Title of Invention

A NEW RAPID UREASE TEST KIT FOR HELICOBACTER PYLORI

Abstract

Rapid urease test is the method of choice for diagnosis of Helicobacter pylori which is the causative agent of gastric ulcers and many other stomach related problems. Although many rapid urease tests are there but none of then is stable at room temperature and there is no good control agent to be used along with these tests so far. This new rapid urease test consisting of urea, potassium dihydrogen phosphate and phenol red in dry filter paper strips is stable at room temperature. Jack bean meal is found to be an ideal control agent to be used along with rapid urease tests.

Full Text

This invention relates to a new rapid urease test -for diagnosis of Helicobacterpylori. In the known art there are many rapid urease tests, but there are many drawbacks of these tests. A review of Helicobacter pylori, rapid urease test and the drawbacks of currently used rapid urease tests are given below, these drawbacks are absent in this new rapid urease test. Spiral bacteria were found in stomach in the early part of the Century. In 1950,Fitzgerald and Murphy noted an association between urease production in the gastric mucous layer and peptic ulcer disease. Gram negative bacteria was found in 80% patients with gastric ulcers by Steer and Colin-Jones in 1975 and they felt these organisms were associated with active gastritis. In 1979,Warren described campylobacter like organisms in almost every gastric biopsy he examined and most of them had histological changes of active chronic gastritis. Subsequently, Warren and Marshall carried out clinical studies of all patients with dyspeptic symptoms requiring upper G.I. endoscopy and described a campylobacter like organism in gastric mucosa in 1983, which was first named as Campylobacter pyloridis and later as Campylobacter pylori However observations over the next few years revealed, this organism has very little resemblance in character with campylobacter. Therefore, In 1989 it was renamed as Helicobacter because of its helical or spiral shape. Association of H.pylori with peptic ulcer is well known, and it is associated with 60-80% of duodenal ulcers. H.pylori also plays a significant role in non-ulcer diseases. Currently, H. pylori are considered to be the major cause of chronic gastritis. These organisms produce a peptidase that can digest gastric mucin. Remission and exacerbation was the accepted natural history of duodenal ulcer but since the possible role of//, pylori was recognized the previous acid hypothesis alone is not sufficient to explain this fact. The organism may be acquired at anytime in life but incidence increases with age. Possible routes of transmission are faeco-oral, oral-oral and through the endoscope. Recently it has been demonstrated that H. pylori may be transmitted by houseflies. Langenberg et al first drew attention to the powerful urease activity of H. pylori. The enzyme urease is a high molecular weight protein and very active in comparison to the urease enzyme produced by other bacteria. A high concentration of urea present in gastric mucosa. Urease reacts with urea in the presence of water molecules and hydrogen ions producing ammonia and bicarbonate. H. pylori can tolerate a pH value even below 3.0 due to rapid formation of ammonia, which raises the pH immediately. The presence of H. pylori in the biopsies taken from gastric mucosa of patients with peptic ulcers and non-ulcer dyspepsia can be demonstrated by in vitro urease tests. Although, H. pylori can be identified during histological examination by Warthin-Starry or by routine haematoxylin and eosin stains and culture but this enzyme test is simpler and accurate. Recent studies also show evidence of definite association of H. pylori with lymphoma and carcinoma of stomach. Tests for gastric urease are specific for H. pylori because mammalian cells do not produce urease and, except for Hpylori, the stomach is usually sterile. 2 At present there are atleast 23 important Helicobacter species- H.. pylori,//, hepaticus, H. muridarumJI. canisji.cinaedi, H.fennelliae, H. nemestrinaejf. acinonychis, H.felis, H. pametensisJJ. mustelae, H. aurati, H. bilis, H. bizzozeronii, H. canadensis, H. cholecystus, H. ganmani,H. mesocricetorum, H. pullorum, H. rodentium, H. salomonis, H. trogontum, H. typhlonius. They can be differentiated by several biochemical and growth requirement factors,PCR(polymerase chain reaction) etc. Most of them are urease test positive. However, almost all isolated Helicobacter species from human stomach belong to the type species H. pylori. BACKGROUND OF THE INVENTION: AVAILABLE RAPID UREASE TEST KITS FOR H. PYLORI : At present all workers consider rapid urease tests as the method of choice since it is very easy to perform and very inexpensive. However, the most popular CLO test costs about USD4 and it should be maintained in a cold chain. In India four kits are available at present for rapid urease tests -The mostly used HELICOCHEK(it is the first commercially available rapid urease test introduced in India) and this was followed by introduction of H-P TEST,PYLORICHEK and H PYLORI TEST. The first three kits are solid gel medium with urea, pH indicator, stabilizing agent and a bacteriostatic agent which do not react with H. pylori. The last kit is also on the same principle except absence of any solidifying agent. Outside India threeiRUT kits for diagnosis of H. pylori are important: Solid urea gel medium with pH indicator,stabilising agent, bacteriostatic agent as mentioned above. The famous CLO test (campylobacter like organism, developed by Delta West LTD, Australia) and PYLORITEK test belongs to this group. The remaining one is a liquid urea medium with pH indicator, stabilizing agent ,bacteristatic agent -MMTTEK broth. Important references of Helicobacter pylori and rapid urease tests are given below: Krienitz W : Uber das Auftren von Mageninhalt bei Carcinoma Ventriculi. Dtsch med Wochenschr 22:872, 1906. Fitzgerald O and Murphy P. Studies on the physiological chemistry and clinical significance of urease and urea with special reference to the human stomach. Irish J. Med. Sci.292:97-159,1950. Steer H W & Colin-Jones D G. Mucosal changes in gastric ulceration and their response to carbenoxolone sodium. Gut,16:590-97,1975. Warren J.R. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet,Letter,i :1273,1983. Marshall B.J. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet,letter,i ,1273,1983. Langenberg M L , Tytgat G N J, Schipper M EI et al. Campylobacter - like organisms in the stomach of patients and healthy individuals. Lancet, i,1348,1984. Morris A & Nicholson G. Ingestion of Campylobacter pyloridis causes gastritis and raised fasting gastric pH . Am J Gastroenterol. 82: 192-99,1987. 3 Goodwin C S, McCulloch R K, Armstrong J A, et al. Unusual cellular fatty acids and distinctive ultrastructure in a new spiral bacterium ( Campylobacterpyloridis ) from the human gastric mucosa. J Med Microbiol 19: 257-67,1985. Grubel P L , Huang L, Masubuchi N et al. Detection of Helicobacter pylori DNA in houseflies ( Musca domestica) on three continents. Lancet,352:9130,32-33,1998. Waghorn D. Campylobacter pyloridis : a new organism to explain an old problem? Postgrad.Medi. Journal.63,533,1987. Marshall B J & Goodwin C S. Revised nomenclature of Campylobacter pyloridis. Inter. J. Syst.Bacterio.,37,68,1987. Coghlan J G, Humphrys H, Dooley C, Keane C et al. Campylobacter pylori and recurrence of duodenal ulcer.- A 12 month study. Lancet, 14:1109-1111,1987. Sloamiany B L, Bilski J, Sarosiek J et al. Campylobacter pyloridis degrades mucin and undermines gastric mucosal integrity. Biochem Biophys Res Commun 144: 307-14, 1987. Marshall B J et al Rapid urease test in the management of Campylobacter pyloridis associated gastritis. Amer. J. Gastro. 82:200-10,1987. Hazell S L, Borody T J, Lee A. Campylobacter pyloridis gastritisl: Detection of urease as a marker of bacterial colonization and gastritis. Am J Gastroenterol 82:292-6,1987. Rauws E , Langenberg W, HoutoffH et al. Campylobacter pylori - associated active chronic antral gastritis.,Gatroenterology,94:33-40,1988. Deltenre M, Glupczynski Y, Nyst J F et al. Assessment of four modalities of urease test in the diagnosis and follow-up of Campylobacter pylori associated gastritis. Gastroenterology,96,117,1989. Delterne M, Glupczynski Y, De Prez C et al, The reliability of urease tests, histology and culture in the diagnosis of Campylobacter pylori infection. Scand. J. Gastro. 24,19- 24,1989. Goodwin C.S. et al, Transfer ofCampylobacter pylori and Campylobacter mustelae to Helicobacter gen. Nov., as Helicobacter pylori comb. nov. and Helicobacter mustelae com. nov. respectively. Int. J. Syst Bacteriol.39,397-405,1989. Calam J. Helicobacter pylori .Medicine Digest,5,1990. Tytgat G N J, Lee A, Graham D Y et al. The role of infectious agents in peptic ulcer disease. Gastroenterol Infect 6: 76-89,1993. Kang, J Y, Tay H H, Wee A et al. Effect of colloidal bismuth subcitrate on symptoms and gastric histology in non-ulcer dyspepsia. A double blind placebo controlled study. Gut 31: 476-80,1990. Graham D Y, Lew G M, Klein P D, et al. Effect of treatment of Helicobacter pylori infection on the long term recurrence of gastric or duodenal ulcer. Ann. Intern. Med. 116,705-8,1992. Borody T, Andrews P. Mancuso N et al. Helicobacter pylori reinfection 4 years post eradication. Lancet,339,1295,1992. The EUROGAST Study Group: An international association between Helicobacter pylori infection and gastric cancer. Lancet,341,1359-62,1993. Wotherspoon A C, Doglioni C, Diss T C et al. Regression of primary low grade B cell lymphoma of mucosa associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet,342,575-7,1993. 4 Stolte M & Eidt S. Healing gastric MALT lymphomas by eradicating H. pylorft Lancet,342,568,1993. Bayerdorffer E, Lehn N, Hatz R et al. Difference in expression of Helicobacter pylori gastritis in antrum and body. Gastroenterology,102: 1575-82, 1992. Details description of the invention: AH urea-based kits available in the market are not stable at room temperature, and they should be transported in cold chain and should be preserved properly in fridges. The doctors also cannot carry these kits with them for study at bedside and these kits are to be kept in a fridge in the endoscopy room. Thus in India as well as in other developing countries this is not practicable and in most of the cases due to nonavailability of these facilities and/or due to loadshedding the test slides often show false positivity and colour change leading to wrong diagnosis and a wrong treatment. Other H. pylori detection kits are costly particularly which are not based on rapid urease tests, thus in developing countries most patients can not afford the cost of such a test. It is the object of this invention to find out a new rapid urease test for obviating the above said prior problems of the available rapid urease tests and to diagnose Helicobacter pylori in an effective way for saving millions human life throughout the world. It is also the object of this invention to have a new rapid urease test which is chemically defined, easy to apply, stable at room temperature, equally good and also available at affordable price. Accordingly the invention provides a new rapid urease test kit details of which are given below: Thin filter paper (Whatman no. 1) strips ( 3.0 CMX 1.5 CM; however, other sizes may also be used according to convenience) are prepared, then they are soaked in a solution which is prepared by mixing 10ml of 10% urea in deionised water, 10 mg KH2PO4 (potassium di-hydrogen phosphate) and 500 l of 1% phenol red in aquous solution( the amounts of different chemicals mentioned here are optimum concentrations for this test; however, other concentrations may also be used.)- The soaked filter papers then dried in incubator at 37°C and kept at room temperature. The stripsshowed a shelf life of more than six months. The indicator in the strip showed mild yellowish colour but changed to red, magenta or pink colour when reagent became alkaline due to urease activity. Addition of KH2PO44 will increase stability of the test reagents because in hot climate some amount of urea may be broken down with formation of ammonia and thus pH of the strip reagents will increase which may lead to a false positive colour formation. Acidic salts like KH2PO4 will diminish this false positive colour formation. Other acidic salts and weak acids may also be used other than KH2PO4. The addition of only acidic salt is completely a new approach which imparts stability of the test. Due to presence of acidic salt, this is thus stable at room temperature and there is no necessity to keep it in a fridge or to transport it in a cold chain. The doctors can easily keep these strips in their bags and can use it at bedside easily. The sensitivity, specificity, positive and negative predictive values and accuracy of this new rapid urease test are 91%, 100%, 100%, 100%, 95% when compared with the 5 histology and these values are comparable/good if we judge this against all available rapid urease tests. Procedure of the test: The test strip should be kept on a clean solid chemically inert surface like glass slide, plastic sheet etc. Then 2-3 drops (100-150 l) of clean water ( sterilization not required) is added on the strip so that it becomes wet. After this the small biopsy material collected by endoscopy or other material to be tested for Helicobacter pylori urease in small amount (in similar amount like endoscopy biopsy material) is to be placed on the wet strip. The strip should be kept at room temperature and it should be observed upto 1 hour for a positive reaction which indicates a colour change from light yellow to magenta or red or pink colour formation. If there is no colour change by this time the test result will be negative. Jack bean meal is used in this test as a positive control. For other rapid urease tests presently available in the market, Proteus group of organisms (these are a group of bacteria producing urease enzyme) are used as positive control, which can only be done in a sophisticated microbiological laboratory. In this strip test Proteus do not give a positive result within one hour. Thus use of Jack bean meal ( contains urease) as positive control in this test is also a new approach. Jack bean meal is also stable at room temperature and when it is added in the wet test strip as described above it gives a positive result within one hour. Thus this invention is very simple and this strip can be used along with the jack bean meal control very efficiently. 6 We claim: 1. A kit for rapid urease test for testing Helicobacter pylori infection in man, comprising thin filter paper strips soaked in a solution which is prepared by mixing 10ml of 10% urea in deionised water, 10 mg KH2PO4 (potassium di- hydrogen phosphate) and 500 pi of 1% phenol red in aqueous solution and then dried in incubator. The indicator in the strip showed mild yellowish colour, which will be changed to red, magenta or pink colour when reagent became alkaline due to urease activity. The kit contains jack bean meal as control. 2. A kit for rapid urease test as claimed in claim 1 wherein the addition of KH2PO4 will increase stability of the test reagents. 3. A kit for rapid urease test as claimed in claim 1 wherein addition of only acidic salt imparts stability of the test. 4. A kit for rapid urease test as claimed in claim 1 wherein due to presence of acidic salt, this becomes stable at room temperature and there is no necessity to keep it in a fridge or to transport it in a cold chain. 5. A kit for rapid urease test as claimed in claim 1 wherein the sensitivity, specificity, positive and negative predictive values and accuracy of this new rapid urease test are 91%, 100%, 100%, 100%, 95% when compared with the histology and these values are cpmparabJe/good if we judge this against all available rapid urease tests. 6. A kit for rapid urease test as claimed in claim 1 wherein Jack bean meal is used in this test as a positive control which is also stable at room temperature and when it is added in the wet test strip it gives a positive result within one hour. Rapid urease test is the method of choice for diagnosis of Helicobacter pylori which is the causative agent of gastric ulcers and many other stomach related problems. Although many rapid urease tests are there but none of then is stable at room temperature and there is no good control agent to be used along with these tests so far. This new rapid urease test consisting of urea, potassium dihydrogen phosphate and phenol red in dry filter paper strips is stable at room temperature. Jack bean meal is found to be an ideal control agent to be used along with rapid urease tests.

Documents:

00480-cal-2002 abstract.pdf

00480-cal-2002 claims.pdf

00480-cal-2002 description(complete).pdf

00480-cal-2002 form-1.pdf

00480-cal-2002 form-18.pdf

00480-cal-2002 form-2.pdf

00480-cal-2002 form-5.pdf

00480-cal-2002 letters patent.pdf

480-cal-2002-granted-abstract.pdf

480-cal-2002-granted-claims.pdf

480-cal-2002-granted-description (complete).pdf

480-cal-2002-granted-form 2.pdf

480-cal-2002-granted-specification.pdf