GB2364778A

GB2364778A - Detection of Helicobacter pylori and apparatus therefor

Info

Publication number: GB2364778A
Application number: GB0017239A
Authority: GB
Inventors: Norman Mark Ratcliffe; Clive Teare; Christopher Dunn; David Charles Cowell; Murdo M Black; Paul Chambers; Caroline Penault
Original assignee: Hypoguard UK Ltd
Current assignee: Arkray Factory Ltd
Priority date: 2000-07-14
Filing date: 2000-07-14
Publication date: 2002-02-06
Also published as: AU2001270817A1; WO2002006822A1; GB0017239D0

Abstract

A method for detecting Helicobacter pylori in a subject's gastroenteral tract involves measuring a change in resistance of an electronic or electrochemical sensor, notably a polypyrrole film, which is caused by ammonia, on exposure to gas from the subject's lungs and/or stomach. Depending on the magnitude of the change (if any) a positive or negative result is indicated visually by electronics means such as LED's 20, 22. In a preferred embodiment, two sensors 16a, 16b are used. One of these receives a sample of gas 24 which has passed through an ammonia-absorbing means 30 such as sodium dihydrogen phosphate or copper sulphate on a porous frit to provide a corrected baseline value for the ammonia. Porous frit 28 balances the gas flow to the two sensors. An antacid or sparkling water may be administered orally to a subject before the test. The chambers 2a, 2b can be expandable and can have an exit vent to assist gas flow. An alkaline desiccant may be provided in the common passageway 32.

Description

2364778 Detection of Relicobacter Pylori

Field of the invention

5 The present invention relates to a device and method for detecting Helicobacter Pylori in human subjects.

Background of the Invention

10 It has been known for some time that inf ection by Helicobacter pylori (H pylori) may increase the risk of a subject suffering from illnesses such as gastritis and duodenitis, and from peptic and duodenal ulcers. Detection of H pylori is therefore desirable to determine 15 whether patients have, or have increased risk of having, such illnesses, and to enable appropriate treatment to be given.

H pylori produces ammonia and carbon dioxide by the action 20 of a urease on urea in bodily fluids, and various tests have been proposed to detect H pylori by detecting the products of this reaction.

A test which is currently in use involves administering 25 13C-labelled urea to the subject and subsequently testing carbon dioxide in the subject's breath for the presence of 13C. However, testing f or 13C requires a sample to be sent away for laboratory testing, which is slow and relatively expensive.

In US 4,947,861 it was proposed to detect the presence of ammonia in a subject's breath following oral administration of urea. The method comprises collecting a sample of alveolar air at least ten minutes after 35 administration of the urea, passing the air over an alkaline hygroscopic material to remove water vapour, and passing the dried alveolar air to a sensor which indicates the presence of ammonia. The sensor described is a glass tube filled with a granular material that changes colour 5 as ammonia is passed through it.

International Patent, Application WO 97/3035 describes various chemical indicators which change colour in the presence of ammonia to provide a visible indicator of 10 ammonia in a subject's breath.

It is desirable to have a detection device and method which is noninvasive, speedy, and which can be used by a patient or other person without medical supervision.

summary of the invention

According to a first aspect of the present invention there is provided a method for detecting the presence of 20 Helicobacter pylori in the gastroenteral tract of a subject, the method comprising the steps of:

a) obtaining a volume of gas from the lungs and/or stomach of the subject; b) causing or permitting at least some of the volume of 25 gas to come into intimate contact with an electronic or electrochemical ammonia sensor connected to means for measuring the electrical resistance of the sensor; c) measuring the resistance of the sensor when in contact with the said gas; 30 d) comparing the said resistance of the sensor with the resistance corresponding to the same or a similar sensor when not in contact with the said gas or when in contact with a portion of the volume of gas which has been treated to reduce the concentration of any ammonia therein, to 35 produce a compared value; and e) producing a visible output signal to indicate a positive or negative diagnosis of Helicobacter pylori infection according to whether or not the compared value exceeds a predetermined threshold value.

The method is non-invasive, and it can be speedy and easy for a patient or other subject to self-adminster. It is not necessary to administer urea to the subject prior to carrying out the method.

An antacid (for example magnesium hydroxide) may be administered orally prior to testing. This will promote conversion of ammonium ions in the stomach to gaseous ammonia. If the antacid is a carbonate or bicarbonate 15 (for example sodium bicarbonate), it will also produce carbon dioxide to facilitate eructation.

The ammonia sensor may be housed in a single chamber through or into which the gas is directed. However, in a 20 preferred embodiment, a pair of similar sensors are provided, each in its own chamber. The gas is distributed substantially equally between the two chambers, but one chamber has an ammonia-absorbing barrier through which gas passes before coming into contact with the sensor.

25 Electronics means compare the difference between or ratio of resistances of the two sensors and express the result as a visible output. The output could be numeric, but is preferably in the form of a signal corresponding to either a positive or a negative diagnosis. For example, a green 30 light or a red light could be illuminated.

To further increase the sensitivity of the device, the gas could be passed through an alkaline desiccant (for example solid sodium hydroxide) in known manner, to remove water 35 vapour (and some carbon dioxide) before the gas enters the chambers.

A preferred sensor comprises a film of polypyrrole, which is connected by electrodes to a suitable meter. Methods 5 of making polypyrrole films suitable for use in the invention are described in GB 2 234 515 and EP 0 206 133. The film preferably has a thickness in the range 50 to 250 /AM.

10 According to another aspect of the present invention there is provided a detection device for measuring ammonia content in gas from a subject's lungs and/or stomach, the device comprising:

a) a chamber having an entrance opening for receiving 15 the said gas and housing an electrical or electrochemical ammonia sensor connected to means for measuring the electrical resistance of the sensor; b) means for comparing the resistance of the sensor before and during exposure to the said gas to produce a 20 compared value; and C) means for producing a visible output signal according to whether the compared value exceeds a predetermined threshold value.

25 According to a further aspect of the present invention there is provided a detection device for measuring ammonia content in gas from a subject's lungs and/or stomach, the device comprising:

a) a first chamber and a second chamber, each of which 30 has an entrance opening for receiving the said gas, and each of which houses an electronic or electrochemical ammonia sensor connected to means for measuring the electrical resistance of the sensor; b) means for comparing the resistance of both sensors to 35 produce a compared value; c) means for producing a visible output signal according to whether the compared value exceeds a predetermined threshold value; and d) wherein the second chamber is provided with means for 5 absorbing ammonia, located between the entrance opening thereto and the sensor therein whereby at least some gas which enters the second chamber through the entrance opening will pass through the ammonia-absorbing means.

10 Although the term "ammonia-absorbing means"' is used herein for convenience, it will be understood that this term includes any means which remove ammonia from the gas. Thus, the term includes ammonia adsorbents and materials which chemically combine with ammonia.

A preferred ammonia sensor comprises a film of polypyrrole, connected by electrodes to a suitable meter.

In a preferred embodiment, the or each chamber is provided 20 with an exit vent to facilitate the passage of gas therethrough.

To reduce the volume of "dead space" in the or each chamber, the chamber(s) may optionally be constructed to 25 be expandable, for example by having elastic walls, by being of telescopic construction, or by having a movable plunger, like a syringe.

Brief Description of the Drawings

The invention will now be further described, by way of example, with reference to the following drawings in 5 which:

Figure 1 is a schematic representation of an ammonia detection device in accordance with one aspect of the present invention; Figure 2 is a graph showing change in resistance of the device of Figure 1, for different subjects; Figure 3 is a graph of response against time for the 15 device of Figure 1; Figure 4 is a schematic representation of an ammonia detection device in accordance with another aspect of the present invention; and Figure 5 shows electrical resistance measurement results for subjects under a defined test protocol.

Detailed Description

The device for detecting gaseous ammonia shown in Figure 1 comprises a chamber 2 in which is housed an ammonia sensor 4. The sensor 4 comprises a polypyrrole f ilm 16, about 50 Am thick, which changes its electrical resistance in 30 the presence of ammonia. The film 16 is carried on a pcbtype conductive board, f or example VeroboardTm, which has been etched to remove conductive material completely across the middle of the sensor 4, so that the two ends 18 of the board are not in electrical contact with each other. An insulating film of PEEK is disposed between the film 16 and the conductive board. The film 16 is in electrical contact at opposed edges with each conductive end portion 18. The end portions 18 are each connected by 5 wires 14 to a meter 6 which measures electrical resistance across the film 16.

The inside of the chamber 2 is maintained at 100% humidity and sealed by clingfilm, in this example NescofilmTm. When 10 the device is used in the method of the invention, a sample of gas 24 from a subject's lungs and/or stomach is collected in a syringe 8 and introduced into the chamber 2 via a needle 10. The meter 6 records the electrical resistance of the polypyrrole film 16 before the gas 24 is 15 introduced into the chamber 2, and again after the gas has been introduced. The meter 6 then compares the resistances to produce a compared value and lights up an LED 20 or 22 according to whether the compared value is above or below a predetermined threshold. The meter 6 may 20 measure the difference in resistance, or a ratio of resistances. The threshold value is calibrated to be just below the value produced by samples from test subjects known to be infected with H pylori. If the LED 22 lights up, showing a value which corresponds to infection, the 25 subject knows to seek appropriate treatment or confirmatory alternative testing.

Figure 2 shows test results for two groups of control subjects, one group known to be H pylori negative and the 30 other H pylori positive. In each case, a 10 ml sample of gas 24 was collected and introduced into a chamber of about 10 to 15 ml volume. The f ilm 16 was 10 mm square. The two sets of results on the left are f or a breath test only, and the two sets of results on the right (the 'belch test') are for gas collected from subjects' stomachs, following ingestion of sodium bicarbonate in water. In each case, there is a clear threshold between the measured resistance for the negative and positive groups.

The same test conditions were used to check the response of sensors over time, but using a known concentration (100 ppm)of ammonia in air. The results are shown in Figure 3, with percentage change in resistance being 10 plotted against the time (days) in which the sensor 4 was maintained in the chamber 2 prior to the measurement being taken. For all times up to 60 days, the percentage change was at least 15%.

15 The device shown in Figure 4 is a preferred embodiment, comprising a first chamber 2a housing a first sensor 16a, and a second chamber 2b housing a second sensor 16b. The chambers 2 and sensors 16 are of identical shape and construction. The first chamber has an entrance opening 20 which is substantially occupied by a first porous frit 28, and the second chamber has an entrance opening which is substantially occupied by a second porous frit 30. The frits 28, 30 are arranged and composed such that each provides substantially the same resistance to the passage 25 of gas 24 which is provided through a common entrance opening 32,for example by a subject breathing through that entrance. Each chamber may optionally be provided with a vent opening (not shown) to facilitate the flow of gas through the chambers. The second frit 30 is provided with 30 means for absorbing ammonia, for example sodium dihydrogen phosphate or copper sulphate crystals, so that at least some of the ammonia (and preferably substantially all of the ammonia) which may be present in gas 24 blown into the second chamber 2b is absorbed in the second frit 30 and does not reach the second sensor 16b. The f irst f rit 28 does not significantly absorb ammonia, so that ammonia which is present in gas 24 blown into the first chamber 2a reaches the first sensor 2a.

Both sensors 16 are connected by wires (not shown) to an integral meter 6. The meter 6 is optionally provided with means (not shown) for detecting gas flow in the chambers 2. A f irst LED 26 on the meter 6 lights up when it 10 detects the passage of gas 24. The meter 6 measures the resistance of both sensors and produces a compared value which is the ratio of the resistances. The meter 6 displays a visible output accordingly, by illuminating (green) LED 20 corresponding to a negative test for H 15 pylori, or (red) LED 22 corresponding to a positive test.

Based on data from in vitro studies, five healthy H.

pyl ori -negative volunteers (determined by the 13C breath test) were studied using the apparatus of Figure 1. In 20 this work, the polypyrrole film was fabricated by dip coating a colloidal suspension of poly(pyrrole), after chemical oxidation of the pyrrole monomer, on an acrylic sheet using known methods- - --(Ratcliffe NR. Poly(pyrrole) based sensor for hydrazine and ammonia. Analytica Chimica 25 Acta 1990; 239: 257-262; Ratcliffe NR. The simple preparation of a conducting and transparent poly(pyrrole) film. Synthetic Metals 1990; 38: 87-92).

The resultant film, approximately 50nm thick, has a surface topography (revealed by transmission electron and 3o atomic force microscopy) composed of spheres in intimate contact with each other. The volunteers were studied twice in random order on two separate days after an overnight fast; once after ingestion of an empty gelatin capsule and once after ingestion of a capsule containing 10 mg of 35 NH4Cl. Three additional volunteers were studied only after ingestion of NH4Cl. Ten minutes after the capsule (a time sufficient for capsule degradation according to pharmacopoeia standards and our own in vitro observations), each subject swallowed a mixture of 15 ml 5 of Milk of Magnesia@ (BCM Ltd, Nottingham: containing 415 mg of Mg(OH)2 per 5 ml) and 50 ml of water and, a further ten minutes later, drank 100 ml of sparkling water to 'drive off' any NH3Mouth air samples (10 ml) were collected into a syringe at baseline (before the capsule); 10 immediately prior to the Milk of Magnesia@/water mixture; and, finally, ten minutes after the 100 ml of sparkling water. These samples were individually expelled into a vial containing the NH3 sensor linked to a multimeter (measuring resistance) as described above. Pilot studies 15 suggested, in contrast to in vitro data, that cold (40C) sparkling water was superior to still water, so the former was used in all in vivo studies.

Five patients (three males and two females) who tested 20 positive for H. pylori with at least one clinicallyvalidated test (e.g. , 13C breath test, serology) underwent the same procedure but without taking NH4Cl- in vivo studies: H. pylori-negative subjects 25 Figure 5 summarises the changes in sensor chemoresistivity of mouth air in H. pylori-negative subjects who had ingested 10 mg NH4Cl or an empty gelatin capsule. On average, NH3 levels detected in mouth air after ingestion of the NH4Cl-containing capsule, but prior to 30 administration of the Milk of Magnesia/water mixture, were almost twice those seen after ingestion of the placebo. Furthermore, these data were obtained without the subjects necessarily belching.

In vivo studies: H. pylori-positive patients Five H. pylori-positive patients underwent the test protocol without taking the KH4Cl-containing capsule. The results are also shown in Figure 5. Pre-protocol NH3 5 levels in the patients' mouths were higher than the baseline levels measured in the H. pylori-negative subjects who ingested NH4Cl. Furthermore, even higher levels were recorded in the four patients in whom the test protocol produced a belch.

None of the healthy volunteers or the H. pylori-positive patients experienced any adverse effects from the study.

The device and method of the present invention can detect 15 sub-ppm concentrations of NH3 in 'endogenous' mouth air, and can provide a,point-of-care diagnostic test for Helicobacter pylori without the need for patients to ingest urea, and with the results being immediately available to the attending physician. Furthermore, the 20 conditions necessary for the bacteria-associated NH4+ to be converted to NH3 and liberated through the oral cavity can be achieved through the use of an established antacid and cold, sparkling water with no adverse reactions amongst the small number of healthy subjects and H.

25 pylori-positive patien ts so far tested.

Studies in the healthy volunteers clearly showed that NH3 levels in mouth air after ingestion of 10 mg NH4Cl were generally higher than in the same subjects tested without 30 ingestion of NH4Cl (Figure 5). This difference was evident irrespective of whether or not the subjects belched. Removing the requirement to belch is seen as a significant advantage for a diagnostic test as, in a study with a larger number of normal subjects, only a proportion were induced to belch reliably under our current protocol.

Given the small number of subjects tested, there is some overlap in the data between those who ingested NH4C1 and 5 those given the placebo. However, the data in Figure 5 show markedly higher levels of mouth NH3 in the overnight fasted H. pylori-positive patients than in either group of controls. Thus, the patients had higher baseline (without the need to belch) NH3 levels than the healthy subjects 10 even after the latter had ingested 10 mg NH4C1Furthermore, four of the five patients did belch and, in each case, this was associated with even higher mouth NH3 levels. All these in vivo data were acquired without any subject or patient being required to ingest urea. The data 15 also suggest that intra-gastric levels of NH3 in patients with H. pylori infection are considerably higher than those attained by the ingestion of 10 mg of NH4Cl- The invention provides a rapid, point-of-care diagnostic 20 test for H. pylori based on the chemiresistive detection of NH3 in mouth air. The proposed test does not require patients to ingest urea, and appears to be possible on endogenous' mouth air without the need for the patient to belch or even to ingest the antacid/water mixture.

25 Additionally, the test method uses neither stable nor radioactive isotopes thus obviating the need to send samples to a central laboratory for analysis, and overcoming difficulties associated with. radioisotopes.

claims 1. A method for detecting the presence of Helicobacter pylori in the gastroenteral tract of a subject, the method 5 comprising the steps of:

a) obtaining a volume of gas from the lungs and/or stomach of the subject; b) causing or permitting at least some of the volume of gas to come into intimate contact with an electronic or 10 electrochemical ammonia sensor connected to means for measuring the electrical resistance of the sensor; C) measuring the resistance of the sensor when in contact with the said gas; d) comparing the said resistance of the sensor with the 15 resistance corresponding to the same or a similar sensor when not in contact with the said gas or when in contact with a portion of the volume of gas which has been treated to reduce the concentration of any ammonia therein, to produce a compared value; and 20 e) producing a visible output signal to indicate a positive or negative diagnosis of Helicobacter pylori infection according to whether or not the compared value exceeds a predetermined threshold value.

25 2. A method as claimed in claim 1, wherein at least some of the volume of gas is divided into two substantially equal portions, one portion of which is caused or permitted to come into intimate contact with the said ammonia sensor, and the other portion of which is caused 30 or permitted to come into intimate contact with another similar ammonia sensor, wherein at least some of the said other portion of gas passes through ammonia absorbing means before coming into contact with the said similar ammonia sensor; and wherein the resistances of both 35 sensors are measured and compared to produce the said compared value.

3. A method as claimed in claim 2, wherein substantially all of the said other portion of gas passes through the 5 said ammonia absorbing means before coming into contact with the said similar ammonia sensor so that substantially all of the ammonia which may be present in the said other portion of gas is absorbed by the ammonia absorbing means.

10 4. A method as claimed in any one of the preceding claims, wherein an antacid is administered orally to the subject prior to obtaining the gas from the subject's lungs and/or stomach.

15 5. A method as claimed in any one of the preceding claims, wherein sparkling water is administered orally to the subject prior to obtaining the gas from the subject's lungs and/or stomach.

20 6. A detection device for measuring ammonia content in gas from a subject's lungs and/or stomach, the device comprising:

a) a chamber having an entrance opening for receiving the said gas and housing an electrical or electrochemical 25 ammonia sensor connected to means for measuring the electrical resistance of the sensor; b) means for comparing the resistance of the sensor before and during exposure to the said gas to produce a compared value; and 30 c) means for producing a visible output signal according to whether the compared value exceeds a predetermined threshold value.

7. A detection device for measuring ammonia content in 35 gas from a subject's lungs and/or stomach, the device comprising:

a) a first chamber and a second chamber, each of which has an entrance opening for receiving the said gas, and each of which houses an electronic or electrochemical 5 ammonia sensor connected to means for measuring the electrical resistance of the sensor; b) means for comparing the resistance of both sensors to produce a compared value; c) means for producing a visible output signal according 10 to whether the compared value exceeds a predetermined threshold value; and d) wherein the second chamber is provided with means for absorbing ammonia, located between the entrance opening thereto and the sensor therein whereby at least some gas 15 which enters the second chamber through the entrance opening will pass through the ammonia-absorbing means.

8. A device as claimed in claim 7, wherein the entrance openings of the chambers are both connected to a common 20 passageway having a single entrance opening for receiving gas from a subject's lungs and/or stomach.

9. A device as claimed in claim 7 or claim 8, wherein an alkaline desiccant is provided in the common passageway.

10. A device as claimed in any one of claims 7 to 9, wherein each chamber is provided with an exit vent to facilitate the passage of gas therethrough.

30 11. A device as claimed in any one of claims 7 to 10, wherein the ammonia-absorbing means comprises sodium dihydrogen phosphate or copper sulphate.

35 12. A device as claimed in any one of claims 7 to 11, further including detection means for detecting the passage of breath one or both chambers, and display means responsive to said detection means.

5 13 A device as claimed in any one of claims 7 to 12, which is arranged and constructed so that substantially all of the gas which comes into contact with the polypyrrole film of the second sensor via the entrance opening of the second chamber will pass through the 10 ammonia-absorbing means.

14. A device or method as claimed in any one of the preceding claims, wherein the or each sensor comprises a film of polypyrrole.

15. A device as claimed in any one of claims 7 to 14, wherein each chamber is expandable.

16. A detection device for measuring ammonia content in 20 gas from a subject's lungs and/or stomach substantially as herein described with reference to or as shown in Figure 1 or Figure 4 of the drawings.

17. A method for detecting the presence of Helicobacter 25 pylori in the gastroenteral tract of a subject, substantially as herein described with reference to the drawings.