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Key methods for laboratory diagnosis of common infections. Diagnostic methods Serological methods for diagnosing viral infections

Table of contents of the topic "Methods for detecting viruses. Methods for diagnosing mycoses (fungal diseases). Methods for detecting protozoa.":

Serological methods for diagnosing viral infections. Inhibition of hemagglutination. Inhibition of the cytopathic effect by viral interference. Direct immunofluorescence. Immunoelectron microscopy.

With a majority viral infections immune reactions develop that are used to diagnostics. Cellular responses are usually assessed in tests of lymphocyte cytotoxicity against infectious agents or target cells infected by them, or the ability of lymphocytes to respond to various Ags and mitogens is determined. In practical laboratories, the severity of cellular reactions is rarely determined. Methods for identifying antiviral ATs have become more widespread.

PH is based on suppression of the cytopathogenic effect after mixing the virus with specific AT. The unknown virus is mixed with known commercial antisera and, after appropriate incubation, added to the cell monolayer. The absence of cell death indicates a discrepancy between the infectious agent and known ATs.

Inhibition of hemagglutination

RTGA is used to identify viruses, capable of agglutinating various red blood cells. To do this, mix the culture medium containing the pathogen with a known commercial antiserum and add it to the cell culture. After incubation, the ability of the culture to hemagglutination is determined and, in its absence, a conclusion is made that the virus does not match the antiserum.

Inhibition of the cytopathic effect by viral interference

The reaction of inhibition of the cytopathic effect due to the interference of viruses used to identify a pathogen that interferes with a known cytopathogenic virus in a culture of sensitive cells. To do this, commercial serum is added to the culture medium containing the virus being studied (for example, to the rubella virus if it is suspected), incubated and a second culture is infected; after 1-2 days, a known cytopathogenic virus (for example, any ECHO virus) is introduced into it. If a cytopathogenic effect is present, it is concluded that the first culture was infected with a virus corresponding to the AT used.

Direct immunofluorescence

Among other tests, the most widely used is direct immunofluorescence reaction(the fastest, most sensitive and reproducible). For example, identification of CMV by its cytopathogenic effect requires at least 2-3 weeks, and when using labeled monoclonal AT, identification is possible within 24 hours. Having a set of such reagents, they can be added to cultures infected with the virus, incubated, washed off the unbound reagent and examine using fluorescence microscopy (allows you to detect the presence of fluorescence of infected cells).

Immunoelectron microscopy

Immunoelectron microscopy(analogous to the previous method) allows you to identify different types of viruses identified by electron microscopy (for example, different types of herpes viruses), which cannot be done based on morphological features. Instead of antisera, ATs labeled in different ways are used for identification, but the complexity and high cost of the method limit its use.

No. 1 Serological reactions used for diagnosis viral infections.

Immune reactions are used in diagnostic and immunological studies in sick and healthy people. For this purpose they use serological methods, i.e., methods for studying antibodies and antigens using antigen-antibody reactions determined in blood serum and other fluids, as well as body tissues.

The detection of antibodies against pathogen antigens in the patient’s blood serum allows a diagnosis of the disease to be made. Serological studies are also used to identify microbial antigens, various biologically active substances, blood groups, tissue and tumor antigens, immune complexes, cell receptors, etc.

When isolating a microbe from a patient, the pathogen is identified by studying its antigenic properties using immune diagnostic sera, i.e. blood sera of hyperimmunized animals containing specific antibodies. This is the so-called serological identification of microorganisms.

In microbiology and immunology, agglutination, precipitation, neutralization reactions, reactions involving complement, using labeled antibodies and antigens (radioimmunological, enzyme immunoassay, immunofluorescent methods) are widely used. The listed reactions differ in the registered effect and production technique, however, they are all based on the interaction reaction of an antigen with an antibody and are used to detect both antibodies and antigens. Immune reactions are characterized by high sensitivity and specificity.

Features of the interaction of antibodies with antigens are the basis of diagnostic reactions in laboratories. The in vitro reaction between antigen and antibody consists of a specific and nonspecific phase. In the specific phase, rapid specific binding of the active center of the antibody to the antigen determinant occurs. Then comes a nonspecific phase - a slower one, which is manifested by visible physical phenomena, for example the formation of flakes (agglutination phenomenon) or precipitate in the form of turbidity. This phase requires the presence of certain conditions (electrolytes, optimal pH of the environment).

The binding of the antigen determinant (epitope) to the active center of the Fab fragment of antibodies is due to van der Waals forces, hydrogen bonds and hydrophobic interaction. The strength and amount of antigen bound by antibodies depend on the affinity, avidity of the antibodies and their valency.

No. 2 Causative agents of leishmaniasis. Taxonomy. Characteristics. Microbiological diagnostics. Treatment.

Taxonomy: phylum Sarcomastigophorae, subphylum Mastigophora - flagella, class Zoomastigophora, order Kinetoplastida, genus Leishmania.

Cultivation: NNN nutrient medium containing defibrinated rabbit blood agar. Leishmania also grows on the chorionic allantoic membrane of the chick embryo and in cell cultures.

Epidemiology: in warm climates. The mechanism of transmission of pathogens is transmissible, through the bite of mosquito vectors. The main sources of pathogens: for cutaneous anthroponotic leishmaniasis - people; for cutaneous zoonotic leishmaniasis - rodents; for visceral leishmaniasis - people; for mucocutaneous leishmaniasis - rodents, wild and domestic animals.

Pathogenesis and clinic. There are two causative agents of cutaneous leishmaniasis: L. tropica - the causative agent of anthroponotic leishmaniasis and L. major - the causative agent of zoonotic cutaneous leishmaniasis.

Anthroponotic cutaneous leishmaniasis is characterized by a long incubation period of several months. At the site of the mosquito bite, a tubercle appears, which enlarges and ulcerates after 3 months. Ulcers are most often located on the face and upper extremities, scarring by the end of the year. Zoonotic cutaneous leishmaniasis (early ulcerating leishmaniasis, Pendinsky ulcer, rural form) is more acute. The incubation period is 2-4 weeks. Weeping ulcers are most often localized on the lower extremities. Mucocutaneous leishmaniasis is caused by Leishmania complex L. braziliensis; granulomatous and ulcerative lesions of the skin of the nose, mucous membranes of the mouth and larynx develop. Anthraponotic visceral leishmaniasis is caused by Leishmania complex L. donovani; In patients, the liver, spleen, lymph nodes, bone marrow and digestive tract are affected.

Immunity: persistent lifelong

In smears (from tubercles, contents of ulcers, punctates from organs), stained according to Romanovsky-Giemsa, intracellularly located small, oval-shaped leishmania (amastigotes) are found. To isolate a pure culture of the pathogen, inoculate on NNN medium: incubation for 3 weeks. Serological methods are not specific enough. It is possible to use RIF, ELISA.

The skin allergy test for HRT to leishmanin is used in epidemiological studies of leishmaniasis.

Treatment: For visceral leishmaniasis, antimony and diamidine preparations (pentamidine) are used. For cutaneous leishmaniasis - quinacrine, amphotericin.

Prevention: destroy sick animals, fight against rodents and mosquitoes. Immunoprophylaxis of cutaneous leishmaniasis is carried out by inoculation of a live culture of L. major.

TICKET#28

No. 1Immunoglobulins, structure and functions.

The nature of immunoglobulins. In response to the introduction of an antigen, the immune system produces antibodies - proteins that can specifically bind to the antigen that caused their formation, and thus participate in immunological reactions. Antibodies refer to β-globulins, i.e., the least mobile fraction of blood serum proteins in the electric field. In the body, β-globulins are produced by special cells - plasma cells. β-globulins that carry the functions of antibodies are called immunoglobulins and are designated by the symbol Ig. Therefore, antibodies are immunoglobulins produced in response to the introduction of an antigen and capable of specifically interacting with the same antigen.

Functions. The primary function is the interaction of their active centers with their complementary antigen determinants. The secondary function is their ability to:

Bind an antigen in order to neutralize it and eliminate it from the body, i.e., take part in the formation of protection against the antigen;

Participate in the recognition of “foreign” antigen;

Ensure cooperation of immunocompetent cells (macrophages, T- and B-lymphocytes);

Participate in various forms of the immune response (phagocytosis, killer function, HNT, HRT, immunological tolerance, immunological memory).

Antibody structure. In terms of their chemical composition, immunoglobulin proteins are classified as glycoproteins, since they consist of protein and sugars; built from 18 amino acids. They have species differences associated mainly with the set of amino acids. Their molecules are cylindrical in shape and are visible in an electron microscope. Up to 80% of immunoglobulins have a sedimentation constant of 7S; resistant to weak acids, alkalis, heating up to 60 ° C. Immunoglobulins can be isolated from blood serum using physical and chemical methods (electrophoresis, isoelectric precipitation with alcohol and acids, salting out, affinity chromatography, etc.). These methods are used in production for the preparation of immunobiological preparations.

Immunoglobulins according to their structure, antigenic and immunobiological properties are divided into five classes: IgM, IgG, IgA, IgE, IgD. Immunoglobulins M, G, A have subclasses. For example, IgG has four subclasses (IgG, IgG 2, IgG 3, IgG 4). All classes and subclasses differ in amino acid sequence.

Molecules of immunoglobulins of all five classes consist of polypeptide chains: two identical heavy chains H and two identical light chains L, interconnected by disulfide bridges. Accordingly, each class of immunoglobulins, i.e. M, G, A, E, D, there are five types of heavy chains: ? (mu), ? (gamma), ? (alpha), ? (epsilon) and? (delta), differing in antigenicity. Light chains of all five classes are common and come in two types: ? (kappa) and? (lambda); The L-chains of immunoglobulins of various classes can combine (recombine) with both homologous and heterologous H-chains. However, in the same molecule there can only be identical L-chains (? or?). Both the H- and L-chains have a variable - V region, in which the sequence of amino acids is not constant, and a constant - C region with a constant set of amino acids. In light and heavy chains, NH 2 - and COOH-terminal groups are distinguished.

During processing? -globulin with mercaptoethanol breaks down the disulfide bonds and the immunoglobulin molecule breaks down into separate chains of polypeptides. When exposed to the proteolytic enzyme papain, immunoglobulin is split into three fragments: two non-crystallizing fragments containing determinant groups for the antigen and called Fab fragments I and II and one crystallizing Fc fragment. FabI and FabII fragments are similar in properties and amino acid composition and differ from the Fc fragment; Fab and Fc fragments are compact formations connected to each other by flexible sections of the H-chain, due to which immunoglobulin molecules have a flexible structure.

Both H-chains and L-chains have distinct, linearly connected compact regions called domains; there are 4 of them in the H-chain, and 2 in the L-chain.

Active centers, or determinants, that form in the V regions occupy approximately 2% of the surface of the immunoglobulin molecule. Each molecule contains two determinants related to the hypervariable regions of the H and L chains, i.e., each immunoglobulin molecule can bind two antigen molecules. Therefore, antibodies are bivalent.

The typical structure of an immunoglobulin molecule is IgG. The remaining classes of immunoglobulins differ from IgG by additional elements of the organization of their molecules.

In response to the introduction of any antigen, antibodies of all five classes can be produced. Usually IgM is produced first, then IgG, the rest a little later.

No. 2 The causative agent of chlamydia. Taxonomy. Characteristics. Microbiological diagnostics. Treatment.

Taxonomy: order Chlamydiales, family Chlamydaceae, genus Chlamydia. The genus is represented by the species C.trachomatis, C.psittaci, C.pneumoniae.

Diseases caused by chlamydia are called chlamydia. Diseases caused by C. trachomatis and C. pneumoniae are anthroponoses. Ornithosis, caused by C. psittaci, is a zooanthroponotic infection.

Morphology of chlamydia: small, gram “-” bacteria, spherical in shape. They do not form spores, there are no flagella or capsules. Cell wall: 2-layer membrane. They have glycolipids. According to Gram - red. The main staining method is Romanovsky-Giemsa.

2 forms of existence: elementary bodies (inactive infectious particles, outside the cell); reticular bodies (inside cells, vegetative form).

Cultivation: Can only be propagated in living cells. In the yolk sac of developing chick embryos, in the body of sensitive animals and in cell culture

Enzyme activity: small. They ferment pyruvic acid and synthesize lipids. They are not able to synthesize high-energy compounds.

Antigenic structure: Antigens of three types: genus-specific thermostable lipopolysaccharide (in the cell wall). Detected using RSC; species-specific antigen of protein nature (in the outer membrane). Detected using RIF; variant-specific antigen of protein nature.

Pathogenicity factors. The proteins of the outer membrane of chlamydia are associated with their adhesive properties. These adhesins are found only in elementary bodies. Chlamydia produces endotoxin. Some chlamydia have a heat shock protein that can cause autoimmune reactions.

Resistance. High to various environmental factors. Resistant to low temperatures and drying. Sensitive to heat.

C. trachomatis is a causative agent of diseases of the human genitourinary system, eyes and respiratory tract.

Trachoma is a chronic infectious disease characterized by damage to the conjunctiva and cornea of the eyes. Anthroponosis. It is transmitted through contact and household contact.

Pathogenesis: affects the mucous membrane of the eyes. It penetrates the epithelium of the conjunctiva and cornea, where it multiplies, destroying cells. Follicular keratoconjunctivitis develops.

Diagnostics: examination of scrapings from the conjunctiva. In the affected cells, Romanovsky-Giemsa staining reveals violet cytoplasmic inclusions located near the nucleus - the Provacek body. To detect specific chlamydial antigen in affected cells, RIF and ELISA are also used. Sometimes they resort to cultivating trachoma chlamydia on chicken embryos or cell culture.

Treatment: antibiotics (tetracycline) and immunostimulants (interferon).

Prevention: Non-specific.

Urogenital chlamydia is a sexually transmitted disease. This is an acute/chronic infectious disease, which is characterized by primary damage to the genitourinary tract.

Human infection occurs through the mucous membranes of the genital tract. The main mechanism of infection is contact, the route of transmission is sexual.

Immunity: cellular, with the serum of infected people - specific antibodies. After an illness, it does not form.

Diagnostics: For eye diseases, the bacterioscopic method is used - intracellular inclusions are detected in scrapings from the conjunctival epithelium. To detect chlamydia antigen in affected cells, RIF is used. In case of damage to the genitourinary tract, a biological method can be used, based on infection of the cell culture with the test material (epithelial scrapings from the urethra, vagina).

RIF and ELISA can detect chlamydia antigens in the test material. Serological method - for the detection of IgM against C. trachomatis in the diagnosis of pneumonia in newborns.

Treatment. antibiotics (azithromycin from the macrolide group), immunomodulators, eubiotics.

Prevention. Only nonspecific (treatment of patients), personal hygiene.

Lymphogranuloma venereum is a sexually transmitted disease characterized by damage to the genital organs and regional lymph nodes. The mechanism of infection is contact, the route of transmission is sexual.

Immunity: persistent, cellular and humoral immunity.

Diagnostics: Material for research - pus, biopsy from affected lymph nodes, blood serum. Bacterioscopic method, biological (cultivation in the yolk sac of a chicken embryo), serological (RSC with paired sera is positive) and allergological (intradermal test with chlamydia allergen) methods.

Treatment. Antibiotics - macrolides and tetracyclines.

Prevention: Non-specific.

C. pneumoniae is the causative agent of respiratory chlamydia, causing acute and chronic bronchitis and pneumonia. Anthroponosis. Infection is by airborne droplets. They enter the lungs through the upper respiratory tract. Cause inflammation.

Diagnostics: staging RSC to detect specific antibodies (serological method). During primary infection, the detection of IgM is taken into account. RIF is also used to detect chlamydial antigen and PCR.

Treatment: This is done using antibiotics (tetracyclines and macrolides).

Prevention: Non-specific.

S. psittaci is the causative agent of ornithosis, an acute infectious disease characterized by damage to the lungs, nervous system and parenchymal organs (liver, spleen) and intoxication.

Zooanthroponosis. Sources of infection are birds. The mechanism of infection is aerogenic, the route of transmission is airborne. The causative agent is through mucus. shells breathe. pathways, into the epithelium of the bronchi, alveoli, multiplies, inflammation.

Diagnostics: Material for research - blood, sputum of the patient, blood serum for serological research.

A biological method is used - cultivating chlamydia in the yolk sac of a chicken embryo, in cell culture. Serological method. RSK, RPGA, ELISA are used using paired patient blood sera. Intradermal allergy test with ornithine.

Treatment: antibiotics (tetracyclines, macrolides).

TICKET#29

No. 1 The causative agent of diphtheria. Taxonomy and characteristics. Conditionally pathogenic corynebacteria. Microbiological diagnostics. Detection of anoxic immunity. Specific prevention and treatment.

Diphtheria is an acute infectious disease characterized by fibrinous inflammation in the pharynx, larynx, and less commonly in other organs and symptoms of intoxication. Its causative agent is Corynebacterium diphtheriae.

Taxonomy. Corynebacterium belongs to the division Firmicutes, genus Corynebacterium.

Morphological and tinctorial properties. The causative agent of diphtheria is characterized by polymorphism: thin, slightly curved rods (the most common) and coccoid and branching forms. Bacteria are often located at an angle to each other. They do not form spores, do not have flagella, and many strains have a microcapsule. A characteristic feature is the presence of volutin grains at the ends of the stick (causing a club-shaped shape). The causative agent of diphtheria stains positive on Gram.

Cultural properties. Facultative anaerobe, optimal. temperature. The microbe grows on special nutrient media, for example, on Clauberg's medium (blood tellurite agar), on which the diphtheria bacillus produces colonies of 3 types: a) large, gray, with uneven edges, radial striations, reminiscent of daisies; b) small, black, convex, with smooth edges; c) similar to the first and second.

Depending on the cultural and enzymatic properties, 3 biological variants of C.diphtheriae are distinguished: gravis, mitis and intermediate intermedius.

Enzyme activity. High. They ferment glucose and maltose into acid formation, but do not decompose sucrose, lactose and mannitol. They do not produce urease and do not form indole. Produces the enzyme cystinase, which breaks down cysteine to H 2 S. Forms catalase, succinate dehydrogenase.

Antigenic properties. O-antigens are thermostable polysaccharides located deep in the cell wall. K-antigens are superficial, thermolabile, grayish-specific. With the help of sera to the K-antigen C.diph. divided into serovars (58).

Pathogenicity factors. An exotoxin that disrupts protein synthesis and therefore affects the cells of the myocardium, adrenal glands, kidneys, and nerve ganglia. The ability to produce exotoxin is due to the presence in the cell of a prophage carrying the tox gene responsible for the formation of the toxin. Aggression enzymes - hyaluronidase, neuraminidase. The microcapsule is also a pathogenicity factor.

Resistance. It is resistant to drying and low temperatures, so it can remain on objects or in water for several days.

Epidemiology. The source of diphtheria is sick people. Infection occurs more often through the respiratory tract. The main route of transmission is airborne; contact route is also possible - through linen and dishes.

Pathogenesis. The entrance gates of infection are the mucous membranes of the pharynx, nose, respiratory tract, eyes, genitals, and wound surface. At the site of the entrance gate, fibrinous inflammation is observed, a characteristic film is formed, which is difficult to separate from the underlying tissues. The bacteria release an exotoxin that enters the bloodstream, causing toxinemia. The toxin affects the myocardium, kidneys, adrenal glands, and nervous system.

Clinic. There are different localization forms of diphtheria: diphtheria of the pharynx, which is observed in 85-90% of cases, diphtheria of the nose, larynx, eyes, external genitalia, skin, wounds. The incubation period ranges from 2 to 10 days. The disease begins with an increase in body temperature, pain when swallowing, the appearance of a film on the tonsils, and enlarged lymph nodes. Swelling of the larynx, diphtheria croup develops, which can lead to asphyxia and death. Other serious complications that can also cause death are toxic myocarditis and paralysis of the respiratory muscles.

Immunity. After the disease - persistent, intense antitoxic immunity. Of particular importance is the formation of antibodies to fragment B. They neutralize diphtheria histotoxin, preventing the latter from attaching to the cell. Antibacterial immunity – unstrengthened, grayish-specific

Microbiological diagnostics. Using a swab, film and mucus are taken from the patient’s throat and nose. To make a preliminary diagnosis, it is possible to use the bacterioscopic method. The main diagnostic method is bacteriological: culture on Klauber II medium (blood tellurite agar), on solid serum medium to detect cystinase production, on Hiss medium, on medium to determine the toxigenicity of the pathogen. Intraspecific identification consists of determining the bio- and serovar. For the accelerated detection of diphtheria toxin, the following are used: IRHA (indirect hemagglutination reaction) with an antibody erythrocyte diagnosticum, antibody neutralization reaction (the presence of the toxin is judged by the effect of preventing hemaggglutination); RIA (radioimmune assay) and ELISA (enzyme immunosorbent assay).

Treatment. The main method of therapy is the immediate administration of specific antitoxic antidiphtheria liquid horse serum. Human anti-diphtheria immunoglobulin for intravenous administration.

Associated vaccines: DPT (absorbed pertussis-tetanus vaccine), ADS (absorbed diphtheria-tetanus toxoid).

No. 2 Classes of immunoglobulins, their characteristics.

Immunoglobulins according to their structure, antigenic and immunobiological properties are divided into five classes: IgM, IgG, IgA, IgE, IgD.

Immunoglobulin class G. Isotype G makes up the bulk of Ig in blood serum. It accounts for 70-80% of all serum Ig, with 50% contained in tissue fluid. The average IgG content in the blood serum of a healthy adult is 12 g/l. The half-life of IgG is 21 days.

IgG is a monomer, has 2 antigen-binding centers (can simultaneously bind 2 antigen molecules, therefore, its valency is 2), a molecular weight of about 160 kDa and a sedimentation constant of 7S. There are subtypes Gl, G2, G3 and G4. Synthesized by mature B lymphocytes and plasma cells. It is well detected in blood serum at the peak of the primary and secondary immune response.

Has high affinity. IgGl and IgG3 bind complement, with G3 being more active than Gl. IgG4, like IgE, has cytophilicity (tropism, or affinity, for mast cells and basophils) and is involved in the development of type I allergic reaction. In immunodiagnostic reactions, IgG can manifest itself as an incomplete antibody.

Easily passes through the placental barrier and provides humoral immunity to the newborn in the first 3-4 months of life. It is also capable of being secreted into the secretions of mucous membranes, including into milk by diffusion.

IgG ensures neutralization, opsonization and marking of the antigen, triggers complement-mediated cytolysis and antibody-dependent cell-mediated cytotoxicity.

Immunoglobulin class M. The largest molecule of all Igs. This is a pentamer that has 10 antigen-binding centers, i.e. its valency is 10. Its molecular weight is about 900 kDa, its sedimentation constant is 19S. There are subtypes Ml and M2. The heavy chains of the IgM molecule, unlike other isotypes, are built from 5 domains. The half-life of IgM is 5 days.

It accounts for about 5-10% of all serum Igs. The average IgM content in the blood serum of a healthy adult is about 1 g/l. This level in humans is reached by the age of 2-4 years.

IgM is phylogenetically the most ancient immunoglobulin. Synthesized by precursors and mature B lymphocytes. It is formed at the beginning of the primary immune response, and is also the first to be synthesized in the body of a newborn - it is determined already at the 20th week of intrauterine development.

It has high avidity and is the most effective complement activator via the classical pathway. Participates in the formation of serum and secretory humoral immunity. Being a polymer molecule containing a J-chain, it can form a secretory form and be secreted into mucous secretions, including milk. Most normal antibodies and isoagglutinins are IgM.

Does not pass through the placenta. The detection of specific antibodies of the M isotype in the blood serum of a newborn indicates a former intrauterine infection or placental defect.

IgM ensures neutralization, opsonization and marking of the antigen, triggers complement-mediated cytolysis and antibody-dependent cell-mediated cytotoxicity.

Class A immunoglobulin. Exists in serum and secretory forms. About 60% of all IgA is contained in mucosal secretions.

Serum IgA: It accounts for about 10-15% of all serum Igs. The blood serum of a healthy adult contains about 2.5 g/l IgA, the maximum is reached by the age of 10. The half-life of IgA is 6 days.

IgA is a monomer, has 2 antigen-binding centers (i.e., 2-valent), a molecular weight of about 170 kDa and a sedimentation constant of 7S. There are subtypes A1 and A2. Synthesized by mature B lymphocytes and plasma cells. It is well detected in blood serum at the peak of the primary and secondary immune response.

Has high affinity. May be an incomplete antibody. Does not bind complement. Does not pass through the placental barrier.

IgA ensures neutralization, opsonization and marking of the antigen, and triggers antibody-dependent cell-mediated cytotoxicity.

Secretory IgA: Unlike serum, secretory sIgA exists in polymeric form in the form of a di- or trimer (4- or 6-valent) and contains J- and S-peptides. Molecular mass 350 kDa and higher, sedimentation constant 13S and higher.

It is synthesized by mature B-lymphocytes and their descendants - plasma cells of the corresponding specialization only within the mucous membranes and is secreted into their secretions. The production volume can reach 5 g per day. The slgA pool is considered the most numerous in the body - its quantity exceeds the total content of IgM and IgG. Not detected in blood serum.

The secretory form of IgA is the main factor in the specific humoral local immunity of the mucous membranes of the gastrointestinal tract, genitourinary system and respiratory tract. Thanks to the S-chain, it is resistant to proteases. slgA does not activate complement, but effectively binds to antigens and neutralizes them. It prevents the adhesion of microbes on epithelial cells and the generalization of infection within the mucous membranes.

Immunoglobulin class E. Also called reagin. The content in blood serum is extremely low - approximately 0.00025 g/l. Detection requires the use of special highly sensitive diagnostic methods. Molecular weight - about 190 kDa, sedimentation constant - approximately 8S, monomer. It accounts for about 0.002% of all circulating Igs. This level is reached by 10-15 years of age.

It is synthesized by mature B lymphocytes and plasma cells mainly in the lymphoid tissue of the bronchopulmonary tree and the gastrointestinal tract.

Does not bind complement. Does not pass through the placental barrier. It has a pronounced cytophilicity - tropism for mast cells and basophils. Participates in the development of immediate type hypersensitivity - type I reaction.

Immunoglobulin class D. There is not much information about Ig of this isotype. Almost completely contained in blood serum at a concentration of about 0.03 g/l (about 0.2% of the total circulating Ig). IgD has a molecular weight of 160 kDa and a sedimentation constant of 7S, monomer.

Does not bind complement. Does not pass through the placental barrier. It is a receptor for B-lymphocyte precursors.

TICKET#30

No. 1 Causative agent of amoebiasis. Taxonomy. Characteristic. Microbiological diagnostics. Specific treatment.

Taxonomy: phylum Sarcomastigophorae, subphylum Sarcodina, class Lobosia, order Amoebida.

Morphology: There are two stages of development of the pathogen: vegetative and cystic. The vegetative stage has several forms: large vegetative (tissue), small vegetative; precystic form, similar to the luminal one, forming cysts.

The cyst (resting stage) has an oval shape. A mature cyst contains 4 nuclei. The luminal form is inactive, lives in the lumen of the upper part of the large intestine as a harmless commensal, feeding on bacteria and detritus.

A large vegetative form is formed, under certain conditions, from a small vegetative form. It is the largest, forms pseudopodia and has movement. Can phagocytose red blood cells. Found in fresh feces during amoebiasis.

Cultivation: on nutrient-rich media.

Resistance: Outside the body, vegetative forms of the pathogen die quickly (within 30 minutes). Cysts are persistent in the environment and persist in feces and water. In food, vegetables and fruits, cysts persist for several days. They die when boiled.

Epidemiology: Amebiasis is an anthroponotic disease; the source of invasion is humans. The transmission mechanism is fecal-oral. Infection occurs when cysts are introduced through food, water, or household items.

Pathogenesis and clinic: Cysts that enter the intestine, and the luminal forms of amoebas that then form from them, can live in the large intestine without causing disease. When the body's resistance decreases, amoebas penetrate the intestinal wall and multiply. Intestinal amebiasis develops.

Tissue form trophozoites are motile due to the formation of pseudopodia. They penetrate the wall of the colon, causing necrosis; able to phagocytose red blood cells; can be found in human feces. With necrosis, ulcers form. Clinically, intestinal amebiasis manifests itself in the form of frequent, loose, bloody stools, accompanied by fever and dehydration. Pus and mucus, sometimes with blood, are found in the stool.

Amoebas with the bloodstream can enter the liver, lungs, and brain, resulting in the development of extraintestinal amoebiasis.

Immunity: Unstable, predominantly the cellular component is activated.

Microbiological diagnostics. The main method is a microscopic examination of the patient’s stool, as well as the contents of abscesses of internal organs. Smears are stained with Lugol's solution or hematoxylin. Serological tests (RNGA, ELISA, RSK): the highest titer of antibodies in the blood serum is detected with extraintestinal amebiasis.

Treatment: Metronidazole and furamide are used.

Prevention: identification and treatment of cyst excretors and amoeba carriers, carrying out general sanitary measures.

No. 2 Interferons. Nature, methods of production. Application.

Interferons are glycoproteins produced by cells in response to viral infection and other stimuli. They block the reproduction of the virus in other cells and participate in the interaction of cells of the immune system. There are two serological groups of interferons: type I - IFN-? and IFN -?; Type II - IFN-.? Type I interferons have antiviral and antitumor effects, while type II interferons regulate the specific immune response and nonspecific resistance.

Interferon (leukocyte) is produced by white blood cells treated with viruses and other agents. β-interferon (fibroblast) is produced by fibroblasts treated with viruses.

Type I IFN, by binding to healthy cells, protects them from viruses. The antiviral effect of type I IFN may also be due to the fact that it is able to inhibit cell proliferation by interfering with the synthesis of amino acids.

IFN-? produced by T lymphocytes and NK. Stimulates the activity of T- and B-lymphocytes, monocytes/macrophages and neutrophils. Induces apoptosis of activated macrophages, keratinocytes, hepatocytes, bone marrow cells, endothelial cells and suppresses apoptosis of peripheral monocytes and herpes-infected neurons.

Genetically engineered leukocyte interferon is produced in prokaryotic systems (Escherichia coli). Biotechnology for producing leukocyte interferon includes the following steps: 1) treatment of leukocyte mass with interferon inducers; 2) isolation of a mixture of mRNA from the treated cells; 3) obtaining total complementary DNA using reverse transcriptase; 4) insertion of cDNA into the E. coli plasmid and its cloning; 5) selection of clones containing interferon genes; 6) inclusion of a strong promoter in the plasmid for successful transcription of the gene; 7) interferon gene expression, i.e. synthesis of the corresponding protein; 8) destruction of prokaryotic cells and purification of interferon using affinity chromatography.

Interferons apply for the prevention and treatment of a number of viral infections. Their effect is determined by the dose of the drug, but high doses of interferon have a toxic effect. Interferons are widely used for influenza and other acute respiratory diseases. The drug is effective in the early stages of the disease and is applied topically. Interferons have a therapeutic effect against hepatitis B, herpes, and also against malignant neoplasms.

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Serological reactions are designated in accordance with the phenomena accompanying the formation of the antigen-antibody complex during the interaction of components with different properties. There are reactions of agglutination, precipitation and lysis.

Agglutination reaction (RA)

The agglutination reaction (RA) is based on the use of a corpuscular antigen (a suspension of bacteria, sensitized erythrocytes, latex particles, etc.) interacting with specific antibodies, as a result of which the resulting antigen-antibody complex precipitates. This reaction is widely used in laboratory practice for the serological diagnosis of bacterial infections and for the identification of isolated microorganisms.

RA is used to diagnose many infectious diseases: brucellosis (Wright, Heddleson reaction), tularemia, leptospirosis (RAL - Leptospira agglutination and lysis reaction), listeriosis, typhus (RAR - Rickettsia agglutination reaction), shigellosis, yersiniosis, pseudotuberculosis, etc.

Indirect or passive agglutination reaction (RIGA or RPGA).

To stage this reaction, red blood cells of animals (sheep, monkey, guinea pigs, some birds) sensitized with antibodies or antigen are used, which is achieved by incubating a suspension of red blood cells and a solution of antigen or immune serum.

Diagnosticums obtained on the basis of erythrocytes sensitized with antigens are called antigen erythrocyte diagnosticums. They are intended for the determination of antibodies in serial dilutions of blood sera, for example, erythrocyte shigella diagnosticums, erythrocyte salmonella O-diagnosticums.

Accordingly, diagnostics based on erythrocytes sensitized with specific immunoglobulins are called antibodies(immunoglobulin) diagnosticums and they serve to detect antigens in various materials, for example, erythrocyte immunoglobulin diphtheria diagnosticum for RIGA, used to detect diphtheria exotoxin of corynebacteria in a liquid nutrient medium when material from the nose and oropharynx is inoculated into it.

The hemagglutination reaction is used to diagnose both bacterial (typhoid fever, paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.) and viral (influenza, adenoviral infections, measles, etc.) infections. In terms of sensitivity and specificity, RIGA is superior to RA.

Hemagglutination inhibition reaction (HAI)

The hemagglutination inhibition reaction (HAI) is used to titrate antiviral antibodies in blood serum, as well as to establish the type of isolated viral cultures. RTGA can be used to diagnose those viral infections whose pathogens have hemagglutinating properties.

The principle of the method is that serum containing antibodies to a specific type of virus suppresses its hemagglutinating activity and the red blood cells remain non-agglutinated.

Passive hemagglutination inhibition (delay) reaction (RPHA).

Three components are involved in RTPGA: immune serum, antigen (test material) and sensitized erythrocytes.

If the test material contains an antigen that specifically reacts with the antibodies of the immune standard serum, then it binds them, and with the subsequent addition of erythrocytes sensitized with an antigen homologous to the serum, hemagglutination does not occur.

RTPHA is used to detect microbial antigens, for their quantitative determination, and also to control the specificity of RTPGA.

Latex agglutination reaction (RLA)

Latex particles are used as a carrier of antibodies (immunoglobulins). RLA is an express method for diagnosing infectious diseases, taking into account the time required (up to 10 minutes) and the ability to detect antigen in a small volume of test material.

RLA is used to indicate antigens of Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis in cerebrospinal fluid, to detect group A streptococci in throat swabs, to diagnose salmonellosis, yersiniosis and other diseases. The sensitivity of the method is 1-10 ng/ml, or 10³ -10⁶ bacterial cells in 1 µl.

Coagglutination reaction (CoA)

The coagglutination reaction (CoA) is based on the ability of protein A of staphylococci to attach specific immunoglobulins. RCA - a method of express diagnostics - serves to identify soluble thermostable antigens in human secretions and in the composition of circulating immune complexes (CIC). Detection of specific antigens in the composition of the CEC requires their preliminary precipitation from blood serum.

Precipitation reaction

In the precipitation reaction (RP), as a result of the interaction of antibodies with highly dispersed soluble antigens (proteins, polysaccharides), complexes are formed with the participation of complement - precipitates. It is a sensitive test used to detect and characterize a variety of antigens and antibodies. The simplest example of high-quality RP is the formation of an opaque precipitation band in a test tube at the boundary of the antigen layering on the immune serum - the ring precipitation reaction. Various types of RP in semi-liquid agar or agarose gels are widely used (double immunodiffusion method, radial immunodiffusion method, immunoelectrophoresis).

Complement fixation reaction (CFR)

The complement fixation reaction (CFR) is based on the phenomenon of hemolysis with the participation of complement, i.e. capable of detecting only complement-fixing antibodies.

RSC is widely used for the diagnosis of many bacterial and viral infections, rickettsial infections, chlamydia, infectious mononucleosis, protozoal infections, and helminthiases. RSC is a complex serological reaction in which two systems are involved: the test (blood serum), represented by the antigen-antibody and complement system, and the hemolytic (sheep red blood cells + hemolytic serum). Hemolytic serum is heat-inactivated rabbit blood serum immunized with sheep erythrocytes. It contains antibodies against sheep red blood cells.

A positive RSC result - the absence of hemolysis - is observed if the test serum contains antibodies homologous to the antigen. In this case, the resulting antigen-antibody complex binds complement, and in the absence of free complement, the addition of the hemolytic system is not accompanied by hemolysis. If there are no antibodies corresponding to the antigen in the serum, the formation of an antigen-antibody complex does not occur, complement remains free and the serum causes hemolysis of red blood cells, i.e. the presence of hemolysis is a negative result of the reaction.

Yushchuk N.D., Vengerov Yu.Ya.

Immune reactions used in diagnostic and immunological studies in sick and healthy people. For this purpose they use serological methods , i.e., methods for studying antibodies and antigens using antigen-antibody reactions determined in blood serum and other fluids, as well as body tissues.

Detection in serum The presence of antibodies against pathogen antigens allows a diagnosis of the disease to be made. Serological studies are also used to identify microbial antigens, various biologically active substances, blood groups, tissue and tumor antigens, immune complexes, cell receptors, etc.

When a microbe is isolated The pathogen is identified from the patient by studying its antigenic properties using immune diagnostic sera, i.e. blood sera of hyperimmunized animals containing specific antibodies. This is the so-called serological identification microorganisms.

Widely used in microbiology and immunology agglutination reactions, precipitation, neutralization, reactions involving complement, using labeled antibodies and antigens (radioimmunological, enzyme immunoassay, immunofluorescence methods). The listed reactions differ in the registered effect and production technique, however, they are all based on the interaction reaction of an antigen with an antibody and are used to detect both antibodies and antigens. Immune reactions are characterized by high sensitivity and specificity.

Features of the interaction of antibodies with antigens are the basis of diagnostic reactions in laboratories. Reaction in vitro between antigen and antibody consists of a specific and nonspecific phase. IN specific phase rapid specific binding of the active center of the antibody to the antigen determinant occurs. Then comes nonspecific phase - slower, which is manifested by visible physical phenomena, for example the formation of flocs (agglutination phenomenon) or precipitate in the form of turbidity. This phase requires the presence of certain conditions (electrolytes, optimal pH of the environment).

Immunodeficiencies, both primary and especially secondary, are widespread among people. They are the cause of many diseases and pathological conditions, and therefore require prevention and treatment with the help of immunotropic drugs.

34. Inactivated (particular) vaccines. Receipt. Application. Advantages. Flaws.

Inactivated (killed, corpuscular or molecular) vaccines– preparations that as an active principle include cultures of pathogenic viruses or bacteria killed by a chemical or physical method (cellular, virion) or antigen complexes extracted from pathogenic microbes containing protective antigens (subcellular, subvirion vaccines).

To isolate antigenic complexes (glycoproteins, LPS, proteins) from bacteria and viruses, trichloroacetic acid, phenol, enzymes, and isoelectric precipitation are used.

They are obtained by growing pathogenic bacteria and viruses on artificial nutrient media, inactivating them, isolating antigenic complexes, purifying them, and constructing them in the form of a liquid or lyophilic preparation.

The advantage of this type of vaccine is its relative ease of production (long study and isolation of strains is not required). Disadvantages include low immunogenicity, the need for three-time use and the high reactogenicity of formalized vaccines. Also, compared to live vaccines, the immunity they produce does not last long.

Currently, the following killed vaccines are used: typhoid, enriched with Vi antigen; cholera vaccine, pertussis vaccine.

To the question about express diagnostics:

1. A culture isolated in its pure form can be diagnosed.
2. In specially equipped laboratories (must have permission)
3. Compliance with strict rules such as: isolated room, required special protective suits, mandatory complete sanitary treatment of the room after working with the pathogen, sanitary treatment of researchers after finishing work.

Methods of expert diagnostics.
1. Bacteriology - combined polytropic nutrient media for quick study of morphs, tinctor, biochemical. properties. Use of enzyme indicator tape, electrophysical method, method of paper disks soaked in various substances (glucose, lactose, etc.)
2. Phage diagnostics.
3. Serodiagnosis - Mancini method, precipitation in gel according to Ascoli, RA, RPGA.
4. Bacterioscopy - direct and indirect RIF.

Express diagnostic methods for:
Cholera - M.Z. Ermolyeva, immobilization station with cholera diagnostic serum, RIF.
Tularemia - RA on glass, RPGA
Chume - phage typing, carbohydrate paper disk method, RPGA.
Sinus ulcer - Ascoli method, RIF, RPGA.

Growth pattern: there are three of them: diffuse (facultative anaerobes), bottom (obligate anaerobes) and surface (obligate aerobes.)

Isolation of a pure culture of anaerobic bacteria

In laboratory practice, you will often have to work with anaerobic microorganisms. They are more demanding of nutrient media than aerobes, more often require special growth additives, require the cessation of oxygen access during their cultivation, and their growth duration is longer. Therefore, working with them is more complex and requires significant attention from bacteriologists and laboratory technicians.

It is important to protect the material that contains anaerobic pathogens from the toxic effects of atmospheric oxygen. Therefore, it is recommended to take material from foci of purulent infection during puncture using a syringe; the time between taking the material and inoculating it on a nutrient medium should be as short as possible.

Since special nutrient media are used for cultivating anaerobic bacteria, which should not contain oxygen and have a low redox potential (-20 -150 mV), indicators are added to their composition - resazurin, methylene blue, etc., which react to a change in this potential. As it grows, the colorless forms of the indicators are restored and change their color: resazurin turns the medium pink, and methylene blue turns the medium blue. Such changes indicate the impossibility of using media for the cultivation of anaerobic microbes.

The introduction of at least 0.05% agar into the medium helps reduce the redox potential, which, by increasing its viscosity, helps reduce the supply of oxygen. This, in turn, is also achieved by using fresh (no later than two hours after production) and reduced nutrient media.

It should be taken into account that due to the peculiarities of the fermentative type of metabolism of anaerobic bacteria, they require environments richer in nutritional components and vitamins. The most commonly used are heart-brain and liver infusions, soy and yeast extracts, hydrolytic digest of casein, peptone, tryptone. It is mandatory to add growth factors such as Tween-80, hemin, menadione, whole or hemolyzed blood.

Isolation of a pure culture of aerobic microorganisms consists of a number of stages.
On the first day (stage 1 of the study), pathological material is taken into a sterile container (test tube, flask, bottle). It is studied for its appearance, consistency, color, smell and other characteristics, a smear is prepared, painted and examined under a microscope. In some cases (acute gonorrhea, plague), at this stage it is possible to make a previous diagnosis, and in addition, select the media on which the material will be inoculated. The occupancy is carried out with a bacteriological loop (used most often), using a spatula using the Drigalsky method, and a cotton-gauze swab. The cups are closed, turned upside down, signed with a special pencil and placed in a thermostat at the optimal temperature (37 ° C) for 18-48 years. The purpose of this stage is to obtain isolated colonies of microorganisms.
However, sometimes, in order to accumulate material, it is sown on liquid nutrient media.

Smears are prepared from suspicious colonies, stained using the Gram method to study the morphological and tinctorial properties of pathogens, and motile bacteria in a “hanging” or “crushed” drop are examined. These signs are of extremely great diagnostic value when characterizing certain types of microorganisms.
The remains of the colonies under study are carefully removed from the surface of the medium without touching others and inoculated onto slants of agar or onto sectors of a Petri dish with a nutrient medium to obtain a pure culture. Test tubes or dishes with cultures are placed in a thermostat at the optimal temperature for 18-24 hours.

Bacteria can also grow differently on liquid nutrient media, although the characteristics of growth manifestations are poorer than on solid media.

Bacteria are capable of causing diffuse turbidity of the medium, its color may not change or acquire the color of a pigment. This growth pattern is most often observed in most facultative anaerobic microorganisms.

Sometimes a precipitate forms at the bottom of the test tube. It can be crumbly, homogeneous, viscous, mucous, etc. The medium above it can remain transparent or become cloudy. If the microbes do not form pigment, the sediment has a bluish-blue or yellowish color. As a rule, anaerobic bacteria grow in a similar manner.

Parietal growth is manifested by the formation of flakes and grains attached to the inner walls of the test tube. The medium remains transparent.

Aerobic bacteria tend to grow superficially. A delicate, colorless or bluish film often forms in the form of a barely noticeable coating on the surface, which disappears when the medium is shaken or shaken. The film may be damp, thick, have a stringy, slimy consistency and stick to the loop, pulling behind it. However, there is also a dense, dry, brittle film, the color of which depends on the pigment produced by microorganisms.

If necessary, a smear is made, stained, examined under a microscope, and microorganisms are inoculated with a loop onto the surface of a solid nutrient medium to obtain isolated colonies.

On the third day (stage 3 of the study), the growth pattern of a pure culture of microorganisms is studied and its identification is carried out.

First, they pay attention to the characteristics of the growth of microorganisms on the medium and make a smear, staining it using the Gram method, in order to check the culture for purity. If bacteria of the same type of morphology, size and tinctorial (ability to dye) properties are observed under a microscope, it is concluded that the culture is pure. In some cases, based on the appearance and characteristics of their growth, one can draw a conclusion about the type of pathogens isolated. Determining the species of bacteria based on their morphological characteristics is called morphological identification. Determining the type of pathogen based on its cultural characteristics is called cultural identification.

However, these studies are not enough to make a final conclusion about the type of microbes isolated. Therefore, the biochemical properties of bacteria are studied. They are quite diverse.

Most often, saccharolytic, proteolytic, peptolytic, hemolytic properties, the formation of decarboxylase enzymes, oxidase, catalase, plasmacoagulase, DNase, fibrinolysin, the reduction of nitrates into nitrites, and the like are studied. For this purpose, there are special nutrient media that are inoculated with microorganisms (variegated Hiss series, MPB, curdled whey, milk, etc.).

Determining the type of pathogen based on its biochemical properties is called biochemical identification.

CULTIVATION METHODS
AND ISOLATION OF PURE CULTURE OF BACTERIA

For successful cultivation, in addition to correctly selected media and properly seeded seeds, optimal conditions are required: temperature, humidity, aeration (air supply). Cultivation of anaerobes is more difficult than aerobes; various methods are used to remove air from the nutrient medium.
Isolation of individual types of bacteria (pure culture) from the test material, which usually contains a mixture of various microorganisms, is one of the stages of any bacteriological study. A pure culture of microbes is obtained from an isolated microbial colony.
When isolating a pure culture from blood (hemoculture), it is first “grown” in a liquid medium: 10-15 ml of sterilely taken blood is inoculated into 100-150 ml of liquid medium. The ratio of inoculated blood to nutrient medium of 1:10 is not accidental - this is how blood dilution is achieved (undiluted blood has a detrimental effect on microorganisms).
Stages of isolating a pure culture of bacteria
Stage I (native material)
Microscopy (approximate idea of microflora).
Sowing on solid nutrient media (obtaining colonies).
Stage II (isolated colonies)
Study of colonies (cultural properties of bacteria).
Microscopic examination of microbes in a stained smear
(morphological properties of bacteria).
Sowing on nutrient agar slants to isolate a pure culture.
Stage III (pure culture)
Determination of cultural, morphological, biochemical
and other properties for identifying bacterial cultures
IDENTIFICATION OF BACTERIA

Identification of isolated bacterial cultures is carried out by studying the morphology of bacteria, their cultural, biochemical and other characteristics inherent in each species.

Related information.