of coiled tubing provided with a fan to cool the area in which the coiled tubing is disposed.

The heating device may further comprise a vacuum pressure pump. The pump is capable of pulling the exhaled air sample through the heating element for conversion of the ammonia present in the sample to nitric oxide for detection in the sensor. As the exhaled air sample is pulled through the heating element, a pressure drop develops and so the pump employed in the heating device must be capable of operating in these conditions. Furthermore, the pump is preferably dual-headed because the heating element requires a constant gas flow, to prevent build-up of excessive heat and to increase the consistency of the gas temperature. Suitable pumps include the D type pump and 31 12.120 pump available from the Boxer® pump range by Uno.

In a preferred embodiment of the present invention, the apparatus may comprise a bypass means by which the heating device can be bypassed. The provision of such a means allows the apparatus to be switched between measuring the level of nitric oxide in the air and measuring the ambient level of ammonia in the air by converting the ammonia present to nitric oxide, as described herein. Such a means can further provide a route for calibration of the sensor device with nitric oxide gas. The bypass means may take the form of an inlet allowing air directly into the cursor device or tube bypassing the heating device between the sampling device and the sensor device, which is selectively openable, e.g. via a valve.

As stated above, the sensor device comprises a nitric oxide (NO) sensor for detecting the level of nitric oxide within the heated exhaled air in order to measure the concentration of ammonia in the original exhaled air sample. The amount of NO in the sample will correspond to the amount of ammonia originally present. Preferably, the sensor device further comprises a pump configured to extract a portion of the oxidised exhaled air sample for detection by the sensor. In this embodiment, the extracted portion may be drawn through a solenoid valve to be brought into contact with the sensor, into which it diffuses.

Preferably the sensor is capable of sensitive measurement of

concentrations of NO from parts per billion (ppb) levels to parts per million levels (ppm). In a particularly preferred embodiment of the present invention, the sensor is capable of detecting levels of NO at around 0 to 20ppm. Typically the sensor is an electrochemical gas sensor. It is particularly advantageous to use a sensor that is temperature stable. This negates the need for heating or cooling of the sensor or heated exhaled air during use. Furthermore, it offers space saving opportunities in the apparatus design and simplicity of design and construction of the apparatus.

Preferably, the electrochemical sensor has a filter capable of removing acid gases, such as those formed as a by-product of thermal oxidation, where the ammonia reacts with too much oxygen, and any particulate objects remaining in the sample. The filter may also be capable of acting as a further moisture filter. Such filters are well known in the art.

The electrochemical sensor may have 5% resolution, in that 5% is the smallest change it can detect in the quantity of NO, and may provide an output which is linearly proportional to the NO concentration. The production of a linear output makes the use of an electrochemical sensor advantageous over prior art sensors as it negates the need to linearise the output before the measurement can be determined. Furthermore, such feature of the electrochemical sensor enables to apparatus of the present invention to be portable and to provide real time ammonia measurements.

The electrochemical device may have cross sensitivity to nitrogen (100% N₂ being detected up to a level of -0.05ppb), carbon dioxide (1 .12% CO2 being detected up to a level of 17.3ppb) and carbon monoxide (45ppm CO being detected up to a level of 17.6ppb). In a preferred embodiment of the present invention, the device, e.g. the electrochemical sensor, has an inbuilt CO filter to remove any CO present in the heated exhaled air sample. Typically, the electrochemical sensor has a 350mV bias and an operating pressure of 1 atm +/- 10%. The sensor may be suitable for continuous use at a temperature between 10°C and 30°C and at a relative humidity of 25% to 75%, and intermittent use at a temperature between 0°C and 35°C and at a relative humidity of 0% to 100%.

Preferably the heating device and sensor device assembly contain an independent power source, such as a battery, such that it is portable and can be used when disconnected from the power grid. Preferably the sensor device contains a back-up battery to ensure power to a memory means within the sensor device which contains software and/or to the gas sensor.

The sampling device may be detachable from the heating device and the sensor device. In this embodiment, the heating device and the sampling device each comprise corresponding interface means to allow the devices to be connected together. Advantageously the connection between the sampling device and the heating device is substantially airtight.

In a preferred embodiment of the invention, the connection comprises a plug and socket arrangement. It is preferred that the interface means on the heating device comprises a recess into which the interface means on the sampling device is inserted. Retaining means to hold the devices together, such as a clip, may be provided. However, the interface means are desirably formed such the friction between the two means is sufficient to hold the apparatus together for use. It is preferred that the interface means are shaped such that they can fit together only in one orientation; a D-shaped means is especially preferred. Preferably the exhaled air is air originating at the alveolar interface of the lungs, where ammonia will be present if the patient has a gastrointestinal disease such as H. pylori in the presence of stomach urea. However, the exhaled air may equally be oral exhaled air (often described as lower respiratory tract breath in the field) or nasally exhaled air. Other gases from the subject might also be analysed using the present device (e.g. gastric gases), but this typically is less preferable than measuring exhaled air as it is significantly less convenient.

The sampling device comprises a patient contact means. The patient contact means suitably comprises a mouthpiece, a facemask, a nasal breath sampling means or a combination of one or more of these. A mouthpiece is preferred as it is simple to use.

The sampling device typically comprises a conduit running between the patient contact means and the interface means.

Preferably the sampling device comprises a pressure regulation means for protecting the apparatus from excessive pressure build-up, which can occur due to the pressure of the exhaled air sample being too high, which would otherwise cause damage to the apparatus. Excessive pressure is defined as a pressure too high for the downstream components, i.e. the heating device and the sensor device, to safely operate under. The amount of pressure which would be considered excess is readily apparent to the person skilled in the art. In a preferred embodiment, a vacuum pump within the heating device draws a steady exhaled sample stream from the exhaled patient breath within the mouthpiece through the heating device. The excess exhaled breath needs to be exhausted into the atmosphere. A steady exhaled breath stream rate ensures even heating of the exhaled breath sample in the heating device. However, if the excess exhaled breath is not exhausted prior to the heating device, excessive pressure can build up in the sampling device.

In one embodiment, the pressure regulation means is a T-piece

incorporated in the sampling system which comprises an orifice of predetermined diameter, as will be appreciated by the skilled person, to provide a 'metered leak' of exhaled air out of the sampling device. In this way, the pressure of the exhaled breath sample within the sampling device is prevented from being too high. Alternatively, the T-piece may comprise a pressure dependent one-way value which cracks (i.e. opens) if the pressure within the sampling device exceeds a predetermined limit.

It is desirable that the sampling device comprises infection control means to prevent infectious particles from passing through the sampling device into the heating device and subsequently into the sensor device. Such infection control means may suitably comprise a filter which is able to remove particles such as small as viruses, bacteria and other potentially infective microbes or particles. Such filters are well known in the art. A preferred embodiment of the present invention, the infection control means may comprise a 50mm, 1 micron PTFE hydrophobic filter which protects against cross infection of the apparatus. The infection control means is suitably positioned in the conduit between the patient contact means and the interface means, more preferably, the infection control means is disposed at the interface means. Where the sampling device comprises such an infection control means, it is a significant advantage that the heating device and sensor device can be reused without the need for sterilisation between patients. The present invention thus provides that the two parts of the breath sample test apparatus are separable from one another, such that the sampling device can be removed and/or replaced. This is advantageous as the sampling device, which is the point of contact for the patient can be disposed of after use and replaced with a new clean/sterile sampling device. In this way, the sampling device may be a single use, disposable unit.

It is envisaged that the testing apparatus of the present invention will be shaped and sized such that it is suitable for portable operation. This advantageously allows the unit to be conveniently used in a variety of settings, either with or without a trip to a trained clinician being necessary. This makes the apparatus particularly useful for H. pylori testing as triage prior to an endoscopy procedure, as it does not require the presence of bulky table top equipment. Additionally, smaller apparatus for testing the concentration of ammonia provides general space saving benefits.

The apparatus of the present invention advantageously negates the need to have an additional gas storage chamber and a pumping device to ensure a controlled flow rate to the sensor. This further facilitates a small and simple testing apparatus and reduces the cost of such apparatus, making it attractive in all primary care settings and developing countries where H. pylori is more prevalent.

In a preferred embodiment, the sampling device comprises a flow regulator. The flow regulator ensures that exhaled air reaches the gas sensor in a controlled manner. Such a flow regulator is preferably disposed in the sampling device, adjacent to the interface means.

The flow regulator may take the form of a mechanical device to restrict or otherwise actively regulate the flow of exhaled air. Such mechanical devices are known in the art. However, a preferred flow regulator is a flow indicator which indicates to a patient the rate of flow and allows the patient to adjust the rate of exhalation accordingly. Conveniently the flow indicator comprises indication means to indicate to the patient that a desired flow rate or range of flow rates is being achieved. Such indication means may be visual or aural. Suitably the indicator means is a visual indicator, such as a scale with the desired flow rate indicated thereupon. The flow indicator preferably comprises a body which is adapted to interact with the flow of breath and have its position or orientation influenced according to the flow rate. Suitably the body is located within a conduit through which at least a portion of the flow of exhaled air will pass, and is moved within the conduit depending on the rate of flow. The conduit in which the body is located is suitably part of the conduit running from the patient contact means to the interface means. The conduit may suitably be arranged such that it is vertical during use and, as such, the body is pushed against the force of gravity by the flow of exhaled air; the height thus depending on the flow rate. Alternatively the body may move against the action of a resilient means, such as a spring for example.

Suitably the body is a ball or bead, preferably having a substantially spherical shape.

It will be obvious that the body must not completely obstruct the conduit such that air can flow around the body. Suitably the conduit comprises retaining means to ensure the body remains located within a desired region of the conduit. The retaining means may be one or more narrowings of the conduit to a dimension smaller than the diameter of the body. Suitably the conduit has a suitable profile such that as the body moves further along the conduit, air is able to flow more easily around the body; for example, a tapered profile.

Preferably the flow regulator is adapted to provide a flow rate of from 10 to 70 ml/s, especially from 45 to 55 ml/s.

Suitably the sensor device comprises an inlet to allow ambient air to be drawn into the sensor device. Preferably the sensor device further comprises a filter disposed at the inlet to filter the ambient air being drawn into the sensor device. As the sensor device is intended to detect levels of NO converted from ammonia in the exhaled air, the filter is suitably adapted to "scrub" the ambient air being drawn into the device, to remove any NO present. Suitable filters for removing NO include alumina impregnated with potassium permanganate KMnO₄ and/or carbon beads/charcoal material. The filter is also desirable configured to remove any particulates or other component parts of the ambient air which could adversely affect the operation and/or accuracy of the sensor device.

The provision of such an inlet with a filter to remove NO in the ambient air provides the sensor device with a convenient means of self calibration. This is because the NO-free ambient air drawn into the sensor via inlet (preferably by a pump) and filter passes over the sensor to calibrate it to "zero" to ensure a true measurement of the ammonia present in the exhaled air is achieved.

It is desirable that the sensor device and/or the heating device comprise one or more one-way valves to direct the path of the exhaled air.

Preferably, the one or more one-way valve is disposed at the interface between the heating device and the sensor device to direct ambient air drawn in through the inlet of the sensor device towards the sensor and prevent the patient from inhaling through the apparatus via the inlet. A further one-way valve may be provided at the inlet of the sensor device to prevent the heated exhaled air passing out of the inlet during an

exhalation phase. Such one-way valves are well known in the art and, in one embodiment, may comprise a simple flap and aperture arrangement.

The sensor device or the distal end of the heating device desirably comprises a moisture regulation means to remove water from the heated exhaled air, as water is present in the heated exhaled air sample from exhalation and as a by-product of thermal oxidation of ammonia to nitric oxide. Suitably this comprises one or more humidity filters. In a preferred embodiment, the humidity filter is located within the sensor device between the interface with the heating device and the NO sensor, to remove the water formed during thermal oxidation of the ammonia to produce NO. The moisture regulation means may comprise a further humidity filter positioned within the conduit of the sampling device, between the patient contact means and the interface means, to remove water from the exhaled air. With many sensor devices it is essential that the air does not contain high levels of water as it can interfere with the result and/or damage the sensor itself.

In a particularly preferred embodiment of the invention, it is beneficial that the one or more humidity filters reduce the humidity level of the exhaled air to a pre-determined level, rather than zero.

Preferably, the moisture regulation means comprises a length of Nafion® tubing, e.g. as disclosed in WO 2010/094967. If a second humidity filter is present in the conduit of the sampling device, the infection control means and the humidity filter may conveniently be provided by a multi-function filter.

The sampling device is suitably formed substantially from a plastics material. Conveniently the sampling device may be moulded. Suitably the plastics material is impregnated with an antimicrobial agent.

In one embodiment the sampling device may be designed to take a sample of nasal breath where the mouth of the patient is closed during sampling to substantially exclude orally exhaled air from the sample.

However, preferably the sampling device comprises a flow restriction means which provides sufficient resistance to exhalation such that the nasal vellum of the patient is closed during exhalation, and thus nasally exhaled air is substantially excluded from the tested breath.

The electronics and software required to control and operate a testing apparatus of the present invention are known in the art.

The apparatus of the present invention is preferably used for measuring levels of gaseous ammonia in an oral breath sample. However, the application is not restrictive to an oral breath sample and so may include a nasal breath sample, or other gas forms from a subject.

In a preferred embodiment of the present invention, the apparatus comprises a means by which the user can choose between a number of different modes of operation such as oral breath sample mode, ambient sample mode, nasal breath sample mode and calibration mode. According to a further aspect of the present invention, there is provided a method for measuring the concentration of ammonia in exhaled air using the apparatus set out above, the method comprising the steps of;

- obtaining a sample of exhaled air in the sampling device;

- heating the sample of exhaled air in the heating device to

convert ammonia present in the sample to nitric oxide; and

- detecting the amount of nitric oxide present in the heated air sample in the sensor device to provide a measurement of the concentration of ammonia in the exhaled air sample.

Preferably, the sample of exhaled air is heated 500 to 1000°C, more preferably 500 to 800°C, especially to approximately, but not exceeding 650°C, in the presence of oxygen, in the heating device to bring about thermal oxidation of ammonia to produce nitric oxide.

Suitably, the method comprises the step of extracting a portion of the heated sample air for analysis within the sensor device of the apparatus. This step may involve the use of a pump, optionally contained in the sensor device, to affect extraction.

The method suitably includes the step of providing to the user an indication of the rate of exhalation so that they can alter the exhalation rate such that it falls within a desired range. This may conveniently be achieved using a floating ball or similar arrangement described above. Suitably the rate of exhalation is as defined above for a time sufficient for sampling to occur.

Preferably, the method further comprises the step of drawing a nitric oxide free sample of air and/or ambient air into the sensor device to calibrate the nitric oxide sensor. The method may comprise the step of comparing the result of the method with an expected value. From this a diagnostic or prognostic indication may be derived, e.g. the diagnosis of H. pylori infection.

The method may comprise the step of disposing of the sampling device of the apparatus after use. A new sampling device can be connected to the sensor device prior to another sample being analysed.

According to a third aspect of the present invention there is provided the use of the apparatus of described above in the diagnosis of a

gastrointestinal disease, in particular where the gastrointestinal disease is caused by Helicobacter pylori infection, e.g. gastro-intestinal inflammation, stomach ulcers or stomach cancer.

Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying figures in which:

Figure 1 is a simple block diagram of the heating device and sensor device of the apparatus in accordance with a first embodiment;

Figure 2 is a more detailed block diagram of the apparatus illustrated in Figure 1 ;

Figures 3a and 3b show graphical representations of the calculated results versus the measured results for 1 ppm ammonia diluted with synthetic air at 650°C;

Figures 4a and 4b show graphical representations of the calculated results versus the measured results for 5ppm ammonia diluted with synthetic air at 650°C; Figures 5a and 5b show graphical representations of the calculated results versus the measured results for 19.7ppm ammonia diluted with synthetic air at 650°C;

Figure 6 shows a graphical representation of the results of the

temperature testing with an alternative heating device;

Figure 7 shows a graphical representation of the calculated results versus the measured results for 5ppm ammonia diluted with synthetic air at 650°C for the alternative heating device;

Figure 8 and 9 show graphical representations of the results of clinical studies; and

Figure 10 shows a graphical representation of the different categories of test subjects from the clinical studies.

With regard to Figure 1 , there is shown a schematic of the apparatus of the present invention 100 comprising a heating device 102, a sensor device 104, user interface 106, serial communications 108 and power source 1 10.

In use a breath sample from a patient enters the apparatus 100 via sampling device (not shown) and then passes into heating device 102 via interface means (not shown). In the heating device 102, the breath sample is heated to 650°C by a heating element (not shown) in the presence of oxygen to bring about conversion of ammonia to nitric oxide. The heating element 102 is powered by a mains supply, which also connected or connectable to the power source 1 10, which in the form of a low voltage DC power supply unit. Power source 1 10 in turn powers the electronics of the sensor device 104 (as described in more detail below) and serves to recharge a rechargeable battery (not shown) provided as a backup for when the apparatus is disconnected from the mains supply. Once the ammonia present in the breath sample has been converted to nitric oxide in the heating device 102, at least a portion of the heated sample passes from the heating device 102 into the sensor device 104. As stated previously, it is not necessary that the heating device 102 has a 100% conversion rate but rather that at least 90% of the ammonia present in the breath sample is converted to nitric oxide. The use of a calibration routine for the sensor device 104 (as discussed in greater detail below) enables a conversion rate of less than 100% to lead to an accurate measurement of ammonia in the breath sample.

Sensor device 104 comprises an electrochemical NO sensor which is mounted within the sensor device 104 such that heated sample passing from the heating device 102 into the sensor device 104 passes over the relevant portion of the NO sensor before exiting the device through an exhaust port (not shown). Suitably the NO sensor is arranged such that a gas entry surface faces into a diffusion cavity through which the heated sample passes.

Suitable NO sensors are well known in the art. In particular, suitable electrochemical NO sensors are available that respond specifically to ppb levels of nitric oxide, as discussed in greater detail above.

The amount of NO present in the heated sample is detected by the sensor device 104, which communicates this information to user interface 106. However, preferably the information provided by the NO sensor is processed within the apparatus (as described below) such that an indication of the concentration of ammonia present in the breath sample is provided at user interface 106. User interface 106 comprises a display, and input means to allow a user of the apparatus to operate the apparatus. A touch sensitive LCD display is a desirable system which combines both input and display functions. The display, amongst other functions, indicates the amount of NO in the heated sample, as detected by the sensor device 104, or the

concentration of ammonia present in the original breath sample. However, in alternative embodiments, the display may be in the form of LED indicators, or an LCD.

The sensor device 104 may also communicate the amount of NO detected in the heated sample to serial communications 108, which is connected to any electronic storage device, such as a computer. Preferably the computer compares the amount of NO detected with known values to ascertain whether the patient has a gastrointestinal disease, such as H. pylori infection.

An embodiment of the apparatus of the present invention will now be described in more detail, with reference to Figure 2 which shows a schematic representation thereof.

Figure 2 shows a block diagram of apparatus 100 in accordance with one embodiment of the present invention and an electric circuit suitable for powering apparatus 100.

Apparatus 100 comprises sampling device 202, heating device 102 and sensor device 104. The sampling device 202 is connected to the heating device 102 via a first cooperating interface means (not shown), which is in turn connected to the sensor device via a second cooperating interface means (not shown). Interface means suitably comprise a conduit of D- shaped profile at the end of the sampling means 202, which may be inserted into a correspondingly shaped D-shaped recess at one end of the heating device 102. Similarly, the second interface means comprise the same features at the corresponding ends of the heating device 102 and the sensor device 104. Interface means are suitably tapered to ensure a neat fit. The connection formed is preferably substantially air tight.

Sampling device 202 comprises a patient contact means in the form of a mouthpiece 204, a flow indicator 206 and a bacterial filter 208.

Mouthpiece 204, which can be made of either cardboard or plastic, is shaped and sized such that a patient can put the device into their mouth and for a seal with their lips. A pipe which is circular or elliptical in profile is suitable. Mouthpiece 204 is connected to flow indicator 206 via a conduit (not shown). Preferably the conduit is provided with a first oneway valve which allows exhaled breath to pass through, but does not allow any return of air therethrough.

Flow indicator 206 provides a visual indication that the breath sample is within the required flow rate band. Flow indicator 206 comprises a flow indicator conduit (not shown) oriented vertically, which is transparent and has an indicator marked upon it. The indicator is a scale which indicates the rate of flow. The indicator conduit contains a ball which is of smaller diameter than the conduit, which is capable of moving up and down within the flow indicator conduit. The indicator shows a preferred region within which the ball should be maintained, this region corresponding to a flow of 45 to 55 ml/s. Flow indicator 206 is arranged such that it will be in the line of sight of a patient exhaling into the sampling device 202.

Bacterial filter 208 is located adjacent to flow indicator 206, at the interface with heating device 102. Bacterial filter 208 is capable of filtering out any potentially infectious particles in the exhaled air to ensure that the exhaled air is cleaned before it passes from the sampling device 202 into the heating device 102 and eventually into the sensor device 104. Filters suitable for this purpose are well known in the art. Bacterial filter 208 may also be capable of removing any moisture in the exhaled air to ensure that it is dried before passing into the heating device 102. Suitable filters for each of these purposes are well known in the art.

Heating device 102 comprises a first heat absorber 210, a heating element 212, a second heat absorber 214, a temperature controller 216, a thermocouple 218 and a high temperature cut-out mechanism 220.

First heat absorber 210 and second heat absorber 214 are disposed on either side of the heating element 212, adjacent to the first interface means, at which the heating device 102 is connected to the sampling device 202, and the second interface means, at which the heating device 102 is connected to the sensor device 104, respectively. First heat absorber 210 is configured to cool the inlet to the heating element 212 and second heat absorber 214 is configured to cool the outlet of the heating element 212 to ensure that excess heat does not affect other parts of the equipment or become a safety hazard. First and second heat absorbers 210 and 214 may be in the form of stainless steel tubing connected to one or more peltier coolers to cool the inlet, outlet and the breath sample flowing therethrough. However, the person skilled in the art would understand that any insulating material known in the art is suitable for this purpose.

Heating element 212 is configured to heat the exhaled breath sample to a temperature of 650°C, in the presence of oxygen, to effect conversion of ammonia contained within the exhaled breath sample to nitric oxide for detection. The temperature of the heating element 212 is controlled by temperature controller 216, which is powered by the electric circuit described below and is capable of maintaining the temperature of the heating element 212 at 650°C. Temperature controller 216, together with thermocouple 218, which measures the temperature of the heating element 212, form a temperature control circuit. Thermocouple 218 must be capable of measuring a temperature of at least 650°C, preferably of more. The temperature control circuit forms a closed loop between the temperature controller 216, the heating element 212 and the thermocouple 218 to ensure that the heating element 212 is at the correct temperature for conversion of ammonia to nitric oxide. The exact temperature of the heating element 212 can be displayed at an external surface of the device, by means of a user interface 232 which is connected to the temperature controller 216, so that the user/patient may monitor the temperature to ensure that the heating element 212 is at the correct temperature. User interface 232 may be in the form of any means which displays the temperature in a way which can be easily read; preferably the user interface 232 is a digital display.

The heating device 102 further comprises a high temperature cut-out 220 which is configured to stop the power to the temperature controller 216 if the overall temperature of the heating device 102 exceeds the safe working temperature, typically at a temperature of around 70°C. This is because although the heating element 212 is insulated to protect the electronics within the heating device 102, failure of this insulation can lead to heat escaping, exposing the surrounding electronics to high

temperatures, which would cause them to fail. The high temperature cutout 220 provides a safety feature to reduce the risk of heat damage to the electronics and apparatus 100 or users of the apparatus 100. Such a device may operate automatically to shut off the power to the temperature controller 216 when it detects that the overall temperature of the heating device 102 exceeds the safe working temperature, or is manually operated, such that the user can activate it when the user interface 232 indicates that the temperature of the heating element 212 exceeds a safe level.

The heating element 212, temperature controller 216, thermocouple 218 and high temperature cut-out 220 may be any suitable devices known in the art, which provide the aforementioned properties.

Sensor device 104 comprises a solenoid valve 222, a scrubber 231 , an air filter 224, a pump 226, a moisture regulator and a nitric oxide (NO) sensor 230.

In this embodiment of the invention, the sensor device 104 comprises an inlet (not shown) through which ambient air can enter the sensor device 104 to calibrate the NO sensor 230 to "zero". The inlet is disposed at the scrubber 231 of the sensor device 104. Scrubber 231 is a filter configured to remove any interfering gases such as nitric oxide from the ambient air passing into the sensor device, to ensure that the air used to calibrate the NO sensor 230 is substantially free from NO. A suitable material for the scrubber 231 is KMnO₄ and/or carbon granules. The scrubber 231 is positioned such that ambient air passes through the scrubber 231 before passing through the solenoid valve 222.

Solenoid valve 222 is capable of switching between two positions. In a first position, the solenoid valve 222 allows ambient air to pass through the sensor device 104 to the NO sensor 230 for self calibration, preventing any ambient air passing into the heating device 104 and sampling device 202 to be inhaled by the patient. In a second position, the solenoid valve 222 allows the heated exhaled breath sample to pass from the heating device 102 into the air filter 224, towards the NO sensor 230, without any of the heated sample being lost via the ambient air inlet (not shown).

Air filter 224, which is disposed adjacent to the solenoid valve 222, is capable filtering all air, ambient or the heated exhaled breath, which subsequently passes over the NO sensor 230. This air filter serves to remove infectious particles which would otherwise cause damage to the NO sensor 230 and so prevent such damage to the sensor 230. Air filter 224 may be made of any known material capable of filtering such infectious particles, as discussed above.

Pump 226 is a variable speed pump and is disposed adjacent to the air filter 224 to extract a portion of the heated exhaled breath sample for detection in the NO sensor 230. Pump 226 also serves to draw a portion of ambient air into the sensor device 104 for calibration of the NO sensor 230 via ambient air inlet.

Moisture regulator 228 comprises Nafion® tubing which reduces moisture present in the sample of the heated exhaled breath sample or the NO free ambient air extracted by pump 226, before it passes into the NO sensor 230. With many sensors it is important to regulate moisture present in the extracted sample, which may have been present in the exhaled breath sample or NO free ambient air, or is present as a consequence of heating of the sample in the heating device 102, as the presence of excess moisture in the extracted sample/NO free ambient air can interfere with the result and/or damage the NO sensor 230.

NO sensor 230 is disposed adjacent to the moisture regulator 228 to receive the moisture-reduced extracted sample and detect the concentration of NO present in the exhaled breath sample. NO sensor 230 comprises an electrochemical NO sensor, as discussed in further detail above, with reference to Figure 1 .

Apparatus 100 is powered by an electric circuit comprising a power source, in this case a universal mains power input of 85-265V, 50-60Hz. However, the apparatus 100 may also be powered by one or more rechargeable batteries 252 to make it either a desktop unit, handheld or portable. Battery type may be in the form of Lithium Ion, but is not restricted to this and may also be powered from other technologies such as Nickel Metal Hydride or other.

Mains power can be supplied to the apparatus 100 using an IEC mains receptacle 234, in the form of a plug for insertion into the socket of the mains power supply, where both live and neutral lines are fused, with user accessible fuses 236. An ON/OFF switch 237 is used to allow the user to disconnect mains power from the apparatus 100 without having to remove the mains receptacle 234. Power from the mains power input is supplied to a transformer 238.

A transformer 238 and a low voltage DC conversion circuit 239 are used to supply power to the rest of the apparatus 100. Transformer 238 is connected to the temperature controller 216 of the heating device 102 to supply power thereto. Low voltage DC conversion circuit 239 comprises a switching circuit to allow the universal mains power input of 85-265V, 50- 60Hz to be converted to low voltage DC power suitable for use in the rest of the electric circuit.

A charging circuit 250 may be connected to the low voltage DC conversion circuit 239 for charging up at least one rechargeable battery 252. Such a rechargeable battery 252 can alternatively be charged from a USB rather than from connection to the universal mains input via low voltage conversion circuit 239. The apparatus may be powered in this way from a docking station or standalone (not shown). The use of at least one rechargeable battery is preferred where the apparatus 100 is a hand-held portable unit.

A microcontroller or microprocessor 244 is used as the main controlling means and is connected to the low voltage conversion circuit 239 via oscillators 240, which may be in the form of XTAL or ceramic resonators. The microcontroller/microprocessor 244 provides a variety of different functions including operation mode (oral breath sample, ambient breath sample, nasal breath sample or calibration mode), settings mode (date and time settings, sound on/off, zeroing or system information, such as version of firmware, last recorded value, calibration date, number of tests performed since last calibration etc), management mode (calibration gas value adjuster, adult/child option, NO measurement option - bypassing heating device -, self test and settings adjustments to alter, for example, exhale time and flow rate) and patient information mode (patient name, personal details and previous test results) and provides a means by which the user can make selections within these functions. The options are displayed user interface 106 in a menu from which the user can select the desired function.

A real time clock 242 is connected to the microcontroller/microprocessor 244 to display the time and date to the user of the device and further for use as a time/date stamp, when recording certain time-based data. Time- based data can be stored within the apparatus 100 or externally, for example on a computer, so that the user can check the times and dates of breath measurements and use these for comparative testing. Further information which may be stored, either internally or externally of apparatus 100, is apparatus usage, calibration reminders and reminders for disposal or change of consumables, i.e. the mouthpiece 204. Where the data/information is stored internally, memory means 256 is included in the apparatus 100 for storing data measurements, time and date settings and other miscellaneous information. In this embodiment, the memory means 256 is in the form of EEPROM (Electrically Erasable

Programmable Read-Only Memory).

A battery backup 254 is connected to the memory means 256 to ensure that memory is retained if there is a mains power failure or the apparatus 100 is disconnected from the power supply. Battery backup 254 is charged via charged circuit 250 and may also serve to maintain functionality of the sensor device 104 in the aforementioned

circumstances.

Serial communications 108 are provided to connect

microcontroller/microprocessor 244 to another external data

storage/analysis device such as a computer. The interface of serial communications 108 may be in the form of any serial communications known in the art, for example it may in the form of USB or RS232.

Microcontroller/microprocessor 244 is programmable and may be reprogrammed via a programming port 246, or via the serial

communications 108 interface.

The apparatus 100 further comprises a user interface 106 which comprises a display, and input means to allow a user to operate the apparatus 100, as described above with reference to Figure 1 . User interface 106 is connected to microcontroller/microprocessor 244 to allow communication of information therebetween.

A sounder 248 is connected to the microcontroller/microprocessor 244 for use as an audible indicator to register when an option has been selected and to indicate when the patient should start exhaling and when they should stop to take the sample of exhaled air. Sounder 248 may be in the form of a speaker, or a piezo or electromagnetic sound device.

The electrical flow through the electrical circuit is shown in Figure 2 by the smaller arrows.

In use a sampling device 202 is connected to the heating device 102 and sensor device 104 to form the complete breath test apparatus 100. The electric circuit is then switched on, the required operation mode is selected and the heating element 212 and NO sensor 230 are allowed sufficient time to become fully heated/functional. Accordingly, the temperature of the heating element 212 is raised to and maintained at 650°C and the NO sensor is allowed at least one minute to become functional.

The NO sensor 230 is then calibrated to "zero" with NO free "ambient air". Before the calibration is performed, the solenoid valve 222 is switched to its first position to allow ambient air to pass into the sensor device 104. Calibration is affected by pump 226 drawing a portion of ambient air into the sensor device 104 via the ambient air inlet (not shown). The portion of ambient air is drawn through the inlet into scrubber 231 , where interfering gases are removed from the ambient air, through the solenoid valve 222 and air filter 224, which removes any infectious particles. The portion of ambient air then passes through pump 226 and moisture regulator 228 and into NO sensor 230. In the NO sensor 230, the ambient air passes over the sensor to calibrate it and then is removed via the exhaust. Once calibrated, the apparatus 100 is ready for use.

The patient inhales normally and then exhales into the sampling device 202 at a slow and steady rate. The patient adjusts the rate of exhalation such that the ball (not shown) is in the correct region of the indicator conduit (not shown) of the flow indicator 206.

The flow of the exhaled breath sample through the apparatus 100 is shown by the larger arrows in Figure 2.

The exhaled breath sample passes through bacterial filter 208 before passing into the heating device 102, where it is heated to 650°C within the oxidation chamber comprising a 1 .5 m of 0.32 mm stainless steel tubing by heating element 212 to effect conversion of ammonia in the exhaled breath sample to nitric oxide for detection.

The heated exhaled breath sample then passes into the solenoid valve of the sensor device 104, which is configured in its second position to allow the heated exhaled breath sample to pass through air filter 224 to remove any infectious particles.

Once the heated exhaled breath sample has passed through air filter 224, it passes into variable speed pump 226, which extracts a portion of the heated exhaled air sample for detection at the NO sensor 230 through a further solenoid valve (not shown). This extracted sample comes into contact with the NO sensor 230. The NO sensor 230 detects the level of the NO in the extracted sample and an output is produced. An exhaust is used to pass the extracted gas to ambient once it has passed through the sensor device 104. The output from the NO sensor 230 will increase in direct proportion to the concentration of NO in the sensor cavity. This output is amplified and fed to the microcontroller/microprocessor 244, where it is first digitised and displayed and/or transmitted to computer via serial communications 108, where it is processed by embedded software. Mathematical algorithms within the firmware of the apparatus create a 3-second running average of the rising sensor output, compensating for temperature and humidity effects by virtue of monitoring said parameters within the NO sensor 230. When this averaged value reaches a peak and starts to subside, the software calculates the equivalent ammonia ppb concentration of this peak and transmits this value to the user interface, e.g. an LCD display 106 and to internal memory 256, where it is stored.

The software is also allows a reading to be taken at a designated point of the rise time and then uses a scaling factor to estimate the value of ammonia ppb concentration. The provision of such a function in the software is readily achievable using known techniques available in the art.

The apparatus 100 as described above is used for measuring levels of gaseous ammonia in a breath sample but the application is not restrictive and therefore may include nasal gas, or other gas forms.

Temperature testing

Procedure

An ambient air sample was taken to give a reading of the background ammonia concentration using the apparatus of the present invention, as described above excluding the flow indicator 206. This was read at 170ppb which is a very high baseline. The inlet portion of the sampling device was scrubbed, using a chemical or compound which removes ammonia from the air leaving the downstream air free from ammonia, to determine if high baseline was a characteristic of the ambient air, or if other contaminants were introduced from the sampling device materials. The scrubbed ambient air sample read 1 1 ppb. The scrubber element was removed and the ambient air sample retested to give a reading of 46ppb. It was concluded that there is the possibility of a small offset in the background levels of NOx in the ambient air, which can affect the reading taken by the NO sensor.

1 ppm ammonia was then fed into the instrument directly and then fed into the instrument with a filter inline The filter was present for breath sampling purposes to see whether the filter would make any significant difference to the reading value given a known concentration of ammonia. The measured NO, which corresponds to the level of ammonia present in the air sample was 1021 ppb (no filter) and 1033ppb (with filter), thus showing that as the increase is less than ±2%, and there is no significant difference.

Ambient tests were carried out with 100ppb ammonia measured then scrubbed down to 40ppb, then un-scrubbed measured 66ppb. 1 ppm ammonia was fed into the apparatus and measurements taken (in ppb) at 60 seconds, the ammonia sample was then fed through the filter at 65 seconds and a further measurement taken at 80 seconds.

Results

Table 1 : Ammonia levels detected in a 1 ppm sample at varying

temperatures 650°C 700°C 750°C 800°C

60 second measurement 1010 1093 1 130 1 125

(no filter)

80 second measurement 1041 1 1 16 1 139 1 153

(via filter)

Conclusion

It was noted that although the higher temperatures increased the level of ammonia detected, i.e. measured level of NO, there was a risk of heat damage to either the pump or NO sensor at these higher temperatures. Therefore, the temperature was kept at 650°C, under the understanding that the measured value of ammonia directly into the apparatus is the effective calibration value and therefore the maximum value that could be safely reached.

Dilution testing

Procedure

To perform dilution tests, a glass mixing chamber was created with two inputs and one output. Ammonia was fed into the glass mixing chamber via one input and synthetic air was fed into the chamber via the second input. The resulting mixture of ammonia and synthetic air, at the required dilution is fed out of the mixing chamber via the output into the apparatus of the present invention.

Mass flow meters were used to control the flows of ammonia and synthetic air (measured in ml/s) to ensure that the required dilution is accurately achieved. The resulting mixtures are subsequently fed into the thermal oxidiser of the apparatus described above via the filter and sampling system. The sampling system employed incorporates a T-piece for protection from excessive pressure build-up, a 50mm 1 micron PTFE hydrophobic filter as the protection from cross infection and a disposable mouthpiece. In this embodiment, the disposable mouthpiece is attached to the hydrophobic filter which is downstream of the T-piece to protection the T-piece from cross infection. The sampling device, which is optionally provided with a flow indicator, is attached to the heating device comprising the thermal oxidiser.

Dilution concentrations of ammonia at 1 ppm, 5ppm and 19.7ppm were tested at 650°C and the results are given in Tables 2 to 4 below. The level of ammonia in the samples was measured in ppb at 10 second intervals between 0 and 50 seconds. The certified value given in the table is the accurate concentration of ammonia in ppb and the calculated value is the expected concentration of ammonia based on the dilution of the ammonia flow within the total flow rate.

Table 2: 1 ppm Ammonia diluted with Synthetic air at 650°C

Table 3: 5ppm Ammonia diluted with Synthetic air at 650°C

Table 4: 19.7ppm Ammonia diluted with Synthetic air at 650°C

Conclusion

For 1 ppm ammonia dilutions the measured and calculated values very closely correlate, as illustrated in Figures 3a and 3b, with the exception of the high end and low end dilutions. The level of the 1 ppm ammonia fed directly into the thermal oxidiser measured much less than the calculated value and appears to be an anomaly in the results. This can be observed in Figure 3a where the response appears almost flat for sample number 1 . When zero ammonia was fed into the thermal oxidiser, a measurement of greater than zero was recorded and therefore could be considered an offset, i.e. resulting from the presence of trace ammonia within the sampling device (i.e. the tube surfaces etc) which degasses into the sample stream. Such an offset means that accurate measurements in the low ppbs are difficult to identify accurately. Dilutions at the mid point of 11tr/min air and 11tr/min ammonia appear to be the most accurate.

For 5ppm ammonia cylinder dilutions, the measured and calculated values also correlate very closely and even more closely than the 1 ppm dilutions, as illustrated in Figures 4a and 4b. An offset is still observed in the low ppb region. Dilutions at the mid point of 11tr/min air and 11tr/min ammonia once again appear to be the most accurate.

For 19.7ppm ammonia concentrations, the measured and calculated values show similarities, as illustrated in Figures 5a and 5b, although do not correlate as closely as either the 1 ppm or 5ppm concentrations. For each concentration the maximum measured value was taken as being the dilution concentration. However, with 19.7ppm this value may not be the most suitable. After looking at the results, it was noticed that the 19.7ppm concentration was actually 19.7ppm ammonia in a balance of nitrogen, and therefore possesses no oxygen required for the oxidisation process. Taking this into account, it would be feasible to say that as the air concentrations increase, the more oxidisation can take place and the more accurate the measurements should be. Therefore, working back from the mid point where 11tr/min air and 11tr/min ammonia are mixed, we can derive a new theoretical maximum concentration and recalculate the expected measurements. This is shown with the yellow trace on Figure 5a and this seems to be more realistic. The first few measurements would have less oxidisation and should be the most inaccurate, then as the air to ammonia ratio increases, so should the accuracy of measurement. This appears to be the case with exception of the low ppb readings which show an offset at baseline.

The dilution tests show that the apparatus of the present invention can accurately measure the level of ammonia present in a synthetic air sample at a known concentration. This proves that the thermal oxidiser, sampling system, filters and sensor can determine whether a subject is suffering from H. pylori injection.

Heating device

It was noted that the apparatus used in the dilution testing suffered a fault where the power rating of the transformer of the power source employed was exceeded, causing the apparatus to fail. The fault was identified to be an incompatibility between the power rating of the transformer capability and the load rating of the heating element employed in the heating device. The heating device therefore heated up too quickly causing failure of the device, particularly at operational temperatures over 700°C. Accordingly, a heating device comprising a heating element with a low power rating was employed, as this ensures that the heating element load rating is compatible with the power rating of the transformer capability. Accordingly, the new heating device will take longer to reach operational temperature preventing failure of the device. The heating element has a power rating of 470W at 240V and the transformer has a power rating of 1 15V 1 .5A, therefore providing an output capability of 172.5W.

The heating element of the heating device is provided in association with a thermal oxidiser, which is in the form of a length of 1 .5m stainless steel tube of 1/8 (3.2 mm) inch diameter. The heating element is provided as a heater core around which the stainless steel tube is wound. In use, the stainless steel tube develops a pressure drop across it when pulling the gas sample through it. Therefore, a pump is provided within the heating device to pull the gas sample through the tube. Suitable pumps are therefore classed as the vacuum pressure pumps, for example solenoid pumps. The typical vacuum pressure monitored in the apparatus of the present invention is 95.4mbar. Therefore, a pump with plenty of margin for error is needed, preferably suitable for withstanding twice the

measured pressure, i.e. approximately 200mbar. A dual headed pump is required because the thermal oxidiser requires a consistent flow of gas, to prevent excessive heat build-up and to increase the consistency of gas temperature. Accordingly, from these requirements it would be obvious to the person skilled in the art as to what pumps are suitable for this purpose.

Particularly preferred pumps include a D type pump and a 31 12.120 pump both available from the Boxer® Pump range by Uno. These pumps are dual headed and can pull a vacuum pressure of at least 200mbar. The D type pump is on its limits when used at 200mbar, but the 31 12.120 pump can withstand higher running pressures although the body size is much greater than the D type pump.

The apparatus of the present invention comprising the new heating device and a 31 12.120 Boxer® pump was tested using the same test protocol as the temperature testing described above and a 5ppm ammonia

concentration sample. The results of this test are shown in Table 5 below and illustrated in Figure 6.

Table 5: Temperature testing of new heating device

These results show that the new heating device produces similar results to the initial temperature testing, as shown in Table 1 , and so the same conclusions may be drawn.

The apparatus comprising the new heating device and the 31 12.120 pump was then tested with differing dilutions of 5ppm ammonium in synthetic air at 650°C as per the test protocol described above. The readings were taken after 50 seconds, as shown in Table 6 below.

Table 6: Dilution testing of Ammonia with Synthetic air at 650°C

Conclusion

The improved heating device suffers some of the same symptoms as the previous heating device, in that the first pass of ammonia is always measured as less than the second pass of ammonia. This is not surprising as ammonia will saturate the sampling surfaces of any 'cold' materials (i.e. any prior to the thermal oxidiser inlet) on the first pass, while on the second pass these materials will be saturated at these

concentrations and so more ammonia will pass through into the heating device. However, the improved heating device appears to be converting more ammonia to nitric oxide for measurement in the sensor than the previous heating device, as shown in Figure 7 compared to Figure 4a. Once calibrated on the desired temperature value, the relationship of concentration to its diluted calibration value is much more accurate for the new heating device.

As the gas sample leaves the heating device, it enters PTFE tubing which act as cooling tubes through which the heated air sample passes before entering the sensor device. The PFTE tubing is provided in a coil of approximately three turns with a coil diameter of approximately 15cm, which is attached to the outlet of the stainless steel tubing of the thermal oxidiser. These coils of PTFE tubing are disposed in a space cooled by a fan. The temperature of gas as it leaves the heating device and enters the PTFE tubing was monitored as 26.2°C at all temperature settings except for 800°C, which measured 26.6°C. The temperature of the gas leaving the heating device drops due to an end of the stainless steel tubing which extends from the heating device and it effective at dissipating the heat. This means that the coils of PTFE cooling tubing can be significantly reduced to a third of the original size as less cooling of the gas is required, therefore saving space in the final design.

Clinical Studies

The apparatus of the present invention, as described above with reference to Figures 1 and 2 including the sampling device (as described for the dilution testing) and the new heating device and pump, was used to test breath samples from test subjects in order to determine whether the test subject was suffering from the H. pylori infection.

Test protocol

The test subject was instructed to fast for a minimum of six hours prior to taking the breath test. Where the test subject is taking any medication which may interfere with the test results, they were instructed to stop taking the medication before the breath test as required.

The test subject was then instructed to follow the test procedure set out below which is based on the test protocols employed by the INFAI breath test, an industry standard ¹³C CO2 breath test. The INFAI test protocols were used as a baseline to establish if similar protocols can trigger a significantly measurable breath-ammonia response in Helicobacter pylori positive (HP+ve) subjects using the apparatus of the present invention. The test subjects for this study were asked to provide two sets of breath samples to enable side-by-side testing for the apparatus of the present invention against the proven ¹³C CO2 breath test.

The test protocol is as follows:

1 . Rinse out mouth with water.

2. Gently blow through straw into white capped test tubes (labelled T₀) as instructed. This provided a sample for comparative testing using the INFAI test. The INFAI test is a well known test in the field, and is discussed for example in Megraud et al. The Journal of

Pediactrics 2005, 146(2), 198 and publications from INFAI, see www.infai.co.uk.

3. Using a disposable mouthpiece, take a breath test using the H. pylori apparatus as instructed (T, test).

4. Drink 200ml orange juice (a test meal preparation may be provided as an alternative). This step gives the stomach some contents and so slows the digestive transit to hold the urea solution, taken in step 5, in the stomach for longer.

5. Drink ¹³C Urea solution (75mg) and wait ten minutes. The purpose of drinking the ¹³C Urea solution (75mg) is that it reacts with the H. pylori bacteria in the stomach to produce ammonia, therefore providing an indication of the presence of H. pylori.

6. Immediately rinse mouth with water and take a second test using the H. pylori apparatus of the present invention as instructed (T₀ test).

7. Take a third breath test using the H. pylori apparatus of the present invention as instructed (T10 test).

8. Wait a further ten minutes and take a fourth breath test on the H. pylori apparatus (T₂o test). 9. Wait a further ten minutes and take a final breath on the H. pylori apparatus (T₃o test).

10. Gently blow through straw into blue capped test tubes (labelled T₃₀) as instructed.

1 1 . Test completed.

Altogether, 18 subjects were tested and their results are provided below. In total, 2 test results were void due to procedural error, of the remaining subjects 5 tested positive.

Results

From the results detailed below in Table 7:

Ti = Test taken prior to subject taking urea solution. (Mouth washed with water)

To = Test taken immediately after taking urea solution (mouth washed with plain water immediately after urea ingestion).

Tio = Test taken 10 minutes after urea solution ingestion.

T₂o = Test taken 20 minutes after urea solution ingestion.

T₃₀ = Test taken 30 minutes after urea solution ingestion.

Table 7: Ammonia Breath test results

^* Readings where taken without switching on the pump on the ammonia prototype monitor, hence they are void. These results are summarised in Figures 10 and 1 1 . Q-Factor Calculation

The study report Detection of H. pylori Infection by Breath Ammonia Following Urea Ingestion', Penault, Spanel & Smith, indicates that between 20 to 30 minutes after urea ingestion, there should be a stable level of breath ammonia in the subject and it should be significantly higher than the breath ammonia level before urea ingestion if the subject is H. pylori positive. Accordingly, t he main points of interest in the present studies are the Τ,, T₂o and T₃o periods as they represent the ammonia breath levels before urea ingestion and after the urea administered has been successfully converted to ammonia by the H. pylori bacteria.

Based on this finding, a relative increase factor (Q- Factor) has been calculated to analyse the test subjects' results and establish a baseline below which test subjects are considered H. pylori negative and above which test subjects are considered H. pylori positive.

The Q- Factor was calculated by dividing a subject's breath ammonia reading 30 minutes after urea ingestion (T₃₀) by the initial breath ammonia reading before urea is administered (T,) i.e. Τ₃₀/Τ,.

The Q-factor calculated from the batch of results is displayed in Table 8 below.

The test subjects have been grouped into 3 categories according to their Q-factors and knowledge of their existing conditions:

Category A: Test subjects with Q-Factor < 1 . This group represents H. Pylori negative subjects because there is no significant increase between their breath ammonia at T, and at T₃₀. Category B: Test subjects with Q-Factor 1 < x < 1 .5. This group represents test subjects who are borderline negative and positive i.e. non-conclusive tests. Their Q-Factors indicate a less than significant increase between breath ammonia at T, and at T₃₀.

Category C: test subjects with Q-Factor > 1 .5. This group represents H. Pylori positive subjects. Their Q- Factors indicate a significant increase between breath ammonia at T, and at T₃₀.

Table 8: Ammonia breath readings with calculated Q factors to

differentiate +ve and -ve test subjects

The highlighted tests are the definitely positive results.

* Void tests (see Table 7) ^** H. pylori negative patients whose results appear non-conclusive because of a high Q-factor.

^*** H. pylori positive patients whose results appear to be negative.

Figure 8 shows the different categories of test subjects. Test subjects with Q-factors above the upper dotted line are considered to be H. pylori positive subjects. Test subjects with Q-factors below the lower dotted line are considered to be H. pylori negative subjects. Test subjects with Q- factors between the two dotted lines are considered to be inconclusive.

Conclusions

From the 18 tests carried out, 2 results were invalid because the test procedure was not followed properly. Hence there are only 16 valid tests, 5 of which are H. pylori positive (HP+ve) subjects and 1 1 who are H. Pylori negative (HP-ve).

Results from the apparatus of the present invention confirms that 3 out of the 5 HP+ve subjects are definitely positive and that 9 out the 1 1 HP-ve subjects are definitely negative. 2 negative subjects appear in the borderline region of non-conclusive tests, and 2 positive subjects appear in the negative region.

It is believed that, in contrast to the study report 'Detection of H. pylori Infection by Breath Ammonia Following Urea Ingestion', Penault, Spanel & Smith, tests carried out using the apparatus of the present invention require an increase Q-factor of only 1 .5 to distinguish between H. pylori positive and negative subjects, whereas the study report suggests that H. pylori positive subjects require an increase factor of 2.5 for positive identification.

While the above results for the ammonia breath test using 75mg ¹³C-Urea has shown that a positive determination of whether attest subject is suffering from a H. pylori infection, it is believed that the significant crossover within the Q-factor analysis can be substantially reduced by increasing the ¹³C-Urea dose. Accordingly, the resulting ammonia breath levels will show a more marked change for a HP+ve subject compared to a HP-ve subject and so assist in differentiation of test subject status. A ¹³C-Urea dose of up to 2000mg has been used in previous studies and so the dose may be increased up to 2000mg to achieve a statistically significant response that can reliably be used in the diagnosis of HP+ve subjects. Accordingly, a 75mg to 2000mg provocation ¹³C-Urea dose may be used in the methods of the present invention.

The results of the dilution tests and clinical studies show that the apparatus of the present invention may suitably be used to detect the levels of ammonia present in a breath sample, therefore assisting in detection of the H. pylori infection. Accordingly, the apparatus of the present invention provides rapid and accurate results compared to the prior art devices, therefore, making testing more accessible to the population. This aids earlier detection of gastrointestinal diseases such as H. pylori which will of course lead to a reduction in patients going on to have peptic ulcers and/or gastric cancer.

Claims

1 . Apparatus for measuring the concentration of ammonia in exhaled air comprising a sampling device and a sensor device, the sensor device comprising a nitric oxide sensor,

characterised in that the apparatus further comprises a heating device configured to heat the exhaled air to affect conversion of ammonia in the exhaled air to nitric oxide prior to reaching the sensor device.

2. The apparatus according to claim 1 wherein the heating device comprises a heating element configured to heat the exhaled air sample to a temperature of from 500 to 1000°C in the presence of oxygen.

3. The apparatus according to claim 2 wherein the heating element is configured to heat the exhaled air sample to a temperature of from 500 to 800°C.

4. The apparatus according to claim 3 wherein the heating element is configured to heat the exhaled air sample to a temperature not exceeding 650°C.

5. The apparatus according to any preceding claim wherein the heating device comprises an oxidation chamber which is adapted to be heated to a suitable temperature to correct ammonia to NO.

6. The apparatus of claim 5 wherein the oxidation chamber is defined by a length of tubing.

7. The apparatus according to any one of claims 2 to 6 wherein the oxygen present in the heating device originates from the exhaled air.

8. The apparatus according to claim 7 wherein the oxygen is present at a level of greater than 15wt% of the exhaled air.

9. The apparatus according to claim 8 wherein the oxygen is present at a level of from 15 to 17wt% of the exhaled air.

10. The apparatus according to any of claims 2 to 9 wherein a catalyst is present in the heating device to enhance conversion of ammonia to NO.

1 1 .The apparatus according to claim 8 wherein the catalyst is one or more of platinum, platinum oxide, ruthenium oxide, rhodium oxide, palladium oxide, iridium oxide, cobalt oxide, vitreous carbon or copper oxide.

12. The apparatus according to any preceding claim wherein the temperature of the heating element is controlled by a thermostat.

13. The apparatus according to any preceding claim wherein the temperature of the heating element is displayed at a user interface.

14. The apparatus according to any preceding claim wherein the heating device comprises a high temperature cut-out device.

15. The apparatus according to any preceding claim wherein the heating device is disposed between the sampling device and the nitric oxide sensor device.

16. The apparatus according to any preceding claim wherein the heating device further comprises one or more heat absorbers or cooling devices positioned at the inlet and/or outlet of the heating element.

17. The apparatus according to any preceding claim wherein the heating device further comprises a vacuum pressure pump.

18. The apparatus according to any preceding claim wherein the apparatus comprises a means by which the heating device can be bypassed.

19. The apparatus according to any preceding claim wherein the sensor is capable of sensitive measurement of concentrations of NO from parts per billion (ppb) levels to parts per million levels (ppm).

20. The apparatus according to any preceding claim wherein the sensor is an electrochemical gas sensor.

21 .The apparatus according to any preceding claim wherein the heating device and sensor device assembly contain an independent power source.

22. The apparatus according to any preceding claim wherein the sampling device is detachable from the heating device and the sensor device.

23. The apparatus according to any preceding claim wherein the exhaled air is air originating at the alveolar interface of the lungs.

24. The apparatus according to any preceding claim wherein the sampling device comprises a pressure regulation means for protecting the apparatus from excessive pressure build-up.

25. The apparatus according to any preceding claim wherein the sampling device comprises a flow regulator.

26. The apparatus according to any preceding claim wherein the sensor device comprises a moisture regulation means to remove water from the heated exhaled air.

27. A method for measuring the concentration of ammonia in exhaled air using the apparatus of any of claims 1 to 26, the method comprising the steps of;

- obtaining a sample of exhaled air in the sampling device;

- heating the sample of exhaled air in the heating device to

convert ammonia present in the sample to nitric oxide; and

28. The method according to claim 27 wherein the sample of exhaled air is heated to from 500 to 1000°C in the presence of oxygen.

29. The method according to claim 28 wherein the sample of exhaled air is heated to from 500 to 800°C.

30. The method according to claim 29 wherein the sample of exhaled air is heated to a temperature not exceeding 650°C.

31 .The method according to any one of claims 27 to 30 wherein the method further comprises the step of comparing the result of the method with an expected value.

32. Use of the apparatus of any of claims 1 to 26 in the diagnosis of a gastrointestinal disease.

33. The method according to claim 32 for diagnosis of a gastrointestinal disease.

34. The use according to claim 32 or 33 wherein the gastrointestinal disease is caused by Helicobacter pylori infection.

35. A device according to any one of claims 1 to 26 for use in diagnosis of a disease.

36. The device for use according to claim 35 wherein the disease is a gastro-intestinal disease.

37. The device for use according to claim 35 or 36 wherein the gastrointestinal disease is claimed H. pylori infection.