RU2092855C1 - Method of assay of helicobacter pylori in bioptate at gastroduodenal pathology - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: method involves incubation of bioptate in mixture of 10% urea solution with Nessler reagent at their volume ratio = 0.3:(0.1-0.2), respectively. Bioptate insemination with Helicobacter pylori is weak (appearance of yellow-brown ring around bioptate), moderate (yellow-brown ring converting to yellow-brown deposit) and expressed one (yellow-brown deposit). Method increases reaction specificity and its sensitivity up to 100%. EFFECT: increased precision of diagnosis.

Description

 The invention relates to medicine, and more specifically to medical microbiology, and can be used for laboratory diagnosis of gastroduodenal pathology associated with Helicobacter pylori, based on the detection of these microorganisms in biopsy specimens extracted during endoscopy and determination of their bacterial contamination.

A known method for the detection of H. pylori by urea hydrolysis and color change of the pH indicator in the diagnosis of lesions of the gastro-duodenal region caused by these microorganisms (Hernandez F. Rivera P. Sigaran M. Miranda J. Diaguosis of Helicobacter pylori comparison of ureasa test, histological visualization of curved bacteria and culture // Rew Just. Meg. trop. Sao Paulo, 1991, vol. 33, No. 1, p. 80-82). For this, a biopsy sample was immersed in a test tube with 200 μl of a 10% solution of urea and a 0.2% solution of phenol red and incubated at 37 ^° C for 10 min. After a specified period of time, in the presence of H. pylori in the test material and their cleavage of urea, the medium was alkalized in a test tube with the color of the solution changing to pink. This was regarded as a positive result of the study. The specificity of the test reached 83%. The sensitivity of 72%. The disadvantage of the described method is that the color change of the indicator occurs when the pH changes. The latter, however, can be based not only on H. pylori-related alkalization of the medium as a result of urea hydrolysis and the formation of NH ₃ , but also on a number of other uncontrolled factors, which significantly reduces the specificity of the test described.

 The purpose of the invention is to increase the sensitivity of the test, reducing research time.

 This goal is achieved through the use of Nessler reagent and urea in a certain ratio.

The method for determining Helicobacter pylori biopsy in gastroduodenal pathology involves incubating the biopsy in a mixture of 10% urea and Nessler reagent with a volume ratio of 0.3 (0.1-0.2) in the reaction mixture, respectively, for 5-10 minutes at a temperature of 25 ^o C. In the presence of H. pylori in the test material, urea is cleaved with the formation of ammonia, which causes the appearance of a yellow-brown ring or yellow-brown sediment around the biopsy specimen. The intensity of staining is directly proportional to the degree of bacterial contamination of the material. In the absence of microorganisms, the color of the system does not change. The advantage of the described method is to increase the sensitivity to 100% due to the use of urea, which is a substrate for H. pylori urease and forms NH ₃ as the final product. The latter is easily detected even in small amounts (2.5 • 10 ^-7 g) using Nessler's reagent. The color intensity allows us to differentiate the degree of bacterial contamination: 1 degree (weak) up to 20 microbial cells, 2 degree (moderate) up to 50 microbial cells and 3 degree (expressed) over 50 microorganisms in the test material, which is necessary for an indirect assessment of the severity of the disease. All this is carried out at a temperature of 25 ^o C, and therefore, eliminates the need for additional laboratory equipment and allows you to diagnose directly in the office of endoscopy.

Nessler's reagent is a solution of potassium tetraiodohydrate hydrate and KOH, forming a yellow-brown precipitate with NH ₃ ions.

Urea (x / h) is a white crystalline substance (urea, CO (H ₂ ) ₂ , M.M. 60.1, melting point 135 ^o C, boiling point decomposes), readily soluble in water and alcohols, insoluble in ether , benzene, chloroform.

 27 children were examined, 16 of them showed endoscopic hypertrophic, 9 erosive and 2 superficial gastritis. In 16 cases, gastritis was accompanied by a duodenal ulcer. The control group consisted of 10 children without endoscopic pathology.

 As a result of a biopsy study of children in the experimental and control groups using the method described above, it was found that the material from 10 children in the control group did not cause a color change in the test system. In the experimental group in all cases there was a yellow-brown staining of the medium of varying degrees of intensity. In 5 cases, a yellow-brown “ring” formed around the biopsy specimens, which corresponded to a weak degree of dissemination of H. pylori material. Biopsies of 12 children after 3 min caused the appearance of a yellow-brown “ring” and the formation of a yellow-brown sediment by the 5th minute of incubation, which corresponded to moderate bacterial contamination of the material. Incubation of biopsy samples of 10 children in the proposed reaction mixture caused sedimentation after 2-3 minutes, which corresponded to pronounced microbial contamination of the test material. The results obtained in the experimental and control groups were confirmed histologically and serologically, on the basis of which the etiology of gastroduodenal pathology was established and the corresponding etiotropic therapy was carried out.

Example 1. Patient N. (a boy of 12 years old) came for pain in the epigastrium and nausea. In the process of endoscopy, hypertrophic gastritis was visually detected. A biopsy was performed to determine the etiology of the disease and conduct etiotropic therapy. The biopsy was introduced into a sterile tube, 0.3 ml of a 10% solution of urea in bidistilled water and 0.1-0.2 ml of Nessler's reagent were aseptically added. The mixture was incubated at 25 ^o C. After 2 min, the appearance of a yellow-brown precipitate was established, which corresponded to a pronounced bacterial contamination of the material. A gastroduodenal pathology associated with Helicobacter pylori was diagnosed.

 The proposed method increases the specificity of the reaction / in relation to / products of hydrolysis of the substrate for urease / and its sensitivity up to 100%, while the prototype sensitivity is 72%. The use of the method of determination in a biopsy Helicobacter pylori for gastroduodenal pathology is necessary for the timely detection of carriers for diseases at the preclinical stage in the process of professional examinations and for conducting highly effective etiotropic therapy.

Claims (1)

 A method for determining Helicobacter pylori biopsy in gastroduodenal pathology, including incubating the biopsy in a mixture of a 10% urea solution and indicator, followed by evaluation of its seeding, characterized in that Nessler reagent is used as an indicator with a volume ratio of it and urea in the reaction mixture of 0, 1 0.2 0.3, respectively, and the dissemination of the Helicobacter pylori biopsy specimen is determined by the appearance of a yellow-brown ring around the biopsy specimen as a weak, yellow-brown ring, turning into a yellow-brown sediment, as moderate and yellow-brown sediment as expressed.