RU2478957C2 - BACTERIOLOGICAL DIAGNOSTIC TECHNIQUE FOR Helicobacter pylori IN GASTRIC MUCOSA OF PATIENTS SUFFERING GASTRODUODENAL PATHOLOGY - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: bacteriological diagnostic technique for Helicobacter pylori in gastric mucosa is implemented as follows. The examined material (biopsy material of gastric mucosa) is inoculated on Petri dishes containing standard medium of low sea water and agar. If small colourless colonies (of the diameter of 0.5-1.0 mm) with a smooth surface appeared to grow on the nutrient medium after 72-96 hours of incubation at temperature 36.0±1.0°C and high CO₂; the colonies are microscopically examined. If the smear occurred to contain thin Gram-variable rods or coccal forms, the grown colonies are examined for catalase formation, oxidase formation, urease formation, for an ability to degrade sodium hippurate, the absence of x factor (hemin consumption) and acid resistance (the absence of growth in thioglycollate medium at pH-3.5).

EFFECT: use of the invention reducing the length of examination and provides higher diagnostic reliability for Helicobacter pylori infection.

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Description

The invention relates to medical bacteriology and can be used for bacteriological diagnosis of Helocobacter pylori-associated diseases of the human stomach and duodenum.

In recent years, the clinical significance of Helicobacter pylori-associated diseases of the gastroduodenal zone, which can be considered as a special case of helicobacteriosis, where Helicobacter pylori (H. pylori) is one of the most significant etiopathogenetic factors, has been increasing. This is more true for patients with a gastroenterological profile and can lead to the development of stomach ulcers and duodenal ulcers, acute and chronic gastritis, and esophagitis.

A known method for the diagnosis of H. pylori in the gastric mucosa (coolant) according to the results of cytological (detection of spiral, bent or cocciform bacteria) and biochemical studies (production of the urease enzyme) [Safronova N.V., Zhebun AB Gastritis, peptic ulcer and helicobacteriosis. Recommendations for doctors. - St. Petersburg, 1993. - 38 p.].

The disadvantage of this method is its low specificity due to the presence in the coolant of other opportunistic strains of microorganisms, including those similar to H. pylori in morphological and biochemical characteristics.

The closest analogue of the proposed method is a detailed bacteriological study of the coolant biopsy, which is currently carried out only in complex diagnostic cases [D. Hoult, Krieg N., Snit P. and others. Identifier of bacteria Berge. Ninth Edition. In two volumes. - 1997. - T.1. - S. 46, 51, 65]. This method includes the following steps: inoculation of a biopsy specimen on a cardiac brain (SMB agar) or chocolate agar; determination of the nature of the growth of colonies on culture media morphology study; enzymatic properties; hydrolysis of fats; decarboxylation of amino acids; growth characteristics on selective nutrient media (including in the presence of certain additives, various levels of pH and temperature conditions). A total of 16 differentiating characters are examined.

The developed method of cultural diagnosis of N. pylori did not find wide practical application due to shortcomings: the difficulty of execution due to the large set of biochemical and enzymatic tests, the significant consumption of expensive nutrient media, usually imported, with a long (over 5 days) analysis period. The proposed diagnostic method eliminates the above disadvantages.

Signs of the known technical solution, which coincides with the features of the claimed invention, consist in the operations of determining the cultural and biochemical properties of the pathogen when sowing biological material on nutrient media, a standard incubation regime, establishing the tinctorial and morphological properties of the studied strain, as well as testing for catalase formation, oxidase formation, urease formation .

The problem to which the invention is directed, is to increase the efficiency of laboratory diagnosis of H. pylori.

The technical result that provides the solution of this problem is to reduce the time of the study and increase the reliability of the diagnosis.

The technical result is achieved by the fact that in the method of bacteriological diagnosis of N. pylori infection of the gastric mucosa in patients with gastroduodenal pathology, including the determination of the cultural and biochemical properties of the pathogen when seeding biological material on nutrient media, a standard incubation regime, the establishment of tinctorial and morphological properties of the studied strain as well as testing for catalase formation, oxidase formation, urease formation, according to the invention additionally testing for the decomposition of sodium hippurate, x-factor and acid resistance. Moreover, the use of a combination of distinctive features can significantly reduce the time of diagnosis and increase the reliability of the diagnosis.

Identification of the hallmarks of H. pylori: the ability to decompose sodium hippurate, the absence of the x-factor and acid resistance are diagnostic criteria in determining the pathogen from the closest representatives of the conditionally pathogenic and normoflora (corynebacteria, hemophilic bacillus, lactobacilli). So, corynebacteria (Corynebacterium sp.) Do not distinguish between sodium hippurate, hemophilic bacillus (Haemophilus influenzae) has a high demand for hemin - the presence of x-factor, increased growth of culture is observed around the disk with saponin. Lactobacilli (Lactobacillus sp.) Are acid-resistant (grow on thioglycol medium with a low pH of 3.5) and do not decompose sodium hippurate, streptococcus (Streptococcus sp.) - lack of x-factor and acid resistance (table).

New features of the claimed method are the use of tests for x-factor, acid resistance and decomposition of sodium hippurate. The method is as follows.

The studied material (biopsy specimen of the gastric mucosa) is seeded on plates with standard medium - SMB agar. In the case of growth on a nutrient medium after 72-96 hours of incubation at a temperature of 35-37 ° C and an increased CO ₂ content _of small (with a diameter of 0.5-1.0 mm) colorless colonies with a smooth surface, they are microscopic. If thin gram-variable rods or coccoid forms are found in the smear, the grown colonies are examined for catalase formation, oxidase formation, urease formation, determine the ability to decompose sodium hippurate, the absence of the x-factor (no need for hemin) and acid resistance (no growth in thioglycol medium with pH-3, 5).

If small translucent catalase-positive, oxide-positive, urea-positive colonies with a diameter of 0.5-1.0 mm are found on the SMV agar, polymorphic bacilli or coccobacteria decomposing sodium hippurate, lacking the x-factor and acid resistance in the smears, the microorganism is classified as H. pylori .

Case Studies

Example 1. Patient B., 62 years old (source. Bol. No. 3529) clinical diagnosis: gastroesophageal disease, insufficiency of cardiac pulp, diverticulum of the antrum, duodeno-gastric reflux. Complaints: discomfort in the right half of the abdomen and gastroduodenal zone of a constant nature. Objectively: the condition is satisfactory, the tongue is covered with a white coating. Palpation: the abdomen is soft, painless, the liver is enlarged by 2 cm. Conclusion of fibrogastroscopy: superficial antrum gastritis (slight changes). Unexpressed insufficiency of cardiac pulp. Diverticulum-like formation of the antrum. Cytology from the diverticulum-like formation of the antrum: erythrocytes cover the entire field of view, neutrophils - 2-5 p / v, helicobacteria in large numbers, layers and accumulations of integumentary-fossa epithelial cells without signs of atypia. X-ray of the stomach, conclusion: insufficiency of cardiac pulp, diverticulum 7 × 5 mm, duodenal gastric reflux in the antrum. Laboratory studies: a general blood test - without pathology, a general urinalysis - without pathology. Biochemical analysis of blood: total protein 84 g / l, urea 7.0 mmol / l, cholesterol 7.5 mmol / l, triglycerides 2.67 mmol / l, bilirubin 34.2 mmol / l (bound 4.9 mmol / l , free 29.4 mmol / L), alpha amylase 124 units / L, alkaline phosphatase 129 units / L, thymol sample 2.5. An enzyme-linked immunosorbent assay revealed a titer of antibodies to the CagA antigen of HP 1:80 (a sharply positive result), IgA (helico-express) - a weakly positive result, helico A and G - a positive result. During bacteriological culture of a biopsy specimen of the gastric mucosa on the surface of the medium SMB agar after 72 hours of incubation at a high content of CO ₂ (in the desiccator) and at a temperature of 37 ° C, small colorless colonies were detected; total microbial number 10 ³ CFU / ml. Microscopy - gram-wise thin curved movable sticks. When setting differentiating tests, the X factor is absent. A test with a 3% solution of hydrogen peroxide is positive (test for catalysis), an oxidase test and a test with sodium hippurate are positive. The microorganism actively decomposes urea and does not have acid resistance. Thus, in a patient with a clinical diagnosis of erosive antrum gastritis and indirect signs of H. pylori infection (cytological and immunological method), H. pylori was isolated by a bacteriological method.

Example 2. Patient Sh., 73 years old (source. Bol. No. 931) clinical diagnosis: chronic erosive gastroduodenitis, stage of exacerbation. Chronic secondary pancreatitis. Hypertension 2 degrees. Complaints: periodic pain in the upper abdomen, bloating in the evening, episodes of blood (scarlet) in the feces. Objectively: the condition is satisfactory. The tongue is coated with white plaque. The abdomen is soft, painful in the epigastrium and left hypochondrium, increased in size due to bloating. Conclusion FGS: erosive antrum gastritis, erosive bulbitis. Cytological examination of a biopsy specimen of the gastric mucosa: erythrocytes in large numbers, neutrophils 0-2 in p / vision. Small accumulations of integumentary fossa epithelial cells without signs of atypia. Laboratory tests: complete blood count: red blood cells 4.7 * 10 ¹² / l, hemoglobin 140 g / l, white blood cells 10.35 * 10 ⁹ , of which eosinophils 8%, stab 3%, segmented 54%, lymphocytes 17%, monocytes 17%, ESR 7 mm / h. General urine analysis without pathology. Biochemical blood test without pathology. Blood test for H. pylori: IgA-positive, IgG-positive, antibodies to HP CagA - weakly positive result. When seeding a biopsy specimen of the gastric mucosa on a medium of SMB agar after 72 hours of incubation at a high content of CO ₂ and at a temperature of 37 ° C, small colorless colonies were detected; total microbial number 10 ³ CFU / ml. For species identification, the isolated strain was catalase- and oxide-positive, decomposed urea, did not possess the X-factor and acid resistance. The sodium hyppurate test worked positively.

Thus, in a patient with a clinical diagnosis of erosive antrum gastritis, H. pylori infection was confirmed by a bacteriological method.

Example 3. Patient P., 53 years old (source. Bol. No. 1249). Clinical diagnosis: peptic ulcer of the duodenum, cholecystitis. Objectively: satisfactory condition, temperature 36.6 C. Conclusion of fibrogastroscopy: reflux esophagitis 1 tbsp., Erosive antrum gastritis against the background of hyperplasia of the mucous membrane. Enzyme-linked immunosorbent assay: IgG-positive, antibodies to CagA antigen not detected. Laboratory tests: general blood test - without pathology. Urinalysis: specific gravity 1019, neutral reaction, neg. Protein, phosphates ++++. Blood chemistry: urea 8.6 mmol / L, creatinine 115 mmol / L, total bilirubin 14.7 mmol / L, ALT 56 mmol / h / L, ACT 42 mmol / h / L. When the coolant biopsy was inoculated on SMB agar after 96 hours of incubation, pointless colorless colonies, oxide-positive, catalase-positive, decomposing urea and sodium hippurate and lacking the X-factor were detected. When the culture of the pathogen is inoculated into a thioglycol medium with a pH of 3.5, there is no growth, which indicates the absence of acid resistance. Thus, in a patient with clinical signs of erosive antrum gastritis, H. pylori was isolated during culture diagnosis.

This method was used to study 15 clinical N. pylori strains isolated from patients with "gastric ulcer" and confirmed by other laboratory diagnostic methods (clinical, immunological, advanced microbiological). The control group consisted of 50 patients with various pathologies and practically healthy patients, from which clinical representatives of microflora - Corynebacterium sp., Haemophilus sp., Lactobacillus sp., Streptococcus sp. Generic and species affiliation of these microorganisms was also confirmed by the method of detailed cultural diagnostics.

The proposed method for the diagnosis of N. pylori in gastroduodenal pathology is informative, reliable, shortens the analysis time (up to 3-4 days), is carried out on standard nutrient media, does not require expensive equipment for production, is convenient for setting up, and is simple in terms of methodology. Therefore, without significant material costs and in a short time, it can be introduced into the work of any bacteriological laboratory. The method can be recommended for a more accurate etiological diagnosis of human gastroenterological diseases.

Claims (1)

A method for bacteriological diagnosis of Helicobacter pylori - infection of the gastric mucosa in patients with gastroduodenal pathology, including the determination of the cultural and biochemical properties of the pathogen when sowing biological material on nutrient media, the standard incubation regime, the establishment of tinctorial and morphological properties of the studied strain, as well as testing for catalase formation, oxidase formation , urease formation, characterized in that they additionally test for the x-factor, acid toustoychivost and decomposition of sodium hippurate.