Cultural properties of tuberculosis. Microbiology of Mycobacterium tuberculosis
The occurrence and course of tuberculosis depend on the characteristics of its pathogen, the reactivity of the body and sanitary conditions. The current name of the pathogen is Mycobacterium tuberculosis. The old name is Koch bacterium (KB). On March 24, 1882, R. Koch demonstrated a pure culture of the pathogen under a microscope, and he also proved its infectious nature by infecting animals. That's why the microbe is named after him. It should be noted that on March 18, 1882, Baumgarten, also a German scientist, showed the tuberculosis bacillus isolated from the organs of a rabbit affected by tuberculosis, but only under a microscope.
The causative agent of tuberculosis belongs to the genus of Mycobacteria, the family of Actinomycetes and the class of schizomycetes. The genus of mycobacteria also includes the causative agent of leprosy and a group of saprophytes, which are found in discharge from the ears, in sputum during bronchiectasis, as well as acid-resistant microorganisms that grow on human mucous membranes, in butter, milk, on plants, in water, soil, etc. d.
Division of mycobacteria by pathogenicity
Based on their pathogenicity for humans and for individual species, mycobacteria are divided into 2 groups. The first group is the actual pathogenic mycobacterium tuberculosis, of which three types are distinguished. The second group is atypical mycobacteria, among which there are saprophytes - non-pathogenic for humans and animals and opportunistic mycobacteria - under certain conditions they can cause mycobacteriosis, which resembles tuberculosis.
Atypical mycobacteria
According to one classification, they are divided into four groups (depending on growth rate and pigment formation).
- Group I - photochromogenic mycobacteria - form a lemon-yellow pigment during exposure of the culture to light; colonies grow within 2-3 weeks. The source of infection can be cattle, milk and other dairy products.
- Group II - scotochromogenic mycobacteria, which form an orange-yellow pigment in the dark. Distributed in water and soil.
- Group III - non-photochromogenic. Cultures are slightly pigmented or non-pigmented; visible growth appears within 5-10 days. Various in virulence and optimal growth temperature. Occurs in soil, water, and various animals (pigs, sheep).
- Group IV - mycobacteria that grow quickly on nutrient media. Growth is achieved in 2-5 days.
Atypical mycobacteria are detected in 0.3-3% of cultures, most often due to environmental contamination. Their etiological role is considered proven if they are re-sown from pathological material and their growth is characterized by a large number of colonies, and there are no other pathogens of the disease.
The disease caused by atypical strains of Mycobacterium tuberculosis is called mycobacteriosis. The product of their vital activity, sensitin, was obtained from strains of atypical mycobacteria. When sensitin is administered intradermally, a positive reaction occurs in patients with mycobacteriosis. The clinical course of mycobacteriosis resembles tuberculosis, sometimes accompanied by tuberculosis, and progresses rapidly.
Types of mycobacteriosis
There are three types of mycobacteriosis, which depend on the type of mycobacteria and the immune status of the body:
1. Generalized infection with the development of pathological changes visible to the naked eye externally resemble tuberculosis, but histologically they are somewhat different from them. Diffuse interstitial changes without granulomas and decay cavities are found in the lungs. The main signs are fever, bilateral dissemination in the middle and lower parts of the lungs, anemia, neutropenia, chronic diarrhea and abdominal pain. The diagnosis is confirmed by the presence of the pathogen in sputum, stool or biopsy. The effectiveness of treatment is low, the mortality rate is high and reaches 20%. Effective for the treatment of mycobacteriosis are ethambutol, kanamycin, rifampicin and partially streptomycin.
2. Localized infection - characterized by the presence of macro- and microscopic lesions detected in certain areas of the body.
3. An infection that occurs without the development of visible lesions; The pathogen is located in the lymph nodes.
Tuberculosis in humans predominantly (95-97%) occurs as a result of infection with human, less often (3-5%) bovine and, incidentally, avian species of Mycobacterium tuberculosis. M. africanum causes tuberculosis in humans in tropical Africa.
Mycobacterium tuberculosis has the form of thin, long or short, straight or curved rods, 1.0-4.0 µm long and 0.3-0.6 µm in diameter; nonmotile, do not form spores or capsules, gram-positive, have high polymorphism.
Mycobacterium tuberculosis of the human species is thinner and longer than that of the bovine species. Mycobacteria of the bovine species are less pathogenic for humans, and the disease caused by them is much less common. To determine the MBT of the human species, the niacin test is used. It is based on the fact that MBT of this type produces more (nicotinic acid).
Young bacteria are homogeneous; during their aging, granularity (Much grains) is formed, which is studied in more detail by electron microscopy. The granular form of Mycobacterium tuberculosis is also formed under the influence of antimycobacterial drugs. After introducing grains to animals, they develop cachexia, enlarged lymph nodes or tuberculosis with the development of typical strains of Mycobacterium tuberculosis. Described splintered forms of Mycobacterium tuberculosis. The causative agent of tuberculosis can also exist in the form of filterable forms.
Under the influence of anti-tuberculosis drugs, the morphological and physico-chemical properties of Mycobacterium tuberculosis change. Mycobacteria become short, approaching cocobacillus, their acid resistance decreases, therefore, when stained according to Ziehl-Neelsen, they become discolored and are not detected.
Reproduction of Mycobacterium tuberculosis
Mycobacterium tuberculosis reproduces by transverse division, branching or budding of individual grains. Mycobacterium tuberculosis grows on nutrient media in the presence of oxygen. But they are facultative aerobes, i.e. They grow even when there is no access to air - they get oxygen from carbohydrates. Therefore, growing mycobacteria requires a nutrient medium rich in carbohydrates.
Dense media containing eggs, milk, potatoes, and glycerin are effective. The most commonly used environments are Levenshtein-Jensen, Gelberg, Finn-2, Middlebrook, and Ogawa.
Mycobacterium tuberculosis grows slowly. The first colonies appear on the 12-30th day, and sometimes after 2 months. To ensure the growth of Mycobacterium tuberculosis, 3-6% glycerol is added to the nutrient media. Mycobacteria grow better in a slightly alkaline environment, although they can also grow in a neutral environment.
Adding bile to the nutrient medium slows down their growth. This circumstance was used by Calmette and Guerin when developing a vaccine. On liquid nutrient media with the addition of glycerol, Mycobacterium tuberculosis grows in the form of a film. Colonies of mycobacteria can be rough (K.-variants) and less often - smooth, merging with each other (8-variants). K.-variants of mycobacteria are virulent for humans and animals, and 8-variants are often non-virulent.
Composition of mycobacteria
Mycobacteria consists of a cell membrane and cytoplasm. The cell membrane is three-layered and consists of outer, middle and inner layers. In virulent mycobacteria it has a thickness of 230-250 nm.
The outer layer surrounding the cell is called the microcapsule. It is formed by polysaccharides and contains fibrils. The microcapsule can surround an entire population of mycobacteria, and can also be placed where mycobacteria adhere to each other. The absence or presence of growth, its intensity and the composition of the microcapsule depend on how much cord factor is extracted from the cytoplasm into the cell wall. The more cord factor is extracted, the better the microcapsule is expressed in Mycobacterium tuberculosis.
The cell membrane is involved in the regulation of metabolic processes. It contains species-specific antigens, due to which the cell wall is the locus where delayed allergic hypersensitivity reactions and the formation of antibodies occur, since it, as the actual surface structure of the bacterial cell, is the first to contact the tissues of the macroorganism.
Under the cell membrane there is a three-layer cytoplasmic membrane, closely adjacent to the cytoplasm. It consists of lipoprotein complexes. Processes occur in it that determine the specificity of the reaction of mycobacteria to environmental factors.
The cytoplasmic membrane of Mycobacterium tuberculosis, through its centripetal invagination, forms an intracytoplasmic membrane system in the cytoplasm - mesos. Mesosomes are semi-functional structures. They contain many enzyme systems. They participate in the synthesis and formation of the cell wall and act as an intermediary between the nucleus and cytoplasm of the bacterial cell.
The cytoplasm of mycobacteria consists of granules and inclusions. In young Mycobacterium tuberculosis, the cytoplasm is more homogeneous and compact than in old ones, which have more vacuoles and cavities in the cytoplasm. The bulk of granular inclusions are made up of ribosomes, located in the cytoplasm in a free state or forming polysomes - an accumulation of ribosomes. Ribosomes consist of RNA and protein and synthesize specific proteins.
The immunogenicity of Mycobacterium tuberculosis is mainly due to antigenic complexes contained in the membranes of mycobacterial cells. Ribosomes, ribosomal protein and cytoplasm of mycobacteria have antigenic activity in delayed reactions.
Chemical composition of Mycobacterium tuberculosis
The chemical composition of Mycobacterium tuberculosis has been studied quite well. They contain 80% water and 2-3% ash. Half of the dry residue consists of proteins, mainly tuberculoproteins, lipids - from 8 to 40%, and the same amount of polysaccharides. It is assumed that tuberculoproteins are full-fledged antigens and can cause a state of anaphylaxis in animals. The lipid fraction leads to resistance of the tuberculosis pathogen, and the polysaccharide fraction is involved in immunogenesis.
Tuberculoproteins and lipid fractions determine the toxicity of Mycobacterium tuberculosis, which is inherent not only in living but also in killed microorganisms. Three lipid fractions have been identified: phosphatidic, fatty and waxy. The high lipid content distinguishes Mycobacterium tuberculosis from other types of microorganisms and leads to the following properties:
1. Resistance to acids, alkalis and alcohols (mainly due to the presence of mycolic acid).
2. Resistant against common disinfectants.
3. Pathogenicity of tuberculous mycobacteria.
Exotoxins have not been identified, but the mycobacterial cells themselves are toxic - they lead to partial or complete breakdown of leukocytes. In the inorganic residue of mycobacterium tuberculosis, iron, magnesium, manganese, potassium, sodium, and cobalt salts are determined. The antigenic structure of mycobacteria is complex and not yet fully understood.
Antigens
Mycobacteria have specific species and interspecific and even intergeneric antigenic relationships. Different antigens have been identified in individual strains. However, all mycobacteria, without exception, contain substances that are resistant to heat and proteolytic enzymes - polysaccharides, which are a common antigen.
Besides, different kinds mycobacteria have their own specific antigens. A. P. Lysenko (1987) proved that all strains of M. bovis have an identical antigenic spectrum with 8 antigens, of which 5-6 were generic and reacted with antisera to mycobacteria of other species: 6 - with M. tuberculosis, 3-5 - M. kansasii, etc.
Pathogenicity of Mycobacterium tuberculosis
Pathogenicity is a species property of Mycobacterium tuberculosis that turns out to be able to cause disease. The main pathogenicity factor is toxic glycolipids - cord factor. This is a substance that glues virulent mycobacteria together, so that they grow on nutrient media in the form of ropes. Cord factor causes a toxic effect on tissue and protects tuberculosis bacilli from phagocytosis by blocking oxidative phosphorylation in macrophage mitochondria. Therefore, when absorbed by phagocytes, they multiply in them and cause their death. Acid-resistant saprophytes do not form a cord factor.
Virulence— degree of pathogenicity; the possibility of growth and reproduction of mycobacteria in a certain macroorganism and the ability to cause specific pathological changes in organs. A strain of mycobacteria is considered virulent when it causes tuberculosis in a dose of 0.1-0.01 mg, and after 2 months - the death of a Guinea pig weighing 250-300 g. When, after administration of this dose, the animal dies after 5-6 months, then this strain is considered weakly virulent. Virulence is not an immutable property of mycobacteria. It decreases with aging of the culture or cultivation on artificial nutrient media and during the treatment of patients. During passages on animals or in cases of exacerbation of the tuberculosis process, virulence increases.
Genetics and variability of mycobacteria
The carriers of genetic information of Mycobacterium tuberculosis are chromosomes and extrachromosomal elements - plasmids. The main difference between chromosomes and plasmids is their size. A plasmid is much smaller than a chromosome and therefore carries less genetic information. It is due to its small size that the plasmid is well adapted to transfer genetic information from one mycobacterial cell to another.
Plasmids can interact with the chromosome. The resistance genes of Mycobacterium tuberculosis against chemotherapy are localized both in chromosomes and in plasmids.
Mycobacteria have DNA that functions as the main carrier of genetic information. The sequence of nucleotides in a DNA molecule is a gene. The genetic information carried by DNA is not something stable and unchanging. It is changeable and evolving, improving. Single mutations are usually not accompanied by large changes in the information contained in the genome. A single strain can produce several different phenotypes (or traits that result from the action of genes under certain conditions) that are resistant to a particular antimycobacterial drug.
The mutation can also manifest itself in changes in the morphology of the colonies. Thus, if the virulence of Mycobacterium tuberculosis is changed, the morphology of the mutant colonies may also change.
Transduction is the transfer of genetic material (DNA particles) from one mycobacterium (donor) to another (recipient), which leads to a change in the genotype of the recipient mycobacterium.
Transformation is the inclusion of a DNA fragment of another mycobacterium (donor) into the chromosome or plasmid of a mycobacterium (recipient) as a result of the transfer of isolated DNA.
Conjugation is a contact between Mycobacterium tuberculosis cells, during which the transfer of genetic material (DNA) from one cell to another occurs.
Transfection is the reproduction of the viral form of Mycobacterium tuberculosis in a cell that is infected with isolated viral nucleic acid.
The hypothetical pathways outlined for the transfer of genetic information have not yet been studied. However, there is no doubt that these genetic processes are the basis for the emergence of drug resistance both in individual mycobacteria and in the entire bacterial population present in the patient’s body.
Variability of mycobacteria
Variabilitymycobacteria- this is their ability to acquire new and/or lose old characteristics. Due to the fact that Mycobacterium tuberculosis has a short generation period, a high frequency of mutations and recombinations, and the exchange of genetic information, the variability in them is very high and frequent (N. A. Vasiliev et al., 1990).
There are phenotypic and genotypic variability. A phenotypic mutation is also called a modification mutation, which is characterized by a high frequency of changes and their frequent reversion to the original form, adaptation to changes in the external environment, and no changes in the genetic code. It is not hereditary.
Genotypic mutation occurs due to mutations and recombinations.
Mutations- these are stable inherited changes in the nucleotide composition of the mycobacterial genome, including plasmids. They can be spontaneous or induced. Spontaneous mutations occur at a gene-specific rate. Most of them are the result of errors in DNA replication and repair. Induced mutations are possible as a result of exposure to mutagens (ultraviolet; ionizing radiation, chemicals, etc.). Mutations often lead to the appearance of a new trait in the phenotype or the loss of an old trait (compared to the parental form).
Recombinationsgenetic- this is the process of producing offspring containing donor characteristics; and the recipient.
One of the types of variability of Mycobacterium tuberculosis is the formation of filterableforms. These are very small forms, invisible under ordinary microscopy, having very weak virulence; they can only be detected during reversion, using repeated passages on guinea pigs. In these cases, acid-fast bacilli with very low virulence are sometimes found.
Filterable forms are small fragments of Mycobacterium tuberculosis, formed under unfavorable living conditions and capable of reversion. The nature of these forms, their structure, as well as their significance in the pathogenesis of tuberculosis have not yet been fully established.
L-forms of Mycobacterium tuberculosis
L-forms of Mycobacterium tuberculosis have either defects or absence of the cell wall. They are characterized by dramatically altered bacterial cell morphology and reduced metabolism. They have low virulence and are quickly destroyed in the environment. Due to the absence or damage to the shell of Mycobacterium tuberculosis, L-forms are painted with conventional dyes, so they cannot be detected bacterioscopically in smears. The transformation of Mycobacterium tuberculosis into L-forms occurs under the influence of anti-tuberculosis drugs, under the influence of the protective forces of the macroorganism and other factors.
L-forms of Mycobacterium tuberculosis can be in a stable or unstable state in the macroorganism, that is, revert to the original microbial form with restoration of virulence. The virulence properties of stable L-forms of mycobacteria are sharply reduced compared to the virulence of unstable forms.
Unstable L-forms of Mycobacterium tuberculosis cause generalized tuberculosis in guinea pigs, and stable L-forms cause only morphological changes close to the vaccine process. Stable L-forms of mycobacteria are predominantly found in inactive tuberculosis lesions. These foci contribute to the development of anti-tuberculosis in healthy infected people.
For effective treatment of patients with tuberculosis, it is necessary to determine the sensitivity of the pathogen, because resistance to antimycobacterial drugs complicates treatment. Usually, in the patient’s body, mycobacteria resistance against drugs can be stored for 1-2 years after their discontinuation.
Drug resistance of Mycobacterium tuberculosis is the resistance of MBT against another antimycobacterial drug or more.
Types of drug resistance
Primary drug resistance is resistance found in newly diagnosed patients who have never taken anti-TB drugs.
Initial drug resistance is MBT resistance detected in newly diagnosed patients treated with anti-tuberculosis drugs for no more than 4 weeks or in patients with no data on previous treatment. Secondary (acquired) drug resistance - MBT resistance, was found in patients who were prescribed anti-tuberculosis drugs for more than 4 weeks. Monoresistance is the resistance of MBT against 1 of 5 first-line drugs (isoniazid, streptomycin, rifampicin, ethambutol, pyrazinamide).
In Ukraine, the incidence of primary resistance of the tuberculosis pathogen against first-line drugs is observed in 23-25%, and secondary resistance in 55-56% of cases. Multidrug resistance is the resistance of MBT against two or more drugs. Multidrug resistance is a type of multidrug resistance, and namely, the resistance of the pathogen only against the combination of isoniazid + rifampicin or other drugs.
The result of determining the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis drugs is called an antibiogram.
Causesdrug resistance:
1. Biological - insufficient concentration of the drug, individual characteristics of the patient’s body (rate of drug inactivation)
2. Reasons caused by the patient - contact with patients with chemoresistant tuberculosis, irregular intake of drugs, premature cessation of medication, unsatisfactory tolerability of drugs, inadequate treatment.
3. Factors caused by the disease - when changing the doses of drugs, with a large amount of MBT in the areas of the affected organ, a certain pH may arise that interferes with the active action of the drugs, treatment with one drug, insufficient dose or duration of treatment.
Genome of Mycobacterium tuberculosis
IN last years Genetic studies of the M. tuberculosis strain were intensively carried out. The amount of guanine cytosine bases that are distributed on the deoxyribonucleic acid (DNA) helix is 65.5%. The genome contains many insertion sequences, multigene families, amplified (duplicated) sites of its own metabolism.
RNA molecules encode about 50 genes, in particular:
- three types of ribosomal RNAs, which are synthesized from a unique ribosomal operon;
- genes encoding 108-RNA are included in the process of protein destruction (it has been revealed that these 108-RNAs are encoded by so-called abnormal and RNA messengers);
- genes encoding the RNA component RNase P;
- transfer RNA genes.
M. tuberculosis has 11 receptor-dependent histidine kinases, several cytoplasmic kinases, and few genes involved in regulatory cascades. M. tuberculosis is a group of eukaryotic serine thyreonine protein kinases responsible for phosphorylation in the bacterial cell.
To carry out lipid metabolism, approximately 250 enzymes are synthesized in M. tuberculosis. The oxidation of fatty acids is ensured by the following enzyme systems:
1. RabA / RabB-R-oxidase complexes.
2. Thirty-six acyl-CoA synthetases and a group of thirty-six acyl-CoA synthetases-linked proteins.
3. Five enzymes complete the oxidation cycle (thiolysis reaction of 3 ketoesters).
4. Four hydroxyacyl-CoA dehydrogenases.
5. Twenty-one types of proteins of the enoyl-CoA-hydratase isomerase group.
6. Acetyl-CoA-C-acetyltransferases.
PathogenicityM. tuberculosis is also caused by such factors as:
1) antioxidase catalase-peroxidase system;
2) sigma factor;
3) MSE operon, encoding intracellular invasion proteins;
4) phospholipase C;
5) enzymes producing cell wall components;
6) hematoglobin-like P-binding proteins, which ensure long-term anaerobic existence of mycobacteria;
7) esterases and lipases;
8) significant antigenic lability;
9) the presence of various ways to ensure antibiotic resistance;
10) the presence of acteriocins with a cytotoxic effect (some polyketins).
Stability of the tuberculosis pathogen in the external environment
The causative agent of tuberculosis is resistant to environmental factors. On the pages of a book, mycobacteria persist for 2-3 months, in street dust - about 2 weeks, in cheese and butter - from 200 to 250 days, in raw milk - 18 days (milk souring does not cause the death of mycobacteria), in a room with diffuse in daylight - 1-5 months, and in damp basements and in garbage pits - up to 6 months.
The optimal growth temperature for the pathogen is 37-38 ° C; at a temperature of 42-43 ° C and below 22 ° C, its growth and reproduction stop. For the avian species of mycobacterium tuberculosis, the optimal growth temperature is 42 ° C. At a temperature of 50 ° C, mycobacteria tuberculosis die after 12 hours, at 70 ° C - after 1 minute. In a protein environment, their stability increases significantly. Thus, mycobacterium tuberculosis in milk can withstand temperatures of 55 ° C for 4 hours, 60 ° C for 1 hour, 70 ° C for 30 minutes, 90 95 ° C for 3 to 5 minutes.
The resistance of Mycobacterium tuberculosis especially increases in dried sputum. To neutralize liquid sputum, they need to be boiled for 5 minutes. In dried sputum, Mycobacterium tuberculosis is killed at 100 ° C after 45 minutes. In a thin layer of liquid sputum, under the influence of ultraviolet rays, Mycobacterium tuberculosis dies in 2-3 minutes, and in dried sputum and in a dark place they can remain viable for 6-12 months. However, when exposed to direct or diffuse solar radiation for 4 hours, dried sputum loses its ability to cause tuberculosis infection in animals. Mycobacterium tuberculosis is not detected in sun-dried sputum.
If sputum enters wastewater or irrigation fields, Mycobacterium tuberculosis retains its virulence for more than 30 days. At a distance of 100 m from the discharge site Wastewater Mycobacterium tuberculosis was not detected from the anti-tuberculosis sanatorium.
Mycobacterium tuberculosis is unequally resistant to various disinfectants. Thus, a double amount of 5% chloramine solution kills mycobacteria in sputum after 6 hours, 2% bleach solution - after 24-48 hours.
Microbiology: lecture notes Ksenia Viktorovna Tkachenko
LECTURE No. 21. Tuberculosis
LECTURE No. 21. Tuberculosis
1. Morphology and cultural properties
The causative agent belongs to the genus Mycobakterium, species M. tuberculesis.
These are thin rods, slightly curved, and do not form spores or capsules. The cell wall is surrounded by a layer of glycopeptides called mycosides (microcapsules).
The tuberculosis bacillus is difficult to perceive conventional dyes (it takes 24–30 hours to stain according to Gram). Gram positive.
The tuberculosis bacillus has structural features and chemical composition of the cell wall, which are reflected in all biological properties. The main feature is that the cell wall contains a large amount of lipids (up to 60%). Most of them are mycolic acids, which are included in the framework of the cell wall, where they are found in the form of free glycopeptides that are part of the cord factors. Cord factors determine the growth pattern in the form of cords.
The cell wall contains lipoarabinomanan. Its terminal fragments, the cap, determine the ability of the pathogen to specifically bind to macrophage receptors.
Mycobacterium tuberculosis is stained using the Ziehl-Neelsen method. This method is based on the acid resistance of mycobacteria, which is determined by the characteristics of the chemical composition of the cell wall.
As a result of treatment with anti-tuberculosis drugs, the pathogen may lose acid resistance.
Mycobacterium tuberculosis is characterized by pronounced polymorphism. In their cytoplasmic membrane, characteristic inclusions are found - Mukha grains. Mycobacteria in the human body can transform into L-forms.
By type of energy production aerobes. According to temperature requirements - mesophiles.
Their reproduction occurs very slowly, generation time is 14–16 hours. This is due to pronounced hydrophobicity, which is due to the high lipid content. This makes it difficult to supply nutrients into the cell, which reduces the metabolic activity of the cell. Visible growth on media is 21–28 days.
Mycobacteria are demanding on nutrient media. Growth factors – glycerol, amino acids. They grow on potato-glycerin, egg-glycerin and synthetic media. In all these media it is necessary to add substances that inhibit the growth of contaminating flora.
On dense nutrient media, characteristic colonies are formed: wrinkled, dry, with uneven edges, and do not merge with each other.
In liquid media they grow in the form of a film. The film is initially tender and dry, but over time it thickens and becomes lumpy and wrinkled with a yellowish tint. The environment is opaque.
Tuberculosis bacteria have a certain biochemical activity, and the study of it is used to differentiate the causative agent of tuberculosis from other representatives of the group.
Pathogenicity factors:
1) mycolic acids;
2) cord factor;
3) sulfatides;
4) mycosides;
5) lipoarabinomanan.
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Morphology of Mycobacterium tuberculosis (red rods) in sputum. Ziehl-Neelsen staining. Gram-positive thin straight or slightly curved rods; - The cell wall contains a large amount of waxes and lipids (mycolic acid), which determines hydrophobicity, resistance to acids, alkalis, and alcohols; - Stained according to Ziehl-Neelsen; - Motile, does not form spores or capsules; - Can be converted to filterable and L-forms
Cultural properties Aerobes; Grow on media containing eggs, glycerin, potatoes, asparagine, vitamins, salts; The most commonly used are Lowenstein-Jensen egg medium and Soton synthetic medium; grow slowly (growth is detected after 2-3 weeks and later); Colonies are dry, wrinkled, grayish; They have biochemical activity that makes it possible to differentiate species. The main test is the niacin test (accumulation of nicotinic acid in a liquid medium. Lowenstein-Jensen medium and the growth of mycobacteria.
Pathogenicity factors Adhesion factor - cord - factor = ester of trehalose and diresidues of mycolic acid; Antiphagocytic factors - waxes (especially wax D), sulfates and some other compounds that prevent the fusion of phago- and lysosomes; Sulfolipids inhibit the activity of lysosomal enzymes; Fosatide and wax fractions of lipids cause sensitization of the body; Acetone-soluble lipids enhance the immunosuppressive properties of mycobacteria and modify host cell membranes; Lipids provide resistance to complement and free radicals of phagocytes. The main factor, tuberculin, has toxic and allergic properties
The interaction of Mycobacterium tuberculosis with the human body begins when the pathogen enters the lungs. After adhesion with the help of a cord factor, they are captured by alveolar macrophages; The events that occur next (macrophages either restrain the proliferation of mycobacteria or not) are determined by the relationship between the bactericidal activity of macrophages and the virulence of mycobacteria. After multiplication inside the macrophage, mycobacteria destroy it. Monocytes, emerging from the bloodstream under the influence of chemotaxis factors, capture mycobacteria released from destroyed macrophages. Macrophages transfer mycobacteria to the nearest lymph nodes, where they persist for a long time due to incomplete phagocytosis. Thus, the initial entry of the pathogen into the lungs or other organs causes development of minor or nonspecific inflammation with macrophage infiltration
Pathogenesis (continued) 2-4 weeks after infection, the next stage of interaction between mycobacteria and the macroorganism begins. In this case, two processes are observed - a reaction of tissue damage like HRT (specific inflammatory reaction) and a reaction of activation of macrophages. With the development of immunity and the accumulation of a large number of activated macrophages in the primary focus, a tuberculous granuloma is formed. Granuloma Granulomas consist of lymphocytes and activated macrophages, that is, epithelioid and giant cells. Epithelioid giant cells The development of a tissue damage reaction leads to the formation of a focus of caseous necrosis in the center of the granuloma. In case of healing of the lesion, necrotic masses become denser, calcified as a result of the deposition of calcium salts, a connective tissue capsule is formed around the lesion - Gon's lesion, but mycobacteria in the form of L-forms retain viability in such a lesion long years With a decrease in the resistance of the macroorganism, activation of the focus occurs with the development of secondary tuberculosis
Immunity Anti-tuberculosis immunity is formed in response to the penetration of mycobacteria into the body during infection or after vaccination and is non-sterile, infectious in nature, which is due to the long-term persistence of L-forms. The decisive role belongs to cellular immunity. The outcome of the disease is determined by the activity of T-helpers, which activate phagocytic activity macrophages and T-killer activity
Epidemiology The main source of infection is a patient with respiratory tuberculosis. Transmission routes are airborne, less often nutritional, contact Mycobacterium tuberculosis is very resistant during external environment. In running water they can remain viable for up to 1 year, in soil and manure for 6 months, on various objects for up to 3 months, in library dust for 18 months, in dried pus and sputum for up to 10 months. When boiled, Koch's bacillus dies after 5 minutes, in gastric juice after 6 hours, when pasteurized after 30 minutes, direct sunlight kills mycobacteria within an hour and a half, and ultraviolet rays within 2-3 minutes. Disinfectants containing chlorine kill mycobacteria within 5 hours.
Epidemiology (continued) Tuberculosis is widespread Socio-economic factors contribute to the increase in incidence (the main factor is starvation) Since 1990, a sharp rise in incidence has been recorded worldwide Human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome have caused a noticeable increase in the number of cases of tuberculosis in some countries C on the other hand, the problem is the spread of multidrug-resistant mycobacteria
Treatment Currently, according to the degree of effectiveness, anti-tuberculosis drugs are divided into 3 groups: Group A - isoniazid, rifampicin and their derivatives (rifabutin, rifater) Group B - streptomycin, kanamycin, ethionamide, cycloserine, fluoroquinolones, etc. Group C - PAS and thioacetozone
BCG vaccine (BCG - bacillus Calmette and Guerin) - contains live avirulent mycobacteria obtained from M. bovis through long-term passages on media containing bile Post-vaccination immunity is associated with the formation of hypersensitivity hypersensitivity (delayed type hypersensitivity) Specific prevention
Laboratory diagnostics Clinical material: pus, sputum, blood, bronchial exudate, cerebrospinal fluid, pleural fluid, urine, etc. Methods: 1. Bacterioscopic: direct staining of a sputum smear using the Ziehl-Neelsen method or a smear after enrichment (concentration by flotation or homogenization methods)
Laboratory diagnostics 4. The bacteriological (culture) method is used to test the effectiveness of treatment (2-8 weeks are necessary for the growth of colonies on Lowenstein-Jensen medium and some more time to assess the effect of drugs introduced into the growth medium); 5. Serological method (RSK, ELISA, radioimmune, etc.); 6. Biological method(infection of guinea pigs and rabbits with subsequent isolation of a pure culture of the pathogen); 7. Mantoux tuberculin test (see below); 8.Molecular genetic method (PCR)
Mantoux skin allergy test Intradermal administration of highly purified tuberculin (PPD = Purified Protein Derivative) causes a local inflammatory reaction in the form of infiltration and redness (HRT reaction) in people infected with mycobacteria. Uninfected people do not show any reaction to the introduction of tuberculin. This test is used to identify infected, sensitized people.
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Mycobacterium tuberculosis
The causative agents of tuberculosis in humans are M. tuberculosis (more than 90% of all cases of tuberculosis infection, M. bovis (5%) and M. africanum (about 3%, mainly among the population of tropical African countries). M. tuberculosis was discovered by R. Koch in 1882Morphology and physiology
Mycobacterium tuberculosis is a gram-positive straight or slightly curved rod. The composition of mycobacteria includes lipids (10-40%), mycolic, phthionic, tuberculostearic and other fatty acids. The structural skeleton of the mycobacterial cell wall consists of two covalently bound polymers - arabinogalactasan mycolate and peptidoglycan, to which proteins, polysaccharides, and lipids are attached. Such a complex chemical complex with a high lipid content gives the cells of Mycobacterium tuberculosis a number of characteristic properties: resistance to acids, alkalis and alcohol, as well as hydrophobicity. The Ziehl-Neelsen method is used for staining tuberculosis bacilli. In crops there are granular forms, branching, Mukha grains - spherical, acid-compliant, easily stained by Gram. Transition to filterable and L-forms is possible. They are immobile, do not form spores or capsules. To cultivate tuberculous mycobacteria in laboratory conditions, special nutrient media containing eggs, glycerin, potatoes, asparagine, vitamins, and salts are used. The most commonly used are Lowenstein-Jensen egg medium and Soton synthetic medium. Mycobacterium tuberculosis multiplies slowly. Under optimal conditions, the generation time is about 15 hours, while bacteria of many other genera divide every 20-30 minutes. The growth of tuberculous mycobacteria can be detected after 2-3 weeks and later - up to 2-3 months, especially in the first generations. On dense media, wrinkled, dry colonies with uneven edges are formed; in liquid media, a delicate film forms on the surface, which thickens and falls to the bottom, while the medium remains transparent. To obtain more homogeneous growth of mycobacteria, Tween - 80 (surfactant) is added to the nutrient media.Antigens
Mycobacterium antigens contain proteins, polysaccharides, lipids, and phosphatides. Antibodies to them are determined by RSK, RNGA, and gel precipitation. There are general and specific antigens in M. tuberculosis, M. bovis and other mycobacteria, including saprophytic species. The pathogenicity of tuberculous mycobacteria is associated with the direct or immunologically mediated damaging effect of lipids (wax D, muramindipeptide, phthionic acids), as well as tuberculin. Their action is expressed in the development of specific granulomas and tissue damage. Virulent strains are characterized by the presence of a so-called cord factor - a glycolipid consisting of trehalose and dimycolate. It destroys the mitochondria of the cells of the infected organism, thereby disrupting the respiratory function. Mycobacteria do not produce exotoxin.Pathogenesis
In the zone of penetration and reproduction of mycobacteria, a specific inflammatory focus appears - an infectious granuloma (primary effect). Then a specific inflammatory process develops in the regional lymph nodes and sensitization of the body is observed. Thus, the so-called primary tuberculosis complex is formed. In the vast majority of cases, the primary lesion has a benign course. It dissolves, the affected area calcifies and scars. However, this process does not end with the complete liberation of the body from the pathogen. In the primary focus and lymph nodes, tuberculosis bacteria can persist for many years, sometimes throughout life. Such people, while remaining infected, acquire immunity to tuberculosis. In case of unfavorable diseases, especially against the background of poor social factors (insufficient and inadequate nutrition, unsatisfactory living conditions, concomitant diseases), activation of the pathogen and generalization of the process may occur. Pulmonary tuberculosis is the most common. Generalization of infection leads to the development of extrapulmonary forms of tuberculosis: skin, bones and joints, kidneys and other organs. The localization of the process to a certain extent depends on the routes of penetration of mycobacteria into the human body and the type of pathogen. Pathogenetically important is the effect of tuberculin on the body of an infected person. This substance was first obtained by R. Koch in 1890, and the drug he studied was called “old tuberculin.” Purified tuberculin (PPD - purified protein derivative) is a protein. Intradermal administration of tuberculin causes a local inflammatory reaction in the form of infiltration and redness (Mantoux reaction) in people infected with mycobacteria. Uninfected people do not show any reaction to the introduction of tuberculin. This test is used to identify infected, sensitized people.Immunity
In tuberculosis, immunity is formed against the background of primary infection of the body with mycobacteria, which remain in it for a long time. This form of immunity is called non-sterile and is expressed in the body’s resistance to superinfection. In addition, long-term persistence of mycobacteria in the body is associated with L-transformation of the tuberculosis pathogen, as well as widespread use live avirulent BCG vaccine for population vaccination. In tuberculosis, antibodies belonging to different classes of immunoglobulins are detected. Antibodies can be detected using various serological reactions (RSK, RPGA, etc.). Their significance in the formation of anti-tuberculosis immunity still remains unclear. It is believed that antibodies to mycobacterium tuberculosis are only “witnesses” of immunity, do not have an inhibitory effect on the pathogen and do not reflect its intensity. Great importance has cellular immunity. The indicators of its changes, judging by the reaction of blast transformation of lymphocytes, the cytotoxic effect of lymphocytes on “target” cells containing mycobacterial antigens, and the severity of the reaction of inhibition of macrophage migration, are adequate to the course of the disease. T-lymphocytes, after contact with mycobacterial antigens, produce immunocytokines that enhance the phagocytic activity of macrophages. When the function of T-lymphocytes is suppressed, the tuberculosis process occurs in a more severe form. Phagocytosis in tuberculosis is incomplete, since mycobacteria can multiply in macrophages and partially destroy them. The preservation of living mycobacteria in tissues provides increased resistance to superinfection, as well as “immunological memory”. Allergy, which develops like HRT, is important in the formation of immunity in tuberculosis. The protective role of HRT is manifested in limiting the proliferation of mycobacteria, fixing them in foci of infection, and the formation of infectious granulomas with the participation of T-lymphocytes, macrophages and other cells.Ecology and epidemiology
Under natural conditions, M. tuberculosis lives in the body of its hosts - humans and some animals (cattle, pigs). M. africanum causes tuberculosis in humans in tropical Africa. Thus, the source of infection is sick people and animals. With active tuberculosis with the presence of inflammatory-destructive changes, they release mycobacteria into the environment. More than 80% of the population is infected with mycobacterium tuberculosis at an early age. The most common route of infection is airborne, in which the pathogen enters the body through the upper respiratory tract, sometimes through the mucous membranes of the digestive tract or through damaged skin. Once in the environment, Mycobacterium tuberculosis retains its viability for a long time. Thus, in dried sputum they survive for several weeks, on objects surrounding the patient (linen, books) for more than 3 months, in water for more than a year, in soil for up to 6 months, and are preserved for a long time in dairy products. Mycobacterium tuberculosis is more resistant to the action of disinfectants than other bacteria; higher concentrations and longer exposure times are required to destroy them. When boiled, they die instantly and are sensitive to direct sunlight.Tuberculosis
Tuberculosis- a primary chronic infectious disease of humans and animals caused by pathogenic mycobacteria. Depending on the location of the lesion, tuberculosis of the lungs, skin, lymph nodes, meninges, bones and joints, genitourinary system and abdominal cavity is distinguished. The modern period is characterized by an increase in morbidity, a severe course, an increase in mortality and the emergence of a significant number of strains resistant to many anti-tuberculosis drugs. In Ukraine, tuberculosis in humans is most often caused by Mycobacterium tuberculosis MI. bovis. Very rarely this disease is caused by avium. In Africa, America and other continents, tuberculosis-like diseases are caused by M africanum, M. asiaticum, M. kansasii, M. fortuitum, M. ulcerans, etc. These diseases are called mycobacteriosis. Another representative of the genus of mycobacteria, M leprae, is the causative agent of leprosy. In total, there are more than 40 types of mycobacteria, 24 of them are pathogenic and potentially pathogenic. Laboratory diagnosis of tuberculosis and mycobacteria includes microscopic, bacteriological, biological, serological studies and allergy tests. The main method is the isolation of a pure culture of the pathogen, its identification and determination of sensitivity to antimicrobial drugs.Taking material for research
The pathological material is sputum, mucus with back wall pharynx, pleural exudate, pus, cerebrospinal fluid, bronchial and stomach lavages, urine, feces, punctates, less often blood, etc. The patient collects sputum in a jar or pocket spittoon. The best results are obtained when examining sputum collected within 12 to 24 hours. Other materials are collected in sterile jars or tubes. They are pasted with a label with the patient’s surname and initials, the dispensary registration group, the purpose of the study and sent to the laboratory. The collected clinical material is considered potentially pathogenic.Bacterioscopic method
Smears are prepared directly from sputum or sediment obtained after centrifugation of homogenized material. The sputum is transferred to a Petri dish placed on a dark background. Using tweezers, mucopurulent beads are selected, transferred to the middle of a glass slide, covered with a second glass slide, and the material is ground between the glasses. Smears are also prepared from manure and punctates. The cerebrospinal fluid is allowed to settle for 18-20 hours in the refrigerator and the resulting delicate fibrin network is carefully straightened onto a glass slide. The urine is centrifuged and smears are prepared from the sediment. It is these smears that are decolorized not only with acid, but with alcohol to differentiate tuberculosis bacteria from M. smegmatis, which can be found in the urine of healthy people. Dried smears are fixed with dry heat, stained using the Ziehl-Neelsen method or with auramine-rhodamine or another fluorochrome. In preparations stained by Ziehl-Neelsen, the causative agent of tuberculosis has the appearance of thin solid rods of ruby-red color, located singly or in groups, mainly outside the cells. Smears are stained with auramine-rhodamine for 15 minutes with heating, then washed with water, and immersed in hydrochloric acid and again thoroughly washed with water. If, during microscopy, the background of the smear has strong fluorescence, it is necessary to quench it somewhat with a 0.25% aqueous solution of methylene blue, rinse with water, dry and examine under a fluorescent microscope. TB bacilli glow golden against a dark green background. Auramine, acridine orange, etc. are also used as fluorochromes. To identify L-forms of mycobacteria, phase-contrast microscopy is used primarily. A positive answer is given when tuberculosis bacilli are detected in a smear after viewing at least 100 fields of view, be sure to indicate the number of bacteria in each field of view . A negative microscopy result does not give the right to exclude the diagnosis. A significant disadvantage of the bacterioscopic method is its low sensitivity: mycobacteria can be detected in a smear only if there are 50-100 thousand microbial bodies in 1 ml of pathological material. In addition, this method cannot distinguish the causative agent of tuberculosis from other mycobacteria and determine its sensitivity to chemotherapy. To increase the frequency of occurrence of Mycobacterium tuberculosis in the test material (especially in sputum), enrichment methods - homogenization and flotation - are used.Homogenization method
A daily portion of sputum is added to a bottle, an equal volume of 1% NaOH solution is added, tightly closed with a rubber stopper and shaken in a Schüttel apparatus for 10-15 minutes until completely liquefied. The homogenized liquid is centrifuged, the decanted solution is poured into a chloramine solution, the precipitate is neutralized with 2-3 drops of a 10% solution of hydrochloric or 30% acetic acid. Smears are prepared from the sediment, stained with Ziehl-Neelsen, and examined under a microscope.Flotation method
A portion of sputum (10-15 ml) is homogenized as described above. A flask or bottle with diluted material is placed on water bath at 55 ° C for 30 minutes, then add 0.5-1 ml of xylene (benzene, gasoline, toluene), shake for 10 minutes and leave for half an hour. Xylene, together with adsorbed mycobacteria, floats to the surface and forms a top-like layer. Add distilled water so that this layer rises into the neck of the flask or bottle. Using a sterile Pasteur pipette, part of the xylene layer is transferred to a glass slide, heated on a glass plate lying in a water bath at a temperature of 60 ° C. The dried smear is covered with a new portion of vershkopodibny material, dried again and this is repeated until the entire flotation layer is transferred to smear. The preparation is washed with ether, dried, fixed with dry heat, stained with Ziehl-Neelsen and microscopically examined. Homogenization and flotation methods increase the presence of Mycobacterium tuberculosis in the test material by 10%. Moreover, they can be identified if there are more than a thousand microbial bodies in 1 ml of sputum. Washing waters of the bronchi and stomach can be examined using additional homogenization or flotation.Bacteriological diagnostic method
The bacteriological diagnostic method is much more effective than the bacterioscopic one. It allows you to identify 20-100 or more mycobacteria in 1 ml of the test material. It is used not only to diagnose the disease, but also to monitor the effectiveness of chemotherapy, determine the virulence and resistance of mycobacteria to antibiotics and other anti-tuberculosis drugs, and identify altered variants, especially L-forms. Almost all studied materials are from tuberculosis patients (except blood, cerebrospinal fluid ) contain accompanying microflora. Therefore, it is impossible to isolate a pure culture of mycobacteria without pre-treatment. To destroy foreign microorganisms, sputum, pus, rinsing water and other materials are treated for 20 minutes at room temperature with a double volume of 6% sulfuric acid solution or 10% trisodium phosphate solution at 37 ° C for 18-20 hours. Then the treated material is centrifuged, the liquid part is drained, and the precipitate is neutralized by adding 1-2 drops of a 3% NaOH solution, or washed three times from the acid with an isotonic sodium chloride solution. After neutralization, the material is inoculated into 3-6 tubes with a dense Lowenstein-Jensen medium (potato starch with glycerin, salts, egg suspension and malachite green) and Finn-2, which has the same composition as the previous one, but in it asparagine is replaced by monosodium glutamate. These media are recommended by WHO as standard in all countries of the world for the primary cultivation of Mycobacterium tuberculosis and determination of their resistance to chemotherapy. For other needs, you can use glycerin broth, Petragnani, Pavlovsky, Soton medium. Cerebrospinal fluid, exudate, blood, punctate are pipetted onto the nutrient medium without pre-treatment. All cotton plugs are cut off at the level of the edges of the test tube, pushed inside 1-1.5 cm and filled with melted paraffin to prevent the medium from drying out. The inoculated test tubes are incubated in a thermostat at 37 ° C for 3-4 weeks. Those tubes with media on which materials were sown with a loop and rubbed into the surface of the agar are placed vertically. If the crops were done with a Pasteur pipette, the test tubes are placed in a thermostat in an inclined position for 2-3 days, then vertically. Crops are examined every 5-7 days. All tubes with early growth of foreign microflora are removed. In cases of early growth of characteristic colonies (before the 5th day), a conclusion is made about the presence of rapidly growing mycobacteria, i.e. negative answer for tuberculosis. The growth of tuberculosis bacteria most often appears after 3 weeks, but there are cases after 2-3 months. On dense media, rough, rough, dry, pigmentless colonies grow, having a wrinkled surface, a thickened center and thin, uneven edges. In appearance, they resemble cauliflower slices or warts. These are typical R-form colonies characteristic of pathogenic strains. The S-shape of yellow or orange colonies is usually characteristic of other types of mycobacteria. But under the influence of antibacterial drugs, M tuberculosis can also form soft, moist, pigmented S-form colonies. It is necessary to note the speed and abundance of growth. Much less often, the studied material is sown in glycerin broth, liquid media with blood plasma, bovine serum or synthetic Soton's medium. On them, Mycobacterium tuberculosis grows somewhat faster, has the appearance of a delicate film, which over time thickens, coarsens, becomes fragile and precipitates. Liquid media are inoculated into test tubes with a dense medium, which increases the number positive results, but the research lasts longer. To increase the frequency of seeding of the tuberculosis pathogen, the test material is treated with detergents that have a bactericidal effect on other microflora (laurosept, sodium laurisulfate, rhodolan, theapol, cetavlon, etc.). This improves the homogenization of the material, eliminates centrifugation, and allows colonies to grow faster.Accelerated cultivation methods
To obtain the growth of Mycobacterium tuberculosis much faster, microculture methods of Price and schoolchildren have been proposed. Price's method involves spreading sputum, pus, urine sediment, rinsing fluids, and other material in a thick layer onto several narrow slides (regular slides are cut in half lengthwise). Dried smears are taken with sterile tweezers and immersed for 15-20 minutes in test tubes with a 2% sulfuric acid solution, and then washed three times with a sterile sodium chloride solution to remove the acid. After this, the drug is placed in test tubes or vials with liquid Soton’s medium or citrated blood. The smear must be completely covered with the nutrient medium. To suppress foreign microflora, which can sometimes remain after treating smears with acid, 10 U/ml penicillin is added to the medium. The crops are grown in a thermostat at 37-38 ° C. After 3-4 days, the glasses with smears are removed, fixed with dry heat, stained with Ziehl-Neelsen or rhodamine and examined microscopically. Virulent microcultures in the preparations form strands or “Skosy”, which are formed under the influence of the cord factor. Maximum growth of microcultures is observed on days 7-10. Deep cultivation of mycobacteria in hemolyzed blood using the Shkolnikov method is obtained by inoculating material treated, as usual, with sulfuric acid and washed with a 0.85% sodium chloride solution. After 6-8 days of cultivation, the culture is centrifuged, smears are made from the sediment, stained using the Ziehl-Neelsen method or with fluorochrome and microscopically examined. Typical microcultures with a characteristic arrangement in the form of braids and ropes are detected in the preparations. L-forms of Mycobacterium tuberculosis, seeding of the material after appropriate treatment with acid is carried out in a special semi-liquid medium, poured into test tubes in the form of a column. The growth of L-forms resembles a cloud with small inclusions. that similar semolina smears are examined under a phase-contrast microscope, with the help of which G-forms of different morphology are better detected. they can also be detected by serial passages on Guinea pigs or using an immunofluorescent method using sera containing labeled antibodies against L-form antigens. Many signs are used to identify isolated cultures of tuberculosis pathogens and differentiate them from other types of mycobacteria. The main ones are virulence, growth rate, colony shape, pigment formation, catalase, urease, nicotinamidase, nitrate reductase. The most important sign of M tuberculosis is the niacin test - the ability to synthesize a significant amount of nicotinic acid (niacin). Catalase activity is relatively weak and is lost at 68 ° C. For rapid differentiation of human tuberculosis pathogens, inoculation on Lowenstein-Jensen medium with 500 µg/ml and 1000 µg/ml salicylic sodium is recommended. Mycobacterium tuberculosis does not grow on this medium under such conditions. The question of the virulence of mycobacteria is resolved on the basis of biological samples and the detection of the cord factor. The latter is determined by the Price microculture method, as well as on the basis of strong binding of dyes such as neutral red or Nile blue. When NaOH solution is applied to a smear, tuberculosis bacilli retain the color of the dye, while non-virulent mycobacteria change the corresponding color. For reliable identification of mycobacterium species in modern conditions, progressive, fast and very precise methods determinations in such studied materials as sputum, pleural fluid, cerebrospinal fluid, pus, blood serum, stomach contents, tissues, etc. These objects are disinfected by heating, stored indefinitely and can be examined at any time. Among them, the molecular genetic method of polymerase chain reaction deserves the most attention. It is based on the detection of mycobacterial DNA in biological material. Even if the material contains only 5-10 microbial cells, using oligonucleotide primers, the synthesis of specific DNA fragments is triggered in a cyclothermostat, which can then be identified by gel electrophoresis. Research results are obtained in 3-4 hours. Specific antigens in the same materials can be quickly detected using enzyme-linked immunosorbent assay, if you have the appropriate antibodies adsorbed on the solid phase in polystyrene tablets.Determination of resistance of mycobacteria to chemotherapy drugs
Drug resistance of tuberculosis pathogens is determined by the method of serial dilutions before treatment, after 3 months, and subsequently with continued isolation of tuberculosis bacilli every 6 months. This is done by growing cultures on media with different concentrations of tuberculostatic agents. There are two ways to determine the resistance of mycobacteria: direct and indirect. In the direct method, appropriately treated material is directly sown on media with various concentrations of antibiotics or other chemotherapy drugs. It is effective, but it can only be used when at least 5 mycobacteria are detected in the material in each field of view. In the indirect method, previously isolated cultures of mycobacteria are sown on the medium with anti-tuberculosis drugs. In both cases, control is required - inoculation on the same medium without tuberculostatic agents. In modern conditions, the most common methods for determining the drug resistance of mycobacteria are: 1) cultivation on a dense Levenpgein-Jensen medium; 2) microculture on Price glasses; 3) deep inoculation in semi-synthetic environment. A suspension of the isolated culture (500 million microbial bodies in 1 ml) is inoculated into test tubes containing different concentrations of drugs and into one control tube (without tuberculostatics). The culture is considered sensitive if less than 20 colonies have grown in the test tube with the drug, with abundant growth in the control. If more than 20 colonies have grown, the culture is considered stable. The resistance of a given strain is expressed by the maximum concentration of the antibacterial drug at which growth still occurs, close to that in the control.Biological method
The biological method for diagnosing tuberculosis - infection of Guinea pigs - is sensitive. The infectious dose of the pathogen for these animals is only a few bacterial cells. The test material is treated with a 2% sulfuric acid solution for 20 minutes, including centrifugation. Then the sediment is washed three times with a 0.85% sodium chloride solution and emulsified in 1-2 ml of an isotonic solution. The emulsified sediment is injected subcutaneously in the groin area of two Guinea pigs weighing 250-300 g with a negative Mantoux test. If the amount of material is small, it is injected into the abdominal cavity or testicular parenchyma. This method of infection increases the sensitivity of the bioassay, especially in cases where the material contains low-virulent tuberculosis bacilli that are resistant to isoniazid and other chemotherapy drugs. After 2-3 weeks, the infected animals are weighed, the size of the enlarged lymph nodes is determined and a Mantoux test is performed, which is repeated after 6 weeks Local pathological changes and a decrease in body weight provide the basis for opening Guinea pigs and examining their internal organs. If the results are negative, the animals are killed after 3-4 months, the parenchymal organs are examined histologically and cultures are done on selective media. Guinea pigs are also used to detect L-forms of mycobacterium tuberculosis. In such cases, it is necessary to make several successive infections, since L-forms have less virulence and cause a benign course of tuberculosis in animals; with reversion, L-forms can become a generalized process. Recently, a bioassay on white mice has been increasingly used, infecting them intracerebrally. Biological tests on laboratory animals are a kind of “gold standard” for diagnosing tuberculosis.Serological diagnosis
To detect anti-tuberculosis antibodies, agglutination, precipitation and complement fixation reactions were initially proposed. Now they are rarely used. But they began to widely practice the indirect hemagglutination reaction. It uses sensitized sheep or human O-group erythrocytes as an antigen. they are loaded with an extract from tuberculosis bacteria or purified tuberculin. 20 ml of mycobacterial extract are added to 1 ml of erythrocyte sediment, kept at 37 ° C for 2 hours and washed by centrifugation to remove excess antigen. Before performing RNGA, the patient’s serum is depleted of red blood cells to eliminate the nonspecific reaction. Then the serum is diluted from 1:2 to 1:272. A dilution of 1:8 is considered a diagnostic titer. In tuberculosis, RNGA is positive in 70-90% of cases. Enzyme immunoassay, immunoblotting and hemagglutination aggregate reaction for determining circulating immune complexes also give good results. Even more precisely, there is a radioimmune method, but due to the high cost of diagnostics and the lack of radiometric equipment, it is rarely used in medical institutions. To improve the serological diagnosis of tuberculosis, it is important to establish the production of monoclonal antibodies to various mycobacterial antigens. With their help, it would be possible to identify specific epitopes of bacteria, as well as the corresponding antibodies. Detection of such antibodies will have important diagnostic value.Allergic method
The tuberculin intradermal Mantoux test is a specific diagnostic test. It is used to determine the level of infection of the population with tuberculosis, mass screening of children and adolescents for tuberculosis, selection of persons who need revaccination, checking its effectiveness, as well as for the purpose of diagnosing tuberculosis and determining the activity of the process. Tuberculin is used for testing. In 1890, Robert Koch proposed the first drug, the so-called old Koch tuberculin (Alttuberkulin Koch or ATK). It is made from a mixture of human and bovine mycobacterial cultures grown in glycerin broth for 5-6 weeks. The culture is sterilized with flowing steam for 30 min, evaporated at 70 ° C to 1 10 of the original volume, filtered through a bacterial filter and poured into ampoules. Koch's tuberculin contains a number of ballast substances and is difficult to standardize. Beginning in 1934, Seibert proposed a highly purified tuberculin preparation, which was called PPD-S (Purified protein derivative - Seibert), to perform allergy tests. After 5 years M.A. Linnikov produced purified tuberculin called PPD-L. It is dosed in tuberculin units (TU). 1 TO contains 0.00006 mg of dry drug. PPD-L is produced in two forms: dry purified tuberculin in ampoules of 50,000 TO and PPD-L in standard dilution in 3 ml ampoules. 0.1 ml of solution contains one dose (2 TU). The drug is administered intradermally with a one-gram tuberculin syringe in a volume of 0.1 ml in the middle third of the forearm. Before injection, the skin is wiped with 70% alcohol. A thin needle, cut upward, is inserted into the surface layer of the skin at an angle of 15 ° to its surface. The results of the allergy test are assessed after 72 hours according to the following scheme: negative test - complete absence of papule; doubtful - a papule measuring 2-4 mm or only hyperemia of any size; positive - a papule with a diameter of 5 mm or more; hyperergic - in children and adolescents a papule with a diameter of 17 mm or more, in adults - 21 mm or more.Prevention and treatment
For specific prevention, the live BCG vaccine (Bacille Calmette-Guerin) is used. The BCG strain was selected by A. Calmette and C. Guerin by long-term passage of bovine tuberculosis bacteria (M.bovis) on a potato-glycerol medium with the addition of bile. They made 230 subcultures over 13 years and isolated a mutant with reduced virulence. In our country, all newborns are vaccinated against tuberculosis on the 5-7th day of life. Revaccination is carried out for persons with a negative tuberculin test at intervals of 5-7 years until the age of 30. This creates infectious immunity with delayed-type hypersensitivity. To treat tuberculosis, antibiotics and chemotherapy drugs are used, to which the pathogens are sensitive. These are first-line drugs: dehydrostreptomycin, PAS and GINK (isonicotinic acid hydrazides - isoniazid, tubazid, ftivazid) and second-line drugs: ethionamide, cycloserine, kanamycin, etc. Due to the current spread of drug-resistant strains of tuberculous mycobacteria, the International Union against tuberculosis proposed a new classification of anti-tuberculosis drugs: I series - the most effective (isoniazid and rifampicin), II series - drugs of average effectiveness (ethambutol, streptomycin, ethionamide, pyrazinamide, kanamycin, cycloserine), III series - minor anti-tuberculosis drugs (PAS and Tibon). The complex of therapeutic measures uses desensitizing therapy and stimulation of the body's natural defense mechanisms.The textbook consists of seven parts. Part one – “General Microbiology” – contains information about the morphology and physiology of bacteria. Part two is devoted to the genetics of bacteria. Part three – “Microflora of the Biosphere” – examines the microflora of the environment, its role in the cycle of substances in nature, as well as the human microflora and its significance. Part four – “The Study of Infection” – is devoted to the pathogenic properties of microorganisms, their role in the infectious process, and also contains information about antibiotics and their mechanisms of action. Part five – “The Doctrine of Immunity” – contains modern ideas about immunity. The sixth part – “Viruses and the diseases they cause” – provides information about the basic biological properties of viruses and the diseases they cause. Part seven – “Private medical microbiology” – contains information about the morphology, physiology, pathogenic properties of pathogens of many infectious diseases, as well as modern methods of their diagnosis, specific prevention and therapy.
The textbook is intended for students, graduate students and teachers of higher medical educational institutions, universities, microbiologists of all specialties and practicing doctors.
5th edition, revised and expanded
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Tuberculosis (lat. . tuberculum- tubercle) is an infectious disease of humans and animals with a tendency towards chronicity, characterized by the formation of specific inflammatory changes, often in the form of small tubercles, with a predominant localization in the lungs and lymph nodes. Tuberculosis is widespread. Social and living conditions are of decisive importance in the incidence of tuberculosis and its spread, since both innate resistance and acquired immunity to it are determined by these conditions.
The causative agent of tuberculosis - Mycobacterium tuberculosis- was discovered in 1882 by R. Koch. He belongs to the family Mycobacterium families Mycobacteriaceae. Mycobacteria are widespread in nature: they are found in soil, water, and in the bodies of warm- and cold-blooded animals. Morphologically they are characterized by the ability to form filamentous and branching forms, especially in old cultures. In addition, they differ from other microorganisms in their higher resistance to acids, alkalis and alcohol, which is due to the peculiarities of the chemical composition of their cells.
M. tuberculosis has the shape of thin, slender, short or long, straight or curved rods, 1.0 – 4.0 µm long and 0.3 – 0.6 µm in diameter; motionless; spores, do not form capsules, gram-positive; have great polymorphism. In old cultures, filamentous, branching forms, often granular forms (Mukha grains), are observed, both in the form of freely lying grains and in the form of grains contained intracellularly. In the body of patients under the influence of chemotherapy drugs, ultra-small forms are often formed that can pass through finely porous bacterial filters (“filterable forms”). M. tuberculosis– aerobic, optimal temperature for growth is 37 °C, optimal pH is in the range of 6.4 – 7.0. The content of G + C in DNA is 62 – 70 mol% (for the genus). Growth at 37 °C is stimulated by incubation in air containing 5–10% CO 2 and the addition of 0.5% glycerol to the medium. Mycobacterium tuberculosis is capable of synthesizing niacin; catalase activity is relatively weak and is lost at 68 °C. Many biological properties of mycobacteria are explained by the high content of lipids, constituting up to 40% of the dry cell residue. Three fractions of lipids were detected: phosphatidic (soluble in ether), fatty (soluble in ether and acetone) and waxy (soluble in ether and chloroform). The lipids contain various acid-resistant fatty acids, including tuberculostearic, phthioid, mycolic, etc. The high lipid content determines the following properties of tubercle bacilli.
1. Resistant to acids, alkalis and alcohol.
2. Difficult to paint with dyes. Intensive methods are used to color them. For example, according to the Ziehl-Neelsen method, they are stained with a concentrated solution of carbolic fuchsin when heated. Having perceived the color, tuberculosis bacteria, unlike other cells, are not discolored by alcohol, acid, or alkali, therefore, when stained with methylene blue in a smear, all bacteria, cellular elements and mucus turn blue, and tuberculosis bacilli retain their original red color ( see color on, fig. 107.1). This method allows them to be differentiated from some non-pathogenic mycobacteria, for example M. smegmatis, contained on the mucous membrane of the urethra, but discolored by alcohol. At the same time, it is necessary to keep in mind that there are also acid-compliant (“blue” when stained by Ziehl-Neelsen) forms of tuberculosis bacteria (including rod-shaped, filamentous and granular).
3. Relatively high resistance to drying and exposure to sunlight. Scattered sunlight kills them only after 8–10 days. When sputum is boiled, death occurs within 5–7 minutes. In dried sputum, viability remains for many weeks.
4. Resistance to the action of conventional disinfectants: a 5% solution of phenol, when added in an equal volume to sputum, causes the death of tuberculosis bacilli after 6 hours, but a 0.05% solution of benzylchlorophenol kills after 15 minutes.
5. High hydrophobicity, which is reflected in cultural properties: on glycerin broth, growth is in the form of a yellowish film, which gradually thickens, becomes brittle and takes on a bumpy-wrinkled appearance, while the broth remains transparent. On glycerin agar, after 7 - 10 days, a dry scaly coating forms, gradually turning into coarse warty formations (see color on, Fig. 107.2 and see Fig. 107.3). On alkaline albuminate (or on a glass placed in citrated lysed blood), the growth of tuberculosis bacteria containing a surface glycolipid - cord factor, serpentine: the multiplying cells are located, forming a structure resembling a snake, a tourniquet, a rope or a woman's braid.
6. The pathogenicity of tuberculosis bacteria is also associated with a high lipid content. Phthioid, mycolic and other fatty acids contained in lipids have a peculiar toxic effect on tissue cells. For example, the phosphatidic fraction, the most active of all lipids, has the ability to cause a specific tissue reaction in a normal body with the formation of epithelioid cells, and the fat fraction - tuberculoid tissue. These properties of these lipid fractions are associated with the presence of phthioid acid in their composition. The waxy fraction containing mycolic acid causes reactions with the formation of numerous giant cells. Thus, the pathogenic properties of the tuberculosis bacillus and the biological reactions with which tissues respond to their implementation. The main pathogenicity factor is a toxic glycolipid (cord factor), which is located on the surface and in the thickness of the cell wall. By chemical nature, it is a polymer consisting of one molecule of trehalose disaccharide and associated with it in equivalent proportions of mycolic and mycolic high-molecular fatty acids - trehalose-6,6 "-dimycolate (C 186 H 366 O 117). The cord factor is not not only has a toxic effect on tissues, but also protects tuberculosis bacilli from phagocytosis, blocking oxidative phosphorylation in the mitochondria of macrophages. Being absorbed by phagocytes, they multiply in them and cause their death. Cord factor has two characteristic properties, indicating its important role as the main one. pathogenicity factor.
1. When white mice are infected intraperitoneally, it causes their death (after several repeated injections of 0.005 mg) after 1 - 2 weeks. after the first injection with symptoms of widespread pulmonary hyperemia. No other fraction of the tuberculosis bacillus has a similar effect.
2. It suppresses the migration of leukocytes of a person suffering from tuberculosis (in vivo and in vitro).
M. tuberculosis, lacking the cord factor, are non-pathogenic or slightly pathogenic for humans and guinea pigs. With the unusual chemical composition tuberculosis cells are also associated with their ability to cause a delayed-type hypersensitivity reaction characteristic of tuberculosis, detected using a tuberculin test.
Besides M. tuberculosis, human diseases can cause M. bovis– causative agent of bovine tuberculosis, and M. avium– causative agent of avian tuberculosis.
M. bovis– short and moderately long thick sticks. M. avium They are characterized by great polymorphism (short and long rods, sometimes filaments), the optimal temperature for their growth is 42 – 43 °C.
Main difference M. bovis from M. tuberculosis lies in their high pathogenicity for rabbits and other mammals. In case of intravenous infection M. bovis in doses of 0.1 and 0.01 mg of culture, rabbits die from generalized tuberculosis after 3–6 weeks. Infection of rabbits M. tuberculosis even at a dose of 0.1 mg does not cause their death; they develop local benign, non-progressive lesions in the lungs. For intravenous infection of rabbits M. avium animals die after 1.5 - 2 weeks. from septicopyemia.
Genus Mycobacterium includes more than 40 species. As it turns out, many of them are often highlighted in various countries the world from people, warm-blooded and cold-blooded animals suffering from diseases of the lungs, skin, soft tissues and lymph nodes. These diseases are called mycobacteriosis. There are three types of mycobacteriosis, depending on the type of mycobacteria and the immune status of the body.
I. Generalized infections with the development of pathological changes visible to the naked eye, externally resembling tuberculosis, but histologically slightly different from them.
II. Localized infections, characterized by the presence of macro- and microscopic lesions detected in certain areas of the body.
III. Infections that occur without the development of visible lesions; The pathogen is found in the lymph nodes intracellularly or extracellularly.
According to pathogenic properties, the genus Mycobacterium are divided into two groups: 1) pathogenic and opportunistic (potentially pathogenic) and 2) saprophytes. For their accelerated preliminary differentiation, three characteristics are taken into account: a) the speed and conditions of growth; b) ability to form pigment; c) the ability to synthesize nicotinic acid (niacin).
According to the growth rate of the genus Mycobacterium divided into three groups:
1. Fast-growing - large visible colonies appear before the 7th day of incubation (18 species).
2. Slow-growing - large visible colonies appear after 7 or more days of incubation (20 species).
3. Mycobacteria that require special conditions for growth or do not grow on artificial nutrient media. This group includes two types: M. leprae And M. lepraemurium.
Differentiation of mycobacteria species among fast- and slow-growing ones is carried out taking into account a number of their biochemical characteristics: reduction of nitrates, tellurite; the presence of catalase, urease, nicotine and pyrazinamidase, the ability to synthesize niacin; as well as pigment formation (see Table 46).
According to their ability to form pigment, mycobacteria are also divided into 3 groups:
1. Photochromogenic - form a lemon-yellow pigment when growing in the light.
2. Scotochromogenic - form an orange-yellow pigment when incubated in the dark.
3. Non-photochromogenic - do not form pigment (regardless of the presence of light); sometimes cultures have a light yellowish color.
There are 24 species classified as pathogenic and potentially pathogenic.
1. Slow growing:
3. Not growing extracellularly or requiring special conditions for growth:
The most common pathogens of tuberculosis and mycobacteriosis include:
M. tuberculosis M. bovis M. ulcerans
All of them are slow growing, non-photochromogenic (except M. kansasii) mycobacteria. The main differences between them are indicated in table. 49.
In Russia main role plays in the etiology and epidemiology of tuberculosis M. tuberculosis, to a share M. bovis accounts for 2–3% (in the world this pathogen accounted for 4–20%) of diseases. However, in African countries, the USA and a number of other countries, mycobacteriosis caused by other species accounts for up to 30% of all diseases classified as tuberculosis.
Table 49
Differential characters of some slow-growing species of the genus Mycobacterium
Note. (+) – positive sign; V – variable sign; (–) – negative sign; f – photochromogenic.
Various nutrient media have been proposed for the cultivation of tuberculosis bacteria: glycerin, potato with bile, egg, semi-synthetic and synthetic. The Levenshtein-Jensen egg medium is considered the best. In addition, a special semi-liquid medium has been proposed for isolating L-forms M. tuberculosis. The effectiveness of obtaining mycobacterial cultures depends on strict adherence to a number of conditions: acidic pH, optimal temperature, high quality nutrient medium, sufficient supply of O 2, appropriate seed dose, especially taking into account the possible presence of altered forms of the pathogen.
Antigenic structure M. tuberculosis. Antigenically, this species is homogeneous (no serovars have been identified) and is very similar to M. bovis And M. microti, but differs significantly from other species. However, a microbial cell has a complex and mosaic set of antigens that can cause the formation of antipolysaccharide, antiphosphatidic, antiprotein and other antibodies in the body of humans and animals that differ in their specificity. Live and killed bacteria can induce the development of delayed-type hypersensitivity. Neither proteins nor any of the lipid fractions of mycobacteria have this property.
For intraspecific differentiation M. tuberculosis a classification system has been developed based on phage typing of strains using a set of ten mycobacteriophages: 4 main and 6 auxiliary.
Pathogenicity for laboratory animals. Most susceptible to M. tuberculosis Guinea pigs. With any method of infection, the tuberculosis bacillus causes a generalized form of tuberculosis in them, from which the mumps dies after 4 - 6 weeks. For subcutaneous infection after 1.5 - 2 weeks. An infiltrate forms at the injection site, turning into an ulcer that does not heal until the animal dies. Regional lymph nodes enlarge, become dense and undergo caseous decay. Numerous tubercles are formed in the liver, spleen, lungs and other organs, in which bacterioscopy reveals M. tuberculosis.
Epidemiology. The source of infection is a person with tuberculosis, less often animals. From a sick person, the pathogen is released mainly through sputum, as well as urine, feces and pus. The tuberculosis bacillus enters the body most often through the respiratory tract - by airborne droplets and, especially often, by airborne dust. However, the entry gate can be any mucous membrane and any damaged area of skin. Infection M. bovis from cattle occurs mainly through the nutritional route through infected milk and dairy products. Tuberculosis caused by M. bovis, is observed most often in children, since milk is the main food product for them. However, infection M. bovis from sick animals and possibly aerogenically.
Features of pathogenesis. Depending on the two main methods of infection, the primary tuberculosis focus is localized either in the lungs or in the mesenteric lymph nodes. However, some experts believe that the pathogen first spreads lymphohematogenously in both cases of infection, and then it selectively affects the lungs or other organs and tissues. When entering through the respiratory tract (or in another way) into the alveoli and bronchial glands, tubercle bacilli cause the formation of a primary affect in the form of a bronchopneumonic focus, from which they penetrate through the lymphatic vessels into the regional lymph node, causing specific inflammation. All this together: bronchopneumonic focus + lymphangitis + lymphadenitis - forms the primary tuberculosis complex (primary focus of tuberculosis). The tuberculosis bacillus, due to the presence of various fatty acids and other antigens in its cells, causes a certain biological reaction in the tissues, which leads to the formation of a specific granuloma - a tubercle. In its center there are usually giant Pirogov-Langhans cells with many nuclei. Tuberculosis bacilli are found in them. The center of the tubercle is surrounded by epithelioid cells, which make up the main mass of the tubercle. Lymphoid cells are located along its periphery. The fate of the primary focus may be different. In cases where the child’s general resistance is reduced for a number of reasons, the lesion may enlarge and undergo cheesy (caseous) decomposition as a result of the action of toxic products of the tuberculosis bacillus and the absence of blood vessels in the tubercles. Such caseous pneumonia can cause severe primary pulmonary consumption, and if the pathogen enters the blood, generalized tuberculosis, leading to the death of the child. In most cases, if there is a sufficiently high natural resistance of the body, the primary focus after some time is surrounded by a connective tissue capsule, wrinkles and becomes saturated with calcium salts (calcified), which is considered as the completion of the body’s protective reaction to the introduction of the tuberculosis bacillus and means the formation of already acquired non-sterile (infectious) immunity to tuberculosis, since mycobacteria can remain viable in the primary focus for many years.
In case of infection through the alimentary route, tuberculosis bacilli enter the intestine, are captured by phagocytes of the mucous membrane and are carried along the lymphatic tract to the regional intestinal lymph nodes, causing their characteristic lesions. According to some experts, tuberculosis bacilli in this case through ductus thoracicus and the right side of the heart can also penetrate into the lungs and cause pulmonary tuberculosis.
The tuberculosis bacillus can affect almost any organ and any tissue with the development of the corresponding clinical picture of the disease.
The clinical picture of pulmonary tuberculosis is characterized by alternating periods of recovery that occur after effective chemotherapy and frequent relapses, which are caused by the persistence of tuberculosis bacilli in the body, especially in the form of L-forms, and changes in the patient’s immune status. L-forms of mycobacteria are slightly virulent, but, returning to their original form, they restore virulence and are able to cause exacerbations of the process again and again.
Features of immunity. The human body has high natural resistance to the causative agent of tuberculosis. This is the reason that in most cases, primary infection does not lead to the development of the disease, but to the formation of a focus, its delimitation and calcification. Natural resistance is largely determined by social and living conditions, therefore, in children living in difficult living conditions, it can be easily undermined, and then primary infection will lead to the development of a severe tuberculosis process. The deterioration of the living conditions of adults can also lead to a weakening of both natural resistance and acquired immunity. From 1991 to 1996, the incidence rate of tuberculosis in Russia increased from 30.6 to 42.2, and the mortality rate increased from 7.9 to 15.0 per 100,000 population.
Acquired post-infectious immunity in tuberculosis has a number of features. Although patients and those who have recovered from the disease exhibit antibodies to various antigens of the tuberculosis bacillus, they do not play a decisive role in the formation of acquired immunity. To understand its nature in tuberculosis, the following observations by R. Koch were very important. He showed that if tuberculosis bacilli are injected into a healthy guinea pig, a limited infiltrate forms at the site of infection after 10–14 days, and then an ulcer stubbornly does not heal until the death of the pig. At the same time, the pathogen spreads through the lymphatic tract, which leads to a generalized process and death of the animal. If you inject live tuberculosis bacilli into a guinea pig infected with tuberculosis a week before, the reaction develops faster: inflammation appears after 2–3 days, leads to necrosis, and the resulting ulcer heals quickly. In this case, the process is limited to the place of new infection and the pathogen does not spread from it. The Koch phenomenon indicates that an organism infected with the tuberculosis bacillus responds to re-infection in a completely different way than a healthy one, since it has developed increased sensitivity (sensitization) to the pathogen, due to which it has acquired the ability to quickly bind a new dose of the pathogen and remove it from the body. Sensitization manifests itself in the form of delayed-type hypersensitivity, it is mediated by the T-lymphocyte system. T-lymphocytes, with the help of their receptors and with the participation of MHC class I proteins, recognize cells infected with tuberculosis bacilli, attack them and destroy them. Specific antimicrobial antibodies, binding to various microbial antigens, form circulating immune complexes (CIC) and promote the removal of antigens from the body. At the same time, interacting with microbial cells, antibodies to cord factor and other virulence factors can have a toxic effect on mycobacteria; antibodies to polysaccharide antigens - enhance phagocytosis, activate the complement system, etc.
Allergic restructuring of the body plays a large role in the pathogenesis of tuberculosis. The disease in adults already infected with the tuberculosis bacillus, in most cases, occurs in a relatively benign form of a local process in the lungs, and not in the form of a generalized process, as in children with primary infection. The appearance of a delayed hypersensitivity reaction to the tuberculosis bacillus indicates the formation of acquired post-infectious (and post-vaccination) immunity to it. This type of delayed hypersensitivity was first identified by R. Koch using a tuberculin test.
Tuberculin test and its significance. R. Koch received his tuberculin preparation in the following way. He sterilized a 5-6 week culture of tuberculosis bacillus in glycerin broth with flowing steam at 100 °C for 30 minutes, and then evaporated it at a temperature of 70 °C to 1/10 of the volume and filtered. Persons infected with tuberculosis bacilli respond to the administration of small doses of tuberculin characteristic reaction: at the site of intradermal injection no earlier than 6–8 hours later, a small compaction appears; the maximum development of the reaction occurs within 24–48 hours—a well-demarcated papule with a diameter of at least 0.5 cm with a hemorrhagic or necrotic center is formed. The tuberculin allergic reaction is very specific. Such sensitization can only be caused by whole live or killed tubercle bacilli; it is detected by tuberculin, but it itself does not cause such sensitization. A positive tuberculin test specifically indicates infection of the body with a tuberculosis bacillus and, therefore, the presence of acquired immunity to it. The tuberculin test had an important diagnostic value for identifying primary tuberculosis infection in children at a time when mandatory mass vaccination against tuberculosis was not carried out, but not in adults, since in most cases they are infected with the tuberculosis bacillus. Nowadays, the tuberculin test is widely used to monitor the effectiveness of anti-tuberculosis vaccination. Due to the fact that the old Koch tuberculin contains various foreign substances and is difficult to standardize, since 1934, a highly purified tuberculin preparation obtained by F. Seibert - PPD-S (purified protein derivative-Seibert) - has been used for tuberculin tests. International Standard Unit of Tuberculin 0.000028 mg dry powder. To determine tuberculin sensitivity, 0.0001 mg PPDS is used. In our country, they produce old Koch tuberculin (ATK - Koch alt-tuberculin), containing 10,000 TU (tuberculin units) in 1 ml (it is used for a skin test and a graduated skin test according to Pirke), and a purified PPD preparation containing or 5 TE in 0.1 ml or 100 TE in 0.1 ml. Purified PPD containing 5 TU/0.1 ml is used for intradermal Mantoux test to select individuals for revaccination. Persons who react negatively to intradermal administration of 5 TE PPD are subject to revaccination. In addition, there are sensitin preparations to detect hypersensitivity to other pathogenic mycobacteria.
Laboratory diagnostics. All methods are used to diagnose tuberculosis: bacterioscopic, bacteriological, serological, biological, allergy tests, PCR. When bacterioscopic examination of the source material (sputum, urine, pus, cerebrospinal fluid, feces), it is necessary to take into account that the content of mycobacteria in it may be insignificant, their isolation is episodic and it may contain altered variants of the pathogen, including L-forms. Therefore, to increase the likelihood of detecting Mycobacterium tuberculosis, methods are used to concentrate them using centrifugation or flotation, as well as phase-contrast (to detect L-forms) and fluorescence microscopy (auramine, auramine-rhodamine, acridine orange, etc. are used as fluorochromes).
The biological method - infection of guinea pigs - is one of the most sensitive. It is believed that the infectious dose of the pathogen for them is several cells. Guinea pigs can also be used to detect L-forms of tuberculosis bacteria, but in this case it is necessary to make several sequential infections, since L-forms are less virulent and cause a benign form of tuberculosis in pigs, which, in the case of reversion of L-forms to their original state may develop into a generalized process.
The significance of the tuberculin test is discussed above.
Among the serological reactions for the diagnosis of tuberculosis, RSK, RPGA, precipitation reactions, enzyme-linked immunosorbent assay methods (including spot tests), radioimmunoassay, immunoblotting, aggregate-hemagglutination reaction (for detecting CIC), etc. have been proposed. The use of various antigens makes it possible to detect the presence of certain antibodies . To improve serological methods for diagnosing tuberculosis, it is important to obtain monoclonal antibodies to various antigens of mycobacteria. This will make it possible to identify those specific epitopes of tuberculosis bacteria and, accordingly, those antibodies to them, the detection of which has the greatest diagnostic significance, and will also make it possible to create commercial test systems for the immunodiagnosis of tuberculosis.
Among all methods microbiological diagnostics In tuberculosis, the decisive factor is still bacteriological. It is necessary not only for diagnosing the disease, but also for monitoring the effectiveness of chemotherapy, timely assessment of the sensitivity of mycobacteria to antibiotics and chemotherapy, diagnosis of relapses of tuberculosis, the degree of cleansing of the diseased organism from the pathogen and identification of its altered variants, especially L-forms. Before sowing, the material under study must be treated with a weak solution of sulfuric acid (6 - 12%) to eliminate the accompanying microflora. Isolation of pure cultures of mycobacteria is carried out taking into account their growth rate, pigment formation and niacin synthesis. Differentiation between certain types mycobacteria are carried out on the basis of their biological properties, as indicated above. The question of the virulence of mycobacteria is resolved using biological samples and based on the detection of the cord factor. For this purpose, cytochemical reactions have been proposed. They are based on the fact that virulent mycobacteria (containing cord factor) firmly bind dyes - neutral red or Nile blue - and when alkali is added, they retain the color of the paint, while the solution and non-virulent mycobacteria change their color.
For faster isolation of the tuberculosis pathogen, a microculture method has been proposed. Its essence is that the test material is applied to a glass slide, treated with sulfuric acid, washed, the glass is placed in citrated lysed blood and incubated at a temperature of 37 °C. Within 3 – 4 days. the growth of mycobacteria on glass manifests itself in the form of microcolonies, which reach their maximum development by 7–10 days, and mycobacteria are clearly detected by microscopy. In this case, virulent mycobacteria form serpentine colonies, and non-virulent ones grow in the form of amorphous clusters.