Laboratory diagnosis of tuberculosis

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

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Clinical blood test

In patients with tuberculosis, changes in the general blood test are not pathognomonic. In limited and low-active forms of tuberculosis, hypochromia of erythrocytes with a normal number is characteristic. In massive infiltrates or caseous pneumonia, with widespread caseous lymphadenitis, specific intestinal damage, as well as with large pulmonary or postoperative hemorrhages, erythropenia and microcytosis, oligochromasia, polychromasia are noted. Macrocytosis, and especially poikilocytosis, are encountered much less often, usually with severe anemia. The number of reticulocytes in the compensated stage of tuberculosis varies from 0.1 to 0.6%, in the subcompensated stage - from 0.6 to 1.0%, and for the decompensated stage, 1% of reticulocytes are characteristic.

In some cases of tuberculosis, moderate leukocytosis (up to 15 thousand leukocytes) may be observed, less often leukopenia, which occurs in 2-7% of cases in patients with limited and mild forms of the process and in 12.5% in destructive and progressive pulmonary tuberculosis.

Most often, shifts occur in the leukocyte formula. Both relative and absolute neutrophilia are noted, a moderate shift in the leukocyte formula to the left to promyelocytes. Myelocytes are very rarely encountered in cases 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 a tuberculosis outbreak subsides, the nuclear shift returns to normal relatively quickly. Pathological granularity of neutrophils usually persists longer than other changes in the hemogram.

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

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

Among the tests for assessing the tuberculosis process, a special place is occupied by determining the erythrocyte sedimentation rate (ESR), which is important in assessing the course of the tuberculosis process and identifying its active forms. An increase in ESR indicates the presence of a pathological process (infectious and inflammatory, purulent, septic, hemoblastosis, lymphogranulomatosis, etc.) and serves as an indicator of its severity, but normal ESR values 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 blood viscosity. Slowing down of erythrocyte sedimentation is characteristic of conditions accompanied by hemoconcentration, an increase in the content of albumins and bile acids.

The hemogram of tuberculosis patients changes during treatment. The more successful the therapeutic intervention, the faster the hematological changes disappear. At the same time, the effect of various antibacterial drugs on hematopoiesis should be kept in mind. They often cause eosinophilia, in some cases - leukocytosis, and more often leukopenia up to agranulocytosis and lymphoid-reticular reaction. Systematic hematological monitoring and correct analysis of the data obtained are essential for assessing the clinical condition of the patient, the dynamics of the process and the effectiveness of the treatment.

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Clinical urine analysis

In case of urinary tract tuberculosis, urine analysis is the main laboratory diagnostic method. Leukocyturia, erythrocyturia, proteinuria, hypoisosthenuria, tuberculous mycobacteriuria, non-specific bacteriuria can be observed.

Leukocyturia is the most common symptom of urinary tract tuberculosis before specific chemotherapy and is absent only in exceptional cases, such as complete obliteration of the ureter 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 to detect it with a normal general urine analysis. However, it should be taken into account that leukocyturia can occur in acute and chronic pyelonephritis, cystitis, urethritis, kidney stones and ureters.

Erythrocyturia, like leukocyturia, is considered one of the most common laboratory signs of genitourinary tuberculosis. The frequency of hematuria depends on the prevalence of the process; it increases as the destructive tuberculous process in the kidney develops. Erythrocyturia without leukocyturia is more typical for the early stages of renal tuberculosis. Hematuria, prevailing over leukocyturia, is an important argument in favor of renal tuberculosis when differentiating it from nonspecific pyelonephritis.

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Biochemical blood test

In tuberculosis, changes in some biochemical indices 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 the blood serum are not changed and determine their normal content.

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

Of significant importance in assessing the functional state and organic damage to the liver in tuberculosis and its complications is the determination of direct and total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) in the blood serum. Dynamic determination of the level of aminotransferases. bilirubin in the treatment of patients with tuberculosis, especially in its severe forms, is an obligatory component of the biochemical examination of patients with tuberculosis and is carried out monthly.

Evaluation of the functional state of the kidneys includes determination of serum creatinine and calculation of the glomerular filtration rate using the Cockcroft-Gault formula. Calculation of the glomerular filtration rate using the Reberg test gives less accurate results.

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

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Application of biochemical research methods in extrapulmonary tuberculosis

The most informative indicator is considered to be the content of tuberculostearic acid in biological fluids, however, its determination is associated with technical difficulties (the need to use gas chromatography and mass spectrometry).

It is promising to measure the activity of adenosine deaminase - an enzyme determined in fluids: synovial, pericardial, ascitic or cerebrospinal. The main producers of adenosine deaminase are lymphocytes and monocytes. Determination of the activity of adenosine deaminase in biological fluids facilitates the diagnosis of tuberculous synovitis, tuberculosis of the lymph nodes, tuberculous meningitis, tuberculous serositis.

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

Optimally, the activity of the organ-specific enzyme transamidinase is determined in urine; its appearance is noted in kidney damage of various origins. The study of transamidinase is justified only in conditions of subcutaneous administration of tuberculin in order to exacerbate the local inflammatory process. Transamidinase activity is determined in urine initially and 24-72 hours after the administration of 50 TE tuberculin. An increase in fermenturia by 2 times or more allows in 82% of cases to differentiate active tuberculosis of the kidneys from exacerbation of chronic pyelonephritis.

In case of tuberculosis of female genital organs, the concentrations of haptoglobin and malondialdehyde in the blood are determined under the conditions of the provocative tuberculin test. Tuberculin is administered subcutaneously at a dose of 50 TE and a repeat biochemical study is performed after 72 hours. In case of tuberculous etiology, the degree of increase in the haptoglobin level is at least 28%, and the level of malondialdehyde is 39% or more. Determination of the activity of adenosine deaminase in the peritoneal fluid obtained from the Douglas pouch is also used. The puncture is examined again 72 hours after the intradermal administration of tuberculin at doses of 0.1 TE and 0.01 TE in the area of projection of the internal genital organs on the anterior abdominal wall. An increase in the activity of adenosine deaminase by 10% or more compared to the initial value indicates a tuberculous process.

In case of eye damage, the focal reaction occurring in the eye in response to antigen stimulation is examined. In this case, the development of a sharply expressed response accompanied by a decrease in visual functions is undesirable. Since the assessment of minimal focal reactions is often difficult, it is recommended to focus in parallel on the degree of increase in haptoglobin or adenosine deaminase in the blood serum to objectify the conclusion.

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

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Study of the blood coagulation system

The relevance of studying the state of the blood coagulation system in phthisiology is due to the presence of hemoptysis or pulmonary hemorrhages in a number of patients with tuberculosis of the lungs, as well as hemocoagulation complications in the surgical treatment of tuberculosis. In addition, the naturally accompanying latent intravascular hemocoagulation affects the course of the disease and the effectiveness of chemotherapy.

In patients with pulmonary tuberculosis with a predominant exudative component of inflammation, a decrease in the anticoagulant activity of the blood is observed. In patients with a low prevalence of specific lung damage with a predominant productive component of inflammation, intravascular hemocoagulation is expressed insignificantly. In patients with pulmonary tuberculosis with hemoptysis and pulmonary hemorrhages, the state of the blood coagulation system is different: in patients with minor blood loss at the height of hemoptoea or immediately after its cessation, a sharp increase in the coagulation capacity of the blood is observed due to a pronounced intensification of thrombin formation processes while maintaining increased "structural" coagulability. 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, and platelet count. At the stage of surgical treatment in patients with limited forms of pulmonary tuberculosis, significant disturbances in the homeostasis system do not occur. In patients with widespread processes, when performing pneumonectomy or pleuropneumonectomy, DIC syndrome often develops, which can take the form of a “second disease”.

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

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Hormonal studies

Modern experimental and clinical observations indicate the presence of changes in hormonal status in specific tuberculous inflammation of the lungs. It has been proven that the correction of dysfunction of the pituitary-adrenal, pituitary-thyroid systems and pancreatic function in combination with anti-tuberculosis therapy contribute to the activation of fibrogenesis and reparation processes in the focus of specific inflammation.

The functional state of the pituitary-thyroid system is judged by the content of triiodothyronine (T3), thyroxine (T4), and pituitary thyroid-stimulating hormone (TSH) in the blood serum _. It _has been established that subclinical hypothyroidism is detected in 38-45% of patients with pulmonary tuberculosis, and it is most often diagnosed in disseminated and fibrous-cavernous forms of the process. In these forms, the levels of both T3 and T4 are most sharply reduced _, and _an imbalance of these hormones occurs in the form of an increase in the T4 _/ T3 _ratio.

The function of the adrenal cortex is assessed by the serum cortisol level, and the endocrine function of the pancreas is assessed by the concentration of immunoreactive insulin. In the acute phase of an infectious disease, the need for endogenous cortisol and insulin increases. Hyperinsulinemia also indicates insulin resistance of body tissues, which is typical for any active inflammatory process, in particular a specific one. Determination of the glucocorticoid function of the adrenal glands in active pulmonary tuberculosis allows us to detect the presence of hypercorticism in most patients. Normal blood cortisol concentrations in a patient with infectious inflammation in the acute period should be regarded as a relative insufficiency of the glucocorticoid function of the adrenal cortex, which can serve as a basis for replacement therapy with adequate doses of glucocorticoids.

Almost a third of patients with pulmonary tuberculosis have a low insulinemia level approaching the lower limit of the norm, while 13-20% have significant hyperinsulinism. Both relative hypo- and hyperinsulinism are high risk factors for the development of carbohydrate metabolism disorders of varying severity. These changes in the functional activity of pancreatic B-cells require regular glycemia monitoring in patients with tuberculosis and timely prevention of diabetes mellitus. In addition, this serves as an additional justification for the appropriateness of using physiological doses of insulin in the complex therapy of tuberculosis.

In general, the decrease in thyroid hormone levels, their imbalance, hypercortisolemia and hyperinsulinism are most pronounced in patients with a severe course of the tuberculosis process, with extensive lung lesions and pronounced symptoms of tuberculosis intoxication.

Microbiological diagnostics of tuberculosis

Microbiological studies are necessary for identifying patients with tuberculosis, verifying the diagnosis, monitoring and correcting chemotherapy, assessing treatment outcomes, in other words, from the moment a patient with tuberculosis is registered until he is removed from the register.

All epidemiological programs and projects are based on the assessment of the number of bacteria excretors, which is impossible to do without the use of laboratory methods for detecting tuberculosis mycobacteria. When examining the appeal of the so-called unorganized population, the percentage of bacteria excretors reaches 70 or more, which makes laboratory methods a fairly effective means of identifying tuberculosis patients among this population group.

Traditional microbiological methods of diagnosing tuberculosis are bacterioscopic and cultural studies. Modern methods include culturing tuberculosis mycobacteria in automated systems and PCR. However, all these methods are necessarily combined with classical bacteriological methods.

Collection of diagnostic material

The effectiveness of laboratory tests largely depends on the quality of the diagnostic material. Compliance with the rules for collecting, storing and transporting diagnostic material and the precise implementation of the patient examination algorithm directly affects the result and ensures biological safety.

A variety of materials are used to test for tuberculosis. Since pulmonary tuberculosis is the most common form of tuberculous infection, the main material for testing is considered to be sputum and other types of tracheobronchial tree discharge: upper respiratory tract discharge obtained after aerosol inhalations: bronchial lavage waters; bronchoalveolar lavages; material obtained during bronchoscopy, transtracheal and intrapulmonary biopsy: bronchial aspirate, laryngeal smears, exudates, wound smears, etc.

The effectiveness of research increases if controlled collection of material from the patient is carried out. For this purpose, a specially equipped room is allocated or special booths are purchased. Collection of material is a dangerous procedure, therefore, material for research must be collected in compliance with infection safety rules.

Material for testing for Mycobacterium tuberculosis is collected in sterile vials with tightly screwed caps to prevent contamination of the environment and protect the collected material from contamination.

Vials for collecting diagnostic material must meet the following requirements:

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

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

• collection of material should be carried out before the start of chemotherapy;
• the material for the study must be collected before eating or taking medications in the morning;
• For the study, it is advisable to collect at least 3 morning sputum samples. Sputum is collected for 3 consecutive days;
• The collected material must be delivered to the laboratory as quickly as possible:
• in cases where it is impossible to deliver the material to the laboratory immediately, it is stored in a refrigerator at an air temperature of 4 °C for no more than 48 hours;
• When transporting the material, it is necessary to pay special attention to the integrity of the bottles.

Correctly collected sputum has a mucous or mucopurulent character. The optimal volume of the examined portion of sputum is 3-5 ml.

Sputum is collected under the supervision of a health worker. Persons responsible for collecting sputum must ensure that certain rules are followed:

• It is necessary to explain to the patient the purpose of the examination and the need to cough up not saliva or nasopharyngeal mucus, but the contents of the deep sections of the 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 must first rinse his mouth with boiled water to remove the main part of the microflora vegetating in the oral cavity and food debris that complicate the examination of sputum;
• The medical worker involved in collecting sputum, in addition to a gown and cap, must wear a mask, rubber gloves and a rubber apron;
• standing behind the patient, he is advised to hold the bottle as close to his lips as possible and immediately separate the sputum into it as he coughs it up, while it is necessary to ensure that the air flow is directed away from the health worker:
• Once the sputum collection is complete, the health worker should carefully close the bottle with the lid and assess the quantity and quality of the collected sputum. The bottle is then labeled and placed in a special box for transportation to the laboratory.

If the patient does not produce sputum, then the night before and early in the morning on the day of collecting the material, he should be given an expectorant: extract of the roots of marshmallow (mucaltin), bromhexine, ambroxol, etc. - or an irritating inhalation should be used, using equipment installed in the room for collecting sputum. The material collected in this way is not subject to preservation and should be examined on the day of collection. To avoid its "rejection" in the laboratory, a special note should be made in the referral.

If microbiological studies are not performed in a given institution, the collected diagnostic material must be delivered to the laboratory centrally, provided that the material is kept in the refrigerator or with preservatives between deliveries. The material is delivered to the laboratory in transport boxes that can be easily disinfected. Each sample must be provided with an appropriate label, and the entire batch must have a completed accompanying form.

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Modes and frequency of examination of patients

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

Primary screening for tuberculosis should be carried out by all medical and diagnostic institutions of the healthcare system. Recently, in order to increase the effectiveness of primary examination, so-called microscopy centers have been organized on the basis of clinical diagnostic laboratories, equipped with modern microscopes and equipment to ensure epidemic safety.

Anti-tuberculosis institutions use a survey scheme that provides for at least 3-fold examination of sputum or other diagnostic material within 3 days. During treatment, microbiological studies are carried out regularly at least once a month in the intensive chemotherapy phase. When moving to the follow-up phase, studies are carried out less frequently - at intervals of 2-3 months, while the frequency of studies is reduced to two.

Features of collecting diagnostic material for extrapulmonary tuberculosis

A feature of pathological material in extrapulmonary forms of tuberculosis is the low concentration of mycobacteria tuberculosis in it, which requires more sensitive methods of microbiological research, primarily methods of sowing on a nutrient medium.

In case of genitourinary tuberculosis, urine is the most accessible material for examination. Urine collection should be performed by a specially trained nurse.

The external genitals are washed with water and soap or a weak solution of potassium permanganate. The external opening of the urethra is carefully treated. The middle portion of morning urine is collected in a sterile bottle: in men - naturally, in women - using a catheter. Urine from the renal pelvis is collected in sterile test tubes during 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, testicular punctures, and prostate secretions are centrifuged to obtain a sediment. With any localization of a specific process in the genital area in men, prostate massage can promote the release of secretions containing tuberculosis mycobacteria.

Menstrual blood is collected from women by suction or using a Kafka cap. The resulting material is freed from erythrocytes by washing it with distilled water and then centrifuging. The sediment is examined.

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

Material obtained during surgical interventions on the kidneys, genitals, biopsies, endometrial scrapings, is homogenized. To do this, it is placed in a sterile mortar and thoroughly crushed with sterile scissors. Sterile river sand is added to the resulting suspension in an amount equal to its mass, then 0.5-1.0 ml of isotonic sodium chloride solution is added and everything is ground until a mushy mass is formed 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 puncture (pus from abscesses) obtained with a sterile syringe is placed in a sterile container and immediately delivered to the laboratory. Using a sterile pipette, previously moistened with sterile isotonic sodium chloride solution, 2-5 ml of pus is taken, transferred to a bottle with beads and another 2-3 ml of isotonic sodium chloride solution is added. The bottle is closed with a stopper and shaken in a shaker for 8-10 minutes. The homogenized suspension is examined.

In fistulous forms of osteoarticular tuberculosis, pus is taken from the fistula. Abundant discharge is collected directly into a test tube. In cases of scanty pus discharge, the fistula tract is washed with a sterile isotonic sodium chloride solution, and the washings collected in a test tube or a piece of tampon soaked in pus are sent for examination.

Surgical material obtained during surgical interventions on bones and joints may consist of purulent-necrotic masses, granulations, scar tissue, bone tissue, synovial membrane tissue and other substrates. Its processing is performed as in the case of renal tuberculosis.

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

Tuberculosis of the lymph nodes. Pus extracted during puncture of the lymph nodes is examined in the same way as pus from abscesses. Lymph node tissue obtained during surgical interventions and biopsies is examined as in other forms of tuberculosis.

The study of feces for Mycobacterium tuberculosis is performed extremely rarely due to the almost complete absence of positive results.

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Microscopy of mycobacteria

Sputum microscopy is a relatively quick, simple and inexpensive method that should be used in all cases where tuberculosis is suspected. In addition, this study is performed to assess the effectiveness of chemotherapy and to confirm recovery or treatment failure in the absence of culture results.

Two methods of microscopic examination are used:

• direct microscopy method, when a smear is prepared directly from the diagnostic material;
• a method of microscopy of sediment prepared from material treated with decontaminants for cultural research.

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

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

In order to detect Mycobacterium tuberculosis with a 50% probability by microscopy, 1 ml of sputum must contain more than 5,000 microbial cells. Sputum from patients with pulmonary forms of tuberculosis usually contains a significant number of acid-fast bacteria, which allows them to be reliably detected by bacterioscopy. The diagnostic sensitivity of this method can be increased by examining several sputum samples from one patient. A negative bacterioscopic examination result does not exclude the diagnosis of tuberculosis, since the sputum of some patients contains fewer Mycobacterium than can be detected by microscopy. Poor preparation of sputum smears can also be the cause of a negative bacterioscopic examination result.

The most common method for detecting acid-fast mycobacteria in a smear is Ziehl-Neelsen staining. The method is based on the penetration of carbol fuchsin into a microbial cell through a membrane that includes a wax-lipid layer, with the simultaneous effect of heating and the strong etching action of phenol. Subsequent decolorization of the smear with a 25% solution of sulfuric acid or 3% hydrochloric alcohol leads to the decolorization of all non-acid-fast structures. The decolorized elements of the smear are stained with a 0.3% solution of methylene blue. Mycobacteria do not perceive conventional aniline dyes, as a result of which acid-fast mycobacteria are stained raspberry-red, and other microbes and cellular elements are stained blue.

To examine smears stained according to Ziehl-Neelsen, use a light binocular microscope with an immersion objective (90- or 100-fold magnification) and an eyepiece with 7- or 10-fold magnification. 100 fields of view are examined, which is sufficient to detect single mycobacteria in the smear. If the result of such an examination is negative, it is recommended to examine another 200 fields of view for confirmation. The results are recorded, indicating the number of acid-fast mycobacteria (AFB) detected.

In addition to this method, fluorochrome staining is used for luminescent microscopy, which allows achieving the best results. The use of this method increases the efficiency of microscopy by 10-15%. When mycobacteria are treated with luminescent dyes (auramine, rhodamine, etc.), these substances also bind to the wax-like structures of the microbial cell. When stained cells are irradiated with an exciting light source (a certain spectrum of ultraviolet radiation), they begin to glow orange or bright red against 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 by 4-10 times, which expands the field of view and reduces the viewing time of the preparation. Along with this, due to the significantly greater depth of field, the comfort of the study can be increased.

When using fluorescence microscopy, viewing the same area of a smear takes significantly less time than light microscopy of smears stained according to Ziehl-Neelsen. If a microscopist views approximately 20-25 such smears during a working day, then with the help of fluorescence microscopy he can examine more than 60-80 samples in the same time. Experienced microscopists know that staining cells with a mixture of auramine and rhodamine is in some way specific for acid-fast mycobacteria, which in this case have the appearance of golden rods. Saprophytes are stained greenish.

Another important advantage of the fluorescence microscopy method is the ability to detect altered mycobacteria that have lost their acid-resistant properties under the influence of a number of unfavorable factors, in particular intensive chemotherapy, and are therefore not detected by Ziehl-Neelsen staining.

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

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

It should be noted that microscopic examination of a smear of pathological material does not allow one to determine the species of the detected acid-resistant mycobacteria. The microscopic method allows one to draw a conclusion only about the presence or absence of acid-resistant microorganisms in the preparation, which is explained by the existence in nature of a large number of non-tuberculous acid-resistant microorganisms morphologically similar to mycobacteria of the tuberculosis complex.

The evaluation of microscopy results is performed in semi-quantitative units.

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

Features of microscopy in extrapulmonary tuberculosis

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

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Culture method for diagnosing tuberculosis

The seeding method, or culture method, is more sensitive than smear microscopy and has a number of advantages over the latter. It allows detecting several dozen viable mycobacteria in the material being examined and has a high diagnostic value. This is especially important when examining material from newly diagnosed or treated patients who excrete a small number of mycobacteria.

Compared with microscopy, culture research allows to increase the number of detected tuberculosis patients by more than 15-25%, as well as to verify tuberculosis at earlier stages, when the disease is still easily treatable. A very important advantage of culture research is considered to be the possibility of obtaining a pathogen culture, which can be identified and studied in relation to drug sensitivity, virulence and other biological properties.

The disadvantages of cultivation methods include their duration (the waiting period for materials reaches 10 weeks), higher cost, and the complexity of processing diagnostic material.

Principles of pre-sowing treatment of diagnostic material

Conventional microbiological methods cannot be used to conduct tuberculosis tests. This is due to the fact that tuberculosis mycobacteria grow very slowly, and most clinical samples contain fast-growing pyogenic and putrefactive microorganisms and fungi. Their rapid growth on rich nutrient media interferes with the development of mycobacteria and does not allow the tuberculosis pathogen to be isolated, so the diagnostic material must be pre-treated before sowing. In addition, mycobacteria released from the patient's respiratory tract are usually surrounded by a large amount of mucus, which makes it difficult to concentrate them. In this regard, before sowing sputum and other similar materials, they must be liquefied and decontaminated.

All detergents and decontaminants have a more or less pronounced toxic effect on mycobacteria. As a result of treatment, up to 90% of mycobacteria may die. In order to preserve a sufficient portion of the mycobacterial population, it is necessary to use gentle treatment methods that allow, on the one hand, to suppress fast-growing pyogenic and putrefactive microorganisms, and on the other hand, to maximally preserve the viability of mycobacteria present in the material.

Depending on the material, its homogeneity and contamination level, various decontaminants are used for pre-seeding treatment: for sputum - 4% sodium hydroxide solution, 10% trisodium phosphate solutions, 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 and surfactants (detergents) are used. The use of Tween and some other detergents is accompanied by lesser death of mycobacterial cells (40-50% survive). However, they can only be used for liquid materials. NALC-NaOH, produced in kits, is the most widely used in the world. This method allows to isolate more than 85% of the mycobacterial cell population. Decontamination of tissue-containing solid materials is more difficult, since it is difficult to guess the degree of dispersion of the material during homogenization. For example, processing of lymph node biopsies is often accompanied by an increased frequency of contamination with foreign flora. In this case, 1% etonium can be used.

Non-homogeneous material is homogenized using glass beads in the presence of decontaminants. Liquid materials are pre-centrifuged and only the sediment is processed.

Technique of sowing and incubation

After preliminary processing, the material is centrifuged, which precipitates the mycobacteria and increases their content in the sediment ("sediment enrichment"). The resulting sediment is neutralized and inoculated onto the surface of dense nutrient media or test tubes with liquid (semi-liquid) media. Smears for microscopic examination are prepared from the remaining sediment. The seeding technique must prevent cross-contamination of the diagnostic material.

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

The inoculated tubes are placed in a thermostat at 37 ^o C for 2 days in a horizontal position. This ensures a more uniform absorption of the material into the nutrient medium. After 2 days, the tubes are moved to a vertical position and hermetically sealed with rubber or silicone stoppers to prevent the seeded media from drying out.

The crops are kept in a thermostat at 37 ^° C for 10-12 weeks with regular weekly inspection. The following parameters are recorded at each control inspection:

• period of visually observable growth from the day of sowing;
• growth rate (number of CFU);
• contamination of the culture with foreign microbial flora or fungi (such test tubes are removed);
• no visible growth. The tubes are left in the thermostat until the next inspection.

Nutrient media

Various nutrient media are used to cultivate mycobacteria: solid, semi-liquid, liquid. However, none of the known nutrient media has properties that ensure the growth of all mycobacterial cells. In this regard, to improve the efficiency, it is recommended to use 2-3 nutrient media of different compositions simultaneously.

As a standard medium for the primary isolation of the tuberculosis pathogen and determination of its drug sensitivity, the WHO recommends the Lowenstein-Jensen medium. This is a dense egg medium on which mycobacteria growth is obtained on the 20th-25th day after sowing bacterioscopically positive material. Sowing bacterioscopically negative material requires a longer incubation period (up to 10-12 weeks).

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

To increase the efficiency of bacteriological diagnostics of extrapulmonary tuberculosis, it is advisable to include modified Finn-II media in the complex of nutrient media. To accelerate growth, 0.05% sodium thioglycolate is additionally introduced into the Finn-II nutrient medium, which reduces the oxygen concentration. To protect the enzyme systems of mycobacteria from toxic products of lipid peroxidation, the antioxidant α-tocopherol acetate is introduced into the Finn-II nutrient medium at a concentration of 0.001 μg/ml. The diagnostic material is seeded using the standard method.

In anti-tuberculosis laboratories in Russia, other modifications of dense nutrient media are also used: the nutrient medium "Novaya" proposed by G.G. Mordovsky, the nutrient media A-6 and A-9 developed by V.A. Anikin, etc.

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

Evaluation and recording of the results of diagnostic material culture

Some strains and types of mycobacteria grow slowly, growth may appear even by the 90th day. The number of such cultures is small, but this forces the sowings to be kept in a thermostat for 2.5-3 months.

Virulent cultures of Mycobacterium tuberculosis usually grow on solid egg media as R-form colonies of varying size and appearance. The colonies are dry, wrinkled, ivory-colored, and slightly pigmented. On other media, Mycobacterium tuberculosis colonies may be more moist. After a course of chemotherapy or during treatment, smooth colonies with moist growth (S-forms) may be isolated.

When isolating cultures, a set of special studies is used to distinguish tuberculosis mycobacteria from non-tuberculous mycobacteria and acid-fast saprophytes.

A positive answer is given after a mandatory microscopic examination of a smear from the grown colonies stained according to Ziehl-Neelsen. In the case of mycobacteria growth, bright red rods are found in smears, lying singly or in groups, forming clusters in the form of felt or braids. In young cultures, especially those isolated from patients treated with chemotherapy for a long time, mycobacteria are distinguished by pronounced polymorphism, up to the presence of short, almost coccoid or elongated variants resembling fungal mycelium, along with rod-shaped forms.

The intensity of mycobacterial growth is designated according to the following scheme: (+) - 1-20 CFU in a test tube (low bacterial excretion); (++) - 20-100 CFU in a test tube (moderate bacterial excretion); (+++) - >100 CFU in a test tube (abundant bacterial excretion). In laboratory diagnostics of tuberculosis, it is not enough to give an answer indicating whether or not mycobacteria have been detected by a particular method. It is also necessary to have a detailed idea of the volume and nature of the mycobacterial population, its composition and properties. It is these data that allow one to correctly interpret the state of the process, plan tactics and promptly adjust treatment.

In recent years, agar-based nutrient media with various growth additives and the use of a special gas mixture have been proposed to accelerate the growth of mycobacteria. To obtain the growth of mycobacteria on these media, an atmosphere with an increased content of carbon dioxide (4-7%) is created during cultivation. For this purpose, special CO2 incubators are used _. However, automated mycobacteria cultivation systems have received the greatest development: MGIT-BACTEC-960 and MB/Bact.

One of such systems is the MGIT (mycobacteria growth indicating tube) system, which is a high-tech development and is designed for accelerated bacteriological diagnostics of tuberculosis and determination of mycobacteria sensitivity to first-line drugs and some second-line drugs. MGIT is designed for use as part of the VASTEC-960 device. Microorganisms are cultivated in special test tubes with a liquid nutrient medium based on a modified Middlebrook-7H9 medium. To stimulate the growth of mycobacteria and suppress the growth of foreign microflora, MGIT Growth Supplement and a mixture of PANTA antibacterial drugs are used.

Microorganism growth is registered optically. It is based on fluorescence, which occurs when mycobacteria consume oxygen during their growth. An oxygen-dependent fluorochrome dye is contained at the bottom of a special test tube and covered with a layer of silicone. Mycobacteria reproduction leads to a decrease in the amount of oxygen in the test tube and a decrease in its concentration, which causes an increase in fluorescence, which becomes visible when the test tube is irradiated with ultraviolet light and is automatically registered by photosensors built into the VASTES-960 device. The luminescence intensity is registered in growth units (GU). Growth data are automatically entered into a computer, where they can be saved. Computer analysis of growth curves can provide information on the presence of various pools of mycobacteria, including non-tuberculous ones, and also helps to evaluate the growth properties of mycobacteria.

As a result of the introduction of such systems, the time of mycobacteria growth has been significantly reduced, averaging 11 days on VASTEC-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. Sowing of material on liquid media is necessarily accompanied by sowing on the Lowenstein-Jensen medium, which plays the role of a backup in cases where tuberculosis mycobacteria do not grow on other media.

[ 38 ], [ 39 ], [ 40 ], [ 41 ], [ 42 ], [ 43 ]

Determination of drug susceptibility of mycobacteria

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 assessment of the spread of drug-resistant tuberculosis. In addition, monitoring drug resistance allows us to assess the effectiveness of the anti-tuberculosis program as a whole, being an integral indicator of the work of all components of anti-tuberculosis measures.

Frequency and timing of drug susceptibility testing:

• before the start of treatment, once to determine the treatment strategy and tactics:
• When isolating cultures from various materials from a patient (sputum, BAL, urine, exudates, cerebrospinal fluid, etc.), all 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 event of:
  • absence of sputum negativity;
  • re-culture after sputum negativity;
  • a sharp increase in the amount of AFB in a smear after an initial decrease. It is well known that strains of Mycobacterium tuberculosis with different drug sensitivity are isolated from material from a patient with tuberculosis. The sensitivity of strains to anti-tuberculosis drugs may differ in the spectrum of drugs, degree, frequency and speed of resistance development.

The degree of drug resistance of Mycobacterium tuberculosis is determined in accordance with established criteria, which are focused on the clinical significance of resistance and depend on the anti-tuberculosis activity of the drug, its pharmacokinetics, concentration in the lesion, the maximum therapeutic dose, etc.

Determination of drug susceptibility of mycobacteria is currently carried out using microbiological methods:

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

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

The absolute concentration method recommended by the WHO Chemotherapy Committee has become widespread in Russia as a unified method. From a methodological point of view, it is the simplest, but requires high standardization and precision of laboratory procedures. The drug susceptibility test consists of a set of test tubes with a nutrient medium modified with anti-tuberculosis drugs. The set consists of 2-3 test tubes with different concentrations of each of the drugs used, one control test tube with a medium without the drug, and one test tube containing 1000 μg/ml sodium salicylate or 500 μg/ml paranitrobenzoic acid to detect the growth of non-tuberculous mycobacteria.

To prepare a set of media with preparations, use a modified Lowenstein-Jensen medium (without starch), which is poured into flasks. A certain volume of the corresponding dilution of the anti-tuberculosis drug is added to each of the flasks. The contents of the flasks are thoroughly mixed, poured into test tubes and coagulated in an inclined position for 40 minutes at a temperature of 85 ° C. It is recommended to coagulate the medium in an electric coagulator with automatic temperature control. Medium with anti-tuberculosis drugs

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

The principle of the method is to determine the concentration of an anti-tuberculosis drug that suppresses the growth of a significant portion of the mycobacteria population. When performed correctly, this method has good reliability.

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

Since the time it takes to isolate the pathogen from clinical material on nutrient media is at least 1-1.5 months, the results of determining drug susceptibility using this method can be obtained no earlier than 2-2.5 months after seeding the material. This is one of the main disadvantages of the method.

The results of mycobacterial drug susceptibility testing are interpreted based on certain criteria. On solid media, a culture is considered sensitive to the concentration of the drug contained in the medium if the number of mycobacterial colonies grown on a given test tube with the drug does not exceed 20 with abundant growth on a control test tube without drugs. Only if there are more than 20 colonies is the culture considered resistant to a given concentration. In practice, when growth results in test tubes close to 20 CFU are obtained, it is necessary to notify the clinical unit that the sensitivity or resistance in this case is borderline, since this can sometimes explain the unclear dynamics of clinical indicators.

For various preparations, a certain concentration is established at which the reproduction of a critical proportion of the mycobacterial population is observed. These concentrations are called "critical". The magnitude of the growth of the mycobacterial population on a nutrient medium with the preparation in a critical concentration is used as a criterion for stability.

In domestic phthisiology practice, when determining 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 chemotherapy tactics, using knowledge of the potentiating effect of drug combinations, to anticipate cross-resistance or to use more effective drugs of the used group of anti-tuberculosis drugs.

The absolute concentration method is the simplest, but also the most sensitive to errors made in its implementation. More reliable, especially when determining sensitivity to second-line drugs, and widespread outside Russia is the proportion method. It takes into account the shortcomings of the absolute concentration method, but it is more labor-intensive to implement.

The method is very similar to the absolute concentration method. Preparation of test tubes with drugs is the same as in the absolute concentration method. However, the seed dose of the tuberculosis mycobacterium suspension is reduced by 10 times, which eliminates the frequency of spontaneous resistance of some tuberculosis mycobacterium strains to drugs such as Ethambutol, prothionamide, capreomycin. As controls, 2 or 3 tubes with a seed dose equal to that in the test tubes, successively diluted 10 and 100 times, are used. The criterion for resistance is the proportion of visually observed growth of tuberculosis mycobacterium. For 1st-line drugs, the criterion for resistance is excess growth of 1% of the initial population, for 2nd-line drugs - growth of 1 or more than 10% of the initial, depending on the selected critical concentration.

In 1997, the WHO and International Union Against Tuberculosis working group on the detection of anti-tuberculosis drug resistance made adjustments to these criteria, proposing to consider mycobacteria growing on the dense Lowenstein-Jensen egg medium at the following concentrations as resistant:

• dihydrostreptomycin - 4 μg/ml;
• isoniazid - 0.2 µg/ml:
• rifampicin - 40 mcg/ml:
• Ethambutol - 2 mcg/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 μg/ml;
• cycloserine - 40 mcg/ml;
• aminosalicylic acid - 0.5 mcg/ml;
• ofloxacin - 2 mcg/ml.

The growth results are assessed after 4 weeks as preliminary and after 6 weeks of cultivation as final.

For determining drug susceptibility to pyrazinamide, which is widely used in modern tuberculosis chemotherapy, the recommended critical concentration is 200 μg/ml. However, there is still no generally accepted method for determining drug resistance to this drug on solid nutrient media, since its antibacterial activity is manifested only in an acidic environment (pH <6), which is technically difficult to maintain. In addition, many clinical cultures of Mycobacterium tuberculosis are reluctant to grow on egg media with an acidic environment.

In order to assess the quality of the results of determining the drug susceptibility of mycobacteria, it is recommended to control each new batch of the Lowenstein-Jensen medium by parallel determination of the susceptibility of the standard museum strain H37Rv. In addition, there are certain microbiological criteria that must be met so that the methods give a well-reproducible and correctly interpreted result. These include the viability of the tuberculosis mycobacteria culture, the rules for obtaining a homogeneous suspension and suspension, the rules for selecting tuberculosis mycobacteria cultures, and the representativeness of the selected bacterial mass. The reliability of determining drug resistance decreases with extremely poor bacterial excretion.

Recently, the method of determining drug susceptibility using automated systems has been recognized as promising. The most advanced in this area are developments based on VASTEC MGIT-960. In this case, drug susceptibility of tuberculosis mycobacteria is determined based on a modified proportion method. During the determination, the growth rate of tuberculosis mycobacteria in the control tube and in tubes with drugs is compared. To determine susceptibility to streptomycin, isoniazid, rifampicin and ethambutol, enriching additives and antibiotics included in the SIRE kit are used. To determine susceptibility to pyrazinamide, the PZA kit is used. During the test, test tubes with drugs are inoculated with a suspension of tuberculosis mycobacteria, as well as control tubes with a 100-fold dilution of the suspension for all drugs, with the exception of pyrazinamide, where the suspension dilution is 10 times. The criterion for stability is the mycobacteria growth indicator of 100 GU when the growth in the control tube reaches 400 GU (see "Cultural methods for isolating mycobacteria"). The results are recorded and interpreted automatically and are set by the entered or selected program.

The final concentrations in the test tube with liquid nutrient medium are used as critical concentrations. Currently, critical concentrations have been developed for both first-line drugs and some second-line drugs. It should be noted that the determination of the sensitivity of tuberculosis mycobacteria to cycloserine and aminosalicylic acid is performed only on egg nutrient media.

A detailed protocol for working with the described system allows for drug susceptibility testing both on an isolated culture (with a dense nutrient medium) and using the primary growth of mycobacteria in an MGIT test tube. The latter option significantly reduces the time required for conducting cultural studies, allowing for full results on the culture of tuberculosis mycobacteria (including information on drug susceptibility) to be obtained within 3 weeks of collecting the material, while the traditional method can only provide this by the 3rd month. Timely results, when the patient is in the intensive phase of treatment, can compensate for the relative high cost of the studies.

[ 44 ], [ 45 ], [ 46 ], [ 47 ], [ 48 ], [ 49 ], [ 50 ]

Differentiation of mycobacteria

Considering that the nutrient media used are not strictly selective, subsequent differentiation of the isolated mycobacteria is considered mandatory. The need for differentiation of mycobacteria is due to a number of features of pathological processes caused by representatives of the genus: 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 the M. tuberculosis complex from non-tuberculous mycobacteria is carried out according to the following characteristics: growth rate on dense nutrient media, pigment formation, colony morphology, the presence of acid resistance and the temperature optimum for growth.

Unfortunately, there is no single laboratory method that can reliably distinguish mycobacteria of the M. tuberculosis complex from other acid-fast mycobacteria; however, a combination of the above-described signs with the results of a number of biochemical tests given below allows for the identification of mycobacteria of the M. tuberculosis complex with a probability of up to 95%.

To differentiate mycobacteria of the M. tuberculosis complex (M. tuberculosis, M. bovis, M. bovisBCG, M. africanum, M. microti, M. canettii and others) from slowly growing non-tuberculous mycobacteria, basic biochemical tests are used to detect the presence of the following signs:

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

As an additional test, growth tests on a medium containing 500 μg/ml para-nitrobenzoic acid or 5% sodium chloride can also be used.

Many bacteriological laboratories identify these microorganisms only at the complex level, 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 to differentiate M. tuberculosis and M. bovis: niacin, nitrate reductase, pyrazinamidase, and growth registration on a medium containing 2 μg/ml thiophene-2-carboxylic acid hydrazide. It is taken into account that mycobacteria of the M. tuberculosis complex are characterized by the following set of features:

• slow growth (more than 3 weeks);
• growth temperature within 35-37 ^o C;
• absence of pigmentation (ivory color);
• pronounced acid-fast coloration;
• positive niacin test;
• positive nitrate reductase test;
• absence of thermostable catalase (68 ^o C).
• lack of growth on Lowenstein-Jensen medium containing:
  • 1000 µg/ml sodium salicylic acid,
  • 500 mcg/ml para-nitrobenzoic 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 significantly with the growth of the frequency of registration of 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.

[ 51 ], [ 52 ], [ 53 ], [ 54 ], [ 55 ], [ 56 ], [ 57 ], [ 58 ]

Immunological diagnostics of tuberculosis

There are a number of universal phenomena, preparations and immunological tests that were initially discovered specifically in tuberculosis or in the model of immune response to mycobacteria. These include BCG and tuberculin, such a phenomenon as skin DST (tuberculin tests - Pirquet and Mantoux reactions), the reaction to subcutaneous administration of tuberculin to sensitized animals (Koch phenomenon). Some of the first antibodies in infectious diseases were also discovered in tuberculosis. Of course, the deeper the understanding of the mechanisms of anti-tuberculosis immunity and their genetic control, the wider the use of immunological methods and preparations affecting immunity can be for solving practical problems of phthisiology.

The most important and complex practical problem at present is considered to be the detection of tuberculosis in the process of mass screening of the population. However, despite numerous reports of "successes" (on limited material), there is no immunological method (reproducible in "any hands") or drug suitable for these purposes.

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

Serological methods, which determine antigens and antibodies in different environments of the body, are in first place among immunological studies used in differential diagnostics.

The specificity of the determination of antibodies to mycobacteria tuberculosis depends on the antigens used in the immune analysis. A significant number of antigens have been proposed, the first of which is tuberculin PPD:

• PPD and other complex preparations from culture fluid;
• ultrasonic disintegrant;
• Triton extract and other complex cell wall preparations;
• 5-antigen (Daniel);
• 60-antigen (Coccito);
• lipoarabinomannan;
• cord factor (trehalose-6,6-di-mycolate);
• phenolic and other glycolipids;
• lipopolysaccharides;
• fibronectin-binding antigen;
• proteins (most often recombinant); 81,65,38,34,30,19,18,16,15.12 KDA, 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 diagnostics of tuberculosis were identified: the more complex the antigen, the higher the sensitivity and the lower the specificity of the tests. Specificity varies in different countries depending on the infection of the population with M. tuberculosis and non-tuberculous mycobacteria, on the BCG vaccination, etc. In children, the informativeness of serodiagnostics is lower than in adults. In primary tuberculosis (more often children), the determination of IgM is more informative; in secondary tuberculosis - IgG. In HIV-infected people, the informativeness of serodiagnostics in determining antibodies is reduced. The effectiveness of antibody determination depends on a number of "clinical moments": the activity of the process (the presence or absence of "isolation" 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 enzyme immunoassay (EIA) method is about 70%. The insufficient effectiveness of the study is due to its low specificity. Previously, the possibilities of using serological screening in high-risk groups were considered, in particular among people with post-tuberculosis changes in the lungs.

To increase the specificity of ELISA, more specific antigens are being sought, including those obtained by genetic engineering: ESAT-6, etc. (see above). The use of strictly specific antigens (38 kDa, ESAT) increases specificity, but significantly reduces the sensitivity of the analysis. Along with ELISA (experimental laboratory test systems, such as Pathozyme ELISA kit), immunochromatographic kits with lateral filtration (Mycodot) are also offered, as well as other similar tests (membrane dot analysis) with visual assessment of the test result. When carrying out these tests, the analysis takes 10-30 minutes; they do not require special equipment, they require visual assessment 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 traditional ELISA.

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

Induction of gamma interferon synthesis in vitro

Gamma interferon (IFN-γ) is a factor of specific immune protection, realized by activating the enzyme systems of macrophages. Induction of IFN-γ synthesis by sensitized T-lymphocytes is caused by their interaction with mycobacterial antigens.

Both tuberculin PPD and specific antigens obtained by genetic engineering are used as antigens, in particular ESAT-6 antigen (early secreted antigen with a molecular weight of 6 kDa) and CFP-10 (culture filtrate protein, 10 kDa). Genetically engineered or recombinant antigens are absent in the cells of the BCG vaccine and other mycobacteria. When using tuberculin, the results of the IFN-γ induction test 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 previous BCG vaccination. When examining vaccinated individuals who have not had contact with tuberculosis infection, the specificity of the test is 99%. The sensitivity of the test among tuberculosis patients ranges from 81 to 89%.

Tests and diagnostics have been developed based on short-term cultivation of whole blood cells or mononuclear cells isolated from blood with tuberculosis mycobacteria antigens in vitro, followed by determination of the IFN-γ concentration or counting the number of T-lymphocytes synthesizing IFN-γ. The concentration of interferon synthesized in a test tube is determined by ELISA using monoclonal antibodies that bind IFN-γ. Then, using calibration of standard IFN-γ, its concentration in the test tube or plate wells is determined.

In the Elispot test, the number of T cells synthesizing IFN-γ is counted on the surface of a dish coated with antibodies to IFN-γ.

The developers of the in vitro IFN-γ induction diagnostic, which is approved by the US Food and Drug Administration, claim that the test cannot differentiate latent tuberculosis infection from active tuberculosis. Therefore, in regions with a high infection rate, the test has no direct diagnostic value. However, in our country, it can be used to differentiate tuberculosis infection in children from post-vaccination allergy, as well as to assess the level of specific immunity during treatment.

Currently, a domestic test system for determining the induction of IFN-γ synthesis by specific tuberculosis antigens in vitro is undergoing study.

Immune status and course of tuberculosis, immunocorrection

During the treatment of tuberculosis, changes in antigenemia and the state of the immune system occur in people.

The data on changes in exudates and tissues are largely contradictory. The only thing that can be noted with full justification is that tuberculous granulomas, as a rule, contain a significant number of activated T-lymphocytes.

It makes sense to dwell on two more points 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 developing multiple drug resistance;
• In case of multiple drug resistance (and in the absence of HIV infection), immune disorders (primarily T-cell immunity) are particularly significant.

In tuberculosis, various methods of immunocorrection are widely used: first of all, these are drugs that act mainly on T-cell immunity and the mononuclear phagocyte system (thymus hormones, isophone, licopid, polyoxidonium, etc.), as well as whole (attenuated) mycobacteria and their components.

Molecular biological diagnostics of tuberculosis

Molecular biology methods in infectious disease diagnostics mainly include methods based on manipulation of genomic materials of bacterial and viral pathogens in order to identify specific genetic material - DNA sections with a nucleotide sequence specific to a given species or strain of pathogen, for analyzing specific DNA sequences in genes that determine the sensitivity of the pathogen to certain drugs, as well as for analyzing the functional activity of certain genes of the pathogen. Molecular biological methods have become widespread in scientific research and practical application in diagnostics and monitoring of various bacterial and viral infections after the discovery of the polymerase chain reaction in 1985 by Carrie Mullis (Nobel Prize laureate. 1989).

Principles and capabilities of the polymerase chain reaction method

PCR allows amplification (multiplication) of a nucleotide sequence (a fragment of pathogen DNA) in a test tube in a few hours by millions of times. Conducting the reaction in the presence of single DNA chains determines the exceptionally high sensitivity of the analysis.

The nucleotide sequence of certain sections of the DNA chain determines the genetic uniqueness of the microorganism, which explains the high specificity of PCR.

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

The principle of the PCR method is amplification - multiple, millions of times multiplication of sections of a specific DNA sequence in a test tube microvolume with cyclic repetition of the following three reaction stages, each of which takes place in a different temperature regime:

• Stage I - denaturation of double-stranded DNA upon heating with divergence of its chains;
• Stage II - complementary binding (hybridization) of primers (priming oligonucleotides) with the end sections of the chains of a strictly specific DNA fragment selected for amplification;
• Stage III – completion of the DNA fragment chain using thermostable DNA polymerase.

For amplification, the test tube must contain molecules of matrix DNA. Four types of deoxynucleoside triphosphates (nucleotides) containing the corresponding nitrogenous bases: adenine (A), thymine (T), guanine (G), cytosine (C); artificially synthesized priming oligonucleotides (primers) consisting of 18-20 base pairs; a thermostable enzyme, DNA polymerase, with a temperature optimum of 68-72 ^° C, and magnesium ions.

The specificity of PCR depends on the choice of DNA fragment. In accordance with it, flanking primer oligonucleotides are synthesized. The specificity of hybridization and completion of the DNA chain is determined by the principle of complementarity of the following pairs of nitrogenous bases: adenine-thymine, guanine-cytosine.

To determine the genome of the tuberculosis complex mycobacteria, the most effective amplification target in most test systems is the IS6110 DNA fragment, which in most tuberculosis mycobacteria strains has a significant number (10-20) of repeats in the genome, which ensures, along with specificity, high sensitivity of the analysis. At the same time, tuberculosis mycobacteria strains with a small number of repeats or the absence of the IS6110 fragment have been described.

Extraction of DNA molecules from a biological sample

To perform PCR, the DNA molecules of the pathogen must be isolated from the biological material in a minimal volume, with a minimal amount of non-specific DNA and various inhibitors of the enzyme - DNA polymerase.

Sample preparation must be carried out under conditions that prevent cross-contamination of the samples being studied with isolated DNA molecules. This requires preliminary treatment of the room with ultraviolet light, floors and work surfaces of tables and devices - with chlorine-containing solutions. It is also necessary to use clean gloves, disposable test tubes and tips for automatic pipettes.

To isolate the DNA of Mycobacterium tuberculosis from clinical samples (cerebrospinal fluid, bronchial lavage) that do not contain a large number of leukocytes, cellular debris or salts, it is sufficient to centrifuge the sample at 3-4 thousand revolutions per minute, add 20-30 µl of a 2% solution of Triton X-100 to the sediment and heat at 90 ^o C for 30 minutes.

Sputum sample preparation requires efficient liquefaction, typically using 4% sodium hydroxide and N-acetyl-L-cysteine (NALC) at 50-80 mg per sample, depending on sample viscosity. The NALC solution should be prepared ex tempore, or NALC powder can be added dry directly to the sample. After liquefaction, samples should be centrifuged for 15 min at 3,500-4,000 rpm (3,000 g) in 50 ml screw-cap tubes, i.e. under the same conditions recommended for pre-culture sputum preparation.

To extract DNA from sediment, a method based on the use of a 5-6 molar solution of guanidine isothiocyanate as a lysing reagent and microporous silicon oxide particles ("diatomaceous earth") that sorb DNA molecules is most often used. Non-specific substances, including possible inhibitors, are then washed in a 2.5 molar solution of guanidine isothiocyanate and an ethanol solution, after which the DNA molecules are desorbed in water, and these samples are used to perform 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 to precipitate the particles instead of centrifugation.

An original method of immunomagnetic separation of mycobacteria with subsequent extraction of pathogen DNA has been developed in Russia. For immunomagnetic separation of tuberculosis mycobacteria, ferroparticles 3-5 μm in size, coated with silicon oxide, are used, to which polyclonal (rabbit) antibodies to tuberculosis mycobacteria are attached by means of a chemical bond. Sputum samples after alkaline lysis are neutralized with an acidic Tris-HCl solution and incubated with an immunomagnetic sorbent. Then the immunoferroparticles are collected using a magnetic rod with a replaceable tip, transferred to a microtube, and precipitated. 20-30 μl of a 2% Triton X-100 solution are added and heated for 30 minutes at 90 ^o C. The supernatant is used as a DNA matrix for PCR analysis.

A difficult problem is the extraction of tuberculosis mycobacterium DNA from biopsy specimens. For biopsy lysis, the enzyme proteinase K is used at a final concentration of 200-500 mg/l at a temperature of 56 ^o C overnight. Then, it is extracted using one of the known methods. Excess non-specific DNA in PCR analysis of biopsy specimens often causes inhibition of the reaction, which requires repeated DNA extraction.

Results detection methods

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

The gel electrophoresis method is well known. In this case, the obtained DNA fragment is identified by a positive control containing the desired specific DNA fragment, or by a previously 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, which is included in double-stranded DNA, the synthesized DNA fragment is revealed as a band that glows under the influence of ultraviolet light.

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

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

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 samples after the enzymatic reaction has occurred.

The disadvantages of these methods include the possibility of intralaboratory contamination with fairly short fragments of DNA molecules. When these molecules enter newly tested samples, they become a matrix for PCR and lead to false positive results.

In this regard, to prevent false positive results, strict rules are introduced for separating and isolating rooms: for DNA extraction from biological samples; rooms for detection of results (electrophoresis) from the clean zone. These rooms represent a zone of probable contamination. Another isolated zone is a clean room for introducing the DNA samples under study into test tubes with the reaction mixture for PCR. And finally, it is assumed that the main device - the DNA amplifier - should be taken out to a separate, possibly office, room.

To prevent contamination by the products of previous reactions - amplicons, some PCR test systems contain deoxynucleoside uridine instead of deoxynucleoside thymidine, which is built in the corresponding position during in vitro chain synthesis, i.e. the nitrogenous base thymine present in native DNA is replaced by uracil. Uracil DNA glycosylase added to the reaction mixture of the analyzed material destroys only contaminating fragments with deoxyuridine, but not the native analyzed DNA containing deoxythymidine. Subsequent heating at 94 ^o 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 reverse transcription and synthesis of DNA molecules are first performed. which in turn are the matrix for subsequent synthesis of RNA molecules. RNA amplicons are detected using an acridine-stained DNA probe during hybridization in a reaction tube solution. This method, in addition to high sensitivity, has the advantage of conducting the analysis in one tube, which prevents contamination. According to the authors, the 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 test tube. This not only technologically simplifies the analysis method, but also prevents contamination of laboratory premises and samples by products of previous PCR.

In real-time PCR, results are detected by fluorescence arising from 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 an enzymatic reaction or distances itself from the fluorescence quencher molecule only upon specific hybridization with the desired DNA molecule amplified during PCR. As the number of molecules hybridized with the probe increases, the increase in fluorescence to a detectable level is proportional to the number of molecules of the amplified product. Since the number of DNA fragment molecules is doubled during each PCR cycle, the cycle number from which fluorescence is detected and increases is inversely proportional to the number of DNA molecules in the original sample. If several different known concentrations of molecules of the corresponding fragment of tuberculosis mycobacterium DNA are introduced into the reaction as a calibrator, then the number of DNA genomes in the material being studied can be calculated using a computer program.

Each standard sample is duplicated. The quantitative criterion is the minimum number of PCR cycles required for the onset and growth of detectable fluorescence. The abscissa axis is the number of cycles; the ordinate axis is the fluorescence value. DNA concentrations are inversely proportional to the number of cycles required for fluorescence to appear. The windows in the right column (21-32) show the cycle numbers for the corresponding concentrations. The differences between 10-fold concentrations of DNA fragments 10 ² -10 ⁶ ml are 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 repeats (6-20) of the analyzed fragments in the genome of Mycobacterium tuberculosis, the number of Mycobacterium tuberculosis in the clinical samples is about 100 and 1000 cells, respectively.

Application of PCR in the diagnosis of tuberculosis

The PCR method is used to the greatest extent for the rapid diagnosis of tuberculosis - detection of Mycobacterium tuberculosis in clinical samples: sputum, bronchial washings, pleural exudate, urine, cerebrospinal fluid, osteolysis punctures, aspirates of the female genital tract and various biopsies. In a study in Holland of about 500 samples of sputum and bronchial washings from 340 patients with a confirmed diagnosis of pulmonary tuberculosis, the comparative sensitivity of the PCR, culture and smear microscopy methods was studied. The sensitivity of the analysis was 92.6, 88.9 and 52.4%, respectively. The specificity of all methods was about 99%.

A comparison was made of the efficiency of detection of Mycobacterium tuberculosis using smear microscopy, sowing on the Lowenstein-Jensen medium, the VASTES test system and PCR analysis. PCR demonstrated a sensitivity of 74.4%, microscopy - 33.8%, sowing on a solid medium - 48.9% and VASTES - 55.8%. The average detection time for sowing on the Lowenstein-Jensen medium is 24 days. VASTES - 13 days, PCR - 1 day.

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

Detection of Mycobacterium tuberculosis DNA by the PCR method with effective chemotherapy is determined over a longer period of time - on average 1.7 months compared to bacterial excretion determined by fluorescent microscopy, and 2.5 months compared to bacteriological examination.

Diagnosis of extrapulmonary forms of tuberculosis

The importance of PCR as a sensitive method is especially great for extrapulmonary forms, since it is precisely in these forms that clinical and radiological methods and traditional bacteriological methods for determining Mycobacterium tuberculosis in diagnostic materials are ineffective.

When examining urine samples, the PCR analysis results 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 efficiency of PCR analysis in the study of bone marrow aspirates in patients with fever of unknown genesis with suspected tuberculous nature of the disease was demonstrated. For the diagnosis of tuberculous lymphadenitis in children, 102 puncture aspirates and biopsy samples of 67 children with suspected tuberculous lymphadenitis were studied. Positive results were obtained: by real-time PCR - 71.6%, fluorescence microscopy - 46.3%, culture study - 41.8%. In the study of 50 lymph node biopsies in patients with cat-scratch disease, all results were negative. Thus, 100% specificity of PCR analysis was demonstrated. In the same work, the possibility of detecting M. avium was shown in puncture biopsy of lymph nodes.

Diagnosis of female genital tuberculosis in infertility is known to be one of the most difficult diagnostic problems. Positive results were obtained in PCR studies of endometrial biopsies, endometrial aspirates, and Douglas pouch fluid samples in 14 (56%) of 25 patients examined laparoscopically with suspected tuberculosis. Smear microscopy and culture studies yielded 1 and 2 positive results, respectively. These cases were also PCR-positive. Most PCR-positive results were in cases with characteristic features of tuberculosis according to histological examination; a smaller number were in cases with suspected tuberculosis according to laparoscopy. Only one positive PCR result 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 identifying the pathogen when examining blood samples using the PCR method. Literature data indicate that the detection of Mycobacterium tuberculosis DNA from blood samples is possible in advanced forms of HIV infection. Mycobacterium tuberculosis DNA was detected only in generalized tuberculosis of various organs in patients with a transplanted kidney and immunosuppression.

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Species identification of mycobacteria

The PCR method can be quite effective for rapid identification of mycobacteria of the tuberculosis complex and some types of non-tuberculous mycobacteria after obtaining their primary growth. In this case, the use of PCR can save 7-10 days required for subsequent cultural identification of a positive result. The PCR study is technically very simple, since it does not require complex sample preparation of clinical material to achieve high sensitivity. When examining 80 positive cultures in such a test system (MB BacT. by Organon), all positive PCR analysis results were strictly specific and were carried out within 1 day. To identify other types of mycobacteria when they are obtained in culture, the pathogen DNA is hybridized with specific DNA probes labeled with acridine, and the strains are detected by the appearance of chemiluminescence using a chemiluminometer or on nitrocellulose strips with visual assessment after hybridization. This kit identifies a limited number of species: Mycobacterium tuberculosis complex, M. avium, M. avium complex, M. kansasii, and M. gordonae.

A. Telenti et al. also developed a relatively simple and inexpensive method for species identification of clinically important mycobacteria based on PCR and subsequent treatment with two restriction enzymes (enzymes that have the ability to cut a DNA molecule at specific points). In this case, a DNA fragment encoding a heat shock protein (65 kDa) is amplified, after which the DNA fragment obtained in PCR, 439 nucleotide pairs in size, is treated separately with two enzymes - Bste II and Нае III. Then, using agarose gel electrophoresis, the two products obtained are analyzed, determining their sizes (number of nucleotide pairs) using a set of standard DNA fragments (molecular DNA markers) from 100 to 1000 nucleotide pairs in length. In each of the defined species (M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M.fortuitum) 2 or 3 DNA fragments of different sizes are found for each restriction enzyme. The combination of the resulting DNA fragments of different sizes allows these species to be differentiated from each other.

A technology for biological DNA microarrays is being developed that will help identify more than 100 species of mycobacteria in a single study.

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

PCR can also be used to identify species within the tuberculosis mycobacterium complex, including differentiation between M. bovis and M. bovis BCG. This is done by analyzing the presence or absence of certain genes in the genomic regions RD1, RD9, and RD10. RD1 is absent in M. bovis BCG, but is present in virulent species, including M. bovis.

Determination of drug susceptibility of Mycobacterium tuberculosis using PCR

The tasks of molecular genetic methods for determining drug sensitivity or resistance of Mycobacterium tuberculosis are reduced to identifying 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 options involve identifying substitutions in nucleotide sequences that, when using DNA probes, lead to the absence or incomplete hybridization on a nitrocellulose membrane using an enzyme conjugate (streptavidin-alkaline phosphatase) - the LIPA-Rif-TB method.

The method of measuring fluorescence in locally fixed DNA probes on microregions complementary to known mutations in PCR-amplified gene regions responsible for drug sensitivity or resistance is called the microbiochips method. The basic algorithm for conducting this study is as follows. After DNA is isolated from a clinical sample or mycobacterial culture, PCR must be performed to amplify the corresponding fragments of the groB gene responsible for drug sensitivity to rifampicin, or the katG and inhA genes encoding mycobacterial proteins responsible for sensitivity to isoniazid. The PCR results are assessed using agarose gel electrophoresis, which confirms the receipt of the corresponding DNA fragments of the desired length. Then, a 2nd round of PCR is performed to introduce a fluorescent label into the DNA. The PCR results are again confirmed by gel electrophoresis. After this, hybridization is carried out (incubation overnight) with subsequent washing of the obtained material on a biochip, which is a large number of short DNA chains (probes) fixed on a small glass plate, complementary to the nucleotide sequences of the drug-sensitive type of tuberculosis mycobacteria at the points of possible mutations. as well as to mutant sequences responsible for drug resistance. The location of the DNA probes on the plate is strictly defined, and the level of fluorescence observed during hybridization for determining the result using a special reading device is established. In this regard, the results of the analysis are determined using a special computer program.

In recent years, alternative methods for determining the drug sensitivity of Mycobacterium tuberculosis have been developed based on real-time PCR technology, allowing these studies to be carried out in a closed test tube mode.

Figure 13-13 shows the result of the analysis of clinical cultures of Mycobacterium tuberculosis in determining drug resistance to rifampicin using real-time PCR: 218 - control sample (sensitive to rifampicin); 93 - positive control for the Ser-Trp TCG-TGG mutation; 4482 - positive control for the Ser-Leu TCG-TTG mutation; 162-322 - experimental samples. The result of calculating the kinetic curves of amplification for 4 channels: channel 1: 393 - positive control for the Ser-Trp TCG-TGG mutation; channel 2: 4482 - positive control for the Ser-Leu TCG-TTG mutation; 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: The analysis revealed the following mutations that determine resistance to rifampicin: in samples 162,163,172,295 - Ser-Leu TCG-TTG. The same principle was used to determine drug resistance to isoniazid by the katG and inhA genes, which determine the most frequent mutations.

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Strain identification of Mycobacterium tuberculosis

The most studied method of strain identification of Mycobacterium tuberculosis is a technology called restriction fragment length polymorphism (RFLP) and which is based on fragmentation (restriction) of Mycobacterium tuberculosis DNA by the enzyme Pvu II and subsequent hybridization of the obtained fragments with certain specific sequences on the DNA of its repeat element IS6110. Intraspecific variability is realized due to the different number of IS6110 repeats and their location on the DNA, as well as the diversity of distances between certain points of attack of the restriction enzyme (restriction sites) and the IS6110 element. This technology is very complex and labor-intensive. After treating the DNA isolated from the tuberculosis mycobacterium culture with restriction enzyme, gel electrophoresis is performed, then DNA fragments of different lengths are transferred to a nitrocellulose membrane, hybridized with fragments of the IS6110 element, and the results are detected using an enzymatic reaction. The resulting specific band pattern characterizes the DNA of a specific tuberculosis mycobacterium strain. Computer analysis reveals the identity or relationship of the strains. Despite the fact that the RFLP method is the most discriminatory, i.e. it reveals the greatest number of differences in the analyzed strains, it is ineffective with a small number (less than 5) of IS6110 repeats, observed in some strains. Figures 13-14 show the results of RFLP typing of the strains.

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

Obviously, a more effective and technologically accessible method is VNTR (abbreviation of English words), or the method of 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 manipulations. Since the number of tandem repeats in different strains and in different loci is different, fragments of different sizes are determined and analyzed on the resulting electrophoregram of PCR products. According to researchers, with the help of VNTR, a greater degree of discrimination of strains is achieved than with the RFLP method.

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

Basic requirements for the quality of molecular biological research

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Main regulatory documents for conducting 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. Guidelines: 1.3.1888-04 "Organization of work during PCR research of material infected with pathogenic biological agents of III-IV pathogenicity groups"; 1.3.1794-03 "Organization of work during PCR research of material infected with microorganisms of I-II pathogenicity groups". 2003; 3.5.5.1034-01 “Disinfection of test material infected with bacteria of pathogenicity groups I-IV when working with the PCR method”, 2001. Appendix 11 to the Instructions on unified methods of microbiological research in the detection, diagnosis and treatment of tuberculosis.

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Staff

Molecular biological studies may be carried out by clinical laboratory diagnostics doctors, bacteriologists, virologists, clinical diagnostic laboratory biologists, as well as specialists with secondary medical education who have undergone specialization and advanced training in the established manner.

Arrangement of laboratory premises

The following laboratory facilities are required:

• Sample processing area - a laboratory adapted for working with infectious agents of pathogenicity groups III-IV, in accordance with Methodological Guidelines 13.1888-04.
• The area for preparing PCR reaction mixtures is a laboratory room that provides protection from internal laboratory contamination - a “clean” area.
• • If electrophoresis or hybridization is used to analyze PCR products, the laboratory room in which the amplified DNA fragments are extracted from the amplification tube and, accordingly, can enter the environment, in accordance with the requirements for PCR laboratories (Methodological Guidelines 1.3.1794-03, Methodological Guidelines 1.3.1888-04) must be completely isolated from the rooms specified in the previous paragraphs. Movement of any personnel, equipment, any materials and objects from the electrophoresis area to the sample processing area and the “clean” area, as well as air transfer through the ventilation system or as a result of drafts, must be excluded. This area is not required for fluorimetric detection of PCR products.
• The room for documentation and processing of results is equipped with computers and the necessary office equipment. This room may contain equipment that ensures 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 processing of sputum are similar to the standard microbiological requirements for working with Mycobacterium tuberculosis.

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Complete set of laboratory equipment for PCR diagnostics

The laboratory kit includes equipment for the following rooms.

• sample preparation room, contains the following equipment: laminar flow hood of protection class II "SP-1.2": solid-state thermostat with a heated lid for Eppendorf test tubes; microcentrifuge at 13,000 rpm; centrifuge ("Vortex"); refrigerator with a temperature range from -20 ^° C to +10 ^° C; variable volume pipettes of the "Proline" series; pump with a trap flask OM-1; pipette rack; work station rack 200x0.5 ml; work station rack 50x1.5 ml; racks 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; pipette rack; workstation rack 200x0.2 ml; racks for storing test tubes 80x1.5 ml; refrigerator with a temperature range from -20 ^° C to +10 ^° C;
• Electrophoresis room: horizontal electrophoresis chamber; power source; transilluminator;
• DNA amplifier or nucleic acid analyzer (real-time PCR) with computer and software; can be placed in any available room. If real-time PCR technology is used, an electrophoresis room is not needed.

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External quality control

To ensure that they obtain objectively reliable results, laboratories must participate in a system of external assessment 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, 3 ampoules with tuberculosis mycobacteria (avirulent strain) at a concentration of 10 ² /ml; 3 ampoules with cells of a similar strain at a concentration of 10 ⁴ /ml; 2 ampoules each with non-tuberculous mycobacteria M. avium-intracellulare and M. kansasii at a concentration of 10 ⁵ /ml.

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

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