Method and test kit for detecting helicobacter pylori

Abstract

Provided herein are methods and test kits for detecting urease producing microorganism such as Helicobacter pylori (H. pylori). In some embodiments, the methods provided herein are for determining the presence of H. pylori in a buccal mucosa biopsy specimen.

Description

METHOD AND TEST KIT FOR DETECTING HELICOBACTER PYLORIPRIORITY

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 623,171, filed January 19, 2024, which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Provided herein are methods and test kits for detecting urease producing microorganism such as Helicobacter pylori (H. pylori). In some embodiments, the methods provided herein are for determining the presence of H. pylori in a buccal mucosa biopsy specimen.

[0003] In the diagnosis and management of many pathologies including, but not limited to gastrointestinal disorders and cancers, the determination of Helicobacter pylori (formerly known as Campylobacter) is very important. In 1994, H. pylori was recognized as a type I carcinogen, and is now considered the most common etiologic agent of infection-related cancers. About 10% of H. pylori individuals develop peptic ulcer disease, 1% to 3% develop gastric adenocarcinoma, and less than 0.1% mucosa associated lymphoid tissue lymphoma (Peterson, W. L., 1991; Wroblewski et al., 2010; Plummer et al., 2016).

[0004] H. pylori grows on the gastric epithelium and does not penetrate the tissues. It is also found on tissue from the esophagus and duodenum. Its ability to proliferate and survive in the gastric environment is due to factors such as its production of urease and its motility and ability to adhere to the gastric epithelium. The urease has very high specific activity and is an important virulence factor involved in a series of processes that allow H. pylori to colonize and induce a strong inflammatory response in the gastric epithelium. Urease produced by H. pylori releases ammonia through hydrolysis of urea, which neutralizes stomach acid.

[0005] There are several means to diagnose H. pylori infections including stool antigen test, stool polymerase chain reaction (PCR) test, urea breath test, upper endoscopy exam and tissue sample biopsies.

[0006] Methods for diagnosis of H. pylori currently available include both invasive and noninvasive tests. The invasive tests (pathological evaluation of biopsies obtained via endoscopy) offer high sensitivity and specificity as well as the option of genoty ping the H. pylori strain by culturing from the biopsy. However, these procedures include risk ofesophageal and / or gastric perforation and bleeding and risks from the medications used for patient sedation, as well as substantial costs from the endoscopic procedure and pathology review of the biopsy specimens.

[0007] Non-invasive tests for diagnosis of H. pylori include the serology test, the stool antigen test, and the urea breath test. Both of these tests have low sensitivity and therefore are not accurate in diagnosing H. pylori. The urea test utilizes H. pylori urease activity. A mucosal biopsy specimen is incubated in a medium containing urea and a pH sensitive dye (Owen, R. J. et al., 1985). Urease produced by H. pylori releases ammonia through hydrolysis of urea and when enough ammonia is produced to raise the pH of the medium the dye changes color.

[0008] A commercial test for H. pylori, named CLOtest, described and claimed in U.S. Pat. No. 4,748,113, detects urease activity on biopsy specimens. The incubation medium is solidified by including a gelling agent. This medium also contains phenol red, which turns pink when ammonia released from urea raises the pH above 6.0. The system is buffered so that specimens contaminated by fluids from the intestine do not raise the pH and cause a false positive result. When specimens are first inserted into the CLOtest gel they may have a slight pink tinge if blood or alkaline bile is present. The analyst is required to record the initial appearance of the specimen. The test is positive only if the pink color increases in intensity or area.

[0009] CLOtest requires three hours incubation at 30° C and up to 21 hours additional incubation at room temperature. About 75% of biopsy specimens infected with H. pylori give positive results in 20 minutes and 90% are positive by 3 hours. Twenty-four hours are required to verify negative results because 5% of infected specimens become positive between 3 and 24 hours.

[0010] The traditional liquid urease tests use a few hundred microliters of medium and ammonia produced by a positive specimen becomes mixed with the medium by diffusion and mechanical stirring. The CLOtest reduces mechanical stirring by using gelled medium and specimens with high urease activity will produce a red color near the specimen in a short time. However, ammonia produced slowly by weakly positive specimens has time to diffuse throughout the gel and a larger amount of ammonia must be produced to give a color change. Thus, the incubation time required for a color change increases disproportionately as the urease content of the specimen decreases. In addition, the buffer included in the test mediumto consume acid or base on the specimen inhibits pH changes due to production of ammonia and this reduces assay sensitivity and increases incubation times.

[0011] Pyloritek is another testing device U.S. Pat. Nos: 5,314, 804 and 5,420,016. The Pyloritek test kit includes three components: Firstly, Pyloritek contains reagent strips which have, in separate dry reagent matrices, the substate urea (substrate pad) and pH indicator (reaction pad). The reaction pad containing the pH indicator is covered by a semipermeable membrane, which allows passage of gaseous ammonia but prevents passage of gastric tissue fluid or hydration reagent form the substrate pad. Secondly, the Pyloritek test kit also contains a hydration reagent that has a Tris buffer that is dispensed onto the substrate pad just prior to performing the test. Thirdly, the Pyloritek test kit contains disposable reaction pouches that provide solid contact between the gastric biopsy and the substrate pad. These ensure that the ammonia gas generated is directed through the membrane to the pH indicator.

[0012] However, there remains a need in the art for efficient, cost effective and non-invasive test for detecting H. pylori infection in patients. The present invention meets this need by providing such testing methods.BRIEF SUMMARY OF THE INVENTION

[0013] Provided herein are methods and test kits for detecting for detecting Helicobacter pylori infection in a buccal mucosa biopsy specimen and / or endoscopic biopsy by detecting the presence of urease produced by the H. pylori using a system for separating and optimizing various reactions involved. In some embodiments, the buccal mucosa biopsy specimen is first positioned on one side of a diffusion element permeable to ammonia. The buccal mucosa biopsy specimen is then put in contact with a urea substrate. If urease producing H. pylori is present in the specimen, the urea is converted into ammonia. The ammonia then permeates through the diffusion element to contact a reaction pad located on the other side of the diffusion element. The reaction pad includes a pH indicator that, upon contact with ammonia, produces a detectable reaction (e.g.. a change in color).

[0014] In one aspect, the present invention provides a method for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising:(a) positioning the buccal mucosa biopsy specimen on one side of a diffusion element permeable to ammonia;(b) contacting the buccal mucosa biopsy specimen with a substrate pad comprising urea, said contacting producing ammonia;(c) allowing the ammonia to permeate through the diffusion element to contact a reaction pad located on the other side of the diffusion element; and(d) observing the reaction of the ammonia with the reaction pad.

[0015] In some embodiments, said substrate pad further comprises a hydration reagent.

[0016] In some embodiments, said the diffusion element is a semi-permeable membrane that allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

[0017] In some embodiments, said substrate pad is contained in a matrix.

[0018] In some embodiments, said reaction pad comprises a pH indicator.

[0019] In some embodiments, said pH indicator comprises bromophenol blue.

[0020] In some embodiments, said pH indicator turns yellow to blue when the ammonia contacts the pH indicator.

[0021] In some embodiments, said buccal mucosa biopsy specimen is placed on the diffusion element, and the substrate pad is placed over the buccal mucosa biopsy specimen to form a multi-layered structure.

[0022] In some embodiments, said hydration reagent is a Tris buffer.

[0023] In some embodiments, said Tris buffer is dispensed onto the substrate pad.

[0024] In some embodiments, said matrix is absorbent paper.

[0025] In some embodiments, said urea is contained in the matrix.

[0026] In another aspect, the present invention provides a method for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising:(a) positioning the buccal mucosa biopsy specimen on one side of a diffusion element permeable to ammonia;(b) contacting the buccal mucosa biopsy specimen with a substrate pad comprising urea, wherein said contacting produces ammonia in the presence of H. pylori; and(c) observing the presence of a reaction of the ammonia with a reaction pad located on the other side of the diffusion element, wherein a reaction occurs when the ammonia permeates through the diffusion element to contact the reaction pad, and wherein the reaction indicates H. pylori infection.

[0027] In one aspect, the present invention provides a test kit for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising:(a) a hydration reagent;(b) a substrate pad comprising urea;(c) a diffusion element;(d) a reaction pad; and(e) means for placing and maintaining the buccal mucosa biopsy specimen between the substrate pad and the diffusion element.

[0028] In some embodiments, said diffusion element is a semi-permeable membrane which allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

[0029] In some embodiments, said substrate pad is contained in a matrix.

[0030] In some embodiments, said reaction pad comprises a pH indicator.

[0031] In some embodiments, said pH indicator comprises bromophenol blue.

[0032] In some embodiments, said pH indicator turns yellow to blue when the ammonia contacts the pH indicator.

[0033] In some embodiments, said hydration reagent is a Tris buffer.

[0034] In some embodiments, said Tris buffer is dispensed onto the substrate pad.

[0035] In some embodiments, said matrix is absorbent paper.

[0036] In some embodiments, said urea is contained in the matrix.

[0037] In some embodiments, the method or test kit as described herein is capable of more accurately detecting and / or detecting with greater sensitivity7H. pylori infection as comparedto a standard histological examination, for example using Warthin- Starry silver stain or hematoxylin and eosin, or as compared to an endoscopic procedure.

[0038] In some embodiments, the method or test kit results in increased accuracy of detection of H. pylori infection as compared to a standard histological examination, for example using Warthin-Starry silver stain or hematoxylin and eosin, or as compared to an endoscopic procedure.

[0039] In some embodiments of the method or test kit, the increase in accuracy and / or greater sensitivity is an increase of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more as compared to a standard histological examination, for example using Warthin- Starry silver stain or hematoxylin and eosin, or as compared to an endoscopic procedure.

[0040] In some embodiments, the method or test kit finds use in the prevention and / or reduction of the risk of stomach cancer, wherein in the reduction of risk is a reduction of about 1%, 2%, 3%, 4%, 5%, 6%. 7%. 8%, 9%, 10%, 15%, 20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more.

[0041] In some embodiments of the methods, the H. pylori infection if further confirmed by histological examination, for example, Warthin- Starry silver stain or hematoxylin and eosin, or an endoscopic procedure.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Fig. 1 shows CLO test patterns across 1602 total patients. Of the total of 1602 patients evaluated, 53.6 % of patients were positive for CLO in both the antrum and duodenum;41.1% of the patients were positive for CLO in just the duodenum and negative in the antrum; 2.7 % of the patients were negative for CLO in both the antrum and duodenum; and 2.6% of the patients were negative in the duodenum but positive in the antrum.

[0043] Fig. 2 shows CLO test duodenum + antrum - versus positive pathology.

[0044] Fig. 3 shows CLO test duodenum - antrum + versus positive pathology.

[0045] Fig. 4 shows CLO test duodenum + antrum + versus positive pathology.

[0046] Fig. 5 shows CLO test duodenum - antrum - versus positive pathology7.

[0047] Fig. 6 shows a summary of the percentage of patients identified as being positive for CLO in either the antrum, duodenum, or both (antrum and duodenum).DETAILED DESCRIPTION OF THE INVENTIONI. Introduction

[0048] H. pylori is a spiral-shaped gram-negative and microaerophilic pathogen that infects about 50% of the world’s population. Helicobacter pylori (H. pylori) is the organism responsible for diseases such as, but not limited to atrophic gastritis, chronic gastritis, duodenal ulcers, gastric mucosa-associated lymphoid tissue lymphoma, and gastric cancers, but may be protective against other diseases including esophageal cancer and asthma. Both human and bacterial genetic variability appear to contribute to differences in disease outcome (Kargar, Mohammad, et al. 2011). H. pylori exhibits extensive inter-strain genetic diversity as well as intra-strain genetic diversification during the infection.

[0049] H. pylori grows on the gastric epithelium and does not penetrate the tissues. It is also found on tissue from the esophagus and duodenum. Its ability to proliferate and survive in the gastric environment is due to factors such as its production of urease and its motility and ability to adhere to the gastric epithelium. The urease has very high specific activity and is an important virulence factor involved in a series of processes that allow H. pylori to colonize and induce a strong inflammatory response in the gastric epithelium.A. Method And Test Kit

[0050] Provided herein are methods and test kits for detecting urease producing microorganism such as Helicobacter pylori (H. pylori). In some embodiments, the methods provided herein are for determining the presence of H. pylori in a buccal mucosa biopsy specimen. In some embodiments, the presence of H. pylori is detected by the presence of its urease activity, which converts a urea substrate into ammonia. The ammonia is then detected by a detectable reaction upon contact with a reaction pad. Without being bound by any particular theory of operation, it is believed that the methods and test kits provided herein allow for non-invasive detection of H. pylori infection in a subject.

[0051] The methods and test kits provided herein utilize several components that are fabricated as a multilayered structure. The several components of the multi-layered structure comprise: 1. a diffusion element to separate the various reactions and allow the ammonia to pass, 2. a substrate pad to be used to contact the buccal mucosa biopsy specimen with urea and produce ammonia upon the presence of urease generating H. pylori, 3. a reaction pad located on the other side of the diffusion element, and 4. a pH indicator to produce a detectable response to a positive result. Each of these components and the assembly thereof is further described in detail below.1. Diffusion element

[0052] In some embodiments, the method and test kit can include a diffusion element. In some embodiments, the diffusion element is a membrane. In exemplar}' embodiments, the membrane is a hydrophobic membrane that minimizes or prevents passage of liquid water and solutes. In some embodiments, the membrane allows for diffusion of water vapor at an amount that will not neutralize the indicator dye. In some embodiments, the diffusion membranes is thin as possible in order to minimize the length of the diffusion path for ammonia. The practical lower limit for membrane thickness is determined by the quantity of material required to maintain mechanical integrity of the membrane. In some embodiments, the membrane can range from 50 to 250 microns thick. In some embodiments, the membrane is about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 50 microns thick Mean pore diameters for commercial membranes range from 0.05 to 10 microns. In some embodiments, the mean pose diameter for commercial membranes is about 0.10, 0.15, 0.20, 0.25, 0.30. 0.35. 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75. 0.80. 0.85, 0.90, 0.95, 1.0, 1.05, 1.10, 1.15, 1.20, 1.25, 1.30. 1.35. 1.40. 1.45. 1.50, 1.55, 1.60, 1.65, 1.70, 1.75, 1.80, 1.85, 1.90, 1.95, 2.0, 2.05, 2.10, 2.15, 2.20, 2.25, 2.30, 2.35, 2.40,2.45, 2.50, 2.55, 2.60, 2.65, 2.70, 2.75, 2.80, 2.85, 2.90, 2.95, 3.0, 3.05, 3.10, 3.15, 3.20, 3.25,3.30, 3.35, 3.40, 3.45, 3.50, 3.55, 3.60, 3.65, 3.70, 3.75, 3.80, 3.85, 3.90, 3.95, 4.0, 4.05, 4.10,4.15, 4.20, 4.25, 4.30, 4.35, 4.40, 4.45, 4.50, 4.55. 4.60. 4.65. 4.70, 4.75, 4.80, 4.85, 4.90,4.95, 5.0, 5.05, 5.10, 5.15, 5.20, 5.25, 5.30, 5.35, 5.40, 5.45, 5.50, 5.55, 5.60, 5.65, 5.70, 5.75,5.80, 5.85, 5.90, 5.95, 6.0, 6.05, 6.10, 6.15, 6.20, 6.25, 6.30, 6.35, 6.40, 6.45, 6.50, 6.55, 6.60,6.65, 6.70, 6.75, 6.80, 6.85, 6.90, 6.95, 7.0, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45,7.50, 7.55, 7.60, 7.65, 7.70, 7.75, 7.80, 7.85, 7.90. 7.95. 8.0, 8.05. 8.10. 8.15, 8.20, 8.25, 8.30,8.35, 8.40, 8.45, 8.50, 8.55, 8.60, 8.65, 8.70, 8.75, 8.80, 8.85, 8.90, 8.95, 9.0, 9.05, 9.10, 9.15,9.20, 9.25, 9.30, 9.35, 9.40, 9.45, 9.50, 9.55, 9.60, 9.65, 9.70, 9.75, 9.80, 9.85, 9.90, 9.95, or 10 microns. In some embodiments, the membranes with the larger pore diameters offer less resistance to diffusion of ammonia. In some embodiments, pore diameters of 1.0 to 10 microns find use with for the present invention. Hydrophobic membranes can be cast with a variety of polymers. Such polymer include, but are not limited to poly vinylidene difluoride, poly trifluoroethylene, polyvinyl chloride, polypropylene, polyvinylidene dichloride and polytetrafluoroethylene.2. Buccal mucosa biopsy specimen

[0053] In some embodiments, the subject methods and test kits are carried out using a “biopsy”, which is a biological material, e.g., a subject or a specimen derived from a subject.

[0054] In some embodiments, the “subject” is a human or a non-human animal. The subject is alive or dead. If the method is earned out on a living subject, then it may be referred to as an in vivo method. If the method is carried out on a specimen, then it may be referred to as an in vitro or ex vivo method.

[0055] In some embodiments, the animal is a mammal. In some embodiments, the animal includes but is not limited to any livestock, domestic or laboratory' animal, such as, mice, guinea pigs, hamsters, rats, goats, pigs, cats. dogs, sheep, rabbits, cows, horses, camels, donkeys, buffalos, lamas, chickens, ducks, geese, and / or monkeys. In some further embodiments, the animal is an insect, bird, or fish. In some yet further embodiments, the animal is a fly or a worm.

[0056] In some embodiments, the method is carried out on an in vivo biopsy, i.e., on a living subject.

[0057] In some embodiments, the method is carried out on a dead subj ect, for example as part of an autopsy or a necropathy.

[0058] In some embodiments, the method, and the test kit is carried out using an ex vivo or in vitro biopsy, e.g., on a specimen. In some embodiments, the specimen may optionally be a provided specimen, i.e., a specimen that is previously obtained or removed from a subject. In some embodiments, the method may include a step of obtaining a specimen from a subject.

[0059] In some embodiments, the method and the test kit are carried out using a specimen, which may optionally be selected, for example, from a surgical resection specimen, a biopsyspecimen, a xenograft specimen, a swab, a smear, a body fluid specimen and / or a fecal specimen.

[0060] In exemplary embodiments, the specimen is a buccal mucosa biopsy. In some embodiments, the buccal mucosa biopsy is obtained from a subject using a swab.

[0061] In some embodiments, a biopsy specimen comprises cells and is optionally a tissue specimen, for example, comprising diseased and / or non-diseased tissue.

[0062] In some embodiments, the specimen is sectioned and / or sequentially disassociated, e.g., mechanically and / or enzymatically. In some embodiments the dissociation of the specimen is achieved with trypsin. In some embodiments, different layers of the specimen are obtained by dissociation. In some embodiments, cells are derived from different layers of a specimen. For example, this may be of interest if the specimen is a tissue, e.g., a xenograft tissue. Different layers, or cells derived from different layers, of the specimen may then be analyzed.

[0063] In some embodiments, the method is optionally carried out on a biopsy that is native. In some embodiments, the native biopsy has not been modified prior to performing the method of the invention. In some embodiments, the biopsy is native in that the tissue or cells present in the biopsy are not subjected to a step of lysis or extraction, e.g., lipid extraction, prior to performance of the method of the invention. In some embodiments, a biopsy may be native in that it comprises intact cells. In some embodiments, the biopsy has not been chemically or physically modified and is thus chemically and physically native. In some embodiments, the biopsy may be chemically native, i.e., it may be chemically unmodified. In some embodiments, the biopsy has not been contacted with a chemical agent to change its chemistry. In some embodiments, a chemical modification comprises contacting a biopsy with a matrix.

[0064] In some embodiments, the biopsy is physically native, i.e., it may be physically unmodified. In some embodiments, freezing, thawing, and / or sectioning are examples of physical modifications. A skilled person will appreciate that although physical actions, such as, freezing, may affect a specimen’s chemistry, for the purpose of this invention such an action is not considered to be a chemical modification.

[0065] In some embodiments, the biopsy is chemically native, but not physically native, e.g., because it has been frozen and / or sectioned.

[0066] In some embodiments, the biopsy is frozen, previously frozen, and then thawed, fixed, sectioned, and / or otherwise prepared, as disclosed herein with regard to specimen preparation.

[0067] In some embodiments, specimen preparation (for any of the methods of the invention and / or any of the analytical methods disclosed herein) involves one or more of the following.

[0068] In some embodiments, the specimen or part thereof is deposited on a solid surface, such as, a glass or plastic slide.

[0069] In some embodiments, the specimen is fixed chemically, or via a frozen section procedure, e.g., to preserve tissue from degradation, and to maintain the structure of the cell and of sub-cellular components such as cell organelles, e.g., nucleus, endoplasmic reticulum, and / or mitochondria. In some embodiments, the fixative may, for example, be 10% neutral buffered formalin.

[0070] In some embodiments, freezing of the specimen comprises e.g., contacting the specimen with a suitable cooling medium, including but not limited to dry ice, liquid nitrogen, or an agent that has been cooled in dry ice or liquid nitrogen, e.g., isopentane (2- methyl butane). In some embodiments, frozen specimens are stored at, e.g., between about -80 and -4 degrees Celsius, e.g., at -70 or -20 degrees Celsius.3. Substrate pad

[0071] In some embodiments of the present invention, the method and test kit include a substrate pad. In some embodiments, the purpose of the substrate pad is to expose the buccal mucosa biopsy specimen to urea. In some embodiments, a second function of the substrate pad is to adjust the pH of the buccal mucosa biopsy specimen to the optimum for urease activity and for formation of ammonia through the ammonia / ammonium ion equilibrium. In exemplary embodiments, the buccal mucosa biopsy specimen is obtained from a subject and the specimen containing swab is contacted with the substrate pad.

[0072] In some embodiments, the substrate pad contains a buffer, urea and one or more reagents that enhance the activity of urease such as EDTA.

[0073] In some embodiments, the substrate pad comprises a matrix. Exemplary substrate pad matrices for use in the subject methods and kits provided herein include, but are not limited to: cellulose based papers, the hydrophilic membranes mentioned above or a woven or nonwoven fabric. In some embodiments, the reagents are incorporated by dipping the matrixinto an aqueous solution of the reagents and drying. In some embodiments, the buccal mucosa biopsy specimens contain enough water to dissolve the dry reagents in the substrate pad and give maximum enzyme activity. In this situation, the substrate pad can be hydrated by applying a few drops of water. In some embodiments, the buccal mucosa biopsy specimens are placed on the diffusion membrane and a wet substrate pad is placed on the specimens and diffusion membrane to form an assembly. In some embodiments, the assembly is held together with a clamp. In some embodiments, the assembly is held together by a holder that exerts an even overall pressure on the folded device (and the components thereof). In some embodiments, the assembly is held together by any suitable technique known to a skilled person in the art.

[0074] In some embodiments, the substrate pad comprises a hydration reagent. In some embodiments, the hydration reagent can be prepared by dipping the matrix in a urea solution and drying. In embodiments, the substrate pad is hydrated with a buffer solution which will maintain the proper pH and other reaction conditions.

[0075] In some embodiments, test kits can also be assembled with a thin membrane as substrate pad mounted on the diffusion membrane. In some embodiments, the buccal mucosa biopsy specimen is placed on the diffusion element, and the substrate pad is placed over the buccal mucosa biopsy specimen to form a multi-layered structure. In some embodiments, the substrate pad can be a thin, porous membrane because ammonia must diffuse through it to reach the reaction pad.

[0076] In some embodiments, the substrate pad contains a buffer at pH 7.0 to 9.0. In some embodiments, the substrate pad buffer pH is about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. In some embodiments, the pH optimum for H. pylori urease is 8.2 (Mobley, et al., 1988). In some embodiments, the pKa for ammonium ion is 9.3 and higher pH favors formation of ammonia from ammonium ion. In some embodiments, a pH between 8.0 and 8.5 would be optimal for maximum urease activity and ammonia diffusion. In some embodiments, the pH for maximum urease activity and ammonia diffusion is about 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5.4. Reaction pad

[0077] In some embodiments, the method and test kit include a reaction pad. In some embodiments, the reaction pad can be located on the other side of the diffusion element from the substrate pad. In some embodiments, the reaction pad includes one or more immobilized reagents capable of reacting with the biopsy and producing a detectable response to indicate the presence or absence the ammonia.

[0078] In some embodiments of the present invention, the method and test kit can include a pH indicator. In some embodiments, the pH indicator is located on the reaction pad. In some embodiments, a pH indicator for inclusion in the indicator element would change from a colorless to a colored form when a proton is removed by ammonia. However, most indicators change from one color in the acid form to another color in the deprotonated form. In embodiments, the indicator changes from a weak color (e.g., yellow) in the protonated form to a strong color (e.g., blue).5. pH indicator element

[0079] In some embodiments, the method and test kit includes a pH indicator element. In embodiments, the pH indicator is a dye. In some embodiments the indicator dye is capable of trapping ammonia as ammonium ion. In exemplary embodiments, the pKa of the dye is low compared to that for ammonium ion, pKa = 9.3. thereby allowing for trapping ammonium ion.

[0080] In some embodiments, the indicator dye used is bromophenol blue (pKa= 4.0). Bromophenol blue responds to ammonia without glycerol present and the color remains stable for at least a few hours if the test device is not opened to the atmosphere. In some embodiments, the pKa of the indicator dye is less than 8.0, within a range of about from 2.0 to 6.0.

[0081] Examples of pH sensitive, ammonia / amine (base) reactive pigments and dyes include, but are not limited to, Bromophenol blue, Bromo chlorophenol blue, Phloxine B, Methyl yellow, Congo red, Methyl Orange, Ethyl orange, Fluorescein, Bromocresol green, Chrysoidin, Methyl red, Alizarin red, Cochineal, Chlorophenol red. Bromocresol purple, 4- Nitrophenol, Alizarin, Nitrazine yellow. Bromothymol blue, Brilliant yellow, Neutral red, Rosolic acid, Phenol red, m-Cresol purple. Thymol blue, Xylenol blue, Cresol red, or any variants thereof.II. Assembly of Devices

[0082] The devices and test kit can be assembled and used in several ways. In some embodiments, the device is assembled in accordance with the embodiments disclosed herein.

[0083] In some embodiments, the diffusion element is positioned between the substrate pad and the reaction pad. If H. pylori is present in the buccal mucosa biopsy specimen, urease is produced when H. pylori reacts with the substrate pad (containing urea) to generate ammonia. In some embodiments, ammonia contacts the pH indicator.

[0084] In some embodiments, the buccal mucosa biopsy specimen is placed on the diffusion element, the substrate pad is placed over the buccal mucosa biopsy specimen; and the reaction pad is placed on the other side of the diffusion element to form a multi-layered structure. In some embodiments, the multi-layered structure comprises: a diffusion element to separate the various reactions and allow the ammonia to pass; a substrate pad to be used to contact the buccal mucosa biopsy specimen with urea and produce ammonia upon the detection of H. pylori in the buccal mucosa biopsy specimen; a reaction pad with a pH indicator located on the other side of the diffusion element. In some embodiments, the pH indicator located on the reaction pad changes color when the test produces a detectable response to a positive result.

[0085] In some embodiments the diffusion element, the reaction pad and the substrate pad are placed in a contiguous relationship. In some embodiments, the substrate pad is located opposite to the reaction pad while connected to each other in a contiguous relationship. In some embodiments, the buccal mucosa biopsy specimen can be added directly to the diffusion element. In some embodiments, rehydrating fluid is added to the substrate pad. In some embodiments, the diffusion element containing the buccal mucosa biopsy is folded over on itself to form a multilayered multielement device where the buccal mucosa biopsy specimen is sandwiched between the diffusion element, the substrate pad, and the reaction pad. In some embodiments, in the presence of H. pylori, the reaction between the buccal mucosa biopsy specimen and the substrate pad comprising urea will cause the production of ammonia.

[0086] In some embodiments, means, such as a clamp or holder which exerts an even overall pressure on the folded device (and the components thereof), may be utilized to keep thesubstrate pad and the diffusion element in intimate contact with the buccal mucosa biopsy specimen included therebetween which allows the reaction of the various components with the buccal mucosa biopsy specimen to proceed in an expeditious manner.

[0087] In some embodiments, the sensitivity of the test kit provided herein needs adjusting. For example, there may be ammonia or other ammonia producing substances in or accompanying the fluids associated with the buccal mucosa biopsy specimens undergoing testing for urease. In some embodiments, the adjustment comprises adding to the diffusion element and / or pH indicator, predetermined amounts of chemicals or adsorbents which react quickly with or tie up the initial surge of ammonia through the diffusion element. By doing this, only the ammonia generated by the urease in the buccal mucosa biopsy specimen will react with the pH indicator in the reaction pad to give a detectable response.

[0088] In some embodiments of the present invention, the various system components are assembled into a test kit, which makes its use more convenient and facile for the test operator. In some embodiments, such a test kit advantageously comprises the test device as well as a rehydrating solution for the substrate pad and a holder for retaining the test device in a folded position during the reaction of the urease in the buccal mucosa biopsy specimen with the various test reagent components. In some embodiments, the rehydrating solution is purified water or a buffer solution for optimizing the reaction of the urease in the biopsy with the urea in the substrate pad. In some embodiments, placement of the buffer in the rehydrating solution obviates the need for including the buffer in the substrate pad along with the urea. In some embodiments, the various components of the test system may be constructed as separate items and the individual components assembled at the time of use. In some embodiments, the substrate pad, the diffusion element and the pH indicator are separate components, which upon use, are assembled into a test device as described herein as opposed to unitizing the various components. In some embodiments, the rehydrating solution may be simply water or may be an aqueous buffer solution having a pH of from about 7.0 to 9.0. In some embodiments, the rehydrating solution pH is about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6. 8.7, 8.8, 8.9 or 9.0. In some embodiments, when water is used as the rehydrating fluid, the buffer is included as a component of the substrate pad.

[0089] In one aspect, some embodiments of the present invention include a test kit for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising: a hydration reagent, a substrate pad comprising urea; a diffusion element; a reaction pad; and a means forplacing and maintaining the buccal mucosa biopsy specimen between the substrate pad and the diffusion element.

[0090] In some embodiments, the test kit includes a diffusion element that is a semi- permeable membrane, which allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

[0091] In some embodiments, the test kit includes substrate pad contained in a matrix.

[0092] In some embodiments, the test kit includes a reaction pad that comprises a pH indicator.

[0093] In some embodiments, the test kit includes a pH indicator that comprises bromophenol blue. In some embodiments, the test kit the pH indicator turn yellow to blue when the ammonia contacts the pH indicator.

[0094] In some embodiments, the test kit includes a hydration reagent that is Tris buffer. In some embodiments, the Tris buffer is dispensed onto the substrate pad.

[0095] In some embodiments, the test kit includes a matrix is absorbent paper.

[0096] In some embodiments, the test kit includes urea in the matrix.III. METHOD OF USE

[0097] In another aspect of the present invention, provided herein is a method for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising positioning the buccal mucosa biopsy specimen on one side of a diffusion element permeable to ammonia; contacting the buccal mucosa biopsy specimen with a substrate pad comprising urea, said contacting producing ammonia in the presence of urease producing H. pylori; allowing the ammonia to permeate through the diffusion element to contact a reaction pad located on the other side of the diffusion element; and observing the presence of a reaction of the ammonia with the reaction pad, thereby detecting a H. pylori infection.

[0098] In some embodiments, the substrate pad further comprises a hydration reagent.

[0099] In some embodiments, the diffusion element is a semi-permeable membrane which allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

[0100] In some embodiments, the substrate pad is contained in a matrix.

[0101] In some embodiments, the reaction pad comprises a pH indicator.

[0102] In some embodiments, the pH indicator comprises bromophenol blue.

[0103] In some embodiments, the pH indicator turns yellow to blue when the ammonia contacts the pH indicator.

[0104] In some embodiments, the buccal mucosa biopsy specimen is placed on the diffusion element, and the substrate pad is placed over the buccal mucosa biopsy specimen to form a multi-layered structure.

[0105] In some embodiments, the hydration reagent is a Tris buffer.

[0106] In some embodiments, the Tris buffer is dispensed onto the substrate pad.

[0107] In some embodiments, the matrix is absorbent paper.

[0108] In some embodiments, the urea is contained in the matrix.

[0109] The following example is illustrative of the present invention.EXAMPLE 1

[0110] The buccal mucosa biopsy specimen is positioned on one side of the diffusion element with a semi-permeable membrane to ammonia. The semi-permeable membrane allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

[0111] The substrate pad comprises a hydration reagent such as Tris buffer. Tris buffer is dispensed onto the substrate pad. The substrate pad is contained in a matrix of absorbent paper that contains urea in the matrix. The buccal mucosa biopsy specimen is put in contact with the substrate pad containing urea to form a multi-layered structure. The contact of the buccal mucosa biopsy specimen and the substrate pad containing urea produces ammonia. The ammonia permeates through the diffusion element to contact a reaction pad located on the other side of the diffusion element. The reaction pad comprises a pH indicator comprised of bromophenol blue. The reaction of the ammonia with the reaction pad is observed. The pH indicator turns yellow to blue when the ammonia contacts the pH indicator.For detection of H. pylori infection in a buccal mucosa biopsy specimen, a test kit is developed. The test kit for detecting H. py lori infection in a buccal mucosa biopsy specimen comprises: a hydration reagent, a substrate pad comprising urea, a diffusion element, a reaction pad. and a means for placing and maintaining the buccal mucosa biopsy specimen between the substrate pad and the diffusion element. The diffusion element is a semi- permeable membrane which allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent (e.g., Tris buffer). The Tris buffer is dispensed onto the substrate pad. The substrate pad is contained in a matrix comprised of absorbent paper that contains urea in the matrix. The reaction pad comprises a pH indicator comprised of bromophenol blue. The pH indicator turns yellow to blue when the ammonia contacts the pH indicator. The pH indicator is yellow due to the protonated form of the indicator dye and positive results with urease impregnated threads appear as blue areas roughly outlining the shape of the underlying thread.EXAMPLE 2Comparative Analysis of Urease testing from antrum and duodenum biopsy for Detection ofH. Pylori Infection compared to histology analysis.

[0112] Helicobacter pylori (H. pylori) is a spiral-shaped, gram-negative bacterial pathogen, known to colonize the human gastrointestinal (GI) tract and has been identified as a major etiological factor in GI disorders, such as peptic ulcers, gastritis, and gastric malignancies. Given its role in the pathogenesis of these conditions, reliable and accurate diagnosis of H. pylori infection is essential for appropriate treatment and management. The gold standard for H. pylori diagnosis has traditionally been histological examination of gastric biopsy specimens.

[0113] Histopathological diagnosis of H. pylori involves examining gastric biopsy samples obtained through endoscopy. The tissue is typically stained with hematoxylin and eosin (H&E), which allows visualization of the general architecture of the gastric mucosa and any inflammatory changes. Special stains, such as Giemsa, Warthin-Starry, or immunohistochemistry (IHC), can enhance the visibility of H. pylori bacteria, allowing the bactena to be easily identifiable under the microscope.

[0114] Giemsa stain can be used to evaluate H. pylori in gastric biopsy samples, although it is not the most commonly used method. More frequently, H. pylori infection is diagnosed through histological examination using Warthin-Starry silver stain or hematoxylin and eosin.

[0115] Giemsa stain is a Romanowsky -type stain that can highlight bacterial organisms with a characteristic purple-blue appearance. Once stained, the bacteria can be visualized on Giemsa-stained slides as small, curved, or helical rods, typically located in the mucus layer or near gastric epithelial cells. One advantage of Giemsa is that it provides good contrast between the bacteria and the surrounding tissue. It stains the gastric mucosa, cells and inflammatory’ cells in various shades of purple, which allows the H. pylori to be differentiated from the background tissue. Giemsa is simple, inexpensive while providing a clear visualization of both bacterium and the surrounding histopathology. One of the limitations of Giemsa stain is that it is less specific for H. pylori as other microorganisms may also take up the stain.

[0116] The Warthin-Starry stain, is a more specific silver stain for H. pylori, and a preferred method in histological evaluation. Although Hematoxylin and eosin (H&E) is a common stain used for general tissue structure, it is not sensitive in detecting H. pylori due to the limited contrast the stain produces.

[0117] In addition to direct bacterial visualization, histopathology can assess the degree of inflammation, which provides information on the chronicity of infection and the potential for complications such as gastric cancer.

[0118] Bacterial load, sampling site and technician expertise are few factors that influence the sensitivity' of the histopathology in detecting a low bacterial density in the biopsy specimen may reduce the likelihood of detecting the organism, leading to false negatives. The antrum and the corpus of the stomach are the areas most commonly sampled. It is known that the bacteria may be unevenly distributed, which can result in false negatives if the biopsy was not taken from the site populated by bacteria. In this example, the bacteria was identified mostly in the duodenum, which has not been a common site to biopsy. The ability to identify bacterial forms and the surrounding inflammatory changes is dependent on the pathologist.

[0119] Histopathology has been reported to have relatively high sensitivity in detecting H. pylori infection, with estimates ranging from 70% to 90%, depending on the quality of the biopsy sample and the staining technique used. However, its sensitivity' can be lower inpatients with low bacterial loads or when gastric atrophy is present. It has been reported sensitivity7as low as 60% in cases with severe gastric atrophy, where H. pylori may be more diffusely distributed or less visible. Specificity7of histopathology is generally high, often approaching 100%, as the bacterium is distinctive in appearance when properly stained. False positives are rare, however possible if there was cross-contamination of samples or misinterpretation of other bacteria as H. pylori. In this example, the ability to identify H. pylori via histopathology was significantly lower than what has been reported in the literature.

[0120] H. pylori infection is quite common in pediatric populations, though its prevalence varies based on geographic location, socioeconomic factors, and hygiene practices. In general, the global prevalence of H. pylori in children ranges between 20% to 80%, with higher rates observed in developing countries compared to developed nations.

[0121] Although histology has been the gold standard method, it requires general anesthesia ultimately increasing the risk and cost for GI patients. Invasive methods like endoscopies can be prone to sampling errors, leading to higher false negatives. Sampling and experimental errors can be attributed to the uneven distribution of the bacteria throughout the GI tract lining.RESULTS

[0122] A total of 1602 pediatric patients underwent endoscopy with biopsy (Fig. 1). These patients presented with either mid-epigastric pain, vomiting and or nausea. It should be noted that the majority of people with H. pylori are asymptomatic or with very mild symptoms.

[0123] Of the total 1602, 1559 patients w ere identified positive for a campy lobacter-like organism test (CLO) in one or more areas of the (duodenum, antrum or both) (Figs. 2-6). Only 65 of the patients identified as positive on CLO test were also identified by the pathologist on biopsy as having H. pylori. This gap in detection indicates that pathological reports alone are inefficient detection measures in H. Pylori testing. If pathology is relied on alone to identify patients, this example demonstrates the majority of those infected would not be identified. In this particular study, 95.9% of pediatric patients would have been missed if additional testing had not been done and we relied solely on pathological evaluation.CONCLUSION

[0124] Histopathology remains a valuable tool for diagnosing H. pylori infection, particularly due to its high specificity and ability to assess the degree of inflammation. However, its sensitivity is variable and can be affected by several factors, including bacterial load, sampling technique, and pathologist expertise. While histopathology is still considered one of the gold standards, other diagnostic methods such as urease tests, offer higher sensitivity and are more practical in certain clinical settings. In practice, a combination of diagnostic methods may be necessary7to ensure accurate diagnosis, especially in cases where histopathological sensitivity is suboptimal.

[0125] Further research is needed to explore the role of newer techniques, in enhancing the sensitivity of H. pylori diagnosis and to determine optimal diagnostic strategies in different clinical contexts. Pathology alone is inefficient for testing. Using urease detection for campylobacter-like organisms such as H. pylori is not routinely performed during endoscopic procedures. These results strongly suggest that the gold standard of pathology is not efficient to identify H. pylori in the pediatric population.

[0126] An estimated 4.4 billion people are infected with H. pylori, making it the most common chronic bacteria infection worldwide. Given the global prevalence is approximately 50% of the world’s population is estimated to be infected by H. pylori. Although the prevalence can vary depending on the region. In developed countries the prevalence can be as high as 80%. In developed countries, the prevalence is approximately 30% in developed countries. In the pediatric population the prevalence is roughly 20% in developed countries.

[0127] The World Health Organization has classified H. pylori as a group 1 carcinogen which indicates there is enough evidence to suggest that it directly contributes to the development of stomach cancer. The cancer risk is roughly 1 -3 percent in those infected with H. pylori. H. pylori, a human carcinogen, accounts for an estimated 80-90% of gastric cancers globally. Accurate diagnosis and subsequently successful eradication is associated with a reduced risk of serious downstream clinical consequences. These numbers are concerning, and lend to the urgent need for more precise diagnostic tools.

Claims

Attorney Docket No.: 137898-5001-WOWHAT IS CLAIMED IS:

1. A method for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising: a. positioning the buccal mucosa biopsy specimen on one side of a diffusion element permeable to ammonia; b. contacting the buccal mucosa biopsy specimen with a substrate pad comprising urea, said contacting produces ammonia in the presence of H. pylori in the buccal mucosa biopsy specimen; c. allowing the ammonia to permeate through the diffusion element to contact a reaction pad located on the other side of the diffusion element; and d. observing the presence of a reaction of the ammonia with the reaction pad. thereby detecting H. pylori.

2. The method of claim 1, wherein the substrate pad further comprises a hydration reagent.

3. The method of claim 1, wherein the diffusion element is a semi-permeable membrane that allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

4. The method of claim 1, wherein the substrate pad is contained in a matrix.

5. The method of claim 1, wherein the reaction pad comprises a pH indicator.

6. The method of claim 5, wherein the pH indicator comprises bromophenol blue.

7. The method of claim 6, wherein the pH indicator turns yellow to blue when the ammonia contacts the pH indicator.

8. The method of claim 1, wherein the buccal mucosa biopsy specimen is placed on the diffusion element, and the substrate pad is placed over the buccal mucosa biopsy specimen to form a multi-layered structure.

9. The method of claim 2, wherein the hydration reagent is a Tris buffer.

10. The method of claim 9, wherein the Tris buffer is dispensed onto the substrate pad.

11. The method of claim 4, wherein the matrix is absorbent paper.

12. The method of claim 4, wherein the urea is contained in the matrix.

13. A test kit for detecting H. pylori infection in a buccal mucosa biopsy specimen comprising: a. a hydration reagent; b. a substrate pad comprising urea; c. a diffusion element; d. a reaction pad: and e. means for placing and maintaining the buccal mucosa biopsy specimen between the substrate pad and the diffusion element, wherein ammonia is produced when urea is in contact with urease in the buccal mucosa biopsy specimen, and wherein the ammonia passes through the diffusion element to contact the reaction pad.

14. The test kit of claim 13. wherein the diffusion element is a semi-permeable membrane which allows passage of ammonia but prevents passage of buccal tissue fluid or the hydration reagent.

15. The test kit of claim 13, wherein the substrate pad is contained in a matrix.

16. The test kit of claim 13, wherein the reaction pad comprises a pH indicator.

17. The test kit of claim 16, wherein the pH indicator comprises bromophenol blue.

18. The test kit of claim 17, wherein the pH indicator turns yellow to blue when the ammonia contacts the pH indicator.

19. The test kit of claim 13. wherein the hydration reagent is a Tris buffer.

20. The test kit of claim 19, wherein the Tris buffer is dispensed onto the substrate pad.

21. The test kit of claim 15, wherein the matrix is absorbent paper.

22. The test kit of claim 15, wherein the urea is contained in the matrix.

23. The method or test kit of any of the preceding claims, wherein the method or test kit is capable of more accurately detecting and / or detecting with greater sensitivity H. pylori infection as compared to a standard histological examination, for example usingWarthin- Starry silver stain or hematoxylin and eosin, or as compared to an endoscopic procedure.

24. The method or test kit of any of the preceding claims, wherein the method or test kit results in increased accuracy of detection of H. pylori infection as compared to a standard histological examination, for example using Warthin-Starry silver stain or hematoxylin and eosin, or as compared to an endoscopic procedure.

25. The method or test kit of claim 23 or 24, wherein the increase in accuracy and / or greater sensitivity is an increase of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more as compared to a standard histological examination, for example using Warthin- Starry silver stain or hematoxylin and eosin, or as compared to an endoscopic procedure.

26. The method or test kit of any of the preceding claims, wherein the method or test kit finds use in the prevention and / or reduction of the risk of stomach cancer, wherein in the reduction of risk is a reduction of about 1%, 2%, 3%, 4%, 5%, 6%. 7%. 8%, 9%, 10%, 15%, 20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% or more.

27. The method of any of the preceding claims, wherein the H. pylori infection if further confirmed by histological examination, for example, Warthin- Starry silver stain or hematoxylin and eosin, or an endoscopic procedure.

Images