Laboratory diagnosis of tuberculosis

Tuberculosis is a disease that is easy to diagnose in modern conditions and scientific achievements. Laboratory diagnosis of tuberculosis is central to other diagnostic methods, second only to X-ray methods of research.

[1], [2], [3], [4],

Clinical blood test

In patients with tuberculosis, changes in the general analysis of blood are not pathognomonic. With limited and inactive forms of tuberculosis, the erythrocyte is hypochromic in normal quantity. With massive infiltrates or caseous pneumonia, with widespread caseous lymphadenitis, specific intestinal lesions, as well as with large pulmonary or postoperative bleeding, erythropenia and microcytosis, oligochromasia, and polychromasia are noted. Macrocytosis, and especially poikilotsitoz meet much less often, usually with severe anemia. The number of reticulocytes with a compensated stage of tuberculosis varies from 0.1 to 0.6%, with a subcompensated - from 0.6 to 1.0%, and for decompensated is characterized by 1% of reticulocytes.

With tuberculosis, in some cases, moderate leukocytosis (up to 15 thousand leukocytes) may occur, less often leukopenia, which is met in 2-7% of cases in patients with limited and easily flowing forms of the process and in 12.5% - with destructive and progressive pulmonary tuberculosis .

The most frequent shifts occur in the leukocyte formula. Mark both relative and absolute neutrophilia, a moderate shift of the leukocyte formula to the left before promyelocytes. Myelocytes are very rare in the case of uncomplicated tuberculosis. An increase in the number of neutrophils with pathological granularity in the hemogram of a patient with tuberculosis always indicates the duration of the process: in patients with severe tuberculosis, almost all neutrophils contain pathological granularity. When the tubercular outbreak ceases, the nuclear shift comparatively quickly approaches normal. The pathological granularity of neutrophils usually persists longer than other changes in the hemogram.

The content of eosinophils in the peripheral blood also varies depending on the phase of the process and the allergic state of the organism. Their amount decreases up to aneosinophilia in severe and protracted outbreaks of the disease and, conversely, increases with resorption of infiltrates and pleural effusions, as well as in the early forms of primary tuberculosis.

Most forms of primary tuberculosis are accompanied by lymphopenia, which is sometimes observed for a number of years, even after scarring of specific changes. Secondary tuberculosis in the phase of exacerbation, depending on the severity of the process, may be accompanied by either a normal number of lymphocytes, or lymphopenia.

Among the tests for evaluating the tuberculosis process, the determination of the erythrocyte sedimentation rate (ESR), which is important in evaluating the course of the tuberculosis process and identifying its active forms, occupies a special place. The increase in ESR indicates the presence of a pathological process (infectious-inflammatory, purulent, septic, hemoblastosis, lymphogranulomatosis, etc.) and serves as an indicator of its severity, however, normal ESR indices do not always indicate the absence of pathology. Acceleration of erythrocyte sedimentation is facilitated by an increase in the content of globulins, fibrinogen, cholesterol in the blood and a decrease in the viscosity of the blood. Slowing down of erythrocyte sedimentation is characteristic for states accompanied by hemoconcentration, an increase in the content of albumins and bile acids.

The hemogram in patients with tuberculosis changes during treatment. Hematologic shifts disappear the faster, the more successful the therapeutic intervention. However, one should keep in mind the effect on hemopoiesis of various antibacterial drugs. They often cause eosinophilia, in some cases - leukocytosis, and more often leukopenia up to agranulocytosis and lymphoid-reticular reaction. Systematic hematological control and correct analysis of the obtained data are essential for assessing the clinical state of the patient, the dynamics of the process and the effectiveness of the treatment used.

[5], [6], [7], [8], [9],

Clinical analysis of urine

With tuberculosis of the urinary system, urinalysis is the main laboratory diagnostic method. You can observe leukocyturia, erythrocyturia, proteinuria, hypoisostenuria, tuberculosis mycobacterium, nonspecific bacteriuria.

Leukocyturia is the most frequent symptom of tuberculosis of the urinary system before specific chemotherapy is performed and is absent only in exceptional cases, for example, with complete obliteration of the ureteral lumen. Nechiporenko's test (determination of the number of leukocytes in 1 ml of urine) helps to more objectively assess the degree of leukocyturia in nephrotuberculosis, and in some cases, and to reveal it in a normal general analysis of urine. However, it must be taken into account that leukocyturia may occur in acute and chronic pyelonephritis, cystitis, urethritis, kidney and ureter stones.

Erythrocyturia. As well as leukocyturia. Are considered one of the most frequent laboratory signs of tuberculosis of the genitourinary system. The frequency of hematuria depends on the prevalence of the process, it increases as the destructive tuberculosis process develops in the kidney. Erythrocyturia without leukocyturia is more typical for early stages of kidney tuberculosis. Hematuria, which predominates over leukocyturia, is an important argument in favor of kidney tuberculosis in its differentiation with nonspecific pyelonephritis.

[10], [11], [12], [13], [14], [15], [16],

Blood chemistry

With tuberculosis, changes in some biochemical parameters depend primarily on the phase of the process, complications and various concomitant diseases. In patients with inactive tuberculosis of the lungs and other organs, the total protein and protein fractions of blood serum are unchanged and determine their normal content.

In acute forms of the disease, as well as with exacerbation and progression of chronic forms of tuberculosis, the albumin-globulin coefficient decreases.

The determination of the direct and total bilirubin, aspartate aminotransferase (ACT), alanine aminotransferase (ALT) in the serum of blood is essential in assessing the functional state and organic liver damage in tuberculosis and its complications. Dynamic determination of the level of aminotransferases. Bilirubin in the treatment of tuberculosis patients, especially in severe forms, is an obligatory component of a biochemical examination of tuberculosis patients and is performed monthly.

Assessment of the functional state of the kidneys includes the determination of serum creatinine and the calculation of the glomerular filtration rate according to the Cockcroft-Gault formula. Calculation of the glomerular filtration rate using Reberg's sample gives less accurate results.

The main goal of dynamic biochemical studies of tuberculosis patients is to monitor the course of the process, timely detection of side effects of drugs and adequate correction of the resulting homeostatic disorders.

[17], [18], [19]

Application of biochemical methods of investigation in extrapulmonary tuberculosis

The most informative indicator is the content of tuberculostearic acid in biological fluids, but its definition is associated with technical difficulties (the need for gas chromatography and mass spectrometry).

Prospective measurement of the activity of adenosine deaminase - an enzyme, determined in liquids: synovial, pericardial, ascitic or spinal. The main producers of adenosine deaminase are lymphocytes and monocytes. Determination of the activity of adenosine deaminase in biological fluids facilitates the diagnosis of tuberculosis synovitis, tuberculosis of lymph nodes, tuberculosis meningitis, tuberculosis serositis.

Some biochemical indicators due to their non-specificity are determined only in biological fluids, close to the lesion focus. Measure the level of indicators in response to subcutaneous or intradermal injection of tuberculin (usually before and 48 and 72 hours after it). After this, the degree of increment of the marker level (in%) is calculated with respect to the initial level.

Optimum determination in the urine of the activity of an organ-specific enzyme of transamnidase, the appearance of which is noted when the kidneys of various nature are affected. The study of transamidinase is justified only under conditions of subcutaneous injection of tuberculin in order to exacerbate the local inflammatory process. Determine the activity of transamidine in the urine initially and 24-72 hours after the introduction of 50 TE tuberculin. Enlargement of the fermentia in 2 times and more allows in 82% of cases to differentiate active tuberculosis of the kidneys from exacerbation of chronic pyelonephritis.

With tuberculosis of female genital organs, concentrations of haptoglobin and malonic dialdehyde in the blood are determined in conditions of a provocative tuberculin test. Subcutaneously injected tuberculin in a dose of 50 TE and 72 hours later performed a second biochemical study. In the case of tuberculosis etiology, the degree of increase in the level of haptoglobin is not less than 28%, and the level of malondialdehyde is 39% or more. The determination of the activity of adenosine deaminase in a peritoneal fluid obtained from the Douglas space is also used. The punctate is re-examined 72 hours after the intradermal injection of tuberculin at doses of 0.1 TE and 0.01 TE into the projection of the internal genital organs on the anterior abdominal wall. In favor of the tuberculosis process, an increase in the activity of adenosine deaminase is 10% or more in comparison with the initial one.

When the eye is affected, the focal reaction that occurs in the eye in response to antigenic stimulation is examined. It is undesirable to develop a pronounced response, accompanied by a decrease in visual functions. Since the assessment of minimal focal reactions is often difficult, for the objectification of the conclusion it is recommended to orientate in parallel and on the degree of growth in the blood serum of haptoglobin or adenosine deaminase.

All biochemical studies should be carried out in conjunction with other methods.

[20], [21], [22], [23],

Blood coagulation system research

The relevance of the study of the state of the blood coagulation system in phthisiology is due to the presence of light hemoptysis or pulmonary hemorrhage in a number of tuberculosis patients, as well as hemocoagulation complications in the surgical treatment of tuberculosis. In addition, the latent flow of intravascular hemocoagulation that naturally accompanies tuberculosis affects the course of the disease and the effectiveness of chemotherapy.

In patients with pulmonary tuberculosis with predominance of the exudative component of inflammation, a decrease in anticoagulant blood activity is observed. In patients with a low prevalence of a specific lesion in the lungs with predominance of the productive component of inflammation, intravascular hemocoagulation is not significantly expressed. In patients with pulmonary tuberculosis with hemoptysis and pulmonary hemorrhage, the state of the blood clotting system is different: in patients with low blood loss at hematopoietic height or immediately after its termination, a sharp increase in the clotting capacity of blood is observed due to pronounced intensification of thrombogenesis processes while maintaining increased "structural" clotting. In patients with massive blood loss, a decrease in the coagulation potential is observed due to a decrease in the concentration of fibrinogen. Factor XIII activity, platelet count. At the stage of surgical treatment, patients with limited forms of tuberculosis do not have mild significant disorders with the homeostasis system. In patients with advanced processes in the performance of pneumon- or pleuropneumonectomy, an ICD syndrome often develops, which can acquire the forms of a "second illness".

To monitor the state of the coagulation system in patients with pulmonary tuberculosis, it is necessary to determine the activated partial thromboplastin time (APTT), fibrinogen, thrombin time, prothrombin index, and bleeding time and blood clotting time.

[24], [25], [26], [27], [28]

Hormonal research

Modern experimental and clinical observations indicate the presence of changes in the hormonal status in a specific tuberculous lung inflammation. It is proved that the correction of hypophyseal-adrenal dysfunction, hypophyseal-thyroid system and pancreatic function in combination with antituberculous therapy promote the activation of fibrogenesis and repair in the focus of specific inflammation.

The functional state of the pituitary-thyroid system is judged by the content of triiodothyronine (T ₃ ), thyroxine (T ₄ ), thyroid-stimulating hormone of the pituitary (TSH) in the blood serum . It is established that subclinical hypothyroidism is detected in 38-45% of patients with pulmonary tuberculosis, and most often it is diagnosed with disseminated and fibrous-cavernous forms of the process. Under these same forms most dramatically reduced levels of both T ₃ and T ₄, and there comes an imbalance of these hormones in the form of increasing the ratio of T ₄ / T _s.

The function of the adrenal cortex is assessed by the level of cortisol in the blood serum, and the incremental function of the pancreas by the concentration of the immune-reactive insulin. In the acute phase of the infectious disease, the need for endogenous cortisol and insulin increases. Hyperinsulinemia also testifies to the insulin resistance of body tissues, which is typical for any active inflammatory process, in particular, specific. Determination of glucocorticoid function of the adrenal glands with active pulmonary tuberculosis allows to reveal the presence of hypercorticism in the majority of patients. Normal blood cortisol concentrations in a patient with infectious inflammation in the acute period should be regarded as a relative deficiency of the glucocorticoid function of the adrenal cortex, which may serve as the basis for conducting substitution therapy with adequate doses of glucocorticoids.

Almost a third of patients with pulmonary tuberculosis can establish that the level of insulinemia in them is quite low and approaches the lower limit of the norm, while 13-20% observe significant hyperinsulinism. Both relative hypo- and hyperinsulinism are high risk factors for the development of violations of carbohydrate metabolism of varying degrees. These changes in the functional activity of B cells of the pancreas require regular monitoring of glycemia in patients with tuberculosis and timely prevention of diabetes mellitus. Also. This serves as an additional justification for the expediency of using physiological doses of insulin in the complex therapy of tuberculosis.

In general, the reduction in thyroid hormone levels, their imbalance, hypercortisolemia and hyperinsulinism is greatest in patients with severe tuberculosis, with extensive lung lesions and severe symptoms of tuberculous intoxication.

Microbiological diagnosis of tuberculosis

Microbiological studies are necessary in identifying patients with tuberculosis, verifying the diagnosis, monitoring and correcting chemotherapy, evaluating treatment outcomes, in other words, from the moment the patient is registered with tuberculosis before taking it off.

All epidemiological programs and projects are based on an assessment of the number of bacterial excretors, which can not be done without using laboratory methods to detect mycobacteria tuberculosis. In a survey of the so-called unorganized population, the percentage of bacterial invaders reaches 70 or more, which makes laboratory methods a sufficiently effective means of identifying tuberculosis patients among this population group.

Traditional microbiological methods for diagnosing tuberculosis are bacterioscopic and culture studies. Modern methods consider the cultivation of mycobacterium tuberculosis in automated systems, the setting of PCR. However, all these methods necessarily combine with classical bacteriological methods.

Collection of diagnostic material

The effectiveness of laboratory studies depends to a large extent on the quality of the diagnostic material. Compliance with the rules for the collection, storage and transportation of diagnostic material and the exact implementation of the patient assessment algorithm directly affects the outcome and ensures biological safety.

For testing on tuberculosis, a variety of materials are used. Due to the fact that pulmonary tuberculosis is the most common form of tuberculosis, the main material for the study is sputum and other types of tracheobronchial tree to be separated: the upper respiratory tract detachment obtained after aerosol inhalation: bronchial flushing; bronchoalveolar rinses; material obtained by bronchoscopy, transtracheal and intrapulmonary biopsy: aspirate from the bronchi, laryngeal strokes, exudates, smears from wounds, etc.

The effectiveness of research increases if a controlled collection of material from a patient is carried out. For this purpose, a specially equipped room is allocated or special cabs are purchased. The collection of material is a dangerous procedure, therefore it is necessary to collect material for research, observing the rules of infectious safety.

The material for testing on mycobacterium tuberculosis is collected in sterile bottles with tightly screwed caps to prevent contamination of the environment and protect the collected material from contamination.

Vials for collection of diagnostic material must meet the following requirements:

• must be made of impact-resistant material;
• should easily melt during autoclaving;
• be of sufficient volume (40-50 ml):
• have a wide opening for sputum collection (diameter not less than 30 mm);
• be easy to handle, transparent or translucent, so that you can assess the quantity and quality of the sample collected without opening the lid.

To obtain optimal research results, the following conditions must be observed:

• Collect the material before the start of chemotherapy;
• material for the study must be collected before the morning intake of food and medicine;
• for research it is desirable to collect at least 3 samples of morning phlegm. Gather sputum for 3 consecutive days;
• the collected material must be delivered to the laboratory as soon as possible:
• in the case where it is immediately impossible to deliver the material to the laboratory, it is stored in the refrigerator at an air temperature of 4 ° C for not more than 48 hours;
• When transporting the material, it is necessary to closely monitor the integrity of the vials.

Correctly collected sputum is mucous or mucopurulent. The optimal volume of the tested sputum is 3-5 ml.

Sputum is collected under the supervision of a medical professional. Persons responsible for sputum collection should follow the implementation of certain rules:

• it is necessary to explain to the patient the purpose of the study and the need to cough up not saliva or nasopharyngeal mucus, but the contents of the deep respiratory tract. This can be achieved as a result of a productive cough that occurs after several (2-3) deep breaths. It is also necessary to warn the patient that he should rinse his mouth with boiled water, to remove the main part of the microflora growing in the oral cavity and food residues that impede the examination of sputum;
• a medical worker participating in sputum collection, in addition to a bathrobe and a hat, must wear a mask, rubber gloves and a rubber apron;
• standing behind the patient, he is recommended to keep the bottle as close as possible to his lips and immediately separate into it phlegm as it coughs, and it must be provided that the air flow is directed away from the health worker:
• Upon completion of the sputum collection, the health worker should carefully close the vial with a lid and assess the quantity and quality of sputum collected. Then the bottle is labeled and placed in a special bix for transportation to the laboratory.

If the patient does not excrete phlegm, then the evening before and early in the morning on the day of collection, give him an expectorant: an extract of althea root medicinal (mucaltin), bromhexine, ambroxol, etc. - or apply irritating inhalation using equipment installed in the collection room phlegm. The material collected in this way is not subject to conservation and should be examined on the day of collection. To avoid its "culling" in the laboratory in the direction should make a special mark.

If microbiological studies are not carried out at this facility, the collected diagnostic material should be centrally delivered to the laboratory, provided that the material is preserved in the intervals between deliveries in the refrigerator or with the use of preservatives. Deliver material to the laboratory in transport boxes, which can be easily disinfected. Each sample should be provided with the appropriate label, and the whole lot should be filled with an accompanying form.

[29], [30], [31],

Modes and frequency of examination of patients

At the initial, so-called diagnostic, examination of the patient for tuberculosis, it is necessary to examine at least 3 sputum portions for 2 or 3 days. Collected under the supervision of medical personnel, which increases the effectiveness of microscopy.

The primary screening for tuberculosis should be carried out by all medical diagnostic institutions of the health care system. Recently, so-called centers of microscopy, equipped with modern microscopes and equipment for epidemic safety, have been organized on the basis of clinical diagnostic laboratories to improve the efficiency of the initial examination.

In anti-TB facilities, a screening test is used that includes sputum examination or other diagnostic material for at least 3-fold for 3 days. During the treatment, microbiological studies are performed regularly at least once a month during the intensive chemotherapy phase. At the transition to the phase of treatment, studies are conducted less often - with an interval of 2-3 months, while the frequency of the study is reduced to two.

Features of the collection of diagnostic material for extrapulmonary tuberculosis

The peculiarity of the pathological material in extrapulmonary forms of tuberculosis is the low concentration of mycobacterium tuberculosis in it, which requires more sensitive methods of microbiological examination, first of all, the methods of sowing on the nutrient medium.

With tuberculosis of the genitourinary system, urine is the most accessible study material. Urine collection should be done by a specially trained nurse.

The external genitalia are washed with water with soap or a weak solution of potassium permanganate. The external opening of the urethra is carefully treated. In a sterile vial, an average portion of the morning urine is collected: in men, naturally, in women, using a catheter. Urine from the renal pelvis is collected in sterile tubes with catheterization of one or two kidneys, in the latter case - necessarily separately from each kidney. A small amount of this urine is centrifuged, the sediment is examined.

In men, sperm, punctate testes, the secret of the prostate is subjected to centrifugation to obtain a precipitate. With any localization of a specific process in the genital area in men, prostate massage can promote the secretion of secretions containing mycobacterium tuberculosis.

Menstrual blood in women is collected by sucking or using a Kafka cap. The obtained material is freed from red blood cells, washing it with distilled water followed by centrifugation. The precipitate is examined.

Allocations from the cervical canal of the uterus are collected in some Kafka capacity or cap, that is, it is desirable to accumulate 1-2 ml of pathological material.

Material obtained from operative interventions on the kidneys, genitals. With biopsies, scrapings from the endometrium, homogenize. To do this, it is placed in a sterile mortar and thoroughly crushed with sterile scissors. To the obtained suspension add sterile river sand in an amount equal to its mass, then add 0.5-1.0 ml of isotonic sodium chloride solution and then grind everything to the formation of a gruel-like mass with the addition of isotonic sodium chloride solution (4-5 ml). Then the mass is allowed to settle for 1-1.5 minutes, the supernatant is examined.

Tuberculosis of bones and joints. The punctate (pus of abscesses) obtained with a sterile syringe is placed in sterile dishes and immediately delivered to the laboratory. Sterile pipette, previously moistened with sterile isotonic sodium chloride solution, take 2-5 ml of pus, transfer it to a bottle of beads and add another 2-3 ml of isotonic sodium chloride solution. The bottle is closed with a cork and shaken in a joker-apparatus for 8-10 minutes. The homogenized suspension is examined.

In fistulous forms of osteoarticular tuberculosis, pus from the fistula is taken. The copious discharge is collected directly into a test tube. In cases of scanty excretion of pus, the fistula is washed with a sterile isotonic sodium chloride solution, and the wash water collected in a test tube or a piece of a tampon impregnated with pus is sent to the study.

The surgical material obtained during surgical interventions on bones and joints can consist of purulent-necrotic masses, granulations, scar tissue, bone tissue, synovial membrane tissue and other substrates. Its treatment is performed, as in tuberculosis of the kidneys.

Microbiological examination of synovial fluid in a 3% solution of sodium citrate (1: 1 ratio) to prevent coagulation is performed immediately after the puncture.

Tuberculosis of the lymph nodes. The pus, extracted during the puncture of the lymph nodes, is also examined. As pus of the abscesses. The tissues of the lymph nodes obtained during surgical interventions, biopsies, are examined, as with other forms of tuberculosis.

The study of stool masses on mycobacterium tuberculosis is extremely rare, due to the almost total lack of positive results.

[32], [33], [34], [35], [36],

Microscopy of mycobacteria

Sputum microscopy is a relatively fast, simple and inexpensive method that should be used in all cases with suspected tuberculosis. In addition, this study is conducted to evaluate the effectiveness of chemotherapy and to ascertain the recovery or failure of treatment if there is no culture test.

Two methods of microscopic examination are used:

• method of direct microscopy, when a smear is prepared directly from the diagnostic material;
• method of microscopy of a sediment prepared from decontaminant treated material for culture.

The first method is used in those laboratories where only microscopic studies are carried out (clinical and diagnostic laboratories of the general medical network).

The best results of microscopic examination are obtained by concentrating the diagnostic material (for example, by centrifugation).

To detect mycobacterium tuberculosis with a probability of 50% when carrying out the microscopy, 1 ml of sputum should contain more than 5000 microbial cells. The sputum of patients with pulmonary forms of tuberculosis usually contains a significant amount of acid-fast bacteria, which allows them to be reliably detected by bacterioscopy. The diagnostic sensitivity of this method can be improved by examining several sputum samples from one patient. A negative result of a bacterioscopic examination does not exclude the diagnosis of tuberculosis, as the sputum of some patients contains less mycobacteria than can be detected with microscopy. Poor preparation of sputum smear can also cause a negative result of a bacterioscopic examination.

The most common method for the detection of acid-fast mycobacteria in the smear is color according to Tsiol-Nelsen. The method is based on the penetration of carbolic fuchsin into a microbial cell through a membrane that includes a waxy-lipid layer, while simultaneously heating and strong etching action of phenol. Subsequent discoloration of the smear with a 25% solution of sulfuric acid or 3% hydrochloric acid leads to a discoloration of all non-acid-fast structures. The discolored elements of the smear are stained with a 0.3% solution of methylene blue. Mycobacteria do not perceive the usual aniline dyes, as a result of which acid-fast mycobacteria are stained crimson-red, and other microbes and cellular elements - in blue.

To study the smears stained by Tsilyu-Nelsen, use a light binocular microscope with an immersion objective (90- or 100-fold magnification) and an eyepiece with a 7- or 10-fold magnification. Explore 100 fields of vision, which is enough to identify single mycobacteria in the smear. In the event that the result of such a study is negative, for confirmation it is recommended to view another 200 fields of vision. Record the results, indicating the number of detected acid-fast bacilli (KUM).

In addition to this technique, color fluorochromes are used for luminescent microscopy, which allows achieving the best results. The use of this method increases the efficiency of microscopy by 10-15%. When treating mycobacteria with luminescent dyes (auramine, rhodamine, etc.), these substances also bind to the wax-like structures of the microbial cell. When irradiating colored cells with an exciting light source (a certain spectrum of ultraviolet radiation), they begin to glow with orange or bright red light on a black or dark green background. Due to the high brightness and contrast of the visible image, the overall magnification of the microscope can be reduced 4-10 times, the field of view widens and the viewing time of the preparation decreases. Along with this, due to the much greater depth of field, you can increase the comfort of the study.

When fluorescence microscopy is used to view the same area, the smear expends significantly less time than with light microscopy of the Tsiol-Nelsen staining. If for a working day a microscope examines about 20-25 such smears, then with the help of fluorescence microscopy, he can examine more than 60-80 samples at the same time. Experienced microscopists know that the coloration of cells with a mixture of auramine and rhodamine is in some way specific to acid-fast bacilli, which in this case look like golden sticks. Saprophytes are painted in a greenish color.

Another important advantage of the method of fluorescence microscopy is the ability to detect altered mycobacteria that have lost the influence of a number of unfavorable factors, in particular intensive chemotherapy, the property of acid-resistivity and which, therefore, do not show up in Tsiol-Nelsen staining.

The disadvantages of the method of fluorescence microscopy include the relatively high cost of the microscope and its operation. However, in centralized or other large laboratories, where the load exceeds the norm of 3 laboratory technicians working with three conventional microscopes, it is cheaper to use one fluorescent microscope instead.

Bacterioscopic methods have rather high specificity (89-100%). About 97% of the positive results obtained by any method of microscopy are unambiguously confirmed by the results of the sowing.

It should be noted that when microscopic examination of the smear of pathological material, it is impossible to determine the species belonging of the identified acid-fast mycobacteria. The method of microscopy allows us to give a conclusion only about the presence or absence of acid-fast microorganisms in the preparation, which is explained by the existence in nature of a large number of tuberculosis-resistant microorganisms that are morphologically similar to mycobacteria.

The results of microscopy are evaluated in semiquantitative units.

In order to be able to compare the results of different methods of microscopy, empirical coefficients are introduced. For example, in order to compare the results of a study of a smear stained with fluorescent dyes with light microscopy (1000x magnification), it is necessary to divide the amount of acid-fast mycobacteria detected with a fluorescent microscope by an appropriate factor at a 250-fold magnification of the microscope by 10, 450-fold - to 4, with 630-fold - to 2.

Features of microscopy for extrapulmonary tuberculosis

Direct microscopy is performed, as well as microscopy of smears prepared after enrichment, followed by Tsiol-Nelsen staining or luminescent dyes. Direct microscopy of smears is ineffective due to a low concentration of mycobacteria in the material, and therefore it is more rational to use methods of enrichment. The most effective is centrifugation. If the biological material is viscous, centrifugation is used with simultaneous homogenization and liquefaction of the material, which is carried out using high-speed centrifuges with a centrifugation force of 3000 g and hypochlorite solutions. Other methods of enrichment, such as micro flotation, are not currently used because of the formation of biologically hazardous aerosols.

[37], [38],

The cultural method of diagnosis of tuberculosis

The method of sowing, or the culture method, is more sensitive than smear microscopy, and has a number of advantages over the latter. It allows to detect several dozens of viable mycobacteria in the test material and has great diagnostic value. This is especially important when examining the material from newly diagnosed or treated patients who release a small amount of mycobacteria.

Compared with microscopy, culture research allows increasing the number of TB patients diagnosed by more than 15-25%, as well as verifying tuberculosis in earlier stages, when the disease is still amenable to treatment. A very important advantage of the culture test is the possibility of obtaining an exciter culture that can be identified and studied with respect to drug sensitivity, virulence and other biological properties.

The disadvantages of methods of cultivation include their duration (the waiting time of materials reaches 10 weeks). Higher cost, complexity of processing of diagnostic material.

Principles of presowing treatment of diagnostic material

Conventional microbiological methods can not be used in conducting studies on tuberculosis. This is due to the fact. That mycobacterium tuberculosis grows very slowly, and most clinical samples contain fast-growing pyogenic and putrefactive microorganisms, fungi. Their rapid growth on rich nutrient media interferes with the development of mycobacteria and does not allow isolating the causative agent of tuberculosis, so the diagnostic material must be pretreated before seeding. In addition, mycobacteria released from the patient's airways are usually surrounded by a large amount of mucus, making it difficult to concentrate. In this regard, before planting sputum and other similar materials, their liquefaction, decontamination is necessary.

All detergents and decontamins have a more or less pronounced toxic effect on mycobacteria. As a result of processing, up to 90% of mycobacteria can die. In order to maintain a sufficient portion of the mycobacterial population, it is necessary to use gentle processing methods that, on the one hand, can suppress fast-growing pyogenic and putrefactive microorganisms, and on the other, maximize the survival of the mycobacteria present in the material.

Depending on the material, the degree of its homogeneity and contamination for presowing treatment, various decontamins are used: for sputum - a solution of sodium hydroxide 4%, solutions of sodium phosphate 10%, benzalkonium chloride trisodium phosphate, NALC-NaOH (N-acetyl-L-cysteine- sodium hydroxide) with a final NaOH concentration of 1%, for urine and other liquid materials - 3% sulfuric acid solution, for contaminated samples, fat-containing materials - oxalic acid solution up to 5%. In addition, in some cases, enzymes, surfactants (detergents) are used. The use of Tween and some other detergents is accompanied by a smaller death of mycobacterial cells (40-50% survive). However, they can only be used for liquid materials. The largest distribution in the world was NALC-NaOH. Produced in sets. This method allows to allocate more than 85% of the population of mycobacterial cells. Decontamination of fabric-containing solid materials is more difficult, since it is difficult to guess the degree of dispersion of the material during the homogenization process. For example, treatment of biopsies of lymph nodes is often accompanied by an increased frequency of contamination by extraneous flora. In this case, 1% of etonium can be used.

Non-homogeneous material is homogenized with glass beads in the presence of decontaminants. The liquid materials are pre-centrifuged and only a precipitate is treated.

Sowing and incubation techniques

After pretreatment, the material is centrifuged, thereby precipitating the mycobacteria and increasing their content in the sediment ("sludge enrichment"). The resulting precipitate is neutralized and inoculated (inoculated) with dense nutrient media or tubes with liquid (semiliquid) media. From the rest of the sediment, smears are prepared for microscopic examination. The seeding technique should prevent cross-contamination of the diagnostic material.

For a reliable clinical interpretation of the results of a microbiological study, the following rule must be observed: microscopic and culture studies must be performed in parallel from the same sample of the diagnostic material.

The inoculated tubes are placed in a thermostat at 37 ^° C for 2 days in a horizontal position. This ensures a more even absorption of the material into the culture medium. After 2 days, the tubes are transferred to a vertical position and hermetically sealed with rubber or silicone plugs to prevent drying of the sown media.

Crops are incubated at 37 ^about C for 10-12 weeks with regular weekly viewing. For each preview, the following parameters are recorded:

• the period visually observed from the day of sowing growth;
• growth rate (number of CFUs);
• contamination of the crop by an extraneous microbial flora or fungi (such tubes are removed);
• lack of visible growth. The tubes are left in the thermostat until the next viewing.

Nutrient media

Various nutrient media are used for the cultivation of mycobacteria; dense, semi-liquid, liquid. However, none of the known nutrient media has properties that ensure the growth of all mycobacterial cells. In connection with this, 2-3 nutrient media of different composition are recommended to be used simultaneously to increase the effectiveness.

WHO recommends the Levenstein-Jensen environment as the standard medium for the primary isolation of the causative agent of tuberculosis and for determining its drug sensitivity. This is a dense egg environment on which the growth of mycobacteria is obtained on the 20th-25th day after seeding a bacterioscopically positive material. Crops of bacterioscopically negative material require a longer incubation period (up to 10-12 weeks).

In our country, the proposed E.R. Finn egg environment Finn-II. It differs in that, instead of L-asparagine, it uses sodium glutamate, which triggers other ways of synthesizing the amino acids of mycobacteria. Growth appears on this medium somewhat earlier, and the frequency of allocation of mycobacteria is 6-8% higher than in the Lowenstein-Jensen medium.

To improve the effectiveness of bacteriological diagnosis of extrapulmonary tuberculosis, it is advisable to include modified Finn-II media in the complex of nutrient media. To accelerate growth, a sodium thioglycolate 0.05%, which reduces the concentration of oxygen, is additionally added to the Finn-II nutrient medium. To protect the enzyme systems of mycobacteria from toxic products of lipid peroxidation in the Finn-II nutrient medium, antioxidant α-tocopherol acetate is added at a concentration of 0.001 μg / ml. The seeding of the diagnostic material is carried out according to a standard procedure.

In Russia's anti-tuberculosis laboratories, other modifications of dense nutrient media are also used; proposed G.G. Mordovian nutrient medium "New", developed by V.A. Anicic nutrient media A-6 and A-9, etc.

Due to the damage to various metabolic systems of the microbial cell during chemotherapy, part of the mycobacterial population loses its ability to develop normally on normal nutrient media and requires osmotically balanced (semi-liquid or liquid) nutrient media.

Assessment and recording of seeding results of diagnostic material

Some strains and species of mycobacteria grow slowly, growth can appear even by the 90th day. The number of such crops is small, but this makes it possible to withstand crops in a thermostat for 2.5-3 months.

Virulent cultures of mycobacterium tuberculosis usually grow on dense egg environments in the form of R-form colonies of various sizes and species. Colonies dry, wrinkled, ivory, slightly pigmented. In other media, the colony of mycobacterium tuberculosis may be more humid. After a course of chemotherapy or during treatment, smooth colonies with moist growth (S-forms) can be allocated.

When isolating crops, a set of special studies is used to distinguish mycobacterium tuberculosis from non-tuberculosis mycobacteria and acid-resistant saprophytes.

A positive response is given after a mandatory microscopic examination of the stained Tsiol-Nelsen smear from the grown colonies. In the case of growth of mycobacteria in smears, bright red sticks are found that lie singly or in groups forming clusters in the form of felt or braids. In young cultures, especially those isolated from long-term chemotherapy drugs, mycobacteria are distinguished by pronounced polymorphism, up to the presence of short, almost coccoid, or elongated variants resembling mycelial fungi along with rod-like forms.

The growth rate of mycobacteria is indicated by the following scheme: (+) - 1-20 cfu in vitro (scant bacterial excretion); (++) - 20-100 CFU in vitro (moderate bacterial excretion); (+++) -> 100 CFU in vitro (abundant bacterial excretion). In the laboratory diagnosis of tuberculosis, it is not enough to answer whether the mycobacterium is detected by one or another method. Have a detailed understanding of the extent and nature of the mycobacterial population, its composition and properties. It is these data that allow us to correctly interpret the state of the process, plan tactics and timely correct the treatment.

In recent years, to accelerate the growth of mycobacteria, nutrient media on an agar basis with various growth additives and the use of a special gas mixture have been proposed. To obtain the growth of mycobacteria on these media, during cultivation, an atmosphere with a high content of carbon dioxide (4-7%) is created. Special CO ₂ -incubators are used for this purpose . However, the most developed automated systems for cultivation of mycobacteria: MGIT-BACTEC-960 and MB / Bact.

One of such systems is the MGIT system (mycobacteria growth indicating tube), which belongs to the development of high technologies and is intended for accelerated bacteriological diagnosis of tuberculosis and determining the sensitivity of mycobacteria to first-line drugs and certain second-line drugs. MGIT is focused on using it as part of the VASTES-960 device. Microorganisms are cultivated in special tubes with a liquid nutrient medium based on the modified Middlebrook-7H9 medium. To stimulate the growth of mycobacteria and suppress the growth of extraneous microflora, MGIT Growth Supplement and a mixture of PANTA antibacterial drugs are used.

The growth of microorganisms is recorded optically. It is based on fluorescence, which occurs when oxygen is consumed by mycobacteria during growth. Oxygen-dependent fluorochromic dye is found on the bottom of a special test tube and covered with a layer of silicone. Propagation of mycobacteria leads to a decrease in the amount of oxygen in the tube and a decrease in its concentration, which causes an increase in fluorescence, which becomes visible when the tube is irradiated with ultraviolet light and automatically recorded by photo sensors built into the VASTES-960 device. The intensity of luminescence is recorded in units of growth (GU-growth units). The growth data is recorded in the computer, where they can be saved automatically. Computer analysis of growth curves can provide information on the presence of various pools of mycobacteria, including non-tuberculosis, and also helps to assess the growth properties of mycobacteria.

As a result of the introduction of such systems, the time for the growth of mycobacteria decreased significantly, averaging 11 days on VASTES-960 and 19 days on MB / Bact versus 33 days on a standard dense nutrient medium. It should be noted that these systems require highly qualified personnel. The sowing of the material on liquid media is necessarily accompanied by sowing on the Levenstein-Jensen medium, which plays the role of a backup in those cases where the mycobacterium tuberculosis does not give rise in other media.

[39], [40], [41], [42], [43], [44],

Determination of drug sensitivity of mycobacteria

The determination of the spectrum and degree of sensitivity of mycobacteria to anti-tuberculosis drugs is of great clinical importance, as well as for the epidemiological evaluation of the spread of tuberculosis with drug resistance. In addition, monitoring of drug resistance makes it possible to assess the effectiveness of the tuberculosis program as a whole, being an integral indicator of the performance of all components of anti-tuberculosis activities.

Multiplicity and timing of drug sensitivity:

• before the start of treatment once for determining the strategy and tactics of treatment:
• when isolated from diseased cultures from various materials (sputum, BAL fluid, urine, exudates, liquor, etc.), all the isolated strains are examined:
• at the end of the intensive phase of treatment in the absence of clinical and radiological dynamics:
• if it is necessary to change the treatment regimen in the following cases:
  • absence of sputum smear;
  • re-isolation of culture after sputum smear-negative;
  • a drastic increase in the number of CMB in the swab after the initial decline. It is well known that strains of mycobacterium tuberculosis, which are heterogeneous in terms of drug sensitivity, are isolated from the material from a patient with tuberculosis. The sensitivity of strains to anti-tuberculosis drugs may differ in the range of drugs, degree, frequency and rate of occurrence of resistance.

The degree of drug resistance of mycobacterium tuberculosis is determined in accordance with established criteria that are oriented toward the clinical significance of resistance and depend on the antituberculous activity of the drug, its pharmacokinetics, concentration in the lesion focus. The maximum therapeutic dose and so on.

The determination of drug sensitivity of mycobacteria is currently carried out by microbiological methods:

• Absolute concentrations (dilution method on dense or liquid nutrient media),
• proportions,
• coefficient of resistance.

Usually, resistance is manifested in the form of visually observed growth of the colonies of mycobacterium tuberculosis, but there are methods that induce growth in the early stages of cell division of mycobacteria in the form of color reactions. These methods shorten the test time from 3-4 to 2 weeks.

As a standard in Russia, the method of absolute concentrations recommended by the WHO Committee on Chemotherapy has been widely spread, which is the simplest from the methodological point of view, but it requires high standardization and accuracy of the laboratory procedures. The drug susceptibility test consists of a set of test tubes with a nutrient medium modified with anti-TB drugs. The kit consists of 2-3 tubes with different concentrations of each of the drugs used, one control tube with medium without preparation and one tube containing 1000 μg / ml salicylic acid sodium or 500 μg / ml paranitrobenzoic acid to detect the growth of nontuberculous mycobacteria.

To prepare a set of media with preparations, a modified Levenstein-Jensen medium (without starch) is used, which is poured into flasks. In each of the flasks, a specific volume of appropriate dilution of the antituberculous preparation is added. The contents of the flasks are thoroughly mixed, poured into tubes and folded in an inclined position for 40 minutes at a temperature of 85 ° C. It is recommended to coil the medium in an electric rewinder with automatic temperature control. Wednesday with anti-TB drugs

1-st series can be stored in the refrigerator at 2-4 ° C for 1 month, with preparations of the second row - no more than 2 weeks. Storage of media with preparations at room temperature is unacceptable. When preparing solutions of anti-tuberculosis drugs, their activity is taken into account, calculating the concentration adjusted for the molecular weight of the nonspecific part of the preparation, purity, etc. To determine drug sensitivity, only chemically pure substances are used.

The principle of the method is to determine the concentration of an antituberculous drug that inhibits the growth of a significant part of the mycobacterial population. If done correctly, this method has good reliability.

Before the test, it is necessary to make sure that the isolated culture of mycobacterium tuberculosis does not have extraneous microflora. From the culture of mycobacteria in 0.9% sodium chloride solution, a homogeneous suspension containing 500 million microbial bodies per ml is prepared (optical turbidity standard of 5 units). The resulting slurry is diluted with 0.9% sodium chloride solution (1:10) and 0.2 ml of slurry is added to each tube of the set of culture media. The seeded tubes are placed in a thermostat at 37 ° C and kept in a horizontal position for 2-3 days so that the sloping surface of the culture medium is uniformly inoculated with the suspension of mycobacterium tuberculosis. The tubes are then transferred to a vertical position and incubated for 3-4 weeks. The results are recorded after 3-4 weeks.

Since the timing of excretory excretion from the clinical material on nutrient media is at least 1-1.5 months, the results of determining the drug sensitivity by this method can be obtained no earlier than 2-2.5 months after the sowing of the material. This is one of the main drawbacks of the method.

Interpret the results of determining the drug sensitivity of mycobacteria on the basis of certain criteria. On dense media, the culture is considered to be sensitive to the concentration of the drug that is contained in the medium if the number of colonies of mycobacteria grown on this tube with the drug does not exceed 20, with abundant growth in a control tube without drugs. Only in the presence of more than 20 colonies is culture regarded as resistant to this concentration. In practice, when obtaining growth results in test tubes close to 20 cfu. It is necessary to notify the clinical unit that sensitivity or resistance in this case is borderline, since sometimes it can explain the fuzzy dynamics of clinical indicators.

For various drugs a certain concentration is established, at which the reproduction of the critical proportion of the mycobacterial population is observed. These concentrations are called "critical". As a criterion of stability, the growth of the population of mycobacteria on a nutrient medium with a preparation at a critical concentration is used.

In domestic TB practice, in determining the drug resistance, they are not limited to determining only critical concentrations. This is due to the fact. That an expanded definition of the level of drug resistance of the pathogen allows the clinician to more correctly formulate the tactics of chemotherapy, using knowledge about the potentiating effects of drug combinations, to anticipate cross-resistance or to use more effective drugs of the group of anti-tuberculosis drugs used.

The absolute concentration method is the simplest, but it is also the most sensitive to the errors made when it is performed. More reliable, especially in determining the sensitivity to second-line drugs, and common outside of Russia is the method of proportions. It takes into account the shortcomings of the method of absolute concentrations, but in execution it is more laborious.

The method is very similar to the absolute concentration method. Preparation of test tubes with medications is carried out in the same way. As in the absolute concentration method. However, the seed dose of the suspension of mycobacterium tuberculosis is reduced by a factor of 10. Which eliminates the frequency of spontaneous resistance of some strains of mycobacterium tuberculosis to such drugs as Etambutol, protionamide, capreomycin. As control, 2 or 3 tubes with a seed dose equal in the test tubes, successively diluted 10 and 100 times, are used. The criterion of stability is the proportion of visually observed growth of mycobacterium tuberculosis. For drugs of the 1st series, the stability criterion is the excess of growth of 1% of the initial population, for the drugs of the second row - an increase of 1 or more than 10% of the initial, depending on the chosen critical concentration.

In 1997, a working group of the WHO and the International Tuberculosis Union on the detection of anti-tuberculosis drug resistance made adjustments to these criteria, suggesting that mycobacteria growing on the dense egg medium of Levenshtein-Jensen are stable, at the following concentrations:

• dihydrostreptomycin - 4 μg / ml;
• isoniazid 0.2 μg / ml:
• rifampicin 40 μg / ml:
• Etambutol is 2 μg / ml.

In 2001, critical concentrations were proposed for the following second-line drugs (for a critical proportion of 1%):

• capreomycin - 40 mcg / ml;
• protionamide - 40 mcg / ml;
• kanamycin - 30 μg / ml;
• viomycin - 30 mcg / ml;
• cycloserine 40 μg / ml;
• aminosalicylic acid - 0.5 μg / ml;
• ofloxacin - 2 μg / ml.

The growth results are evaluated after 4 weeks as a preliminary and after 6 weeks of cultivation - as the final one.

To determine the drug sensitivity to pyrazinamide, which is widely used in modern chemotherapy for tuberculosis, the recommended critical concentration is 200 μg / ml. However, until now there is no generally accepted method for determining drug resistance to this drug on solid nutrient media, since its antibacterial activity is manifested only in acidic medium (pH <6), which is technically difficult to sustain. In addition, many clinical cultures of mycobacteria tuberculosis reluctantly grow on egg environments with an acidic environment.

To assess the quality of the results of determining the drug sensitivity of mycobacteria, it is recommended that each new batch of the Levenstein-Jensen medium be monitored by a parallel determination of the sensitivity of the standard museum strain H37Rv. In addition, there are certain microbiological criteria that must be maintained so that the techniques give a well reproducible and correctly interpreted result. These include the viability of the culture of mycobacterium tuberculosis, the rules for obtaining a homogeneous suspension and suspension, the rules for selecting cultures of mycobacteria tuberculosis, the representativeness of the selected bacterial mass. The reliability of the determination of drug resistance decreases with an extremely scarce bacterial release.

Recently, a method for determining drug sensitivity using automated systems has been considered promising. The most perfect in this area are developments based on VASTES MGIT-960. In this case, the drug sensitivity of mycobacteria tuberculosis is determined on the basis of a modified method of proportions. In the process of determination, the growth rate of mycobacterium tuberculosis in a control tube and in test tubes with drugs is compared. To determine the sensitivity to streptomycin, isoniazid, rifamp-picin and ethambutol, enrichment supplements and antibiotics included in the SIRE kit are used. To determine the sensitivity to pyrazinamide, use the PZA kit. In the course of the test, the tuberculosis mycobacterium suspension inoculum is inoculated with test tubes containing drugs, as well as control tubes with dilution of the suspension 100 times for all preparations, except for pyrazinamide, where the dilution of the suspension is 10 times. The criterion of stability is the growth indicator of mycobacteria of 100 GU when growth is achieved in the control tube 400 GU (see "Culture methods for isolating mycobacteria"). Accounting and interpretation of the results are carried out automatically and are set by the input or the selected program.

As critical concentrations, final concentrations are used in a test tube with a liquid nutrient medium. At present, critical concentrations have been developed for both 1st-line and second-line drugs. It should be noted that the sensitivity of mycobacteria tuberculosis to cycloserine and aminosalicylic acid is determined only on egg nutrient media.

A detailed protocol of work on the described system makes it possible to study drug susceptibility both on an isolated culture (with a dense nutrient medium) and using the primary growth of mycobacterium in a MGIT tube. The latter option significantly reduces the time of cultural studies, allowing to obtain full results about the culture of mycobacterium tuberculosis (including information on drug susceptibility) as early as 3 weeks from the time of collection, while the traditional method can only achieve this by the 3rd month. In time, the results obtained, when the patient is in an intensive phase of treatment, can compensate for the relatively high cost of research.

[45], [46], [47], [48], [49], [50], [51],

Differentiation of mycobacteria

Taking into account that the used nutrient media are not strictly selective. The subsequent differentiation of the isolated mycobacteria is recognized as mandatory. The need for differentiation of mycobacteria is due to a number of features of pathological processes caused by representatives of the genus: the different course and outcome of tuberculosis and mycobacteriosis, the presence of natural drug resistance to some anti-tuberculosis drugs.

It is recognized that the primary identification of mycobacteria of M. Tuberculosis complex from non tuberculosis mycobacteria is carried out according to the following characteristics: growth rate on dense nutrient media, pigmentation, colony morphology, acid resistance and temperature optimum of growth.

Unfortunately, there is no single laboratory method that makes it possible to reliably distinguish the M. Tuberculosis mycobacteria from other acid-fast mycobacteria, nevertheless, the combination of the above characteristics with the results of a number of biochemical tests given below allows the identification of mycobacteria of the M. Tuberculosis complex with a probability of up to 95%.

To differentiate the mycobacteria of the M. Tuberculosis complex (M. Tuberculosis, M. Bovis, M. BovisBCG, M. Africanum, M. Microti, M. Canettii and others) from slowly growing nontuberculous mycobacteria, basic biochemical tests are used to detect the presence of the following signs:

• the ability to produce nicotinic acid (niacin test):
• nitrate reductase activity;
• thermostable catalase;
• growth on medium with sodium salicylate (1 mg / ml).

As additional tests, growth on a medium containing 500 μg / ml of paranitrobenzoic acid or 5% sodium chloride can also be used.

Many bacteriological laboratories identify these microorganisms only at the level of the complex, which is due to the limited capabilities of laboratories and the methodological capabilities of specialists.

In most cases, in practice, the following tests are sufficient for differentiation of M. Tuberculosis and M. Bovis: niacin, for the presence of nitrate reductase, for the presence of pyrazinamidase and recording growth in a medium containing 2 μg / ml thiophene-2-carboxylic acid hydrazide. It is taken into account that the mycobacteria of M. Tuberculosis complex are characterized by the following set of characters:

• slow growth (more than 3 weeks);
• growth temperature in the range of 35-37 ^o C;
• absence of pigmentation (ivory);
• marked acid-fast color;
• a positive niacin test;
• a positive nitrate reductase test;
• absence of thermostable catalase (68 ^° C).
• The absence of growth on a Levenstein-Jensen medium containing:
  • 1000 μg / ml sodium salicylate,
  • 500 μg / ml of paranitrobenzoic acid,
  • 5% sodium chloride:
• growth in the presence of 1-5 μg / ml thiophene-2-carboxylic acid.

The relevance of differentiation of isolated mycobacteria will increase markedly with the increase in the frequency of recording HIV / AIDS cases associated with tuberculosis or mycobacteriosis. At present, there is no absolute certainty of the readiness of practical regional laboratories to correctly perform this volume of work.

[52], [53], [54], [55], [56], [57], [58], [59]

Immunological diagnosis of tuberculosis

There are a number of universal phenomena, drugs and immunological tests that were originally found precisely with tuberculosis or on the model of the immune response to mycobacteria. These include BCG and tuberculin, a phenomenon such as cutaneous GZT (tuberculin tests - reactions of Pirke and Mantoux), a reaction to subcutaneous injection of tuberculin to a sensitized animal (Koch phenomenon). One of the first antibodies in infectious disease was also detected in tuberculosis. Of course, the deeper the understanding of the mechanisms of antituberculous immunity and their genetic control, the wider may be the use of immunological methods and drugs that affect immunity to solve the practical problems of phthisiology.

The most important and difficult practical problem is currently considered the detection of tuberculosis in the process of mass screening of the population. However, despite the numerous reports of "successes" (on limited material), there is no suitable immunological method (reproducible in "any arms") and a drug suitable for this purpose.

Immunological methods, in particular serological studies (determination of antigens, antibodies) and tuberculin-provoking tests are very widely used in clinical practice.

In the first place among the immunological studies used in differential diagnosis, there are serological methods - the determination of antigens and antibodies in different environments of the body.

The specificity of antibodies to mycobacteria tuberculosis depends on the antigens used in the immunoassay. A significant amount of antigens is proposed, the very first of which is tuberculin PPD:

• PAP and other complex preparations from the culture liquid;
• ultrasonic disintegration;
• Triton extract and other complex preparations of cell walls;
• 5-antigen (Daniel);
• 60-antigen (Coccito);
• Lipoarabinomannan;
• cord-factor (trehalose-6,6-di-mycollate);
• phenolic and other glycolipids;
• lipopolysaccharides;
• fibronectin-binding antigen;
• proteins (most often recombinant); 81.65,38,34,30,19,18,16,15.12 CDA, etc.

As a result of many years of research by Russian and foreign scientists, the main patterns of antibody formation and the effectiveness of serological diagnosis of tuberculosis were revealed: the more complex the antigen, the higher the sensitivity and the lower specificity of the tests. Specificity varies from country to country depending on the infection of the population with M. Tuberculosis and non-tuberculosis mycobacteria, from vaccination with BCG, etc. In children, the informative value of serodiagnostics is lower than in adults. In primary tuberculosis (more often children), the definition of IgM is more informative. With secondary IgG. In HIV-infected patients, the informative value of serodiagnosis in determining antibodies is reduced. The effectiveness of antibody determination depends on a number of "clinical moments": the activity of the process (presence or absence of "secretion" of mycobacteria, the presence of decay cavities, the degree of infiltration), the prevalence of the process, the duration of its course.

The sensitivity of the method of enzyme immunoassay (ELISA) is about 70%. Insufficient effectiveness of the study is due to its low specificity. Previously, the possibility of using serological screening in high-risk groups, in particular among persons with post-tuberculosis changes in the lungs, was considered.

To increase the specificity of ELISA, searches for more specific antigens, including those obtained by genetic engineering: ESAT-6, etc. (see above) continue. The use of strictly specific antigens (38 kDa, ESAT) increases the specificity. But significantly reduces the sensitivity of the analysis. Along with ELISA (experimental laboratory test systems, eg Pathozyme ELISA kit), immunochromatographic kits with lateral filtration (Mycodot), as well as other similar tests (dot-membrane assay) with visual evaluation of the study result were also proposed. During these tests, the analysis is carried out for 10-30 minutes; they do not require special equipment, they require a visual evaluation of the results, which is associated with a certain subjectivity. These methods have approximately the same sensitivity and specificity characteristics (70% and 90-93%, respectively) as the traditional ELISA.

The use of methods of immune analysis has a definite value as an additional, taken into account in the complex of methods used, in the differential diagnosis of tuberculosis, especially in the diagnosis of its extrapulmonary forms. The most effective ELISA method is in the diagnosis of tuberculosis meningitis in the study of cerebrospinal fluid. In this case, the sensitivity of the analysis is 80-85%, and the specificity is 97-98%. There are data on the effectiveness of the detection of antibodies to mycobacteria tuberculosis in tear fluid in the diagnosis of tuberculous uveitis.

Induction of gamma interferon synthesis in vitro

Gamma interferon (IFN-γ) is a specific immune defense factor realized by activating macrophage enzyme systems. Induction of IFN-γ synthesis by sensitized T-lymphocytes causes their interaction with antigens of mycobacteria.

As antigens used as tuberculin PPD. And specific antigens obtained genetically engineered, in particular antigens ESAT-6 (early secreted antigen with a molecular weight of 6 kDa) and CFP-10 (a protein of culture filtrate, 10 kDa). Genetic engineering or recombinant antigens are absent in the cells of the BCG vaccine and other mycobacteria. When using tuberculin, the results of induction test IFN-γ are comparable to the results of the tuberculin skin test (direct correlation). When using genetically engineered antigens, the test results are more specific and do not depend on the previous vaccination of BCG. When testing vaccinated persons who did not have contact with tuberculosis infection, the specificity of the test is 99%. The sensitivity of the test among patients with tuberculosis varies from 81 to 89%.

Tests and diagnosticums based on short-term culture of whole blood cells or mononuclear cells isolated from the blood with mycobacterial antigens of tuberculosis in vitro with subsequent determination of the concentration of IFN-γ or calculation of the number of T-lymphocytes synthesizing IFN-γ have been developed. The concentration of interferon synthesized in a test tube is determined by ELISA using monoclonal antibodies binding IFN-γ. Then, by calibrating the standard IFN-γ, its concentration is determined in the test tube or wells of the plate.

When carrying out the Elispot test, the number of T-limocytes synthesizing IFN-γ. Are counted on the surface of a plate coated with antibodies to IFN-γ.

Developers of diagnosticum on the basis of induction of IFN-γ in vitro, which is approved by the US Agency for Medicines and Products, argue that it is impossible to differentiate latent tuberculosis infection from active tuberculosis with the help of the test. Therefore, in regions with a high level of infection, the test is not directly diagnostic. However, in our country it can be used to differentiate tuberculosis infection in children from post-vaccination allergy, and to assess the level of specific immunity in the treatment process.

At present, the domestic test system for determining the induction of IFN-γ synthesis by specific tuberculosis antigens in vitro is being studied.

Immune status and course of tuberculosis, immunocorrection

In the process of treatment of tuberculosis in humans, there are changes in antigenemia and the state of the immune system.

Data on changes in exudates and tissues are largely contradictory. The only thing that can be noted with good reason is that in tubercular granulomas, as a rule, a significant number of activated T-lymphocytes are detected.

It makes sense to dwell on two more provisions that are necessary to understand the role of immunological mechanisms in the treatment of tuberculosis in humans:

• AIDS patients have a particularly high incidence of multiple drug resistance;
• with multiple drug resistance (and in the absence of HIV infection), immunity disorders (especially the T-cell link) are particularly significant.

With tuberculosis, various methods of immunocorrection are widely used: these are primarily drugs that act primarily on T-cell immunity and a system of mononuclear phagocytes (thymic hormones, isophone, lycopide, polyoxidonium, etc.). As well as whole (attenuated) mycobacteria and their components.

Molecular-biological diagnosis of tuberculosis

The methods of molecular biology in the diagnosis of infectious diseases include, basically, methods based on manipulation with genomic materials of bacterial and viral pathogens in order to identify a specific genetic material - DNA segments with a nucleotide sequence specific for this species or strains of the pathogen, for the analysis of specific DNA sequences in genes that determine the sensitivity of the pathogen to certain medicinal substances, and also for functional analysis activity of certain genes of the pathogen. Molecular biological methods have gained wide acceptance in scientific research and practical application in the diagnosis and control of various bacterial and viral infections after the discovery in 1985 by Carrie Mullis (Nobel Prize winner, 1989) of a polymerase chain reaction.

Principles and possibilities of the polymerase chain reaction method

PCR allows to amplify (multiply) in vitro a nucleotide sequence (DNA fragment of the pathogen) for several hours in millions of times. The reaction in the presence of single DNA chains determines the extremely high sensitivity of the assay.

The nucleotide sequence of certain regions in the DNA chain determines the genetic identity of the microorganism, which explains the high specificity of PCR.

The importance of this method for the detection and investigation of the characteristics of mycobacteria tuberculosis is due to the biological characteristics of the microorganism, which has very slow growth: the time of doubling the DNA of mycobacterium tuberculosis during cultivation is 12-24 hours.

The principle of the PCR method consists in amplification - multiple, millions of times. Multiplying the sections of a specific DNA sequence in a tube microvolume during a cyclic recurrence of the following three reaction stages, each of which passes in a different temperature regime:

• Stage I - denaturation of double-stranded DNA upon heating with a divergence of its chains;
• II stage - complementary binding (hybridization) of primers (priming oligonucleotides) with terminal sections of chains of strictly specific, chosen for multiplication of the DNA fragment;
• Stage III - completion of the DNA fragment chain with the help of a thermostable DNA polymerase.

For amplification in vitro, there must be molecules of matrix DNA. Four types of deoxynucleoside triphosphates (nucleotides) containing the corresponding nitrogenous bases: adenine (A), thymine (T), guanine (D), cytosine (C); artificially synthesized priming oligonucleotides (primers) consisting of 18-20 base pairs; thermostable DNA polymerase enzyme having a temperature optimum of 68-72 ^{on the} C, and magnesium ions.

The specificity of PCR depends on the choice of the DNA fragment. In accordance with this, flank seed oligonucleotides are synthesized. Specificity of hybridization and completion of the DNA chain is due to the principle of complementarity of the following pairs of nitrogenous bases: adenine-thymine, guanine-cytosine.

To determine the genome of mycobacteria of the tuberculosis complex, the IS6110 DNA fragment was selected as the most effective target for amplification in most test systems, which in most strains of mycobacterium tuberculosis has a significant number (10-20) of repetitions in the genome, which, along with specificity, provides high sensitivity of the assay. At the same time strains of Mycobacterium tuberculosis with a small number of repeats or the absence of IS6110 fragment are described.

Isolation of DNA molecules from a biological sample

For carrying out PCR, the DNA molecules of the pathogen should be isolated from the biological material in a minimal volume, with a minimum amount of nonspecific DNA and various inhibitors of the enzyme-DNA polymerase.

The preparation of samples should be carried out under conditions that prevent cross contamination of the samples by the isolated DNA molecules. To do this, pre-treatment of the room with ultraviolet, floors and working surfaces of desks and appliances is necessary with chlorine-containing solutions. Also mandatory use of clean gloves, disposable test tubes and tips to automatic pipettes.

To isolate the DNA of mycobacterium tuberculosis from clinical specimens (spinal fluid, bronchial flushing) that do not contain a large number of leukocytes, cellular detritus or salts, it is sufficient to centrifuge the sample at 3-4 thousand rpm, add to the pellet 20-30 μl of 2% solution triton X-100 and warmed at 90 ^about C for 30 min.

For the preparation of sputum samples, effective dilution is necessary, for which a 4% sodium hydroxide solution and N-acetyl-L-cysteine (NALC) are usually used in an amount of 50-80 mg per sample, depending on the viscosity of the sample. The NALC solution must be prepared ex tempore or NALC powder can be added dry to the sample directly. After liquefaction, samples should be centrifuged for 15 minutes at 3.5-4,000 rpm (3000 g) in 50 ml vials with screw caps, i. E. Under the same conditions that are recommended for the presowing preparation of phlegm.

To extract DNA from the precipitate, a method based on the use of a 5-6 molar solution of guanidine isothiocyanate as lysis reagent and microporous silica particles ("diatomaceous earth"), sorbing DNA molecules, is often used. Nonspecific substances, including possible inhibitors, are then washed in a 2.5 molar solution of guanidine isothiocyanate and a solution of ethanol, after which the DNA molecules are desorbed in water, and these samples are used for PCR. To simplify the technology of DNA extraction, "diatomaceous earth" is often replaced by magnetic microparticles coated with silicon oxide. In this case, a special magnetic stand for microtubes is used instead of centrifugation to precipitate the particles.

In Russia, an original method for immunomagnetic separation of mycobacteria was developed, followed by extraction of the DNA of the pathogen. For immunomagnetic separation of mycobacterium tuberculosis, ferrocharticles 3-5 μm in size coated with silicon oxide are used, to which chemically linked polyclonal (rabbit) antibodies to mycobacteria of tuberculosis are connected. Samples of sputum after alkaline lysis are neutralized with an acidic tris-HCl solution and incubated with an immunomagnetic sorbent. Then, the immunoferroparticles are collected with a magnetic rod with a replaceable tip, transferred to a microtube, and precipitated. Add 20-30 μl of a 2% Triton X-100 solution and warm for 30 minutes at 90 ^° C. The supernatant is used as a DNA template for PCR analysis.

A difficult problem is the isolation of mycobacterium tuberculosis DNA from biopsy specimens. For enzyme biopsy, the enzyme proteinase K is used at a final concentration of 200-500 mg / l at a temperature of 56 ^° C overnight. Further, one of the known methods is used. Excess nonspecific DNA in the PCR analysis of biopsies often causes the inhibition of the reaction, which requires repeated extraction of DNA.

Methods for detecting results

After completion of the reaction, the amplified DNA fragments of the pathogen are identified by various methods.

The gel electrophoresis method is well known. The resulting DNA fragment is identified by a positive control containing the desired specific DNA fragment, or according to a known size (number of nucleotide pairs) of the fragment, which is determined using a standard molecular marker.

In the presence of a specific dye, ethidium bromide is included in double-stranded DNA. The synthesized DNA fragment is detected as a band luminous under the action of the ultraviolet.

The size of the DNA fragment, determined by electrophoresis from the distance from the start, must correspond to a known molecular weight marker or positive control.

Other methods for determining the results of PCR are based on the hybridization of single-stranded PCR products with a complementary oligonucleotide-DNA probe labeled with biotin, followed by detection by an enzymatic reaction, for example, by binding to biotin of the streptavidin-alkaline phosphatase conjugate.

Based on this type of detection, PCR analyzers have been created in which the detection of PCR results is carried out automatically as a result of reading the optical density in the samples after the manifestation of the enzymatic reaction.

The disadvantages of these methods are the possibilities of intralaboratory contamination by rather short fragments of DNA molecules. When molecules enter new samples, they become a matrix for PCR and lead to false positive results.

In this regard, to prevent false-positive results, strict rules for the separation and isolation of premises are introduced: to extract DNA from biological samples; premises for the detection of results (electrophoresis) from the clean zone. These premises are a zone of probable contamination. Another isolated area is a clean room for introducing the DNA samples to be tested in tubes with a reaction mixture for PCR. Finally, it is assumed that the main device - the DNA-amplifier - should be placed in a separate, possibly office, room.

To prevent contamination by the products of the previous reactions - amp-lycones, some PCR test systems instead of deoxynucleoside-thymidine contain deoxynucleosiduridine, which, when synthesized in vitro, is inserted in its place in the corresponding position, i.e. The nitrogenous base of thymine present in native DNA is replaced by uracil. Uracil-DNA glycosylase, added to the reaction mixture to the material under analysis, destroys only the contaminant fragments with deoxyuridine, but not native DNA to be analyzed. Lt; / RTI & gt; Subsequent heating at 94 ^° C inactivates this enzyme and does not interfere with amplification in PCR.

There is a test system based on isothermal amplification of rRNA, for which the reverse transcription and synthesis of DNA molecules are carried out first. Which, in turn, is a matrix for the subsequent synthesis of RNA molecules. RNA amplicons are detected using an acridine-stained DNA probe when hybridized in a reaction tube solution. This method, in addition to high sensitivity, has the advantage of analyzing in one tube, which prevents contamination. According to the authors, sensitivity of this method in respiratory samples reaches 90% with a specificity of 99-100%.

New detection methods are implemented in real-time PCR. These methods differ primarily in that PCR and detection of its results are carried out simultaneously in one closed tube. This not only technologically simplifies the technique of analysis, but also prevents the contamination of laboratory rooms and test samples with products of previous PCR.

With real-time PCR, the detection of the results is due to fluorescence arising from the hybridization of a fluorogenic DNA probe with a specific DNA fragment amplified during PCR. The structure of fluorogenic DNA probes is constructed in such a way that the fluorescent marker is released as a result of the enzymatic reaction or distances itself from the molecule of the fluorescence quenching agent only in a specific hybridization with the desired DNA molecule amplified during PCR. With the increase in the number of molecules hybridized with the probe, the increase in fluorescence to the detectable level is proportional to the number of molecules of the amplified product. Since the number of molecules of the DNA fragment is doubled during each PCR cycle, the number of the cycle from which fluorescence is determined and increases is inversely proportional to the number of DNA molecules in the original sample. If several different known molecular concentrations of the corresponding DNA fragment of mycobacterium tuberculosis are introduced into the reaction as a calibrator, then by the computer program the number of DNA genomes in the test material can be calculated.

Each standard sample is duplicated. The quantitative criterion is the minimum number of PCR cycles necessary for the onset and growth of the determined fluorescence. On the abscissa - the number of cycles; the ordinate is the fluorescence value. The DNA concentrations are inversely proportional to the number of cycles required for the appearance of fluorescence. In the right column (21-32), the cycle numbers for the corresponding concentrations are marked. Differences between 10-fold concentrations of DNA fragments 10 ² -10 ⁶ ml - 3.2-3.4 cycles. For two patients, the concentrations of IS6110 fragments were about 10 ³ / ml and 10 ⁴ / ml. Taking into account the number of repetitions (6-20) of the fragments analyzed in the genome of Mycobacterium tuberculosis, the number of myco-bacteria in clinical samples is about 100 and 1000 cells, respectively.

The use of PCR in the diagnosis of tuberculosis

The PCR method is most used for accelerated diagnosis of tuberculosis - detection of mycobacterium tuberculosis in clinical specimens: sputum. Bronchial irrigation, pleural exudate, urine, cerebrospinal fluid, osteolysis punctate, female genital tract aspirates and various biopsy specimens. When studying about 500 samples of sputum and bronchial flushes from 340 patients with confirmed diagnosis of pulmonary tuberculosis in the Netherlands, the comparative sensitivity of PCR, culture and smear microscopy was studied. The sensitivity of the analysis was 92.6.88.9 and 52.4%, respectively. The specificity of all methods was about 99%.

The effectiveness of detection of mycobacterium tuberculosis by smear microscopy, seeding on the Levenstein-Jensen medium, VASTES test system and PCR analysis was compared. PCR showed a sensitivity of 74.4%, microscopy - 33.8%, seeding on a dense medium - 48.9% and VASTES - 55.8%. The average detection time for seeding on a Levenstein-Jensen medium is 24 days. VASTES - 13 days, PCR - 1 day.

The possibilities of using PCR as a sensitive and rapid method for monitoring the effectiveness of tuberculosis treatment are also discussed.

Detection of the DNA of mycobacteria tuberculosis by PCR with effective chemotherapy is determined for a longer time - an average of 1.7 months compared with the bacterial release, determined by luminescence microscopy, and 2.5 months compared to bacteriological study.

Diagnosis of extrapulmonary forms of tuberculosis

The significance of PCR as a sensitive method is especially great for extrapulmonary forms, since it is with these forms that clinico-radiologic methods and traditional bacteriological methods for determining mycobacteria of tuberculosis in diagnostic materials are ineffective.

In the study of urine samples, the results of PCR analysis were positive in 16 of 17 patients with active tuberculosis of the urinary system and negative in 4 patients with inactive renal tuberculosis and 39 patients with non-tuberculous diseases of the urinary system.

The effectiveness of PCR analysis in the study of bone marrow aspirates in patients with fever of unknown origin was demonstrated in cases of suspected tuberculosis. To diagnose tuberculous lymphadenitis, 102 puncture aspirates and a biopsy specimen of 67 children with suspected tuberculous lymphadenitis were studied in children. Positive results were obtained: 71.6% real-time PCR. Fluorescence microscopy - 46.3%. Culture research - 41,8%. In the study of 50 lymph node biopsies in patients with "cat scratch" disease, all the results were negative. Thus, 100% specificity of PCR analysis was demonstrated. In the same work, with puncture biopsy of lymph nodes, the possibility of detection of M. Avium was demonstrated.

Diagnosis of tuberculosis of the female genital area infertility, as is known, is one of the most difficult problems of diagnosis. In a PCR study of endometrial biopsies, endometrial aspirates and fluid samples from the Douglas space, 14 (56%) of 25 patients examined laparoscopically with suspected tuberculosis received positive results. Using smear microscopy and culture, 1 and 2 results were obtained, respectively. These cases were also PCR-positive. Most PCR-positive results were related to cases with characteristic signs of tuberculosis according to the histological study; a smaller number - with suspicion of tuberculosis according to laparoscopy data. Only one positive result of PCR analysis was obtained in the absence of laparoscopic data for tuberculosis.

When diagnosing extrapulmonary forms of tuberculosis, clinicians often have a question about the possibility of detecting a pathogen when testing blood samples with the PCR method. Literary data indicate that the detection of DNA from the mycobacterium tuberculosis from blood samples is possible with far-reaching forms of HIV infection. The DNA of mycobacterium tuberculosis was detected only with generalized tuberculosis of various organs in patients with transplanted kidney and immunosuppression.

[60], [61], [62], [63], [64], [65]

Species identification of mycobacteria

The PCR method can be quite effective for the rapid identification of mycobacteria of the tuberculosis complex and certain types of nontuberculous mycobacteria after obtaining their primary growth. In this case, the use of PCR can save 7-10 days, necessary for the subsequent cultural identification of a positive result. The PCR test is technically very simple, since it does not require complicated sample preparation of the clinical material to achieve high sensitivity. In the study of 80 positive cultures in this test system (MB VaT of Organon) cultures, all positive results of PCR analysis were strictly specific and were performed for 1 day. To identify other species of mycobacteria when they are obtained in culture, the DNA of the pathogen hybridizes with specific DNA probes labeled with acridine, and the strains are detected by the appearance of chemiluminescence with chemiluminescence or on strips of nitrocellulose with a visual evaluation after hybridization. With the help of such a set, a limited number of species are identified: the mycobacterium tuberculosis complex. M. Avium, M. Avium complex, M. Kansasii and M. Gordonae.

A.Telenti et al. Have also developed a relatively simple and inexpensive method for the species identification of clinically important types of mycobacteria based on PCR and subsequent treatment with two restriction enzymes (enzymes with the properties of dissecting the DNA molecule at specific points). The DNA fragment is amplified. Which encodes a heat shock protein (65 kDa), and then the PCR fragment of 439 nucleotide pairs produced by PCR is treated separately with two enzymes, Bste II and Nae III. Then, using the agarose gel electrophoresis, the two products obtained are analyzed by determining their size (number of nucleotide pairs) using a set of standard DNA fragments (molecular DNA markers) with a length of 100 to 1000 nucleotide pairs. In each of the specific species (M. Tuberculosis, M. Avium, M. Intracellulare, M. Kansasii, M.fortuitum), 2 or 3 DNA fragments of different size are detected for each restriction enzyme. The combination of different DNA sizes obtained allows differentiating these species among themselves.

The technology of biological DNA microarrays is being developed. Which will help identify more than 100 species of mycobacteria in one study.

Species identification can also be carried out by PCR amplification of the 16S rRNA variable region followed by amplicon sequencing when compared with the corresponding primary structure, which allows identifying more than 40 species of mycobacteria.

With the help of PCR, a species identification within the mycobacterium tuberculosis complex can also be carried out, including differentiation of M. Bovis and M. Bovis BCG. To do this, the presence or absence of some genes in the genomic regions of RD1 is analyzed. RD9 and RD10. RD1 is absent in M. Bovis BCG, but is present in virulent species, including M. Bovis.

Determination of drug sensitivity of Mycobacterium tuberculosis by PCR

The tasks of molecular genetic methods for determining drug susceptibility or resistance of mycobacterium tuberculosis are reduced to the detection of mutations in certain nucleotide sequences of known genes. The main methods are based either on direct reading (sequencing) of these sequences after amplification, or on hybridization of biotin-labeled DNA fragments amplified during PCR with DNA probes. Both variants involve the detection of substitutions in nucleotide sequences that, when using DNA probes, lead to the absence or inadequate hybridization on the nitrocellulose membrane with the enzyme conjugate (streptavidin-alkaline phosphatase) -the LIPA-Rif-TB method.

The method of measuring fluorescence in locally fixed DNA probes that are complementary to known mutations in PCR amplified regions of genes responsible for drug sensitivity or resistance has been termed the method of microbiochips. The main algorithm for carrying out this research is as follows. After isolation of DNA from a clinical sample or culture of mycobacteria, PCR is necessary to amplify the corresponding fragments of the gene's gene responsible for drug sensitivity to rifampicin, or katG and inhA genes encoding mycobacterial proteins responsible for sensitivity to isoniazid. The PCR results are evaluated by agarose gel electrophoresis, in which the corresponding DNA fragments of the desired length are confirmed. Then, a second round of PCR is performed to introduce a fluorescent label into the DNA. The results of PCR are again confirmed by gel electrophoresis. After this, hybridization is carried out (overnight incubation) followed by washing of the obtained material on the biochip, which is a large number of short chains of DNA (probes) fixed on a small glass plate, complementary to the nucleotide sequences of the drug-sensitive type of tubercle bacilli at the points of possible mutations. As well as to the mutant sequences responsible for drug resistance. The location of the DNA probes on the plate is strictly defined, and the level of the observed fluorescence during hybridization is determined to determine the result by means of a special reader. In this regard, the results of the analysis are determined by means of a special computer program.

In recent years, alternative methods for determining the drug sensitivity of mycobacteria tuberculosis on the basis of real-time PCR technology have been developed, which make it possible to conduct these studies in a closed-tube test.

In Fig. 13-13 presents the result of the analysis of clinical cultures of mycobacterium tuberculosis in the determination of drug resistance to rifampicin by real-time PCR: 218 - a control sample (sensitive to rifampicin); 93 - positive control for the mutation of Ser-Trp TCG-TGG; 4482 - positive control for mutation of Ser-Leu TCG-TTG; 162-322 - experimental samples. Result of calculation of kinetic curves of amplification on 4 channels: channel 1: 393 - positive control for mutation of Ser-Trp TCG-TGG; channel 2: 4482 - positive control for mutation of Ser-Leu TCG-TTG; 162, 163, 172, 295 - experimental samples; channel 4: kinetic curves of amplification of all samples participating in the experiment. Positive control of the amplification reaction. Conclusions: Based on the results of the analysis, the following mutations determining the resistance to rifampicin were revealed: in samples 162,163,172,295-Ser-Leu TCG-TTG. The same principle was used to determine the drug resistance to isoniazid for the genes katG and inhA, which determines the most frequent mutations.

[66], [67], [68], [69], [70], [71],

Strain identification of mycobacterium tuberculosis

The most studied method of strain identification of mycobacterium tuberculosis is a technology called polymorphism of the length of restriction fragments (RFLP, or RFLP in the English version), which is based on fragmentation of the DNA of mycobacterium tuberculosis by Pvu II and subsequent hybridization of the resulting fragments with specific specific DNA sequences its repeated element IS6110. Intraspecific variability is realized due to the different number of repetitions of IS6110 and their location on DNA. As well as the variety of distances between certain enzyme restriction enzyme attack points (restriction sites) and IS6110 element. This technology is very complicated and time consuming. After processing the DNA isolated from the culture of mycobacterium tuberculosis, a gel electrophoresis is performed with restriction enzyme, then DNA fragments of different length are transferred to the nitrocellulose membrane, hybridization with fragments of the IS6110 element is performed, and the results are determined by an enzymatic reaction. The resulting specific pattern of bands characterizes the DNA of a specific strain of Mycobacterium tuberculosis. With the help of computer analysis, the identity or relatedness of the strains is revealed. Despite the fact that the RFLP method is the most discriminatory, i.e. Reveals the greatest number of differences in the strains analyzed, it is ineffective with a small number (less than 5) IS6110-repeats observed in some strains. In Fig. 13-14 shows the results of RFLP-typing of strains.

An alternative may be the method of spoligotyping - analysis of the polymorphism of spacer DNA sequences - intermediate between direct repeats of the DR region. When carrying out spoligotyping of the strains, PCR is carried out with primers bounding the DR region, after which fragments of different length are formed which hybridize with the variable intermediate regions of DNA. Analysis of the spacer sequences of the DR region is presented. According to researchers, more simple, productive and suitable for primary screening of strains and preliminary epidemiological analysis, as well as research directly clinical material.

Obviously, a more effective and technologically accessible method is VNTR (abbreviation of English words), or a method for determining the variable number of exact tandem repeats in the DNA of mycobacterium tuberculosis. This method is based only on the use of PCR and does not require additional manipulation. Since the number of tandem repeats in different strains and in different loci is different, the fragments of different sizes are determined and analyzed on the resulting electrophoregram of PCR products. According to the researchers, using VNTR achieves a greater degree of discrimination of strains than with the RFLP method.

Much attention has been paid in recent years to the distribution of strains of Mycobacterium tuberculosis of the W-Beijing family (sometimes called the Beijing strain), which are largely drug-resistant.

Basic requirements to the quality of molecular biological research

[72], [73], [74], [75],

Basic regulatory documents for PCR

Orders of the Ministry of Health of the Russian Federation: No. 45 of 7.02.2000. No. 109 of 21.03.2003. No. 64 of 21.02.2000. Methodical instructions: 1.3.1888-04 "Organization of work in studies using the PCR method of material infected with pathogenic biological agents of groups III-IV of pathogenicity "; 1.3.1794-03 "Organization of work in the study of PCR material, infected with microorganisms of the I-II pathogenicity groups". 2003; 3.5.5.1034-01 "Decontamination of the study material infected with bacteria of the I-IV pathogenicity groups by PCR", 2001. Appendix 11 to the Instruction on Unified Methods of Microbiological Research for Detection, Diagnosis and Treatment of Tuberculosis.

[76], [77], [78]

Staff

Clinical laboratory diagnostics, bacteriological doctors, virologists, biologists of the clinical diagnostic laboratory, as well as specialists with secondary medical education, who have passed specialization and advanced training in the established order, can carry out molecular biological studies.

Arrangement of laboratory premises

The following laboratory rooms are required:

• Sample handling area is a laboratory adapted to work with infectious agents of III-IV pathogenicity groups, according to the Methodical Instructions 13.1888-04.
• Zone for the preparation of reaction mixtures PCR - laboratory room, which provides protection from internal laboratory contamination - "clean" zone.
• • If electrophoresis or hybridization is used to analyze PCR products. A laboratory room in which the multiplied DNA fragments are extracted from the amplification tube and, accordingly, can enter the environment, in accordance with the requirements for PCR laboratories (Guidelines 1.3.1794-03, Guidelines 1.3.1888-04) should be fully is isolated from the premises indicated in the previous paragraphs. The movement from the electrophoresis area to the sample handling area and the "clean" zone of any personnel, equipment, any materials and objects, as well as air transport through the ventilation system or as a result of drafts, should be avoided. This zone is not required for the fluorimetric detection of PCR products.
• The room for documentation and processing of results is equipped with computers and necessary office equipment. This room may contain equipment that provides detection of PCR products without opening the tube. - fluorescent PCR detectors and thermal cyclers for real-time PCR.

Sanitary and epidemiological requirements for the primary treatment of sputum are similar to the standard microbiological requirements for working with mycobacteria tuberculosis.

[79], [80], [81], [82],

Completion of laboratory equipment for PCR diagnostics

The laboratory includes equipment for the following rooms.

• room for sample preparation, contains the following equipment: laminar of the II class of protection "SP-1.2": solid-state thermostat with a heating cover for test tubes of the "Eppendorf" type; microcentrifuge at 13,000 rpm; a centrifuge (Vortex); refrigerator with a temperature range from -20 ^о С to +10 ^о С; pipettes of variable volume of the "Rroline" series; a pump with a OM-1 trap flask; a tripod for pipettes; tripod workstation 200x0.5 ml; tripod workstation 50x1.5 ml; Stands for storing test tubes 80x1.5 ml;
• Reaction mixture preparation room: protective chamber PCR-box ("Laminar-C. 110 cm); centrifuge - Vortex; Variable volume pipettes of the Proline series; a tripod for pipettes; tripod workstation 200x0.2 ml; Stands for storing test tubes 80x1.5 ml; refrigerator with a temperature range from -20 ^о С to + 10 ^о С;
• room for electrophoresis: camera for horizontal electrophoresis; source of power; transilluminator;
• DNA amplifiers or nucleic acid analyzer (PCR in real time) with a computer and software; can be placed in any spare room. If real-time PCR technology is used. Room for electrophoresis is not needed.

[83], [84], [85], [86]

External quality control

To be confident in obtaining objectively reliable results, laboratories should participate in the system of external evaluation of the quality of laboratory research.

Participants in the quality control system receive; 12 ampoules with lyophilized suspensions of bacterial cells, two of which contain E. Coli E. Coli, 3 ampoules with mycobacterium tuberculosis (avirulent strain) at a concentration of 10 ² / ml; 3 ampoules with cells of a similar strain in a concentration of 10 ⁴ / ml; 2 ampoules with non-tuberculosis mycobacteria M. Avium-intracellulare and M. Kansasii in a concentration of 10 ⁵ / ml.

Distributed tests for external quality assessment are pre-tested in two independent laboratories with extensive experience in this field.

[87], [88]