MXPA95004461A - Test composition for the rapid detection of helicobacter pylori in a biopsiagastr tissue - Google Patents

Abstract

The present invention relates to a novel test composition for the diagnosis of gastrointestinal disorders, this new test composition is particularly useful for the diagnosis of peptic ulcers, including both gastric and duodenal ulcers, detecting the enzyme urease associated with the bacterium Helicobacter pylori in samples of endoscopically obtained biopsy, the test is not intended for in vivo use but only for in vitro diagnosis

Description

COMPOSITION OF TEST FOR THE RETEST DETECTION OF HE ICOBACTER PYLORI IN A GASTRIC BIKPSY TISSUE BACKGROUND AND PREVIOUS TECHNIQUE 5 Peptic ulcers, which were once thought to result from pressures, or excess acid, or a reduction of the mucosal defense factors in the stomach are now the majority of cases, considered as the result of i ^? Bacterial infection by Helicobacter pylori. The mounting evidence for this effect is well documented in Helicobacter pylori in Feptic Ulceration and Sastritis edited by Barry 3. Marshall, Richard W. McCallum and Richard L. Guerraut, Black.well Scientific Publications, Boston, E.U. A., Pertinent Chapters, in this work, chapter four is included, The Epide ilagy of Helicobacter pylori infection by D. N. Taylor and M. 3"Blaser; Chapter Seven, Laboratory Diagnosis and Handling of # Helicobacter pylori, by T. U. We & and chapter twelve, Practical Diagnosis of Helicobacter pylori by B. 3. Marshall.

The history of the discovery of H. pylori and its association with gastrointestinal diseases is described extensively in "Marshall's Hunch" The New Yorker magas ine, page? U-72, September 20, 1993 and "The Doctor Who Would not Accept islo. " Reader s Diqest magas pe, pages J20-12M-, October 1993. 25 The effect of the treatment of the infection Helicobacter pylori in a long-term recurrence of gastric or duodenal ulcer described by David Y. Graham et al. In? ^ Annals of Internal Medicine 1992; 116: No. 9. Helicobacter pylori has now shown to be the causal agent of the ayoria of chronic gastritis cases (1). 5 See the bibliography below. And, it is now known that, in the absence of aspirin, nonsteroidal anti-inflammatory drugs or hypersecretory states, this bacterium is directly involved in the production of peptic ulcer diseases such as duodenal and benign gastric ulcers (2).

I? ÍtfM And now there are epidemiological data correlated with the presence of H. pylori with gastric cancer (3). The eradication of H. pylori gastritis by antibiotics has been shown to cure peptic ulcers and prevent their recurrence (4,5). Having seen that the H. pylori bacterium is present in samples of gastric biopsies endoscopically obtained from patients with both gastric and duodenal ulcers and it being known that the enzyme urease is always associated with ^^ that bacterium, the concept of diagnosing the presence of such ulcers by testing biopsy samples for urease is suggested by itself. Chemical tests for urease are already known in the art. In said test, a broth containing urea provides a positive urease reaction (hydrolysis of urea) urea + HsO urease - > NHx-3 + CO ^ ,, as indicated by a change in the color of the Bacta red indicator Phenol from yellow (pH ß.ß) to red to cherry color at a pH of ≥1 or more alkaline due to the production of ammonia and / or ammonium carbonate by the urea-urease reaction. See Difco Manual, 9a. edition, Difco Laboratories, Detroit, Michigan, (1653). The urea broth described in Difco Manual was apparently used by B. 3. Marshall in the work described in 5 Rapid Diagnosis of Campylobacteria Associates with Gastritis, The Lance. June 22, 1965. This type of urease test has become commercial and clinical use. In the United States, a commercial test product is sold under the brand name of lß4ß "Clotesfc". This product is described in the patent of E.U.A. 4.74-6,113 issued to Bary 3. Marshall, on May 31, 1966. Since this test is based on the reaction of the urease enzyme with urea it will be evident for any chemical that this reaction can be used to detect already is urea or urease, whichever is present in the material to be tested, by the addition of another material in a test solution. The course of the reaction can be MtL followed by the use of known dye indicators that change the color with the pH change of the reaction mixture as the reaction proceeds. Therefore, references to the detection of urea in the blood or elsewhere are also allowed. Such references include Mast patent of E.U.A. 3,395,062 issued July 30, 1966 for a urea test in aqueous fluids; attente of E.U.A. 4,101,362 of Chang issued on June 16, 1976 for a reagent for the determination of urea in biological fluids; and Modrovich patent of E.U.A. 4,262,316 issued August 4, 1961 ^ r for enzymatic solutions stabilized to determine urea. The detection of preformed urease for the diagnosis of gastrointestinal diseases through the use of water, urea, a bactericide and a pH indicator, is described by B.

J. Marshall Derwent Publication C-66-141647 (1966). The use of urease to determine urea is also described in Derwent Publication C64-12670 (1965). A non-aqueous analytical unit for the determination of urease, consisting of urea, a pH regulator with a pH of 5 to 7.5 and a pH indicator is described in C90-066529 (1990). The test composition sold under the trade name "Clotest" as described in the US patent. of Marshall 4,746,113, has the following composition: 15 a) Urea 10-40 g / liter b) Bactericity 1-5 g / liter c) A color indicator a pKa of approximately 6.5-6.5 effective amount 20 d) Water, wherein the one for making the composition has a 1 liter pH of about 5.0-6.5 and wherein the pH is about 25 a pH unit lower than the pKa of the indicator.

The composition of the Marshall patent uses phenol red only as the dye indicator. The method of the Macsha.ll patent is initiated by obtaining a sample of gastric material from the patient by endoscopic biopsy according to the procedures *? well known in the art. The biopsy specimen is then contacted with the test composition set forth above. The test composition is said to be used in liquid form as a rule but it can also be in a gelled form. the change in the color of phenol red from yellow at the initial pH (about 5.O-6.5) to red at a pH of about 6.6-9 by the formation of ammonia and / or ammonium carbonate in the hydrolysis reaction, IHfj indicates the presence of urease and the bacteria Helicophaecter pylori that will be identified in the biopsy specimen. Since the test described by Marshall has proven to be highly useful, it has been recognized that it can be improved in certain aspects. In the biopsy of the gastric mucosa of the Marshall's urease test containing H. pudor i, solution or placed on an agar gel containing urea, an indicator of phenol red and pH regulators. The urease in H. pylori g 'j converts the urea to ammonia, which increases the pH and returns the color of the agar from yellow to red, indicating a positive test. According to the package insert in the commercial available main phenol red test (called CL0testR), it is recommended that the test be incubated at 30-40 ° C for 3 hours and that it may take up to 24 hours to develop a positive test . This test depends on the passive diffusion of urease from the cell wall of the bacteria to the agar gel test solution. In addition, by operating as it does at a pH above 6.5, the test ** can give a positive result with a bacterium other than H. pylori and thus not entirely specific for Helicobacter pylori. Specifically, proteus, Pseudomonas and E. Coli species can cause a color change at this level and give a false positive test. Therefore, it is evident that there is a need in the art for a highly specific test for Helicobacter pylori. which allows to diagnose a gastrointestinal disorder during a single visit of the patient of normal duration to the doctor or to the clinic.

BRIEF DESCRIPTION OF THE INVENTION The present invention resides in an improved test composition for detecting the presence of Hel icobacter pylor i in a biopsy specimen for the purpose of J diagnose gastrointestinal diseases. The greatest improvements in the new composition among others reside in the improved specifity of the test for H. pylori; a color change not masked by the specimen color of the biopsy and which gives an indication of the degree of infection; and increased rapidity of the test allowing diagnosis during an initial visit of the patient. The new preferred test composition is in the form of an aqueous gel containing: Urea about 1% Approximately 1% N-octyl glucose (pyranoside) (NQG) approximately 1% NaHffiP0l + 1. 5 to 3. 5 mM Dye supply material 25% Preservative agent approximately 0.% Deionized water rest 100% The dye supply material contains: Methyl Red 1% Bromotimol Blue Red 0.1% Ionized Water The preservative agent contains: M ilparaben 0.16% Pro ilparaben 0.02% all by weight of the total test composition in deionized water. Preferred compositions as set forth above and below have a pH of about 5.2 to 5.3 at 55 ° C. Other useful compositions may have an initial pH in the range of approximately 4.9 to approximately . 6. The proportions of the various ingredients may vary as follows: Urea from approximately 0.5 to approximately 2.0%; 6 ^ agar from about 0.4 to about 1.4%; '** N-octyl ioglucose ÍNOG) from approximately 0.2 to approximately 1.2%; monobasic sodium phosphate of approximately 1.5 mM to about 3.5 mM; methyl red from about 0.05 to about 0.40% immately; bromothymol blue from approximately 0.005 to approximately 0.040%; preservative agent, sufficient amount, approximately 0.01 to about 0.2% total, eg, a preferred preservative agent being a combination of approximately 0.16% methylphenidate and approximately 0.02% propylparaben; water > 96% to make up to 100% of the composition; all percentages being by weight of the total composition. The color indicators different from those previously mentioned as preferred can be used in adequate quantities. Suitable compositions of J? Dye indicators may include: broccoli purple green with bromocresol green or bromocresol green with red dealizarin 6 and, of course, with phenol red. Bromocresol purple is yellow at a pH of 5.2 and purple at a pH of 6.6 and, therefore, may be the basis for several useful combinations of dyes. A useful combination of three dyes may be composed of, e.g. , 0.133% methyl red, 0.133% bromothymol blue or 0.133% bromocresol purple.

The preservatives, ethylparaben and i ', propylparaben, are well known in the art as effective food preservatives against bacteria, yeasts and molds. Its use is restricted up to 5 percent 0.1% in food. These preservatives are very effective at acid and neutral pH levels. They are of limited solubility in water but this is overcome by using them in combination, such as methylparaben and propylparaben as previously mentioned and as suggested in Antimicrobial iQ ^ ß Preservatives in Pharmaceuticals, p. 701, (reference supplied). Sodium phosphate monobasic is not used as a pH regulator but is used to provide the composition with the desired initial pH level of approximately 5.3 on the scale of approximately 4.9 to 5.6.

The absence of pH regulators in the new test compositions allows the pH to change more rapidly. The color reaction, therefore, is usually available for reading when the patient recovers from endoscopy. In reality, over 95% of all tests become positive in 20 minutes, an occasional test can take as long as 1 hour, but all are positive in 4 hours. Therefore, there is no need to wait 24 hours to ensure a final result with previously available test compositions.

N-actylglucase, a cell wall detergent, is used to help release the urease enzyme from the biopsy specimen as will be explained later. T M "'The test composition is in the form of a soft gel that is capable of receiving the biopsy specimen and contacting it intimately in order to ensure interreaction as quickly as possible.

DETAILED DESCRIPTION OF THE INVENTION The new test composition is formulated by mixing _ ^. Supply solutions prepared as follows: 1.- Solutions of dye supply material: A. 1.0% methyl red (ICN catalog # 151676) in distilled water. B. 1.0% bromothymol blue (ICN catalog # 150524) in distilled water. Procedure: (1) prepare a 1:10 dilution of gt blue bromothymol by mixing one part of the 1% solution with 9 parts of distilled water; (2) prepare a dye mixture consisting of equal parts of 1% of a solution of methyl red and 0.1% of bromothymol blue. 2.- Other solutions of reagent supply material: all prepared in distilled or deionized water.

A. 6.0% NOG (N-oc i 1-glucose) (Sigma) B. Monobasic sodium phosphate 0.25 M C. 10.0% urea D. 2.0% Bacto agar (DIFCO) 3.- To prepare the mixture of final reagent: A. An autoclave with a 2% agar solution (121 ° C for a minimum of 15 minutes at a pressure of 6.61 kg) then keep in a 55 ° C water bath until it is used. B. Prepare 2X (double concentration) of the following mixture. 5.3 ml solution of dye supply material. 2.5 ml of 6% NOG 0.2 ml of a solution of sodium phosphate monobasic 2.0 ml of a solution of 10% urea Then, sterilize with a filter of 2 ml ras and mix with an equal volume of hot agar, passed through the autoclave; distribute while it is liquid (45-50 ° C). For example, the above mixture (10 ml) + 10 ml of agar produces 20 ml of the final reagent solution, which is then used at 200 microliters per "unit" test. 4.- Final concentration of reagents in the preferred rapid urease test composition: A. methyl red: O.1326% (a dilution of 3.77 times of a 0.5% solution), blue of 0 bromothymol: 0.0133% (a dilution of 3.77 vsces of a 0.05% solution) B. 1% of NOG 5 C. Monobasic sodium phosphate 2.5 mM D. 1% urea E. 1% F. 0.2% agar conservative (0.16% ethylparaben and 2% propylparaben). IQ ^ 5.- Final pH of the rapid urease test reagent: * F pH 5.3 ("peach" color) An important advantage of the new test composition lies in the use of the bleaching indicator combination, as opposed to a single dye indicator, which provides a broad spectrum of colors depending on the concentration of H. pylori infection and the resulting change in pH. jájji The colors of the dye spectrum are to vary the pH are as follows: 20 pH Color spectrum (lighter color with smaller volume) ini il 5.3 dark peach 25 5.4 darker peach negative for H. pylori 5.6 yellow-coffee 5.6 greenish-brown ^ 0 ^ oO light green positive for H. pylori 6.2 dark green 5 6.4 darker green 6.6 emerald green 6.6 emerald green, darker 7. 0 aquamarine blue (light blue) 7. 2 blue aquamarine medium Qdß? 7.4 dark blue aqua blue 7.6 dark blue 7-6 dark "blue" ink The preferred test composition has an initial pH of approximately 5.3 and is peach-colored to yellow-brown. At a pH of about 6.0, the test solution becomes light green, which is also considered as a negative or indeterminate. A positive test for H.

The pylori occurs when the test solution becomes dark green at a pH of about 6.2 or greater. This is in contrast to the commercially available test compositions, which remain yellow (negative) at a pH of 6.2. This scale can be further refined, if desired, by reading light green as negative; green a little more 5 dark as probably negative, dark green as weakly positive; darker green and emerald green or blue - light as moderately positive; and blue sea water more * Dark or dark blue as strongly positive. This color extension provides the doctor with faster quantitative and diagnostic information. A diagnosis so regular can be done 2 10 minutes and thus allow the effective treatment at a time in an effective way. As noted above, 95% of the tests, if they are positive, show the result in 1 hour and all in 4 hours; Most tests that show a positive result K? Ggw are from 2 to 10 minutes. In addition, the green and blue colors are clearly different from red and pink, which are used in the phenol red test. In view of the fact that gastric biopsies are pinkish red, it can be difficult to read such tests ep limits cases when the indicated one is only phenol red. Since the optimal urease enzyme reaction for H.

ML pylo i takes place at an alkaline pH, it is a very important advantage of the present invention that a reaction occurs positive on the acid side at a pH as low as 6.2. Of course, the pH can rise as much as 7.6 in a highly positive test. This improves the speci fi city of the improved test since other organisms such as Proteus, Pseudomonas and E. coli. may be present in the sample of biopsy, do not react in acid pH reactions. The test compositions of the prior art only produce positive results on the alkaline side at a pH above 7 and in this way they are less specific for H. pylori and can give false positives due to the presence of Proteus and other organisms. With the test composition of the present, Proteus only produces a very light green test, which is clearly a negative for H. pylor i. This broad appearance of colors from peach to light green, green, light blue, and dark blue also provides a semi-quantification of the degree of infection, which was not the case. possible with prior art compositions employing a single indicator such as phenol red which only turned yellow to red. The initial pH of the test solution of about 5.3 is achieved and maintained during the use of monobasic sodium phosphate (NaHaP01 +), which ensures that the test reaction is initiated at an acidic pH where it is specific for H. pylori. Sodium phosphate monobasic does not J ^. it acts as a pH regulator, but merely reduces the pH to the desired initial level. The purpose of the preservative agent is to ensure that the test solution is and remains sterile, that is, that it does not contain any organisms initially or that an organism grows after inoculation with the biopsy sample, which could react with the medium. test by producing a false positive result. In other words, the preservative agent ensures that the test medium reacts only with any H. pylori present in the biopsy sample. As noted above, this is achieved in a preferred test composition by the addition of 0.16% methyl paraben and 0.02% pro-ilparaben, a combination and concentration of preservatives known in the art which is effective in preventing the growth of bacteria in food and pharmaceutical compositions. As noted above, other preservatives known in the art can also be used. - *! ^. A very important advantage of the new test composition lies in the use of NOG as a cell wall detergent. This agent is known in the art and is effective as a cell wall detergent. Approximately 95% of H. pylori urease is present in the cell wall of the biopsy sample. The NOG, which helps a faster release of the urease enzyme from the biopsy sample makes it more available for the reaction as confirmed by the Jj laboratory and clinical studies in patients. See characterization of Helicobacter pylori Urease and Purification of its Subunits by Evans, D.J.Jr., D.G. irkpatr ick and S.3. Graham, D.Y. Microbiology Pathogenesis 1991, Vol. 10, pp. 15-26. The smoothness of the agar that allows the biopsy test sample to be easily imbibed in the gelled test medium is a significant improvement over the current prior art and commercially available test products wherein the gel is So firm that its rigid makes it difficult for the technician to push the sample towards the test gel since it has the tendency to slip. The milder gel of the test composition of the present invention not only more easily accepts the test specimen when it is pushed, but also allows the gel to run off completely around the biopsy sample and makes immediate intimate contact. of urease within the mucous membrane of the sample with the test medium. This is a significant improvement over the currently available test products where the stiffer gels do not flatten around the sample and tend to leave air cavities limiting contact between the sample and the test composition. It will be noted, therefore that not only the use of NOG but also the use of a softer gel facilitates contact and thus accelerates the test. As previously observed, the initial pH of the ^ p test solution which is approximately 4.9 to 5.6, preferably approximately 5.3, is well on the side scale acid. While the optimum urease enzyme reaction of H. pylori is on the alkaline side of the scale, this reaction remains sufficiently effective under acidic conditions to be entirely satisfactory. In view of other bacteria such as Proteus and Pseudo onas react with urease alone under alkaline conditions, the operation of the new test composition under conditions cides excludes any false positive results due to the ^ presence of bacteria other than H. pylori. Therefore, the new test is specific to the presence of H. pylori. and in this way it is more reliable than the tests of the technique above for the diagnosis of gastric diseases.

COLLECTION AND MANAGEMENT OF BIQPSIA Patient preparation When used in suboptimal therapy of antibiotics and bismuth, they can suppress but not eliminate the H. pylori organism. Therefore, the patient must not have used these agents for several weeks before the test. Following inadequate therapy, the organism can regrow in an irregular manner and may not be detected by random biopsies. Ideally, the patient should not have received the proton pump inhibitor, omeprazole, and ^^ that this drug has been shown to inhibit the body's growth. 20 Evaluation and preparation of the test A test kit containing the test composition on agar must be evaluated before the test is conducted. The agar should have a light peach color. A colour Very light green may indicate contamination and the subsequent result should be interpreted cautiously. A dark green color of the blue agar or agar should be discarded. ™ Heat will increase the speed of most enzyme reactions. Therefore, it is recommended that the test be heated by hand for a few minutes before the test. A small heating unit or incubator (35-40 °) will increase the reaction rate in lightly infected samples.

LIMITATIONS AND PRECAUTIONS False positive Studies indicate that urease in proteus, pseudomonas and E. coli is not active at an acidic pH where the new tests are initiated. This aspect tends to separate the reactivity of urease from H. pylori from others. Patients with complete achlorhydria (pernicious anemia, previous gastric surgery and suppression of long-term acidic drugs) may have gastric bacterial growth due to other urease-containing bacteria. In general, these bacteria have less or less urease and generally do not produce a rapid positive test.

False negative Several factors can produce a false negative result. 5 a. Irregular distribution of infection: The disease has been shown to occur in a distribution ? ^ irregularly so that two biopsies of the cavity are recommended, usually in the immediate pre-pyloric area and on the minor curvature of the proximal cavity. 5 b. Intestinal Metaplasia: H. pylori does not colonize intestinal metaplasia so that if this mucosal change is extensive, a false result may occur. Again, multiple biopsies are recommended. c. Antibiotics; H, pylori is sensitive to many anicrobials including bismuth (Pepto-Bismol). If patients have used an antimicrobial in the past, the organism can be suppressed but usually eliminated.

In this situation, an inadequate number of bacteria containing urease can be obtained. Women who take short courses of metronidazole for vaginal infections may ^ t delete but not delete the. bacterium. If antibiotics have recently been used, multiple biopsies should be done.

In difficult or questionable cases of infection, additional testing should be considered by histology, serolagy, and 14C breath test. The test composition of the present invention is only intended for in vitro diagnostic use.

STORAGE Í The new test compositions should be stored at 2-6 ° C in the shipping container.

STABILITYi The new test composites are stable and provide results that can be reproduced accurately for up to 12 months. In order to further explain the invention, the following clinical examples are presented.

CLINICAL EXAMPLE I Pacien e MAM lOafifc. This 57-year-old woman came to the office with severe symptoms of dyspepsia and nausea. He had a history of peptic ulcer disease dating back 20 years, endoscopic examination of the stomach and duodenum showed severe peptic ulcers of the stomach and duodenum. The stomach biopsies tested during the method of the present invention were positive for the Helicobacter pylori organism containing urease. Two blood energetic tests n. They were also positive (Quick-Vue and Omega Lab H. pylori Ab Index). The histological representation subsequently showed active gastritis with idenification of Helicobacter pylori organisms. Since the diagnosis was made immediately, appropriate therapy for the infection was initiated in the initial visit to the urine.

EXAMPLE II Patient KF This 31-year-old woman complains of nausea and vomiting. An endoscopic examination of the upper gastrointestinal tract was performed. A normal examination was observed, biopsies of the stomach were obtained since it is known that a normal endoscopic examination can continue to contain organisms of Helicobacter pylori and acute gastritis. A test of the lOjßfc. present invention was performed immediately with negative results. Therefore, a diagnosis of ulcer-free dyspepsia was made, and the patient was treated appropriately with safety and acid suppression drugs. Subsequent histological examination in other stomach biopsies demonstrated findings normal. Na gastritis was present or normal findings were demonstrated. Ho gastritis existed to na Helicobacter pylori organisms were found. The result of the test of the preventive invention was, therefore, confirmed. ? EXAMPLE III Patient AO This 44-year-old Hindu woman suffers from anemia.

A previous operation of the stomach was performed (gastroenteritis) for peptic ulcer disease in 1977. As part of their medical research, an endoscopic examination of the stomach was performed. The results of surgery € previous were shown but the test was, otherwise, normal. Since ulcers were diagnosed previously, biopsies were obtained for Helicobacter pylori. The normal commercial urease test (CLOtest tests) and the test of the present invention were both performed. The two initially were negative, but the next morning, the CLOtest test showed a positive result, while the test of this invention was negative. The subsequent analysis of the histology in the biopsies did not show gastritis or Helicobacter organisms. Therefore, the test of the present invention in this case provided the correct diagnosis, whereas the CLOtest test showed a false positive result. Due to these results, no antibiotic therapy was given. There are several methods currently available to diagnose H. pylori gastritis to confirm rapid test results. a) Growing the culture of H. pylo i from biopeia is difficult and takes time. It is generally not available in most hospitals or commercial laboratories. b) Serology: There are several available septic tests that measure the antibodies against H. pylori. The presence of the IGG antibody indicates a recent infection. However, it may take 6 months or more for the antibody level to fall following the eradication of the infection # (6) In this way, the presence of the antibody does not necessarily indicate the presence of infection. c) Histology: Routine histology studies 5 of the biopsy material may indicate the presence of the bacteria as well as implicit gastritis. d. 13C or 14C respite test: Ingestion of urea labeled as 13C or 14C in the presence of urease with H. pylori results in an intragastric conversion to C0S lO ^ fc labeling that can then be detected in exhaled air. The equipment for this technique is expensive, not widely available and has not been tested by the FDA.

BIBLIOGRAPHY 15 1.- Mar shall B.3. , MacGechie D.B. Rogers P.A.R., Glnacty R.G. Pylori Campylobcter infection and gastroduodenal disease. Med 3. Aust 1965; 149 439-444. 2. Graham D.Y. Helicobacter pylori: Its epithemiology 20 and its role in duodenal ulcer disease. 3. Gastroenterol Hepatol 1991; 6: 105-13. 3.- Parsonnet 3. Friedman G.D., Vandersteen D.P., et al: Helicobacter pylori infection and the risk of gastric cancer, N. Engi 3. Med 1991; 325: 1727. 25 4. Hentschel E, Bradstatten G., Dragasics B., et al.

Effect of ranitidine and amoxicillin plus metronidazole on the eradication of Helicobacter pylori and the recurrence of duodenal ulcer. N. Engl 3. Med. 1993; 326: 306-12. 5.- Grabam D.Y., Lew G.M. Klein P.D. et al. Effect of treatment of Helicabacter pylori infection on the long term 5 recurrence of gastric or duodenal ulcer; a rando ized, controlled trial. Ann Intern Med 1992; 116: 705-6. 6. Evans D.3., 3r. Graham D.Y., Lew G.M., Evans D.G., Malaty H. M. Can one use the results of serologic testing to tor the results of the therapy of Helicobacter pylori? lOAj. Gastraenteralagy 1991; 100: A62. 7. Baron C., Thomas T.E. , Biochim Biophy Acta; 1975: 362, 276-265. m

Claims (3)

1. In a test composition for the diagnosis of gastric disease detecting the presence of urease associated with H. pylori in a biopsy sample; said composition contains urea and a dye indicator with which if H. pylori is present in the biopsy sample, the associated dye hydrolyzes the urea to produce ammonia which increases the pH and changes the color of the dye indicator indicating the gastric disease; the improvement being < ? '? characterized in that it comprises: said dye indicator comprises at least two dyes in combination, 15 which together change the color from an initial color to at least one different color indicating the. presence of H. pylori; said color changes indicating the presence of H. pylori that initially occurs at an acidic pH; and said color indicating the presence of H. pylori being different 20 of the color of the biopsy sample.

2. The composition according to claim 1, characterized in that it has an initial pH in the scale of approximately 4.9 to approximately 5.6 and where the color change indicates the presence of. 25 pylari takes place initially at a pH below 7.0.

3. The composition according to 4. The composition according to claim 2, further characterized in that the heat indicator changes the color when the pH is increased over a spectrum of a plurality of colors giving not only an indication of the presence of H. pylori but also, if present, an indication of the degree of infection. 5.- The composition in accordance with the IG Jfc claim 1, further characterized in that a positive test indicating the presence of H. pylori takes place in most cases for approximately 2 to approximately 10 minutes; and in all cases no more than 4 hours. 6.- A test composition for H. pylori in the diagnosis of gastric disease, characterized in that it comprises: a) from about 0.5 to about 2.0% of áj? urea; b) from approximately 0.4 to approximately 1.4% agar; c) from about 0.2 to about 1.2% N-20 octylglucose; d) Approximately 1.5 mm to approximately 3. 5 mm of aH-2P0l +; e) conservative agent; f) a dye indicator composition comprising at least two combination dye indicators, which provide a broad spectrum of colors on a pH scale and indicate a 25 positive result at an acidic pH; and g) water, like the rest; said composition being in the form of a gel 26 ^ soft enough to easily receive and wrap intimately a biopsy sample pushed towards said gel; said composition having an initial acid pH; all percentages being by weight of the total composition. 7. The composition according to claim 6, further characterized in that the dye indicator / composition comprises methyl red and bromotol blue. 6. The composition according to claim 6, further characterized in that the preservative agent comprises methylparaben and propi-1 pair-aben. 9. The test composition according to claim 6, for H. pylori in the diagnosis of gastric disease, characterized in that it comprises: a) Approximately 1% urea; b) about 1% agar; c) / 1% N-opt ilglucose; d) from about 1.5 to about 3.5 mM of NaHaPOl +; e, approximately 0.2% of i? t preservative agent composed of 0.16% methylparaben and 0.02% prapilparaben; f) about 25% of a dye indicator that contains approximately 1% methyl red and approximately 0.1% bromothymol blue; g) water, the rest; said composition being in the form of a gel sufficiently soft to readily receive and intimately wrap a biopsy sample pushed towards said gel; said composition having an initial pH of about 5. 3; all percentages being by weight of the composition ^ tot l. 10. The composition according to claim 6, further characterized in that it has been sterilized by filtration through a filter having pores of approximately 0.10 to approximately 0.5 microns. COMPOSITION OF TEST FOR THE QUICK DETECTION OF HELICQBACTER * PYLORI IN A 6ASTRIC BIOPSY TISSUE SUMMARY OF THE INVENTION The present invention relates to a novel test composition for the diagnosis of gastric-intestinal disorders; This new test composition is particularly useful for the diagnosis of peptic ulcers, lOßhf including both gastric and duodenal ulcers, detecting the enzyme reasa associated with the bacterium Heli obacter pylori in endoscopically obtained biopsy samples, the test is not intended for use in vivo but only for in vitro diagnosis. fifteen F 20 GD / gfr * mvs *