UA34746A - Method for quick tuberculosis diagnostics - Google Patents

Abstract

The invention relates to medical and veterinary microbiology. The method includes complex of studies of microscopy of blood smears, with treatment of pathological material with inhibitor, with inoculation of medium. In the smears one finds specific tuberculosis agent astrospheres, molicutes, segmented stems of molicutes, or one of other stages of tuberculosis pathogen, pathological material is after treatment with inhibitor additionally kept during 12-24 hours in stimulator of mycobacterium growth, this includes (mass %) C - 21,2, Na - 6,1, H - 42,4, O - 30,3 with inoculation of medium.

Description

The invention relates to medical and veterinary microbiology and can be used in medical and veterinary practice.

There are well-known methods of diagnosing tuberculosis by conducting clinical, patho-anatomical, allergic and laboratory tests.

Laboratory diagnostics is based on bacteriological, histological and biological research methods.

The closest method in terms of technical essence to the proposed method is described in "Methodical instructions for the application of unified microbiological studies in tuberculosis" (M., 1978). In accordance with it, microscopy of smears of the test material is carried out, then the samples are treated with an inhibitor (sulfuric acid, sodium trioxygen phosphate, etc.) and sown on a nutrient medium.

The growth of the pathogen on existing nutrient media takes 20-90 days. Then, over a period of 10 days, the sensitivity of the mycobacterial strain to medical drugs is determined. That is, the diagnosis is made after 30-100 days, after which targeted treatment of the patient begins.

The main disadvantage of the prototype is that the method is long-term and its efficiency is low.

The task of this invention is to reduce the time of diagnosis of tuberculosis by using a complex of studies.

The task is solved by the fact that for the tentative diagnosis of tuberculosis, a blood smear, stained according to Romanovsky-Giemz, or the unified method of Pappenheim is carried out and a specific tuberculosis agent is determined, which is one of the forms of development of mycobacteria in accordance with the concept of staged development of mycobacteria developed by the authors (Figure 1).

The hypothesis developed by the authors is that fragmentation occurs in Koch rods under certain conditions, while the plasma inside the cell breaks up into separate parts and several arthrospores are formed. The cell membrane disintegrates and arthrospores are released. Under favorable conditions, mollicutes develop inside the arthrospores. The entire contents of the cell (arthrospores) are spent on the formation of mollicutes. Under unfavorable conditions, arthrospores can significantly decrease to the size of 0.12-0.15 μm in diameter and can pass bacteriological filters.

Mollicutes are not stable. With smear microscopy, we see coccoid cells. Mollicutes germinate in the form of 1-3 stems. After some time, segmentation of the formed stem (filament) occurs, then partitions are formed that divide them into a number of segments (zebra). Each segment breaks up into several particles, which form rods (Koch rod).

In a blood smear at the initial stage of the disease, we detect one of the stages of the development of mycobacteria.

The problem is also solved by the fact that seed treated with inhibitors is treated with a growth stimulator, which includes (wt. 95): C - 21.2; Ma - 6.1; H - 42.4; O - 30.3; kept in a thermostat at a temperature of 37-417C for 12-24 hours and sown on a nutrient medium of the following composition: dry agar-agar - GOStT-1628-70 - 3.0; peptone - GOST-1385-60 - 7.0; calcium chloride - 0.012; potato starch - 1.5; distilled water up to 100 ml; The pH of the medium is 7.3.

According to the concept developed by the authors, tuberculosis mycobacteria under certain conditions go through a series of successive stages of development. (V.V. Vlasenko, Yu.O. Kolos and others, 1998). Figure 1 shows these stages.

Fig. 1 Koch rod. Fig. 2 Fragmentation of sticks, Fig. From Arthrospora, Fig. 4 Formation of mollicutes, Fig. 5 Mollicutes,

Fig. 6 Germination of mollicuta, in the form of a stem, Fig. 7 Segmentation of stems (zebra), Fig. 8 Separation of stem segments. Fig. 9 Disintegration of segments into sticks, Fig. 10 Koch rod.

The Koch rod, stained according to Tziel-Nilson, acquires a uniform ruby color. In the process of further development, as a result of fragmentation, several cocci-shaped formations are formed under the general shell of the rods, which are visible when stained by the Gram-Fly method (Fly grains). Later, under certain conditions, the granular papillae turn into coccoid forms - arthrospores. Under favorable conditions, arthrospores increase in size, acquire a pear-shaped or ovoid shape, in the middle of which several amoeboid cells are formed. During the ripening period, through the narrowed end of the arthrospore, amoeboid cells - mollicutes go outside.

Mollusks do not have a shell, can increase in size and reproduce by fission and budding. Later, a lipid-wax shell is formed in the molluscs. Under certain conditions, 1-3 rays germinate in mollicates. In the process of development, ray segmentation (zebra) occurs. The segments are separated, the shell of sickle-shaped formations decreases, the nuclear substance is divided into small lumps and rods are formed, which are stained by Gram in purple color (Koch's rod).

Example 1.

During the study of 947 human blood smears, a tuberculous agent (zebritis, mollicutes, arthrospores) was found in six cases. Subsequent clinical and bacteriological studies confirmed the diagnosis of "tuberculosis" in all six cases.

Example 2.

The inoculation material was the sputum of patients in whom tuberculosis mycobacteria were observed under microscopy.

The test material was treated with trisubstituted sodium phosphate (10905) according to the existing method, washed with a growth stimulator, the sediment was mixed with a growth stimulator, kept for 12 hours in a thermostat at a temperature of 37-41"C and sown on a nutrient medium with a minimum content of ingredients: dry agar-agar - 2g, peptone - BG, calcium chloride - 0.01g, potato starch - 0.5g, distilled water - up to 100ml, medium pH - 7.2.

The growth of mycobacterium tuberculosis was noted on the 2nd-3rd day of incubation, but the growth was not active enough, the grass-like growth of the colonies appeared on the 4th-5th day. All 10 samples gave growth.

Example 3.

10 samples of sputum from patients with bacterial isolates, prepared in the specified way, were kept for 18 hours in a growth stimulator and transplanted to a nutrient medium with the optimal content of components, i.e.: agar-agar - Zg, penton - 7g, calcium chloride - 0.012g, potato starch - 1.5g , distilled water up to 100 ml, medium pH - 7.3. The cultures were thermostated at 37-21"C. Colony growth appeared after 36 hours, and after 72 hours, linear growth of colonies was observed. Colony growth was noted in all 10 samples.

Example 4.

The material from 10 patients with bacillus isolates, prepared in the above-mentioned way, was kept in a growth stimulator for 24 hours and sown on a nutrient medium with the maximum ratio of ingredients, i.e.: agar-agar - Ag, peptone - 8g, calcium chloride - 0.015g, potato starch - 2 ,0g, distilled water up to 100ml, medium pH - 7.3. The growth of mycobacteria on this medium appeared after 48 hours and only on 7 samples, and after 72 hours it was noted on all sown test media. It should be noted that the growth of the colonies was somewhat suppressed and there was grass growth only for 4-5 days.

On the control medium (Levenshtein-Jensen), the growth of colonies was noted for 25-60 days in 8 out of 10 samples.

Thus, the proposed method of accelerated diagnosis of tuberculosis shortens the time of diagnosis by 10-15 times, is easy to perform, does not require expensive imported components and can be used in medical and veterinary practice. -sh- t h-

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Claims (1)

The method of accelerated diagnosis of tuberculosis, which includes a complex of studies of microscopy of blood smears, treatment of patmaterial with an inhibitor, sowing on a nutrient medium, is distinguished by the fact that in the smears, a specific tuberculosis agent, arthrospores, mollicutes, segmented stems of mollicutes, or one of the other stages of development of the causative agent of tuberculosis, patmaterial is detected after treatment with an inhibitor, they are additionally kept for 12-24 hours in a mycobacterial growth stimulator, which includes (wt. 95): C - 21.2, Ma - 6.1, H - 42.4, O - 30.3 and sown on nutrient environment of the following composition Agar-agar 2.0-40g Peptone 5 60-80 Calcium porous 0.01-0.015 g Potato starch 0.5-20 g Distilled water up to 100 ml medium pH 72-73