HIV infection
HIV infection is a disease caused by the human immunodeficiency virus (HIV), persisting for a long time in lymphocytes, macrophages, cells of the nervous tissue, resulting in a slowly progressive damage to the immune and nervous systems of the body, manifested by secondary infections, tumors, subacute encephalitis and other pathological changes.
The causative agents are human immunodeficiency viruses of the t and 2 types - HIV-1, HIV-2 (HIV-I, HIV-2, Human Immunodeficiency viruses, types I, 11) - belong to the family of retro viruses, the subfamily of slow viruses ... Virions are spherical particles 100-140 nm in diameter. The viral particle has an outer phospholipid membrane containing glycoproteins (structural proteins) with a certain molecular weight, measured in kilodaltons. In HIV-1, these are gp 160, gp 120, gp 41. The inner envelope of the virus covering the nucleus is also represented by proteins with a known molecular weight - p 17, p 24, p 55 (HIV-2 contains gp 140, gp 105, gp 36, p 16, p 25, p 55).
The HIV genome includes RNA and a reverse transcriptase (reverse transcriptase) enzyme. In order for the retrovirus genome to connect with the host cell genome, DNA is first synthesized on the viral RNA template using reverse transcriptase. Then the provirus DNA is inserted into the genome of the host cell. HIV has a pronounced antigenic variability, significantly exceeding that of the influenza virus.
In the human body, the main target of HIV is T-lymphocytes, which carry the largest number of CD4 receptors on the surface. After HIV penetrates into the cell with the help of reverse transcriptase, following the pattern of its RNA, the virus synthesizes DNA, which is integrated into the genetic apparatus of the host cell (T-lymphocytes) and remains there for life in a provirus state. In addition to T-helper lymphocytes, macrophages, B-lymphocytes are affected. cells of neuroglia, intestinal mucosa and some other cells. The reason for the decrease in the number of T-lymphocytes (CD4 cells) is not only the direct cytopathic effect of the virus, but also their fusion with uninfected cells. Along with the defeat of T-lymphocytes in patients with HIV infection, there is a polyclonal activation of B-lymphocytes with an increase in the synthesis of immunoglobulins of all classes, especially IgG and IgA, and the subsequent depletion of this part of the immune system. Impaired regulation of immune processes is also manifested by an increase in the level of alpha-interferon, beta-2-microglobulin a, and a decrease in the level of interleukin-2. As a result of dysfunction of the immune system, especially when the number of T-lymphocytes (CD4) decreases to 400 or less cells in 1 μl of blood, conditions arise for uncontrolled HIV replication with a significant increase in the number of virions in various environments of the body. As a result of the defeat of many parts of the immune system, a person infected with HIV becomes defenseless against pathogens of various infections. In the lobby of increasing immunosuppression, severe progressive diseases develop that do not occur in a person with a normally functioning immune system. These diseases are defined by WHO as AIDS marker (indicator).
The first group - diseases that are inherent only in severe immunodeficiency (CD4 count below 200). Clinical diagnosis is made in the absence of anti-HIV antibodies or HIV antigens.
The second group - diseases that develop both against the background of severe immunodeficiency, and in some cases without it. Therefore, in such cases, laboratory confirmation of the diagnosis is necessary.
Serological methods for the diagnosis of viral infections. Inhibition of hemagglutination. Inhibition of the cytopathic effect by viral interference. Direct immunofluorescence. Immunoelectron microscopy.
With the majority viral infections develop immune responses used to diagnostics... Cellular responses are usually assessed in tests of the cytotoxicity of lymphocytes against infectious agents or target cells infected by them, or the ability of lymphocytes to respond to various Ag and mitogens is determined. In the work of practical laboratories, the severity of cellular reactions is rarely determined. Methods for identifying antiviral AT 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, is introduced into the cell monolayer. The absence of cell death indicates a discrepancy between the infectious agent and the known AT.
Inhibition of hemagglutination
RTGA is used to identify viruses capable of agglutinating various erythrocytes. For this, the culture medium containing the pathogen is mixed with a known commercial antiserum and introduced into the cell culture. After incubation, the ability of the culture to hemagglutination is determined and, in its absence, a conclusion is made about the inconsistency of the virus with 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. For this, commercial serum is introduced into the culture medium containing the virus under study (for example, for rubella virus, if it is suspected), the second culture is incubated and infected; after 1-2 days, a known cytopathogenic virus (for example, any ECHO virus) is introduced into it. In the presence of a cytopathogenic effect, it is concluded that the first culture was infected with a virus corresponding to the applied AT.
Direct immunofluorescence
Among other tests, the most common was found direct immunofluorescence reaction(the fastest, most sensitive and reproducible). For example, identification of CMV by cytopathogenic effect requires at least 2-3 weeks, and when using labeled monoclonal AT, identification is possible after 24 hours. investigate using fluorescent microscopy (allows you to detect the presence of fluorescence of infected cells).
Immunoelectron microscopy
Immunoelectron microscopy(an analogue of the previous method) allows one to identify various types of viruses detected by electron microscopy (for example, various 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 application.
119. Serological tests used to diagnose viral infections.
Detection in serum a patient with antibodies against the antigens of the pathogen makes it possible to diagnose the disease. Serological studies are also used to identify antigens of microbes, 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, that is, blood sera of hyperimmunized animals containing specific antibodies. This is the so-called serological identification microorganisms.
In microbiology and immunology, they are widely used reactions of agglutination, precipitation, neutralization, reactions involving complement, using labeled antibodies and antigens (radioimmunological, enzyme immunoassay, immunofluorescence methods). The listed reactions differ in the recorded effect and the technique of staging, however, they are all based on the reaction of interaction of an antigen with an antibody and are used to detect both antibodies and antigens. Immunity reactions are characterized by high sensitivity and specificity.
Features of the interaction of antibodies with antigen are the basis of diagnostic reactions in laboratories. Reaction in vitro between antigen and antibody consists of a specific and non-specific phase. V specific phase there is a rapid specific binding of the active site of the antibody to the antigen determinant. Then comes nonspecific phase - slower, which manifests itself in visible physical phenomena, for example, the formation of flakes (agglutination phenomenon) or precipitation 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 bound antigen by antibodies depend on the affinity, avidity of antibodies and their valence.
To the question about express diagnostics:
1. The culture isolated in its pure form lends itself to diagnostics. 2. In Specially equipped laboratories (permission must be obtained) 3. Compliance with strict rules such as: an isolated room, the necessary special protective suits, mandatory complete sanitization of the room after working with the pathogen, sanitization of researchers after the end of work. Methods of expert diagnostics. 1. Bacteriology - combined polytropic nutrient media for the rapid study of morphs, tinctor, biochem. properties. Use of enzyme indicator tape, electrophysical method, method of paper disks impregnated with various substances (glucose, lactose, etc.) 2. Phagodiagnostics. 3. Serodiagnostics - Mancini's method, raction of precipitation in gel according to Ascoli, RA, RPGA. 4. Bacterioscopy - direct and indirect RIF. Methods of express diagnostics for: Cholera - M.Z. Ermolyeva, district of immobilization with cholera diagnostic serum, RIF. Tularemia - RA on glass, RPGA Plague - phage typing, method of carbohydrate paper disks, RPGA. Sib.yazva - Ascoli method, RIF, RPGA. Growth pattern: there are three diffuse (facultative anaerobes), bottom (obligate anaerobes) and superficial (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 whimsical to nutrient media than aerobes, they often need special growth supplements, require the cessation of oxygen access during their cultivation, their growth duration is longer. Therefore, working with them is more complicated and requires considerable attention from bacteriologists and laboratory assistants.
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 their puncture with a syringe; the time between taking the material and sowing it on the nutrient medium should be as short as possible.
Since special nutrient media are used for the cultivation of anaerobic bacteria, which should not contain oxygen and have a low redox potential (-20 -150 mV), indicators - resazurin, methylene blue and the like, are introduced to their composition, which react to a change in this potential. With its growth, colorless forms of indicators are renewed and change their color: resazurin stains the medium pink, and methylene blue - blue. Such changes indicate the impossibility of using media for the cultivation of anaerobic microbes.
It helps to reduce the redox potential of the introduction of at least 0.05% agar into the medium, which, by increasing its viscosity, helps to 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 nutrients and vitamins. The most commonly used are cardio-cerebral and hepatic infusions, soy and yeast extracts, hydrolytic digest of casein, peptone, tryptone. It is imperative 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 by its appearance, texture, color, smell and other signs, 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, choose the medium on which the material will be inoculated. It took a bacteriological loop (most often used), using a spatula following the Drygalsky method, with a cotton-gauze swab. The cups are closed, turned upside down, signed with a special pencil and placed in a thermostat at the optimum temperature (37 ° C) for 18-48 years. The goal of this stage is to obtain isolated colonies of microorganisms. However, sometimes in order to pile up the 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 are examined in a “hanging” or “crushed” drop. These signs are of extremely great diagnostic value in the characterization of certain types of microorganisms. The remains of the investigated colonies, carefully, without touching others, are removed from the surface of the medium and inoculated on slant agar or on sectors of Petri dishes with a nutrient medium to obtain a pure culture. Test tubes or culture dishes are placed in a thermostat at the optimum temperature for 18-24 hours.
On liquid culture media, bacteria can also grow in different ways, although the features of the manifestations of growth are poorer than on dense ones.
Bacteria can cause diffuse clouding of the medium, while 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 sediment forms at the bottom of the test tube. It can be crumbly, homogeneous, viscous, slimy, etc. The environment above it can remain transparent or become cloudy. If microbes do not form pigment, the precipitate has a syruvate-bilium or yellowish color. As a rule, anaerobic bacteria grow in a similar rank.
Partial growth is manifested by the formation of flakes, grains attached to the inner walls of the test tube. At the same time, the medium remains transparent.
Aerobic bacteria tend to grow superficially. A delicate, colorless or bluish film is often formed in the form of a barely noticeable coating on the surface, which disappears when the medium is shaken off or agitated. The film can be moisture, thick, have a bundle, a slimy consistency and stick to the loop, stretches behind it. However, there is also a dense, dry, fragile 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 on the surface of a dense nutrient medium to obtain isolated colonies.
On the third day (stage 3 of the study), the nature of the growth of a pure culture of microorganisms is studied and its identification is carried out.
First, they pay attention to the peculiarities of the growth of microorganisms on the medium and make a smear, painting 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 paint) properties are observed under a microscope, it is concluded that the culture is pure. In some cases, already behind the appearance and features of their growth, it is possible to draw a conclusion about the type of pathogens isolated. Determining the type of bacteria for their morphological characteristics is called morphological identification. Determining the type of pathogens for their cultural characteristics is called cultural identification.
However, these studies are not enough to make a final conclusion about the type of isolated microbes. Therefore, they study the biochemical properties of bacteria. They are quite diverse.
Most often, saccharolytic, proteolytic, peptolytic, hemolytic properties, the formation of enzymes of decarboxylases, oxidase, catalase, plasmocoagulase, DNase, fibrinolysin, reduction of nitrates to nitrites, and the like are studied. For this, there are special nutrient media that are inoculated with microorganisms (variegated row of Giss, MPB, curdled whey, milk, etc.).
Determining the type of pathogen by its biochemical properties is called biochemical identification.
METHODS OF CULTIVATION AND ISOLATION OF A PURE BACTERIA CULTURE For successful cultivation, in addition to correctly selected media and correctly sowed, optimal conditions are necessary: temperature, humidity, aeration (air supply). The cultivation of anaerobes is more difficult than aerobes; various methods are used to remove air from the nutrient medium. Isolation of certain types of bacteria (pure culture) from the test material, which, as a rule, 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 a pure culture is isolated from blood (blood culture), it is preliminarily "grown" in a liquid medium: 10-15 ml of sterile blood is inoculated into 100-150 ml of a liquid medium. The ratio of inoculated blood and culture medium 1:10 is not accidental - this is how blood dilution is achieved (undiluted blood has a detrimental effect on microorganisms). Stages of isolation of a pure culture of bacteria Stage I (native material) Microscopy (rough idea of the 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). Inoculate on nutrient agar slant to isolate pure culture. Stage III (pure culture) Determination of cultural, morphological, biochemical and other properties for identification of bacterial culture 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.
It is based on the determination of antiviral antibodies in the patient's blood in serological reactions using specific viral antigens - diagnosticums or special test systems. Serological reactions for viral infections are performed in a liquid medium (RSK, RTGA, RNGA, RONGA, RTONGA, RIA), in a gel (RPG, RRG, RVIEF) or on a solid-phase carrier (for example, on the walls of the well of a polystyrene plate with fixation of one of the components of the immune response - antigen or antibody). Such solid-phase methods as ELISA, IEM, RGadsTO, RIF, RGads, RTGads are known.
Often, due to the presence of natural antiviral antibodies in the blood of most healthy people, serological diagnosis of viral infections is based on research paired serums, taken at the beginning and in the midst of the disease or during the period of convalescence in order to determine the increase in antibody titer. An increase in antibody titer by four times or more is considered diagnostically significant.
Increasing the sensitivity of serological methods is achieved by the adsorption of antigens or antibodies on erythrocytes (RNGA, RONGA, RTONGA, RGadsTO, RRG), labeling with enzymes (ELISA), radioactive isotopes (RIA, RPG) or fluorochromes (RIF), The principle of lysis of erythrocytes is also used ( systems) in the interaction of antigens and antibodies in the presence of complement (RSK, RRG).
Complement fixation reaction (CBC) as a variant of complement binding in the cold (overnight at + 4 ° C) is often used in virology for retrospective diagnosis of a number of viral infections and for the determination of virus-specific antigens in materials from patients.
Radial hemolysis reaction (RHR) in agarose gel is based on the phenomenon of hemolysis of erythrocytes sensitized with an antigen, under the influence of virus-specific antibodies in the presence of complement and is used for serological diagnosis of influenza, ARVI, rubella, mumps, togavirus infections.
To formulate the reaction to lamb erythrocytes (0.3 ml of 10% suspension) add 0.1 ml of undiluted viral antigen and the mixture is kept for 10 minutes at room temperature. 0.3 ml of sensitized erythrocytes and 0.1 ml of complement are added to 1.2% agarose at a temperature of 42 ° C, the mixture is poured onto glass slides or into the wells of polystyrene plates, holes are cut out in the frozen agarose gel using a punch and filled with the investigated and control sera. The glasses or panels are covered with a lid and placed in a humid chamber for 16-18 hours in a thermostat. The reaction is taken into account by the diameter of the hemolysis zone around the holes filled with serum. There is no hemolysis in the control.
Laboratory diagnostics
UDC -078
Laboratory diagnostics of viral infections
N.N. Spout, V.M. Stakhanov
Institute of Virology named after DI. Ivanovsky RAMS, Moscow
Laboratory Diagnosis of Viral Infections
N.N. Nosik, V.M. Stachanova
Introduction
Expanding opportunities in the treatment and prevention of viral diseases using antiviral drugs, immunomodulators and vaccines with different mechanisms of action requires fast and accurate laboratory diagnostics. The narrow specificity of some antiviral drugs also requires a quick and highly specific diagnosis of the infectious agent. There is a need for quantitative methods for the determination of viruses for monitoring antiviral therapy. In addition to establishing the etiology of the disease, laboratory diagnostics is important in organizing anti-epidemic measures.
Early diagnosis of the first cases of epidemic infections allows timely implementation of anti-epidemic measures - quarantine, hospitalization, vaccination, etc. The implementation of programs to eliminate infectious diseases, such as smallpox, has shown that, as they are implemented, the role of laboratory diagnostics increases. Laboratory diagnostics in the blood service and obstetric practice plays an essential role, for example, the identification of donors infected with human immunodeficiency virus(HIV), hepatitis B virus (HBV), diagnosis of rubella and cytomegalovirus infection in pregnant women.
Diagnostic methods
There are three main approaches to laboratory diagnosis of viral infections (Table 1, Table 2):
1) direct examination of the material for the presence of viral antigen or nucleic acids;
2) isolation and identification of the virus from clinical material;
3) serological diagnostics based on the establishment of a significant increase in viral antibodies during the course of the disease.
With any chosen approach to viral diagnostics, one of the most important factors is the quality of the material under study. So, for example, for direct analysis of a sample or for isolation of a virus, the test material must be obtained at the very beginning of the disease, when the pathogen is still excreted in relatively large quantities and is not yet bound by antibodies, and the sample volume must be sufficient for direct research. It is also important to select the material in accordance with the alleged disease, that is, the material in which, based on the pathogenesis of the infection, the probability of the presence of the virus is greatest.
An important role in successful diagnostics is played by the environment in which the material is taken, how it is transported and how it is stored. So, nasopharyngeal or rectal smears, the contents of the vesicles are placed in a medium containing a protein that prevents the rapid loss of the infectivity of the virus (if it is planned to isolate it), or in an appropriate buffer (if it is planned to work with nucleic acids).
Direct methods for the diagnosis of clinical material
Direct methods are methods that detect a virus, viral antigen or viral nucleic acid(NC) directly in the clinical material, that is, they are the fastest (2-24 hours). However, due to a number of peculiarities of pathogens, direct methods have their own limitations (the possibility of obtaining false positive and false negative results). Therefore, they often require confirmation by indirect methods.
Electron microscopy (EM). Using this method, you can detect the actual virus. For successful determination of the virus, its concentration in the sample should be approximately 1 · 10 6 particles in 1 ml. But since the concentration of the pathogen, as a rule, in the material from patients is insignificant, the search for the virus is difficult and requires its preliminary precipitation using high-speed centrifugation followed by negative contrast. In addition, EM does not allow viruses to be typed, since many of them do not have morphological differences within the family. For example, herpes simplex, cytomegaly, or herpes zoster viruses are morphologically virtually indistinguishable.
One of the EM variants used for diagnostic purposes is immune electron microscopy(IEM), which uses specific antibodies to viruses. As a result of the interaction of antibodies with viruses, complexes are formed, which, after negative contrasting, are easier to detect.
IEM is somewhat more sensitive than EM and is used in cases where the virus cannot be cultured in vitro, for example, when searching for pathogens of viral hepatitis.
Immunofluorescence reaction (RIF). The method is based on the use of antibodies bound to a dye, for example, fluorescein isothiocyanate. RIF is widely used to detect viral antigens in patients' material and for rapid diagnosis.
In practice, two RIF options are used: straight and indirect... In the first case, dye-labeled antibodies to viruses are applied, which are applied to infected cells (smear, cell culture). Thus, the reaction proceeds in one step. The disadvantage of this method is the need to have a large set of conjugated specific sera for many viruses.
In the indirect version of RIF, a specific serum is applied to the test material, the antibodies of which bind to the viral antigen contained in the material, and then an anti-species serum is layered to the gamma globulins of the animal in which the specific immune serum was prepared, for example, anti-rabbit, anti-horse, etc. Advantage indirect version of RIF consists in the need for only one type of labeled antibodies.
The RIF method is widely used to quickly decipher the etiology of acute respiratory viral infections in the analysis of smear-prints from the mucous membrane of the upper respiratory tract. The successful use of RIF for the direct detection of a virus in clinical material is possible only if it contains a sufficiently large number of infected cells and a slight contamination with microorganisms that can produce nonspecific luminescence.
Enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assays for the determination of viral antigens are, in principle, similar to RIF, but are based on labeling antibodies with enzymes rather than dyes. The most widely used are horseradish peroxidase and alkaline phosphatase; -galactosidase and -lactamases are also used. The labeled antibodies bind to the antigen, and such a complex is detected by adding a substrate for the enzyme to which the antibodies are conjugated. The end product of the reaction can be in the form of an insoluble precipitate, and then the accounting is carried out using a conventional light microscope, or in the form of a soluble product, which is usually colored (or can fluoresce or luminesce) and is recorded instrumentally.
Since ELISA can measure soluble antigens, there is no need for intact cells in the sample and thus different types of clinical material can be used.
Another important advantage of the ELISA method is the ability to quantify antigens, which makes it possible to use it to assess the clinical course of the disease and the effectiveness of chemotherapy. ELISA, like RIF, can be used both in direct and indirect versions.
Solid-phase ELISA, which gives a soluble colored reaction product, is most widely used. ELISA can be used both to determine antigen (then antibodies are applied to the solid phase - the bottom of the well of a polystyrene plate), and to determine antibodies (then antigens are applied to the solid phase).
Radioimmunoassay (RIA) ... The method is based on the labeling of antibodies with radioisotopes, which provided high sensitivity in the determination of viral antigen. The method became widespread in the 80s, especially for the determination of markers of HBV and other unculturable viruses. The disadvantages of the method include the need to work with radioactive substances and the use of expensive equipment (gamma counters).
Molecular methods. Initially, the classical method for detecting the viral genome was considered a highly specific method of NK hybridization, but nowadays, the isolation of viral genomes using polymerase chain reaction(PCR).
Molecular hybridization of nucleic acids. The method is based on hybridization of complementary strands of DNA or RNA with the formation of double-stranded structures and on their identification using a label. For this purpose, special DNA or RNA probes are used, labeled with an isotope (32 P) or biotin, which detect complementary strands of DNA or RNA. There are several variants of the method: - point hybridization - isolated and denatured NCs are applied to filters and then a labeled probe is added; indication of results - autoradiography when using 32 P or staining - with avidin-biotin; - blot hybridization - a method for isolating NK fragments cut with restriction endonucleases from total DNA and transferred to nitrocellulose filters and tested with labeled probes; used as a confirmatory test for HIV infection; - hybridization in situ- allows the determination of NK in infected cells.
PCR based on the principle of natural DNA replication. The essence of the method lies in the repeated repetition of cycles of synthesis (amplification) of the virus-specific DNA sequence using thermostable Taq DNA polymerase and two specific primers - the so-called primers.
Each cycle consists of three stages with different temperature conditions. In each cycle, the number of copies of the synthesized section is doubled. Newly synthesized DNA fragments serve as a template for the synthesis of new strands in the next amplification cycle, which allows for 25–35 cycles to generate a sufficient number of copies of the selected DNA region for its determination, usually by electrophoresis in agarose gel.
The method is highly specific and very sensitive. It allows you to detect multiple copies of viral DNA in the test material. In recent years, PCR has been increasingly used for the diagnosis and monitoring of viral infections (hepatitis viruses, herpes, cytomegaly, papilloma, etc.).
A variant of quantitative PCR has been developed, which makes it possible to determine the number of copies of the amplified DNA site. The technique is complex, expensive and not yet sufficiently standardized for routine use.
Cytological methods currently have limited diagnostic value, but should still be used for a number of infections. Materials of autopsy, biopsy, smears are examined, which, after appropriate processing, are stained and analyzed under a microscope. In case of cytomegalovirus infection, for example, in tissue sections or in urine, characteristic giant cells - "owl's eye" are found, in case of rabies - inclusions in the cytoplasm of cells (Babesh-Negri's little bodies). In some cases, for example, in the differential diagnosis of chronic hepatitis, it is important to assess the state of the liver tissue.
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