RU2126052C1 - Method of detecting tuberculosis mycobacteria - Google Patents

Abstract

FIELD: phthysiology. SUBSTANCE: paired smears are prepared from material under study (phlegm, smear from epiglottis or from tonsils). One of smears is treated for 24 h with solution provoking osmotic cytolysis, the other being used as reference one. The two smears are stained with fluorochromes and microscope. If typical morphologically unchanged mycobacteria are revealed, preparation is regarded as positive and patient as contagious. Revealing of differences in population detected tuberculosis mycobacteria in reference (not treated with disintegrating solution) and experimental (treated) smears-prints makes it possible to identify modified (L-transformed) cells. As osmotic cytolysis provoking solution, solution with following composition is used, g/l: sucrose, 2.0-16.0; sodium chloride, 0.1-0.8; disodium ethylenediaminetetraacetate, 0.1-0.8; disodium phosphate, 0.1- 0.8; monosodium phosphate, 0.05-0.4; sodium orthophosphate, 0.05-0.4; citric acid, 0.05-0.4 dimethylsulfoxide, 0.02-0.2; bidistilled water, up to 1 l. EFFECT: enabled maximum liberation of mycobacteria from cell pool and so increased efficiency of isolation of bacteria in tuberculosis patient. 2 cl, 2 ex

Description

 The invention relates to the field of biotechnology and medical microbiology and can be used in the diagnosis of tuberculosis.

 Tuberculosis, being an infectious disease, is caused by a specific pathogen - mycobacterium tuberculosis. With the formation of microcolonies of the pathogen in the lungs or other tissues, the clinical form of a specific process develops in the body. Therefore, the detection of mycobacteria in sputum or other material is the main diagnostic criterion for a patient with tuberculosis.

 A known method for the detection of mycobacterium tuberculosis, including taking lymphoid tissue, crushing and processing it with artificial gastric juice to maximize the release of the pathogen from the biomaterial, followed by the study of the enriched material by the bacteriological method (SU 1734699, A 61 B 10/00, 05/23/92).

 However, the known method does not provide high accuracy for the diagnosis of tuberculosis.

 There is also a method for detecting mycobacteria in extracellular forms of tuberculosis, including introducing a hemolyzer into the test material (blood, endometrial scraping), subsequent sonication, preparation of smears from the resulting precipitate and flotation ring, staining with phosphor and detection of mycobacteria by cell luminescence (SU 1286933, A 61 B 10/00, 01.30.87).

 However, the known method is used only for the diagnosis of tuberculosis of the female genital organs.

 The closest analogue is a method for the detection of mycobacterium tuberculosis, which consists in the fact that the native test material or centrifuged inoculum is deposited on a glass slide, fixed, stained with fluorochromes and smear microscopy (Shestochenko MA, Kuzmin N.A. Luminescent analysis in veterinary medicine .-M .: Kolos, 1979, pp. 96-98).

The disadvantages of the analogue include the following:
- in the presence of an abundant cell pool in the preparation, part of mycobacteria either does not stain at all, being in the thickness of the smear, or, staining, is not determined by the microscope, being covered with cell layers;
- a high population density during intracellular reproduction of the pathogen does not allow to identify in detail the form of acid-resistant mycobacteria;
- in a traditionally prepared smear, it is impossible to determine the ratio of the intra- and extracellularly located forms of the pathogen, which reduces the information content of the study.

 The technical result of the invention is to increase the accuracy of the method due to a more complete identification of mycobacteria in samples, including L-transformed ones, as well as the possibility of detailed identification of the shape of the intracellular location of acid-resistant formations and determination of the intracellular and extracellularly located forms of the pathogen.

 The essence of the invention is as follows: from the test material (sputum, smear from the epiglottis or tonsils), paired smears are prepared, one of which is treated for at least 24 hours with a solution that causes osmotic cytolysis, and the other is used as a control. After staining with fluorochromes, both smears are microscopic. Detection of typical, morphologically safe mycobacteria allows us to consider the drug positive and the patient dangerous. The detection of differences in the population of detected tuberculosis mycobacteria in the control (without treatment with a disintegrating solution) and in the experimental (processed) smear-imprints allows the identification of modified (L-transformed) cells.

As a solution that causes osmotic cytolysis, use a solution of the following composition:
Sucrose - 2.0-16.0 g
Sodium chloride - 0.1-0.8 g
Disodium ethylenediaminetetraacetate - 0.1-0.8 g
Disubstituted sodium phosphate - 0.1-0.8 g
Sodium phosphate monosubstituted - 0.05-0.4 g
Sodium orthophosphate - 0.05-0.4 g
Citric acid - 0.05-0.4 g
Dimethyl sulfoxide - 0.02-0.2 ml
Double-distilled water - Up to 1 l
The method allows the maximum release of mycobacteria from the cell pool and thereby increase the effectiveness of bacterial excretion in tuberculosis.

 The invention is illustrated by the following examples.

 Example 1. For the study, sputum and / or smears from the epiglottis and tonsils are taken. A smear collection is performed by a qualified otolaryngologist. Paired smears-prints are prepared from the obtained material by a known method. One of the paired fingerprint smears serves as a control. After fixation, one of the smears (experimental) is applied with 0.1 ml of a solution that causes osmotic cytolysis, and placed in a moist chamber for 24 hours. After that, the smear is dried and re-fixed. The control smear is identical, but without the addition of a disintegrating solution. After staining with fluorochromes, both smears are microscopic. If typical, morphologically safe mycobacteria are detected, the drug is considered positive, and the patient is epidemiologically dangerous. If intracellular or extracellularly located acid-resistant granular or other forms (L-transformed) are detected, “cell” or “tissue” tuberculosis or a combination thereof is identified.

 Example 2. Surveyed 42 patients of the hospital and clinic. The contingent of the examined consisted of both previously confirmed bacilli excipients, who made up 20% of the studies, and those who did not previously have bacillus excretion - 80% of the studies.

 In all patients belonging to the category of bacilli, mycobacterium tuberculosis was found according to the invention both in a smear from the epiglottis, and in a smear from the tonsils. By the known method (Ziel - Nielson), mycobacteria were detected only twice and only in smears from the epiglottis. Among patients not belonging to the category of bacilli, mycobacterium tuberculosis was detected by the claimed invention in 32 smears from tonsils, which accounted for 60% of all studies, in smears from the epiglottis - in 5 cases, in sputum - in one. In this case, the Ziehl-Nielson method did not reveal mycobacteria in the indicated contingent even once.

 Along with the discovery of morphologically intact forms in a number of patients, spherical L-forms were found by the method according to the invention. The luminescence intensity of L-forms was less pronounced in comparison with rod-shaped mycobacteria (bright ruby-orange color). The color of L-forms ranged from moderate lemon to slightly yellow with a greenish tint.

 In two patients with the presence of typical rod-shaped mycobacteria, granular forms located extra- and intracellular were found in smears from the epiglottis in preparations from the tonsil. In two other patients, with repeated studies, acid-resistant granular forms in smears were detected each time from the epiglottis and tonsils.

 Thus, the invention allows to significantly increase the percentage of detection of Mycobacterium tuberculosis in the test material and to identify their L-transformation.

Claims (2)

 1. A method for detecting mycobacterium tuberculosis, including the manufacture of smear prints from the test material, their fixation, staining with fluorochromes, followed by the results of fluorescence microscopy, characterized in that the test material used is sputum, smears from the epiglottis and / or tonsils, prepared from paired fingerprints, one of which is treated for at least 24 hours with a solution that causes osmotic cytolysis, and the other is used as a control, while the results are taken into account the detection of characteristically luminous formations in the processed smear imprint. 2. The method according to claim 1, characterized in that as a solution causing osmotic cytolysis, use a solution of the following composition:
Sucrose - 2.0 - 16.0 g
Sodium chloride - 0.1 - 0.8 g
Disodium ethylenediaminetetraacetate - 0.1 - 0.8 g
Disubstituted sodium phosphate - 0.1 - 0.8 g
Sodium phosphate monosubstituted - 0.05 - 0.4 g
Sodium orthophosphate - 0.05 - 0.4 g
Citric acid - 0.05 - 0.4 g
Dimethyl sulfoxide - 0.02 - 0.2 ml
Double-distilled water - Up to 1 liter.