COMPOSITIONS AND METHODS FOR THE DETECTION OF UREASE FOR THE DIAGNOSIS OF CAMPYLOBACTER PYLORIDIS INFECTION

Abstract: The invention relates to a composition for the detection of preformed urease to provide a diagnosis of gastrointestinal disorders which comprises (a) urea; (b) a bactericide; (c) a pH indicator which undergoes a colour change upon an increase of pH, at an effective concentration; and (d) water; wherein said composition has an acid pH of at least 5.0 and the pH of said composition is at least about one pH unit lower than the pK a of said indicator.

Description: BACKGROUND OF THE INVENTION This invention relates to compositions and methods for the detection of urease as a means of diagnosing the presence of gastrointestinal disease. Factors adversely affecting the function of the gastro­ intestinal system in humans are exceedingly varied in their nature. Such disorders may arise in the upper or lower gastrointestinal tracts or both. There is a broad range of causes of gastrointestinal disorders, including genetic, physiological, environmental, and psychogenic factors. Accordingly, the diagnosis and management of these disorders can be exceptionally difficult. A detailed discussion of gastrointestinal tract functions, disorders, causes, and treatments can be found in Spiro, Clinical Gastroenterology (3d. edition 1983). Among the chronic disorders of the upper gastrointestinal tract are those which fall under the general categories of gastritis and peptic ulcer disease. (The upper gastrointestinal tract is generally defined as including the esophagus, the stomach, the duodenum, the jejunum, and ilium.) Peptic ulcers are lesions of the gastro­ intestinal tract lining, characterized by loss of tissue due to the action of digestive acids and pepsin. It has been generally held that peptic ulcers are caused either by gastric hypersecretion, or (more often) by decreased resistance of the gastric lining to digestive acids and pepsin. Gastritis is, by definition, typified by an inflammation of the stomach mucosa. In practice, though, the disorder is manifested by a broad range of poorly-defined, and heretofore inadequately treated, symptoms such as indigestion, ″heart burn″, dyspepsia and excessive eructation. A general discussion of gastritis appears in B.J. Marshall and J.R. Warren, ″Unidentified Curved Bacilli in the Stomach of Patients with Gastritis and Peptic Ulceration″. The Lancet , 1311-1315 (1984), and in R.Greenlaw, et al., ″Gastroduodenitis, A Broader Concept of Peptic Ulcer Disease″. 25 Digestive Diseases and Sciences 660 672 (1980). As with the management of any disorder, the rapid precise, and accurate diagnosis of gastrointestinal disorders is of paramount importance. However, the diagnostic methods typically employed in the art are often slow, cumbersome, costly and may yield equivocal or inaccurate results. See, e.g., Spiro, supra . It has been discovered that many disorders affecting the upper gastrointestinal tract are mediated by bacteria, such as those of the genus Campylobacter , particularly Campylobacter pyloridis. Presently used methods of detecting campylobacter pyloridis are time consuming and expensive. In particular, it is not possible to complete a test to detect the infection within the time of an appointment of a patient with a physician. Thus, there is a need for further consultation. Campylobacter pyloridis is almost unique in its ability to produce large amounts of urease enzyme. Urease splits urea to form ammonia and carbon dioxide. It has now been discovered that the urease enzyme produced by campylobacter pyloridis is so active that it destroys all urea present in gastric juices of humans. The present invention provides a method for the detection of preformed urease present in gastric tissue infected with campylobacter pyloridis . The presence of such pre­ formed urease in gastric mucosa is pathognomonic of the infection and indicates that gastritis is present. Further, the method of the present invention has general applicability for the detection of preformed urease. For example, it may be used for the detection of preformed urease in the vomitus of patients with campylobacter pyloridis gastritis. Also, it may be used to detect the presence of preformed urease in leguminous plants. The presence of urease in these plants may be an indication of the presence of toxins in pasture which can cause gas production or bloat in animals. The methods and compositions of this invention thus provide a rapid, inexpensive, and accurate diagnosis of the presence of urease resulting from such disorders. SUMMARY OF THE INVENTION In accordance with one aspect of the present invention there is provided a composition for the diagnosis of gastrointestinal disorder in a human or lower animal subject by detection of urease in gastric material of the subject, which comprises (a) urea, preferably at a concentration of from about 10 to about 40 grams per litre; (b) a bactericide which substantially inhibits growth of urease producing organisms, preferably at a concentration of from about 1 to about 5 grams per litre; (c) a pH indicator which undergoes a colour change upon an increase of pH, at an effective concentration; and (d) water; wherein said composition has an acid pH of at least 5.0, and the pH of said composition is at least about one pH unit lower then the pKa of said indicator. Preferably, the composition of the present invention is in gel form, containing a gelling agent at a concentration of from about 5 to about 50 grams per litre. In accordance with another aspect of the present invention there is provided a method for the diagnosis of gastrointestinal disorder in a human or lower animal subject by detection of urease in gastric material of the subject, comprising the steps of obtaining a sample of gastric material from said subject, contacting said sample with a compos­ ition of the present invention, and observing any colour change in said composition. All concentrations herein are by weight of component per volume of total composition. DESCRIPTION OF THE DRAWINGS The accompanying drawings are of a preferred test device useful in the method of the present invention. In particular, Figure 1 is an isometric view of a test device of this invention, with a cutaway exposing a container containing a composition of this invention; Figure 2 is a sectional view of the device of Figure 1; and Figure 3 is an isometric view of the device of Figure 1, showing the device in use. DESCRIPTION OF THE INVENTION As used herein, ″an effective concentration″ of indicator is a concentration of indicator which effects a readily-discernible colour of the composition of this invention when used according to the method of this invention. Typically, the indicator is present at a level of from about 2 to about 150 milligrams per litre, preferably from about 20 to about 150 milligrams per litre. It is found that amounts of urea in the range from about 10 to about 40 grams per litre of the composition of the present invention are sufficient for reaction with the preformed urease present in typical specimens to produce a colour change. When the urease contacts the urea a reaction follows which produces ammonia. The presence of ammonia increases the pH of the adjacent portion of the carrier medium and if sufficient ammonia is produced there is a resultant colour change because of the presence of the pH indicator. More preferably, the composition of the present invention contains urea at a concentration of from about 20 to about 40 grams per litre. As used herein, ″gastrointestinal disorder″ encompasses any disease or other disorder of the gastrointestinal tract of a human or lower animal. Such gastrointestinal disorders include, for example: disorders not manifested by presence of ulcerations in the gastric mucosa (herein ″non-ulcerative gastrointestinal disorder″), including chronic or atrophic gastritis, gastroenteritis, non-ulcer dyspepsia, esophogeal reflux disease and gastric motility disorders; and ″peptic ulcer disease″, i.e., gastric and duodenal ulcers. In particular, ″gastrointestinal disorder″ refers to such disorders of the upper gastro­ intestinal tract caused or mediated by bacteria, including campylobacter-like organisms (herein ″CLO″), e.g., Campylobacter pyloridis . Such CLO include those described in J.R. Warren and B.J. Marshall, ″Unidentified Curved Bacilli on Gastric Epithelium in Active Chronic Gastritis″, The Lancet 1273-1275 (1983). Urea is of the formula H₂NCONH₂, and is a naturally occurring product of protein metabolism. Urea for use in the compositions of this invention is available from a variety of commercial sources. As a basis for this invention, it has been found that gastric materials from humans or other animals having gastrointestinal disorders contain relatively large quantities of urease (urea amidohydrolase), which hydrolyses urea to ammonia and carbon dioxide (or ammonium carbonate). The compositions of this invention serve, in part, to detect the presence of urease through its hydrolysis of urea. The pH indicators useful in this invention are weak acids, with sharply different colours in their dissociated (ionized) and undissociated (neutral) states. The indicators useful herein preferably have pK a values of from about 6.5 to about 8.5, preferably from about 7.0 to about 8.0. The colour exhibited by the indicator in the present composition will depend upon the pH of the composition, the particular indicator used, and the dissociation constant (K a ) for that indicator (i.e., pK a = -log 10 K a ). As the colour exhibited by the indicator changes over a range of pH values (pH = -log 10 [H⁺]), the indicators useful in the present composition change colour over a pH range of from about 5.5 to about 9.0, preferably from about 6.5 to about 8.5. The pH of the composition of the present invention is, accordingly, adjusted to a pH at least about one pH unit lower than the pK a of the indicator used (i.e., having a hydrogen ion concentration [H⁺] ten times less than (10% of) the hydrogen ion concentration in a solution having a pH equal to the pK a of the indicator). Preferably, the pH is adjusted to a pH about two pH units below the pK a of the indicator. Adjustment of the pH of the present compositions can be effected by addition of a base (e.g., sodium hydroxide) or an acid (e.g., hydrochloric acid or citric acid). Thus, preferably, the pH of the composition of this invention is adjusted to a pH of from about 5.0 to about 6.5, more preferably from about 5.0 to about 6.0. Indicators among those useful herein include p-nitro­ phenol, bromothymol blue (dibromothymolsulfonphthalein), phenol red (phenolsulfonphthalein), neutral red (2-methyl-3-amino-6-dimethylaminophenazine), quinoline blue (cyanine), cresol red (o-cresolsulfonphthalein), metacresol purple (m-cresolsulfonphthalein), and thymol blue (thymolsulfonphthalein). Bromothymol blue, phenol red, neutral red and cresol red are preferred indicators for use in the compositions of this invention. Indicators among those useful herein are described in the The Merck Index (9th ed. 1976). The bactericide incorporated in the compositions of this invention is one or more materials which substantially inhibit the growth of urease-producing organisms in the composition. The bactericide is most preferably present in an amount in the range from 2 to 4 grams per litre of the composition. Bactericides useful in this invention include sodium azide and methylhydroxybenzoate. Methylhydroxybenzoate is a particularly preferred bactericide. The specific amount of bactericide to be used in this present composit­ ion depends upon factors well known in the microbiological arts, such as the particular bactericide used, and the bactericidal properties (if any) of the other components in the present compositions. The presence of the bactericide means that in the absence of an excess of alkali the test will only give a positive result when there is preformed urease present in the sample of gastric material. The compositions of this invention may contain optional components which affect the performance or physical characteristics of the compositions. Such additional components must not, however, interfere with the indicator, as by obscuring the colours exhibited by the indicator. The compositions of this invention preferably contain a gelling agent, so that the compositions are in a semi­ solid state at ambient conditions. A particularly preferred gelling agent is agar, preferably present at a level of from about 5 to about 50 grams per litre, more preferably from about 10 to about 20 grams per litre. The agar used in the compositions of this invention is readily available from a variety of commercial sources. Typically, agar (a polysaccharide complex) is extracted from the agarocytes of certain algae. Also, preferably, the agar used in the present composition is nonnutritive, i.e., does not support the growth of microorganisms In fact since the test of the present invention is intended to detect only the presence of preformed bacterial urease, the usual microbial nutrients may be omitted from the compositions. A particularly preferred optional component of the present invention is a buffer. As stated above, the compositions of this invention are adjusted to a pH at least one pH unit below the pK a of the indicator used. Thus, preferably, the pH of the present composition is from about 5.0 to about 6.5, more preferably from about 5.0 to about 6.0. This pH of the final composition is preferably effected by the addition of a suitable buffer. Such buffers are well known in the chemical art, including the use of such weak acid salts as sodium bisulfate, sodium acetate and sodium phosphate. The total amount of buffer incorporated in the present composition will depend upon the total amount of urea incorporated in the composition, such that the buffer does not prevent sufficient change in composition pH (resulting from hydrolysis of the urea present), so as to cause a change in the colour of the indicator used. However, the buffer is preferably present in a concentration sufficient to prevent substantial changes in composition pH, and (thereby) spurious indicator colour changes, due to chemicals other than preformed urease enzyme in the gastric material sample to be analyzed. Typically, then, the buffer is incorporated in the present composition at concentrations of from about 50 to about 2000 milligrams per litre. (As used herein, the buffer concentration includes the concentration of the buffer salt and of the acid used to effect pH adjustment of the composition.) As used herein, ″gastric material″ refers to any material obtained directly or indirectly from the upper gastro­ intestinal tract of a human or other animal. Such materials include, for example, gastric epithelium, gastric mucosa, and digestive fluids. Samples of such materials, for use in the method of this invention, may be obtained by any of a variety of well known methods, according to sound medical practice. Such methods include, for example, obtaining the sample by biopsy of the subject, obtaining the sample from the vomitus of the subject, and obtaining the sample from nasal gastric aspirate. As used herein, ″contacting″ the sample with the compos­ ition of the present invention, refers to any method which effects substantial interface between a composition of this invention and the sample gastric material, for a time sufficiently long so as to allow the hydrolysis of urea by any urease present in the sample to an extent sufficient to produce a detectable colour change. Such time is typically longer than about five minutes and is typically in the range from 5 to 15 minutes. Preferably, though, the gastric material sample is immersed, or substantially immersed, in a composition of this invention. Also, preferably, care is taken so as to avoid contamination of the gastric material sample with organisms from a source other than the stomach of the subject to be diagnosed. In the event that the gastric material used constitutes digestive fluids, then a preferred optional step in the present processes is testing the pH of said gastric material. Preferably, then, the sample is contacted with a pH-test composition which is an aqueous solution of the particular indicator used in said composition of this invention (without urea), adjusted to the same pH as said urea-containing composition of this invention. If the gastric material effects no change in the indicator colour of the pH-test composition, then the material is of an acid pH, and may be used directly in the contacting step of the process of this invention described above. If, however, the colour of the pH-test composition changes, then the pH of the gastric material should be acidified, as by addition of an acid. The observing step of this process entails detection of any colour change in the composition colour, to the colour exhibited by the indicator in its dissociated state. Failure of the composition to change colour after about twenty-four hours reflects a negative test result. In most positive cases the physician will have the results available before the patient leaves, and he may, therefore, prescribe therapy immediately. Further tests may not be required. When small numbers of bacteria are present a delayed reaction may occur which can be read the next day but this occurs in less than 10% of positive cases. As all patients infected with campylobacter pyloridis have gastritis a positive result also indicates the presence of this condition. Also, as indicated above, the present invention may be extended to veterinary use such as testing of pasture to ensure that it is at the right stage of maturity for grazing and does not contain excessive quantities of plant urease. The method of this invention is preferably performed using a device, herein ″diagnostic device″, which contains a quantity of a composition of this invention in an easily-handled container. Such devices may comprise: (a) a container, having an opening aperture area of from about 20 square millimetres to about 200 square millimetres and having a total contained volume of from about 40 cubic millimetres to about 1000 cubic millimetres; and (b) a cover for said container, affixed to said container by a means which allows said cover to be moved so as to open and close said container opening; wherein said container contains from about 0.04 to about 2.0 millilitres of a composition of this invention. Preferably, the container contains from about 0.20 milli­ litres to about 0.40 millilitres of a composition of this invention. One such preferred diagnostic device is pictured in Figures 1 through 3 of the Drawings. Figure 1 and Figure 2 show a container 3 which is formed as a concave form, or well, in a continuous, flat holder 2 , which is a sheet of rigid, or semi-rigid, water-impermeable material. The container contains a composition of this invention 4 . A cover 1 , is affixed to the container, and made of a flexible, water-impermeable material, and is of the same dimensions as the rigid holder sheet 2 . As shown in Figure 1, a label 5 is preferably affixed to or printed directly on the flexible cover 1 . Figure 3 shows the diagnostic device with the flexible cover partially removed, opening the container 3 , thereby allowing a sample of gastric material 6 to be placed in the test composition 4 using a suitable device, such as a forceps 7 . The flexible cover 1 is affixed to the rigid container/holder material 2 by a means allowing its removal, and opening and closing of the container, such as by an adhesive which remains functional (tacky) throughout repeated openings and closings of the container. Preferably the cover material is sufficiently thin so as to allow piercing by a syringe and injection of a liquid gastric material directly into the composition, without opening the cover. The diagnostic device described above enables positive results to be easily seen and the device is flat enough to be carried in the physician's pocket or attached to a patient's endoscopy report which enables the test to be examined for late positive results. The following non-limiting examples illustrate the compositions, methods and devices of the present invention. EXAMPLE 1 A composition, according to this invention, was made comprising the following components in water: *: phenol sulfonphthalein indicator, having pK a = 7.9, exhibiting a yellow colour in undissociated state (below pH 6.4) and red colour in dissociated state (above pH 8.2) The components, except urea, were dissolved in 100 millilitres of water, heated to 100°C, and stirred until the solution was clear. The solution was then cooled to below 45°C, the urea added and stirred, and the solution pH was measured to be 6.0. The composition was then poured into containers, in a device of this invention, each container receiving approximately 0.3 millilitres of the composition. The composition was allowed to cool to ambient temperature, forming a gel in the container and having a deep yellow colour. A composition made as above is used in a process of this invention, by obtaining a sample of gastric mucosa from the stomach of a human subject presenting symptoms of gastritis. The mucosa sample is then inserted into the composition and the colour observed. After approximately 15 minutes, a red colour is observed. indicating the presence of CLO-mediated gastrointestinal disorder. EXAMPLE II A composition is made, according to the present invention, comprising the following components in water: The components are dissolved in 100 ml water, heated to 100°C, and the pH adjusted to pH 5.50. Approximately 0.3 millilitres of the composition is poured into the container of a device of this invention and allowed to cool, forming a gel. The composition and device are used in a process of this invention by obtaining a sample of vomitus from a human infant subject suspected of having gastritis. A sample of the vomitus is drawn into a syringe, and a portion injected into a pH-test composition comprising 0.6 milligrams of phenol red in 10 ml of water, adjusted to pH 5.5. The colour of the pH-test composition does not change. Thereafter, the remainder of the vomitus sample is injected into the composition of this invention, by piercing the cover of the device. The colour is observed for approximately 20 minutes, noting a change in colour from deep yellow to red, and indicating the presence of gastrointestinal disorder. Modifications and variations such as would be apparent to a skilled addressee are deemed within the scope of the present invention.

Claims: 1. A composition for the diagnosis of gastrointestinal disorder in a human or lower animal subject by detection of urease in gastric material of the subject charaterised in that it comprises (a) urea; (b) a bactericide which substantially inhibits growth of urease producing organisms; (c) a pH indicator which undergoes a colour change upon an increase of pH, at an effective concentration; and (d) water; wherein said composition has an acid pH of at least 5.0 and the pH of said composition is at least about one pH unit lower than the pKa of said indicator. 2. An composition according to claim 1, characterised in that it contains urea at a concentration of from about 10 to about 40 grams per litre. 3. A composition according to claim 2, characterised in that it contains urea at a concentration of from about 20 to about 40 grams per litre. 4. A composition according to any one of the preceding claims, characterised in that the bactericide is present at a concentration of from about 1 to about 5 grams per litre. 5. A composition according to claim 4, characterised in that the bactericide is present at a concentration of from about 2 to about 4 grams per litre. 6. A composition according to any one of the preceding claims, characterised in that the pH indicator has a pK a of from about 6.5 to about 8.5. 7. A composition according to claim 6, characterised in that the pH indicator has a pK a of from about 7.0 to about 8.0. 8. A composition according to any one of the preceding claims, characterised in that the pH indicator is present at a concentration of from about 2 to about 150 milligrams per litre. 9. A composition according to claim 8, characterised in that the pH indicator is present at a concentration of from about 20 to about 150 milligrams per litre. 10. A composition according to any one of the preceding claims, characterised in that the pH indicator is phenol red. 11. A composition according to any one of the preceding claims, characterised in that it has a pH of from about 5.0 to about 6.5. 12. A composition according to claim 11, characterised in that it has a pH of from about 5.0 to about 6.0. 13. A composition according to any one of the preceding claims, characterised in that it additionally comprises a pH buffer. 14. A composition according to any one of the preceding claims, characterised in that it additionally comprises a gelling agent. 15. A composition according to claim 14, characterised in that the gelling agent is present at a concentration of from about 5 to 50 grams per litre. 16. A composition according to claim 15, characterised in that the gelling agent is present at a concentration of from about 10 to 20 grams per litre. 17. A composition according to claim 14, 15 or 16, characterised in that the gelling agent is agar. 18. A composition according to any one of the preceding claims, characterised in that it is free from microbial nutrients. 19. A method, for the diagnosis of a gastrointestinal disorder in a human or lower animal subject by detection of urease in gastric material of the subject, characterised in that it comprises the steps of: (a) obtaining a sample of gastric material from said subject; (b) contacting said sample with a composition of any one of claims 1 to 18; and (c) observing the colour of said composition; wherein a change in colour of said composition indicates the existence of a gastrointestinal disorder in said subject. 20. A device, for use in the diagnosis of gastrointestinal disorder in a human or lower animal subject by detection of urease in gastric material of the subject, characterised in that it comprises: (a) a container, having an opening aperture area of from about 20 square millimetres to about 200 square millimetres and having a total contained volume of from about 40 cubic millimetres to about 1000 cubic millimetres; and (b) a cover for said container, affixed to said container by a means which allows said cover to be moved so as to open and close said container opening; wherein said container contains from about 0.04 to about 2.0 millilitres of a composition of any one of claims 1 to 18. 21. A device, according to claim 20, characterised in that said container contains from about 0.20 millilitres to about 0.40 millilitres of a composition of any one of claims 1 to 18.

Inventors: MARSHALL, BARRY JAMES, M.D.
Applicants: MARSHALL, BARRY JAMES, M.D.
Application year: 1986
Main classification: C12Q1
IPC codes: C12Q1, C12M1