RU2648440C1 - Method for determination of species of mycobacteria of tuberculosis in the infected patient - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to clinical diagnostics and can be used for species identification of mycobacterium in patients, infected with mycobacterium tuberculosis. Concept of the method: carrying out the collection of venous blood in a test tube, containing anticoagulant heparin, mixing of blood with the antigenic material of mycobacteria (test sample) and with a salt solution of antigenic material, identical to the component composition of the solvent (control sample), incubation and determination in samples of the amount of CD45+ lymphocytes, gated by morphological characteristics and staining with a fluorescent dye. By reducing the number of labeled cells in the test sample relative to the control, that the organism is infected by that kind of mycobacteria, whose antigenic material was used to incubate with blood in a test tube. Method makes it possible to establish the species belonging of the causative agent of the disease, to confirm the presence of infection, determine the effectiveness of vaccination. Method is rapid-action, can be used as a screening method in the general treatment network and anti-tuberculosis institutions to determine the type of mycobacterium tuberculosis of a human species or a vaccine strain.

EFFECT: as a result of the application of the method, the accuracy of the study is increased, the possibility of performing the study using other dosage forms of allergens.

1 cl, 3 ex

Description

The invention relates to medicine, namely to clinical laboratory diagnostics of tuberculosis, and can be used for species identification of mycobacteria in individuals infected with mycobacterium tuberculosis.

It is known that the establishment of the type of pathogen of tuberculosis determines the option of etiotropic antibacterial therapy. Currently, species identification of Mycobacterium tuberculosis is based on cultural and molecular genetic studies. However, the first group of methods is time-consuming, lengthy in execution, carries the risk of infection of the researcher and requires the organization of a special complex of premises for analysis. The second group of methods involves the availability of expensive equipment, reagents, disposable supplies and special organization of the laboratory space. In both cases, the result will largely depend on the activity of bacterial excretion and the quality of taking the material for research.

A known method for the species identification of mycobacterium tuberculosis in individuals infected with mycobacterium tuberculosis (RU 2491551 C1). The method includes determining in heparinized venous blood in vitro the level of lymphocytes with CD45 receptors⁺ markers labeled with fluoroisotianate after blood incubation with antigenic material of mycobacteria (experimental sample) and saline (control sample), and to reduce the level of fluorescence of labeled CD45⁺ lymphocytes in the experimental sample relative to the control more than 20% make a conclusion about the species identification of mycobacteria. Moreover, in the known method in a control sample, physiological saline is used as a solvent — 0.9% NaCl saline.

It is known that in the experimental test with Diaskintest in terms of 0.2 ml of Diaskintest (2 doses of 0.1 each), in addition to the protein of recombinant CFP10-ESAT6 - 0.4 μg (2 doses of 0.2 μg), sodium chloride - 0 , 92 mg (2 doses of 0.46 mg) and water for injection - up to 0.2 ml (2 doses up to 0.1 ml) contain additional components:

- sodium phosphate disubstituted 2-water - 0.47752 mg (2 doses of 0.3876 mg);

- monosubstituted potassium phosphate - 0.126 mg (2 doses of 0.063 mg each);

- phenol - 0.5 mg (2 doses of 0.25 mg);

- Polysorbate 80 - 0.01 mg (2 doses of 0.005 mg);

In a test sample of a vaccine strain representing bovine mycobacterial antigens (BCG), in terms of 0.2 ml, 20 doses of the drug ((0.1 ml per 10 doses) * 2) are used except 1 mg of BCG microbial cells ((0.5 mg of microbial cells per 10 doses) * 2) and saline up to 0.2 ml ((0.1 ml per 10 doses) * 2), additionally contains 6 mg of monosodium glutamate ((3 mg per 10 doses) * 2).

The disadvantage of this method is the error that occurs when comparing the results of experimental samples with the control due to differences in the component composition of the solvent, which leads to a decrease in the accuracy of the species identification of mycobacterium tuberculosis.

The technical result is an increase in the accuracy of the method, the possibility of species determination of mycobacterium tuberculosis using various dosage forms of allergens.

The technical result is achieved by the fact that for comparison with experimental samples, control samples are used, the solvent composition of which is identical to the solvent composition of the antigenic substance of the compared experimental sample.

So, as a control for Diaskintest, a solution of the following composition is used: 0.92 mg of sodium chloride, 0.47752 mg of sodium phosphate disubstituted 2-aqueous, 0.126 mg of potassium phosphate monosubstituted, 0.5 mg of phenol, 0.01 mg of polysorbate 80 and up to 0 , 2 ml of water for injection, identical to the component composition of Diaskintest solvent.

To control the samples of the vaccine strain representing bovine mycobacterial antigens (BCG), a solution containing 6 mg of sodium glutamate and up to 0.2 ml of physiological saline is used, which corresponds to the solvent components of the vaccine strain representing bovine mycobacterial antigens (BCG).

The proposed method allows to increase the accuracy of the method of species identification of mycobacterium tuberculosis in individuals infected with mycobacterium tuberculosis, due to the creation of identical solvent mixtures of comparable composition, to reduce the effect of the ingredients of the salt mixture on the result of the study. The method also makes it possible to carry out a specific determination of mycobacterium tuberculosis using other dosage forms of allergens, regardless of the component composition of the solvent of Mycobacterium tuberculosis antigens.

The reasons for the development of the proposed method were the following:

1. Diaskintest contains 0.5 mg of phenol as a preservative, creating a 0.25% solution. As you know, back in 1914, A. Fleming was able to prove that phenol (hydroxybenzene, carbolic acid) “kills” white blood cells (CD45 ⁺ cells), which create a natural immune barrier to wounds [1]. It has now been shown that the most damaging effect is exerted by the dissolved form of the compound, which causes an inflammatory reaction, the main participants of which are white blood cells. A change in the functional and metabolic activity of leukocytes under the influence of phenol has been proven [2], which can lead to a decrease in the proportion of cells bearing the CD45 ⁺ marker on their surface.

2. Expression of surface markers on cells (in this case, CD45 ⁺ ) depends on various factors, including the ionic composition of the medium. The drug Diaskintest, which contains a complex of buffer salts of sodium phosphate disubstituted 2-aqueous and potassium phosphate monosubstituted, allows you to create the optimal environment for the existence of in vitro blood cells. The use of non-buffered saline as a control against the use of a buffered experimental sample in the prototype is not correct in the study of the receptor apparatus of the cell.

3. Polysorbate 80 (tween 80), which is part of Diaskintest, is the causative agent of anaphylactoid reactions [3], initiates inflammation of some tissues [4]. Its structure is similar to the proteins that form the membrane-attacking complex, leading to cell tunneling and their subsequent destruction. A comparative analysis of a suspension of cells dissolved in a saline solution containing Polysorbate 80 must be carried out using a liquid containing the same component as a control sample.

4. The experiment proved the ability of glutamate sodium to stimulate an increase in the number of lymphocytes. The number of cells in comparison with the control can increase by 12% or more [5], which will affect the results of ongoing studies. In order to avoid a significant measurement error, it is optimal to introduce sodium glutamate into the control sample.

The proposed method for the specific determination of mycobacterium tuberculosis in an infected patient is new and meets the criterion of "inventive step".

The method is as follows. In a patient subject to aseptic and antiseptic rules, peripheral blood is collected in a standard manner in a test tube containing an anticoagulant (lithium heparin) in a volume of 5-7 ml. In four tubes, 1 ml of blood is added. In each of them add 0.2 ml of a solution containing:

Test tube No. 1 (Diaskintest control sample): 0.92 mg of sodium chloride, 0.47752 mg of sodium phosphate disubstituted 2-aqueous 0.126 mg of potassium phosphate monosubstituted, 0.5 mg of phenol, 0.01 mg of polysorbate 80 and up to 0.2 ml water for injection;

Test tube No. 2 (experimental test of Diaskintest): 0.2 ml of Diaskintest;

Test tube No. 3 (control sample of a vaccine strain representing bovine mycobacterial antigens - BCG): 6 mg of sodium glutamate and up to 0.2 ml of physiological saline;

Test tube No. 4 (experimental test BCG): 0.2 ml of the vaccine strain representing bovine mycobacterial antigens - BCG.

After mixing blood with solutions, the tubes are incubated at 37 ° C for 24 hours. Then, the standard manipulations used in the study by flow cytofluorimetry are performed to evaluate the expression of CD45 ⁺ antigen on the surface of lymphocytes. In particular, 100 μl of a sample is taken from each test tube of the experimental and control samples, the taken volumes are stained with anti-CD45 ⁺ monoclonal antibodies for 30 minutes in the dark by incubation at room temperature, subsequently the red blood cells interfering with the analysis are destroyed with a hemolytic mixture, and the percentage of labeled cells is determined on a flow cytometer lymphocytes during heterogeneous gating of cells (based on morphological characteristics and staining with a fluorescent dye).

To reduce the number of cells labeled with a fluorescent dye in the experimental sample relative to the control, it is concluded that the body is infected with the species of mycobacteria whose antigenic material was used for incubation with blood in the experimental tube. The calculation of the indicator is carried out according to the formula: (control-experience) / control * 100%. A decrease in the calculated indicator by a value of more than 20% is considered as an indicator of infection by the investigated agent, a change by an amount equal to and less than 20% is regarded as the absence of infection by the investigated agent.

The method allows you to more accurately establish the species of the causative agent of the disease, confirm the presence of infection, determine the effectiveness of vaccination. The method is quick. It can be used as a screening method in the general medical network and anti-tuberculosis institutions to determine the type of mycobacterium tuberculosis of a human species or a vaccine strain.

The proposed method is illustrated by the following examples.

Example 1. Patient I., 10 years old. BCG vaccination was carried out in the hospital (post-vaccination scar - 10 mm). Directed for examination in connection with an increase in the post-vaccination mark. The last Mantoux test is a papule size of 14 mm. Blood was taken from a patient for examination. Laboratory manipulations were performed, including mixing 1 ml of heparinized blood with solutions in four test tubes.

Test tube No. 1 (Diaskintest control sample) - 0.2 ml of saline solution containing: 0.92 mg of sodium chloride, 0.47752 mg of sodium phosphate disubstituted 2-aqueous, 0.126 mg of potassium phosphate monosubstituted, 0.5 mg of phenol, 0, 01 mg of polysorbate 80 and up to 0.2 ml of water for injection.

Test tube No. 2 (experimental test of diaskintest) - 0.2 ml of Diaskintest.

Test tube No. 3 (control sample of a vaccine strain representing bovine mycobacterial antigens - BCG) - 0.2 ml of saline solution containing: 6 mg of monosodium glutamate and up to 0.2 ml of physiological saline.

Test tube No. 4 (experimental test BCG) - 0.2 ml of the vaccine strain representing bovine mycobacterial antigens - BCG.

The following results were obtained:

Test tube No. 1 - 58% of CD45 ⁺ lymphocytes (control sample Diaskintest);

Test tube No. 2 - 56% (experimental test of Diaskintest);

Test tube No. 3 - BCG - 61% (control sample of a vaccine strain representing bovine mycobacterial antigens);

Test tube No. 4 - 39% (experimental test BCG).

Calculation:

Test with Diaskintest: (control-experience) / control * 100% = (58-56) / 58 * 100% = 3.4% (decrease less than 20%)

A sample with a vaccine strain representing bovine mycobacterial antigens - BCG: (control-experience) / control * 100% = (61-39) / 61 * 100% = 36.1% (decrease of more than 20%).

Conclusion: the presence of bovine tuberculosis infection with mycobacteria.

Example 2. Patient B., 12 years old. BCG vaccination (7.5 mm post-vaccination scar) was performed in the maternity hospital. The results of the annual Mantoux tuberculin tests performed in the clinic at the place of residence, preschool, comprehensive school according to the vaccination certificate: 9, 7, 7, 8, 9, 7, 8, 9, 8, 11, 11. A slight increase in the result is not allows you to confidently confirm the presence of infection by a pathogenic strain. It is aimed at an additional examination to conduct a laboratory study to establish the species of the pathogen. The subject was taken with an anticoagulant according to the method described above for the study. The following components were added to the tubes:

Test tube No. 1 (Diaskintest control test): 1 ml of blood + 0.2 ml of saline solution containing: 0.92 mg of sodium chloride, 0.47752 mg of sodium phosphate disubstituted 2-aqueous 0.126 mg of potassium phosphate monosubstituted, 0.5 mg of phenol , 0.01 mg of polysorbate 80 and up to 0.2 ml of water for injection;

Test tube No. 2 (experimental test of Diaskintest): 1 ml of blood + 0.2 ml of Diaskintest;

Test tube No. 3 (control sample of the vaccine strain - BCG): 1 ml of blood + 0.2 ml of saline solution containing: 6 mg of monosodium glutamate and up to 0.2 ml of physiological saline;

Test tube No. 4 (experimental test BCG): 1 ml of blood + 0.2 ml of the vaccine strain representing antigens of bovine mycobacteria - BCG. Laboratory studies performed. The number of CD45 + lymphocytes in test tubes: No. 1 - 65%, No. 2 - 41%, No. 3 - 68%, No. 4 - 67%. Calculation:

Test with Diaskintest: (control-experience) / control * 100% = (65-41) / 65 * 100% = 36.9% (decrease over 20%)

Sample with a vaccine strain representing bovine mycobacterial antigens - BCG: (control-experience) / control * 100% = (68-67) / 68 * 100% = 1.5% (decrease less than 20%)

Conclusion: the presence of Mycobacterium tuberculosis infection of the human species.

Example 3. (The use of other dosage forms of allergens for the species determination of mycobacterium tuberculosis, for example, purified tuberculosis allergen in standard dilution).

Patient P., 12 years old. Vaccinated in a maternity hospital (post-vaccination scar 7 mm). Mantoux tuberculin tests were performed annually. The result of the latter was represented by a 10 mm infiltrative seal. Clinical and radiological data correspond to the presence of active tuberculosis. In the family, the father has been suffering from pulmonary tuberculosis for more than three years. A specific determination of mycobacterium tuberculosis in an infected patient was carried out using a laboratory blood test. In four test tubes, 1 ml of blood was mixed and:

Test tube No. 1 (control sample of the tuberculosis allergen purified in standard dilution) - sodium hydrogen phosphate heptahydrate - 1.566 mg, sodium chloride - 0.914 mg, potassium dihydrogen phosphate - 0.126 mg, polysorbate - 80 - 0.01, phenol - 0.5 mg.

Test tube No. 2 (experimental sample of purified tuberculosis allergen in standard dilution) - 0.2 ml of purified tuberculosis allergen in standard dilution containing two doses of the active substance of tuberculoprotein allergen - 2 TE (tuberculin units).

Test tube No. 3 (control sample of a vaccine strain representing bovine mycobacterial antigens - BCG) - 6 mg of sodium glutamate and up to 0.2 ml of physiological saline.

Test tube No. 4 (experimental test BCG) - 0.2 ml of the vaccine strain representing bovine mycobacterial antigens - BCG.

According to the results of immunophenotyping, the number of CD45 + lymphocytes in test tubes was: No. 1 - 58%, No. 2 - 39%, No. 3 - 65%, No. 4 - 62%.

Calculation:

Test with Diaskintest: (control-experience) / control * 100% = (58-39) / 58 * 100% = 32.8% (decrease more than 20%)

Sample with a vaccine strain representing bovine mycobacterial antigens - BCG: (control-experience) / control * 100% = (65-62) / 65 * 100% = 4.6% (decrease less than 20%)

Conclusion: the presence of Mycobacterium tuberculosis infection of the human species.

The advantage of the proposed method over the known lies in the increased accuracy of the study in comparison with the prototype, the ability to perform studies using other dosage forms of allergens (regardless of the component composition of the solvent of Mycobacterium tuberculosis antigens), reducing the time for research in comparison with known analogues. This method can find wide clinical application in the diagnostic laboratories of TB facilities and the general treatment network for the species identification of mycobacterium tuberculosis.

Used Books

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2. Lazarev N.V. Harmful substances in industry / M .: Book on demand, 1977. - 589 p. (S. 404-406).

3. Coors E.A., Seybold N., Merk H.F., Mahler V. Polysorbate 80 in medical products and nonimmunologic anaphylactoid reactions // Ann Allergy Asthma Immunol. - 2005. - No. 95 (6). - P. 593-599.

4. Yuan Z.Y., Hu Y.L., Gao J.Q. Brain localization and neurotoxicity evaluation of polysorbate 80-modified chitosan nanoparticles in rats // PLoS One. - 2015. - No. 10 (8). - e. 0134722.

5. Kumar S., Kumar N., Kumar B. Evaluation of mono sodium glutamate induced nephrotoxicity in adult Wistar albino rats // World journal of pharmacy and pharmaceutical sciences. - 2015. - Vol. 4. - Iss. Number 4. - P. 846-862.

Claims (1)

A method for the specific determination of mycobacterium tuberculosis in an infected patient by comparing the number of lymphocytes expressing on the surface of CD45 + markers labeled with fluorescein isothiocyanate in heparinized venous blood with the antigenic material of mycobacteria - an experimental sample and saline - a control sample, characterized in that the experimental sample is compared with a control , the saline solution of which is identical in composition to the solvent of the biological preparation of the experimental sample.