Cell cultures

Animals

Indication of viruses based on the following phenomena

Developmental delay

death, change

membranes of the embryo, RGA

CPP, plaque formation, RIF, RGads, interference

Illness, death,

histological changes

in tissues, inclusions

Titration of the isolated virus; selection of working dose.

Virus titer- maximum dilution of the virus-containing material, in which the expected effect is still observed (CPE, RGA, death of the animal).

Identification of the isolated virus in neutralization reactions, RTGads, RSC, suppression of plaque formation, etc. with diagnostic sera. Type (type) of virus determined by neutralization of the specific effect of the virus by the appropriate immune serum.

Note: Titration and virus identification are performed using the same phenomenon.

Virus cultivation

KABARDINO-BALKARIAN STATE

UNIVERSITY named after. Kh.M.BERBEKOVA

_________________________________________________________________

FEATURES OF RNA VIRAL AND DNA VIRAL

INFECTIONS
Methodological recommendations for studying theoretical material

on the course “Private medical virology”

for foreign students
For specialty 060101 – General Medicine

Nalchik - 2010

BBK 52.63ya73

Reviewer:

Candidate of Biological Sciences, Senior Lecturer, Department of Microbiology, Hygiene and Sanitation, Kabardino-Balkarian State

agricultural academy

M.H. Pezheva

Compiled by: Blieva Larisa Zaurbekovna

Features of RNA viral and DNA viral infections: Methodological recommendations for studying theoretical material in the course “Private Medical Virology” for foreign students - Nalchik: Kab.-Balk. univ., 2010.- 48 p.

The methodological recommendations present the goals and objectives of each topic, theoretical information on private virology, and requirements for the level of preparedness of students. For each topic, test questions, situational tasks and a list of recommended literature are given. The work contains a glossary of basic concepts and definitions in private virology.

The publication is intended for foreign students studying in the 3rd year of the specialty “General Medicine”.

UDC 576.858(075.8)

BBK 52.63ya73

State University, 2010

The course “Private Medical Virology” is taught to 3rd year students of the Faculty of Medicine and is an integral part of the discipline “Microbiology, Virology, Immunology”. The total volume of classroom hours in the discipline is 184. 11 hours are allocated for medical virology, of which 2 hours for lectures and 3 hours for 3 laboratory classes. These guidelines have been developed on topics relevant to laboratory work. The existing laboratory workshop does not contain all the necessary material for mastering these topics.

It is difficult for foreign students to master a large amount of information in 3 laboratory classes. The author considered it appropriate to present in this publication a brief description of the causative agents of viral infections and the pathogenesis of the diseases they cause.

A list of control questions is given for each topic; situational tasks that will allow students to assess their preparedness on the topic covered; list of recommended literature.

The reference part of the publication contains a description of the basic concepts and terms used in modern virology and the structure of the most common viruses. The alphabetical order of presentation of the terminological dictionary-reference book is convenient for use.
TOPIC 1. RNA viruses

Target– study of the structural features of RNA-containing viruses and the pathogenesis of the diseases they cause.

Tasks:

1 – know the systematic position of each pathogen;

2 – know the morphology and structure of pathogens;

3 – study the pathogenesis of all diseases caused by these pathogens according to the plan: a) source of infection; b) methods of infection; c) entrance gate of infection; d) stages of pathogenesis;

4 – know methods of laboratory diagnosis of the disease;

5 – know specific prevention and specific therapy;

6 – be able to differentiate between different viruses;

7 – be able to solve situational problems on the topic;

8 – master the preparation of situational problems on the topic.
Characteristics of RNA viruses

Lesson 1

Family Orthomyxoviridae (from the Greek orthos - straight, myxa - mucus)

Genus 1 - Influensavirus A, B

Influenza A virus

Influenza B virus

Genus 2 - Influensavirus C

Influenza virus type C

Features of the pathogenesis of influenza

Source – sick person, carrier; The method of infection is airborne. The incubation period is 1-3 days. The prodromal period is general malaise, a feeling of weakness. The main symptoms are a rapid increase in temperature to 37.5-38 0 C with accompanying myalgia, runny nose, cough, headaches; The duration of the febrile period is 3-5 days. The influenza A virus is neurotropic, so the development of neurotoxicosis is possible. Catarrh of the upper respiratory tract develops (dry cough, chest pain, rhinitis). Hemorrhagic pneumonia and pulmonary edema are possible, resulting in rapid death. Rarely and more often in children there is abdominal syndrome (abdominal pain, nausea, vomiting, diarrhea).

Laboratory diagnostics

Express diagnostics. Viral antigens are detected in the test material (nasopharyngeal discharge) using the immunofluorescence reaction (RIF) (direct and indirect versions) and enzyme-linked immunosorbent assay (ELISA). It is possible to detect the genome of viruses in the material using polymerase chain reaction (PCR).

Virological method. HeLa-2 cell cultures are infected, and microscopy within 24 hours reveals finely focal degranulation in the cell culture. Indication of viruses is also carried out by the formation of “plaques”, “color test”, hemagglutination reaction and hemadsorption reaction. Viruses are identified by their antigenic structure. The complement fixation reaction, the hemagglutination inhibition reaction and the biological neutralization reaction of viruses are used.

Serological method. The diagnosis is made by a fourfold increase in antibody titer in paired sera from the patient, obtained with an interval of 10-14 days. When setting up a reaction to neutralize the cytopathic effect, a positive result is noted when adding type A serum. The hemagglutination inhibition reaction, complement fixation reaction, immunofluorescence reaction, and enzyme immunoassay are used. Specific prevention – live attenuated vaccine, killed vaccine, split vaccines, chemical vaccine. Specific therapy is anti-influenza γ-globulin.

Family Paramyxoviridae (from Latin para - about)

Genus 1 – Respirovirus – parainfluenza viruses types 1 and 3

Genus 2 – Rubulavirus – mumps and parainfluenza viruses types 2 and 4

Genus 3 – Morbilivirus – measles virus

Genus 4 – Pneumovirus – respiratory syncytial virus (RS virus)

Features of the pathogenesis of parainfluenza

Source – sick person, carrier; The method of infection is airborne. Incubation period – 3-6 days; in adults - in the form of catarrh of the upper respiratory tract (laryngitis); in children - it is more severe, often with symptoms of intoxication; laryngotracheobronchitis with the development of false croup is most often observed; in children under one year of age – bronchiolitis with pneumonia.

Laboratory diagnostics

Virological method. Mucus or respiratory tract effluent and sputum are taken from the patient and the cell culture is infected. Indication is carried out by the cytopathic effect of viruses and the hemagglutination reaction. Identification is carried out using the hemagglutination inhibition reaction, the complement fixation reaction, and the neutralization reaction.

Serological method. To identify virus antigens and to detect antibodies in paired patient blood sera, a hemagglutination inhibition reaction, a complement fixation reaction, and a neutralization reaction are performed (retrospective diagnostics). Specific prevention and specific therapy are absent.

Features of the pathogenesis of mumps, or “mumps”

The source is a sick person; The method of infection is airborne. The incubation period is 14-21 days. The typical form of the disease manifests itself as unilateral or bilateral parotitis, accompanied by fever. Infection of the parotid salivary glands occurs through hematogenous spread of the virus, which occurs after 3-5 days of the appearance of the first symptoms. Viremia leads to the dissemination of the virus throughout the body; serous meningitis and epididymo-orchitis are possible, resulting in infertility. Immunity is strong.

Laboratory diagnostics

Virological method. The test material (saliva, cerebrospinal fluid, urine, blood serum) is used to infect a chicken fibroblast cell culture or a chicken embryo. The virus is identified using the hemagglutination inhibition reaction, immunofluorescence reaction, neutralization reaction, and complement fixation reaction.

Serological method. Antibodies are determined in paired patient sera using an enzyme immunoassay, complement fixation reaction, and hemagglutination inhibition reaction. Specific diagnostics – live vaccine, associated vaccine (against measles, mumps, rubella). There is no specific therapy.

Features of the pathogenesis of measles

Source - a sick person (usually children 4-5 years old); methods of infection are airborne, less often contact. The incubation period is 8-15 days. Acute respiratory manifestations (rhinitis, pharyngitis, conjunctivitis, photophobia, temperature 38.8-39 0 C). On days 3-4, a maculopapular rash appears on the mucous membranes and skin: first on the face, then on the torso and limbs. A day before the rash appears, small spots appear on the mucous membrane of the cheeks, surrounded by a red halo. The disease lasts 7-9 days, the rash disappears without leaving any traces. Lifelong immunity.

Laboratory diagnostics

Virological method. They examine nasopharyngeal washings, scrapings from elements of the rash, blood, and urine. The virus is detected in pathological material and in infected cell cultures using the immunofluorescence reaction, hemagglutination inhibition reaction and neutralization reaction. Characterized by the presence of multinucleated cells and pathogen antigens in them.

Serological method. For serological diagnosis, the complement fixation reaction, the hemagglutination inhibition reaction and the neutralization reaction are used. Specific prevention is a live attenuated vaccine. There is no specific therapy.

Features of the pathogenesis of diseases caused by respiratory syncytial virus

The source is a sick person; methods of infection – airborne, contact and household. The pathogen penetrates the epithelial cells of the upper respiratory tract, multiplies, causing their death, the pathological process spreads to the lower respiratory tract, secondary immunodeficiency develops, which leads to the development of secondary bacterial infections. The incubation period is 3-5 days. Signs of acute respiratory infections, then tracheobronchitis, pneumonia. Immunity is short-lived, relapses are possible.

Laboratory diagnostics

Virological method. Cell cultures are infected with the test material (nasopharyngeal secretions, lung tissue). Indication of viruses is carried out by the nature of the cytopathic action - the formation of syncytium, and identification of viruses - using the neutralization reaction, the complement fixation reaction.

Serological method. Detection of a specific antigen is carried out using an immunofluorescence reaction, enzyme-linked immunosorbent assay (rapid diagnostics).

Virusoscopic method. Microscopic (histological) examination reveals multinucleated cells and syncytium in the epithelium of the bronchial mucosa. Specific prevention and specific therapy are absent.

Control questions:

1. 
   Influenza virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
2. 
   Parainfluenza virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
3. 
   Mumps virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
4. 
   Measles virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
5. 
   Respiratory syncytial virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.

1. 
   Material (scrapings from elements of the rash) was received from a child with acute respiratory manifestations, photophobia, temperature 38.8-39.0 0 C and a maculopapular rash on the mucous membranes and skin. When cell cultures were infected, multinucleated cells were found. Identify the pathogen.
2. 
   Material (nasopharyngeal discharge) was received from a child with signs of acute respiratory disease. The pathogen was identified using the virological method. Syncytium formation was observed in cell culture. Identify the causative agent of the disease.

FamilyPicornaviridae

Genus 1 – Enterovirus – polio viruses, Coxsackie viruses, ECHO viruses

Features of the pathogenesis of poliomyelitis

The source is a sick person; mode of infection – fecal-oral; airborne; contact. The incubation period is 7-14 days. There are 3 clinical forms of polio: paralytic, meningeal, abortive. The disease begins with increased body temperature, general malaise, headaches, vomiting, and sore throat. The paralytic form is most often caused by polio virus serotype 1. Immunity is lifelong.

Laboratory diagnostics

Virological method. The material for research is feces, nasopharyngeal discharge, and in case of death - pieces of the brain and spinal cord, and lymph nodes. Cell cultures are infected with the test material. The reproduction of viruses is judged by their cytopathic effect. The isolated virus is identified (typed) using type-specific sera in a neutralization reaction in cell culture.

Serological method. Serodiagnosis is based on the use of paired patient sera using reference virus strains as a diagnostic tool. The content of serum immunoglobulins of the classes IgG, IgA, IgM is determined by the method of radial immunodiffusion according to Mancini. Specific prevention is mass immunization of children with an oral live vaccine of 3 serotypes. There is no specific therapy.

Features of the pathogenesis of diseases caused by Coxsackie viruses

The source is a sick person; The method of infection is fecal-oral, contact. Infections are often seen in children. Typically, symptoms of a cold or fever of unknown origin are noted. In rare cases, severe lesions develop - pemphigus of the oral cavity and extremities, epidemic pleurodynia, pericarditis and myocarditis. Acute intestinal viral diseases are caused only by group A viruses.

Laboratory diagnostics

Virological method. The material for research is feces and nasopharyngeal discharge. They infect cultures of HeLa cells or monkey kidneys (Coxsackie B, individual serotypes of Coxsackie A) or suckling mice. The nature of pathological changes in infected mice is taken into account. Viruses are identified by hemagglutination inhibition test, complement fixation test, neutralization test and enzyme immunoassay. Specific prevention and specific therapy are absent.

Features of the pathogenesis of diseases caused by ECHO viruses

The source is a sick person; The mode of infection is fecal-oral, airborne. Viruses cause colds, infectious diseases, aseptic meningitis, which is relatively mild, less commonly, ascending paralysis and encephalitis, a febrile state accompanied by measles-like rashes.

Laboratory diagnostics

Virological method. The virus is isolated from cerebrospinal fluid, feces, and nasopharyngeal discharge. Monkey kidney cell cultures are infected and identified by hemagglutination inhibition test, complement fixation test, neutralization test and enzyme immunoassay.

Serological method. An increase in antibody titer is detected in blood serum using the hemagglutination inhibition reaction, complement fixation reaction, neutralization reaction and enzyme immunoassay. Specific prevention and specific therapy are absent.
FamilyTogaviridae

Genus 1 – Rubivirus – rubella virus

Features of the pathogenesis of rubella

Source – sick person, carrier; methods of infection – airborne, transplacental. There are 2 forms of the disease: 1 – acquired. The incubation period is 11-24 days. The disease begins with a slight increase in temperature and mild catarrhal symptoms, conjunctivitis, as well as enlargement of the posterior cervical and occipital lymph nodes. Subsequently, a maculopapular rash appears throughout the body. Immunity is strong.

2 – congenital – a slow viral infection that develops as a result of intrauterine transplacental infection of the fetus. The disease is characterized by the development of cataracts, deafness and heart defects, as well as other developmental abnormalities. Blindness combined with deafness and damage to the central nervous system leads to mental retardation. Immunity is incredible.

Laboratory diagnostics

Virological method. The virus is isolated from swabs from the mucous membrane of the nose and pharynx, blood, urine, and less commonly, feces, as well as the internal organs of dead children. Sensitive cells are infected with the test material, and viruses are indicated on the basis of interference with cytopathogenic viruses or by detection of a cytopathic effect.

Serological method. To detect antibodies, a neutralization reaction, a complement fixation reaction, a hemagglutination inhibition reaction, and an enzyme-linked immunosorbent assay are used. A fourfold or more increase in antibody titers in the dynamics of the disease, as well as the determination of specific IgM, indicating a recent illness or illness at the time of examination, is of diagnostic importance. Specific prevention – live vaccine, associated vaccine. There is no specific therapy.

Family Rhabdoviridae

Genus 1 – Lessavirus – rabies virus

Features of the pathogenesis of rabies

Sources - wild rabies - foxes, wolves, rodents, bats, urban rabies - dogs, cats; methods of infection - contact through bites, less often - with excessive salivation of damaged integuments, aerogenic is possible in caves inhabited by bats, sometimes alimentary. The virus, having entered the damaged outer integument with the saliva of a sick animal, replicates and persists at the site of introduction. Then it spreads along the axons of peripheral nerves, reaching the cells of the brain and spinal cord, where it multiplies. Babes-Negri bodies are found in the cytoplasm of brain neurons. Cells undergo degenerative changes. Having multiplied, the virus travels from the brain through centrifugal neurons to various tissues, including the salivary glands. The incubation period is from 10 days to 3 months, sometimes up to a year. At the beginning of the disease - malaise, fear, anxiety, insomnia, then reflex excitability and spasmodic contractions of the muscles of the pharynx and larynx develop. The cramps intensify when trying to drink, at the sight of pouring water, from bright light, noise. Hallucinations develop, and at the end of the disease - paralysis of the muscles of the limbs and breathing. Less commonly, the disease develops without agitation and hydrophobia; Paralysis and drooling develop. Mortality – 95%. Post-infectious immunity has not been studied.

Laboratory diagnostics

Virusoscopic method. Postmortem diagnosis includes the detection of Babes-Negri bodies in fingerprint smears or sections of brain tissue. Babes-Negri bodies are identified by staining methods according to Romanovsky-Giemsa, Mann, Turevich, Muromtsev, etc.

Virological method. Pathological material is injected intracerebrally into white mice. Identification of viruses is carried out using an enzyme immunoassay, as well as a neutralization reaction in mice, using rabies immunoglobulin to neutralize the virus.

Serological method. Antibodies are determined in patients using the complement fixation reaction and enzyme immunoassay. Specific prevention and therapy – inactivated Fermi vaccine, genetically engineered vaccine. Immunize people bitten by animals suspected of rabies. In this case, active immunity is formed during the incubation period. In case of multiple bites, passive immunity is created by administering rabies immunoglobulin.

Family Retroviridae

Genus 1 – Lentivirus - HIV

Features of the pathogenesis of HIV infection

Source – sick person, carrier; methods of infection - sexual, parenteral, transplacental. Stages of HIV infection:

1 – incubation period – 2-4 weeks;

2 – stage of primary manifestations: acute fever, lymphadenopathy, diarrhea, the stage ends with an asymptomatic phase, restoration of well-being, can last for years;

3 – stage of secondary diseases, manifested by damage to the respiratory, nervous systems, gastrointestinal tract, and the occurrence of malignant tumors in various combinations;

Stage 4 – AIDS itself, characterized by cachexia, persistent diarrhea, adynamia, anemia, dementia, a decrease in all immune parameters with a fatal outcome.

Laboratory diagnostics

Serological method. To confirm the diagnosis, antibodies to the proteins gp41, gp120, gp160, p24 are determined in HIV-1 and antibodies to the proteins gp36, gp105, gp140 in HIV-2. HIV antibodies appear 2-4 weeks after infection and are detected at all stages of HIV infection and AIDS. For any positive test, an immunoblotting reaction is performed to confirm the results. PCR is also used. Specific prevention and specific therapy are absent.

Control questions:

1. 
   Poliomyelitis virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
2. 
   Coxsackie virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
3. 
   ECHO virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
4. 
   Rubella virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
5. 
   Rabies virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.
6. 
   Human immunodeficiency virus: taxonomy, structure, pathogenesis of the disease, laboratory diagnosis, specific prevention and therapy.

1. 
   Material for research was received (washing from the mucous membrane of the nose and pharynx) from a child with mild catarrhal symptoms and enlarged posterior cervical and occipital lymph nodes. A cell culture was infected, in which CPE was detected after incubation. Identify the pathogen.
2. 
   Material for research (stool) was received from a child with signs of general malaise, headache, and fever. They inoculated cells into a culture in which, after incubation, viral particles resistant to ether were found. Identify the pathogen.

Literature

1. 
   Large dictionary of medical terms / Comp. Fedotov V.D. – M.: ZAO Tsentrpoligraf, 2007.
2. 
   Vorobyov A.A. Medical microbiology, virology and immunology: textbook. – M.: MIA, 2004.
3. 
   Vorobyov A.A., Bykov A.S. Atlas of medical microbiology, virology and immunology. – M.: MIA, 2003.
4. 
   Vorobyov A.A., Krivoshein Yu.S., Shirobokov V.P. Medical and sanitary microbiology. – M.: ASADEMA, 2003.
5. 
   Golubev D.B. Guide to the use of cell cultures in virology. – L., 1986. Electronic textbook.
6. 
   Krasilnikov A.P., Romanovskaya T.R. Microbiological dictionary-reference book./ - 2nd ed., additional. and processed – Mn.: “Asar”, 1999.
7. 
   Korotyaev A.I., Babichev S.A. Medical microbiology, virology and immunology: a textbook for honey. universities – 3rd ed., rev. and additional – St. Petersburg: SpetsLit, 2002.
8. 
   Medical microbiology, virology and immunology: textbook / Ed. A.A. Vorobyova - M.: Medical Information Agency, 2004.
9. 
   General medical virology / N.S. Goryachkina and others; edited by N.S. Goryachkina, L.I. Kafarskaya. – Rostov n/d: Phoenix, 2007.
10. 
    www. infections.ru/rus/all/mv b journals.shtml

The main methods used in the diagnosis of viral diseases are the cultivation and identification of viruses.

To prove the viral etiology of the disease, it is necessary: ​​isolation of the virus from the body of a sick fish, passage of it on a cell culture or sensitive fish, reproduction of the disease in healthy fish of the same or related species, repeated isolation of the same virus from experimental animals.

To identify viruses, several complementary methods are used: electron microscopy of the virus, the study of its physicochemical properties, detection of characteristic morphological changes in infected cells and symptoms in infected animals, various immunological methods.

Viruses are isolated mainly on single-layer primary or continuous cell cultures, selecting in each case cultures that are sensitive to a given virus. To obtain primary cultures of fish cells, the gonads of female carp or crucian carp are most often used. Gonads should be stage II or II-III of maturity according to the Kiselevich scale. Such gonads do not contain eggs visible to the naked eye. Otherwise, the contents of the eggs will negatively affect cell growth. Cell cultures from gonads of carp and crucian carp are prepared according to an approved method.

The following cell lines are most widely used as continuous cultures: FHM - from the tissues of the caudal peduncle of the fathead minnow; RTG - from rainbow trout gonads; EPC - from smallpox growths on the skin of carp. These lines are maintained in specialized laboratories for the study of fish diseases at veterinary and fishery research institutes, where they can be ordered and received.

When diagnosing well-studied viral diseases, organs and tissues where the pathogen is concentrated are examined.

In case of fish diseases, information about which is insufficient, the most affected organs are subjected to virological examination. Scrapings from the skin and gills, pieces of these organs along with mucus are placed in sterile vials with 2-3 ml of sterile physiological or buffer solution. Samples from internal organs are taken under strictly aseptic conditions.

In cases where it is impossible to quickly examine the material, it is stored for no more than a day in the refrigerator at a temperature not exceeding 5°C. Frozen material can be stored for a longer time.

The pathological material intended for research is crushed in a homogenizer or ground in a porcelain mortar with quartz sand. A 10% suspension is prepared from crushed tissues in Hanks, Earle, buffer or saline solutions and centrifuged for 10-15 minutes at 2000-3000 rpm, the supernatant is sucked off with a pipette and placed in sterile vials. If the suspension is not sterile, the prepared materials are filtered through membrane filters with a pore diameter of 0.2-0.45 μm or treated with antibiotics (penicillin 1000 U/ml and streptomycin 1000 μg/ml).

A 20% suspension is prepared from the intestinal contents in sterile distilled water and centrifuged for 10-15 minutes at 2000 rpm. The supernatant is centrifuged again at 4000-5000 rpm for 30 minutes. The supernatant is then aspirated into a sterile vial and treated with antibiotics. Add 1000 μg/ml streptomycin and 1000 U/ml penicillin per 1 ml. The mixture is kept for 2-3 hours at: room temperature. All materials are tested for bacterial sterility by inoculation with MPB and MPA. The prepared materials are immediately used for work or, in extreme cases, stored frozen (at a temperature of minus 20°C).

Infection of cell culture. For infection, tubes with a good cell monolayer or growth zone around the explant are used. The nutrient medium is sucked out and 0.2-0.3 ml of the test suspension is added to each test tube. At the same time, 0.8-0.9 ml of nutrient medium with 2-3% serum is added to the test tubes.

The material prepared from each sample is used to infect tissue culture in 4-6 test tubes. From each series of studies, the same number of test tubes are left as a control, adding 1 ml of nutrient medium to them. The tubes are left at room temperature for 1-2 hours for the virus to be adsorbed on the cells, then the supernatant is sucked off with a pipette and the maintenance nutrient medium is added to the original volume.

Infected and control cell cultures are incubated at a temperature of 22-26°C and examined daily under a low magnification microscope to detect morphological changes in the cells. In case of severe cell degeneration, the culture liquid is sucked out and passages are made, and in the absence of a cytopathogenic effect (CPE), two successive passages are carried out. To do this, a culture liquid is used together with a cell fraction destroyed by repeated freezing and thawing. To infect fresh cultures, use the supernatant of centrifuged cell mass. CPE after the third passage is taken into account as the specific effect of the viral agent.

The degree of damage to the cell monolayer is assessed using the 4-cross system; " + - damage up to 25%, " + + " - up to 50%, "+ + +" - up to 75% and " + + + + " - up to 100% of the monolayer.

In some viral diseases of fish, inclusion bodies appear in the cells (cytoplasmic nucleus) of various organs and tissues. The material for studying viral inclusions are infected tissue cultures, scrapings and impression smears from organs and tissues; before staining, these materials are fixed according to generally accepted methods, using Dubosc-Brazil-Buena, Buena, Carnoy liquids or a 10% solution of neutral formaldehyde.

Viral inclusions are stained using various methods: Muromtsev, Rubina, Mann, Sellex, Klisenko, Romanovsky-Giemsa, May-Grunald-Giemsa, etc.

Virus titration- quantitative determination of viral activity. The virus titer is expressed by the number of infectious units contained per unit volume of virus suspension. An infectious unit of a virus is defined as the dose that causes infection in 50% of sensitive objects infected by it. This dose of the virus is called infectious and is designated ID 50.

Cell cultures are mainly used as sensitive objects when titrating fish viruses. Titration on cell culture is carried out according to the cytopathogenic effect of viruses. In this case, ID 50 is called tissue cytopathogenic dose (TCD 50), and the virus titer is expressed by the amount of TCD 50 in 1 ml of viral suspension. The virus titer is determined by the final dilution method. According to this method, sensitive cell cultures inject a certain volume of a virus suspension in successively increasing dilutions and, taking into account the result of each injection as positive (if there is a CPD) or negative if there is no CPP), the titration end point is calculated - 1 TCD 50.

To titrate viruses that give a pronounced CPE, the plaque method is also used. In this case, a monolayer of cells infected with the virus is poured with a mixture of nutrient medium with agar in order to prevent the transfer of the virus to other cells significantly removed from the initially infected ones, and to be able to infect the initial foci of infection of the plaque).

Each plaque arises from a single infectious unit, which is designated PFU (plaque-forming unit), and the titer of the virus is expressed as the number of PFU per unit volume of suspension.

Neutralization reaction(RN) on cell culture.

The reaction is based on the binding of the antigen by antibodies of a homologous antiserum. The reaction is used to identify pathogens in the diagnosis of diseases of viral etiology. It allows you to determine an unknown viral antigen by known antibodies or by a known (standard) antigen - unknown antibodies in the sera of sick or recovered fish.

Determination of the isolated virus in the neutralization reaction is carried out using a set of diagnostic hyperimmune antisera (antibodies) of antigens (viruses) homologous to them.

Hyperimmune antisera are obtained by infecting laboratory animals (for example, rabbits) with known strains of viruses that cause fish diseases. The titers of specific antibodies in the obtained antisera are determined. For work, take antisera containing antibodies in high titers.

The order of the reaction.

1. Inactivation of normal and hyperimmune serum by heating at 56 o C for 30 minutes.

2. Preparation of dilutions of reaction ingredients. The antigen and serum are diluted with a nutrient medium without serum and antibiotics, starting from 1: 5, 1: 50, 1: 500 and until a dilution is obtained containing less than 1 TCD 50 / 0.2 ml. Hyperimmune serum is diluted 1:2 or 1:5. If the titer is low, the serum is used undiluted. Normal serum is diluted in the same way as hyperimmune serum.

3. Setting up the reaction. Three rows of sterile tubes are placed in a rack. Diluted hyperimmune serum is poured into the first row, diluted normal serum into the second, and nutrient medium into the third. Each ingredient is added in a volume of 0.5 ml.

The prepared dilutions of the virus are transferred in 0.5 ml quantities into the corresponding test tubes of each of the three rows, and the virus of each dilution I is transferred with a separate pipette, starting with the highest dilution. Thus, in each row of test tubes, successive 10-fold dilutions of the virus are obtained: 10-1, 10-2, etc.

To control the toxicity of serums, add 0.5 ml of the prepared dilution of hyperimmune serum into a separate test tube, and then add an equal amount of nutrient medium. The same is done with normal serum.

Test tubes with mixtures are thoroughly shaken and kept at room temperature for 1 hour. Then the cell culture is infected with each dilution of the virus (0.2 ml per tube) with hyperimmune normal serum and nutrient medium, 4 cell culture tubes each. At the same time, controls are placed for the toxicity of the cells used and control cell culture samples.

Tubes with cell culture are incubated in a thermostat at the optimal temperature for the propagation of a given virus, and examined daily under a low-magnification microscope to detect the CPE of the virus. The results are entered into the table (Table 11).

The virus titer is expressed by the number of infectious units contained per unit volume of virus suspension. Find the neutralization index (IN). It corresponds to the maximum amount of ID 50 that can be neutralized by hyperimmune serum. IN is calculated using the formula: logIN = logT 1 -lgT 2, where T 1 is the titer of the virus in the presence of normal serum; T 2 - virus titer in the presence of hyperimmune serum. The value of IN is found from the table of antilogarithms. It is generally accepted that an IN value of up to 10 is negative, from 10 to 49 is doubtful, and 50 or more is a positive result.

The results of the reaction can only be considered reliable if the hyperimmune serum is tested for specific neutralizing activity. To do this, the titer of neutralizing antibodies in this serum or its neutralization index in reaction with a homologous virus is first determined.

Isolation of rhabdoviruses by plaque method. This method is specific; it is used for the isolation, preliminary typing and selection of rhabdoviruses of carp and trout in the presence of specific immune sera for virus identification.

Round colonies (plaques) are formed in cell cultures under an agar coating in the presence of a virus in the test material.

Under aseptic conditions, tissue of the kidneys, liver, spleen and fluid from the abdominal cavity are collected with a Pasteur pipette, transferred to a bottle with a medium containing 500 IU (mcg)/ml of antibiotics in a ratio of 1: 10. Incubated for 60-90 minutes at a temperature of 18-22 °C, then centrifuge at 2-3 thousand rpm for 10 minutes. The supernatant is diluted with nutrient medium 1:10 (dilution 1:100). To infect cell cultures, supernatant liquids of both dilutions are used.

For the study, a 3-day continuous cell culture grown in mattresses with a well-defined monolayer is selected at the rate of 2 mattresses for each dilution of pathological material and 2 mattresses for control. The nutrient medium is removed from the bottles and 2 ml of medium without fetal serum is added. Then 0.2 ml of the test material is added and left to adsorb the virus for 60 minutes at the optimal temperature for viruses that infect fish (for carp viruses - 24-26°C and for trout viruses - 16-18°C).

The control is placed in 2 mattresses with cell cultures of 0.2 ml each, containing 100 TCD 50 / ml of the known virus, and in 2 - 0.2 ml of nutrient medium without the virus.

After 60 minutes, the liquid is removed from the bottles. Along the wall opposite the monolayer, add 5 ml of agar coating heated to 40-42°C into a 50 ml bottle, heated to 40-42°C (when isolating the HCV virus - no more than 38°C). The mattresses are turned monolayer down and covered with black paper. After 15-20 minutes, the mattresses are transferred for incubation at the optimal temperature for the viruses being studied. The mattresses are placed with the agar coating facing up. If the virus is unknown, the cultures are kept at two temperature conditions - 14-18 and 22-24°C.

Infected cell cultures are viewed against a white background. If there is a virus in the cell culture being studied, transparent dots first appear on the pink-matte background of the culture. Subsequently, they increase in size, forming round transparent colonies - plaques, the presence of which indicates the presence of rhabdoviruses in the pathological material.

Using a Pasteur pipette, take a piece of the plaque at the border of the affected and unaffected parts in such a way that not only the agar layer, but also the cell layer is included. The selected piece is placed in a test tube with 1 ml of growth medium, frozen at minus 20°C and kept for 60 minutes. In the case of a large number of plaques (the entire cell layer is transparent), the studies are repeated in dilutions of 10 -3 and 10 -4.

Plaques with a diameter of 3-8 mm of carp rhabdovirus on a continuous culture of EPC and FHM, incubated at a temperature of 24-26°C, appear on the 4-7th day.

In the absence of plaques and the presence of CPD in cell cultures, additional studies are carried out on virus-containing culture fluid in dilutions of 10 -2 -10 -3 (2-3 passages). Additional methods for identifying viruses include: fluorescent antibody method; determination of the sensitivity of the virus to chloroform, ether, pH value, heating; electron microscopic study of the morphology of viruses.

15.1. Characteristics of microbiological and immunological laboratories

All work with microbes is carried out in laboratories, which, depending on the main tasks, can be research, diagnostic or production.

The health care system includes:

Clinical diagnostic laboratories of general or special types (biochemical, bacteriological, immunological, cytological, etc.) that are part of hospitals, clinics, dispensaries and other medical and preventive institutions;

Bacteriological laboratories of the State Sanitary and Epidemiological Supervision (GSN);

Sanitary and bacteriological laboratories of the GSN;

Sanitary and chemical laboratories of the state government;

Central (TsNIL), problem, industry, educational laboratories of universities;

Specialized laboratories (especially dangerous infections, etc.).

Currently, laboratories and larger laboratory institutions (departments, institutes, manufacturing enterprises), as a rule, are specialized and work with one or another group of microbes.

They work with viruses in virology laboratories that have the appropriate equipment and use special research methods. There are mycological and protozoological laboratories. Specialized nature

Bacteriological laboratories are also acquiring, in which work is concentrated on certain groups of bacteria, for example, rickettsial, tuberculosis, leptospirosis, anaerobic, etc. Immunological studies are carried out in immunological laboratories, although certain types of research can also be carried out in microbiological laboratories, for example, serodiagnosis of infectious diseases.

Laboratory work with pathogenic microbes is carried out in specially equipped laboratories that ensure operating conditions and safety precautions, eliminating the possibility of infection of personnel and leakage of microbes outside the laboratory.

The need for clear regulation of working conditions with microbes, which are dangerous to varying degrees for laboratory workers and the surrounding population, led to the development of a classification of microbes, dividing them into 4 groups according to the degree of their biological hazard (WHO classification). In Russia, in accordance with WHO recommendations, pathogenic microbes are also divided into 4 groups: 1st group - pathogens of particularly dangerous infections; 2nd group - pathogens of highly contagious human epidemic diseases; 3rd group - pathogens of infectious diseases, classified into independent nosological groups; Group 4 - opportunistic microbes - causative agents of opportunistic infections. The numbering of microbial groups adopted in Russia differs in reverse order from the WHO classification, where group 1 includes microbes of the lowest pathogenicity, and group 4 includes particularly dangerous ones.

In accordance with the division of microbes into groups according to the degree of biological hazard, laboratories are also divided into categories. According to WHO nomenclature, there are 3 categories of microbiological laboratories:

Basic (main or general type) laboratories, which, due to the specific features of the work, can be equipped with various protective devices;

Security (isolated) laboratories and special regime laboratories (maximum isolation).

The safety of work in laboratories of all categories is ensured by compliance with the routine and rules of work in the laboratory, compliance with the requirements for laboratory premises and their equipment, and provision of laboratories with appropriate equipment.

tion, medical monitoring of the health status of employees, education and training of personnel in laboratory safety procedures.

15.2. Equipment for microbiological and immunological laboratories

The basic laboratory premises must be spacious to ensure the safe conduct of laboratory work. Walls, ceilings, and floors must have a smooth, easy-to-clean surface, impermeable to liquids, and resistant to disinfectants commonly used in the laboratory. The surface of work tables must be waterproof, resistant to disinfectants, acids, alkalis, organic solvents and moderate heat. Laboratory furniture must be durable. The space under tables and between furniture should be easily accessible for cleaning. The laboratory must have an autoclave for waste disinfection.

Basic laboratory equipment must limit or prevent microbiologist contact with infectious material and must be made of durable materials, impervious to liquids, and resistant to corrosion. Equipment must be designed and installed so that it can be easily cleaned, disinfected and inspected.

The laboratory is equipped with a microscope, an autoclave, thermostats, drying and sterilization cabinets, a whey coagulation apparatus, a distiller, centrifuges, laboratory scales, a pH meter, FEC, a magnetic stirrer, and a washing bath.

The working premises of the laboratory must be equipped with cold and hot water supply, electricity, vacuum, oxygen, high pressure air, etc. Some offices are equipped with boxes and fume hoods.

Mandatory premises include laboratories for intestinal and droplet infections, sanitary-bacteriological, serological, as well as auxiliary premises: media preparation, washing, sterilization (clean and dirty), registration, storerooms, bathroom for employees, vivarium. In laboratories with points for testing for the carriage of microorganisms, they will additionally equip a reception area, treatment room, and toilets for collection

material. The premises are located in such a way that dirty and clean flows do not cross or touch.

For the premises of high-security laboratories, the same requirements as those provided for the basic laboratory must be observed. In addition, this type of laboratory should be separated from those parts of the building where the movement of employees is not restricted. Hand-washing facilities must be equipped with a foot pedal or elbow-operated mechanism for opening the water. Windows must be closed and sealed. Entrance doors to laboratory premises must be self-closing and padlockable. Exhaust ventilation is designed so that the lowest pressure is created in rooms with the highest risk of infection. In this case, air movement will occur from auxiliary rooms in the direction of the main working room. Exhaust air is released into the environment only after being filtered through bacterial filters. When equipping high-security laboratories with equipment, they are guided by the recommendations developed for basic laboratories, with the addition that all work with infectious material in them is carried out in protective boxes. In the mode of maximum isolated laboratories, there are a number of features to ensure maximum biological safety of personnel, the public and the environment. Entry into and exit from the laboratory is carried out through a sanitary checkpoint. Upon entry, a complete change of clothes into special clothing is required; upon exit, before changing clothes, targeted sanitization (shower, disinfectants) of personnel is required. To reduce the risk of infectious material entering the environment, boxing is used. By using boxes (desktop, laminar) create physical barriers to prevent possible contact of working personnel with infectious material.

15.3. Rules for working in a microbiological laboratory

Basic rules for working in a basic laboratory include:

Prohibition of working with a pipette using the mouth;

Prohibition of eating, drinking, smoking, storing food and using cosmetics in work areas;

Maintaining cleanliness and order;

Disinfect work surfaces at least once a day and after each contact with infectious material;

Personnel washing their hands after working with infectious material, animals, and before leaving the laboratory;

Carrying out all work in such a way as to minimize the possibility of aerosol formation;

Decontaminate all contaminated materials before disposal or reuse.

15.4. Principles of microbiological diagnosis of infectious diseases

The most important place in the laboratory diagnosis of infectious diseases is occupied by specific microbiological diagnostics, which are carried out in bacteriological, virological, immunological and other laboratories. It consists of three stages: preanalytical, analytical and postanalytical.

The first stage of microbiological diagnostics is preanalytical, including taking material for research. The choice of material to be studied is determined by the pathogenesis and clinical picture of the infectious disease. The test material is taken, if possible, under aseptic conditions, placed in sterile containers and delivered to the laboratory as quickly as possible (preferably within 1 hour). In some cases, seeding of material is carried out at the patient's bedside. Sometimes short-term storage of material under regulated conditions is allowed. The material being studied is accompanied by a document that must indicate the time of collection, the nature of the material, its source and precisely define the purpose of the study.

The material for research in medical microbiology is various biological and pathological body fluids (blood, pus, urine, sputum, liquor, feces, vomit, washing water, etc.) and tissue - biopsy material from a living person or autopsy from a corpse. In sanitary microbiology, environmental objects (air, water, food products, etc.) or swabs from them are taken for research. When collecting material

For microbiological research, the following rules must be observed:

The material is taken directly from the source of infection or the corresponding discharge is examined (pus, urine, bile, etc.);

The amount of material should be sufficient to conduct the study and repeat it if necessary;

The material is taken, if possible, in the initial period of the disease, since it is during this period that pathogens are isolated more often, there are more of them, they have a more typical localization;

The material is taken before the start of antimicrobial chemotherapy or after a certain period of time after taking the antibacterial drug, necessary for its removal from the body;

The possibility of antimicrobial agents (disinfectants, antiseptics, antibiotics) getting into the material should be prevented;

Transportation of the material to the laboratory should be carried out as quickly as possible, under conditions that exclude the death of unstable species of microbes, or it should be placed in special transport media;

During transportation, all biological safety regulations must be observed;

An accompanying document is attached to the material, containing the basic information necessary for conducting a microbiological study (last name, first name, patronymic of the patient, medical history number, clinical diagnosis, etc.).

15.5. Microbiological diagnostic methods

The analytical stage includes microscopic, cultural, biological, serological and allergological methods of microbiological diagnostics.

Microscopic method consists in the preparation of preparations (native or stained by simple or complex methods) from the material under study and their microscopy using various types of microscopic equipment (light, dark-field, phase-contrast, fluorescent, electronic, etc.). In bacteriology, the microscopic method is called bacterioscopic, in virology - virusoscopic.

Culture method consists of inoculating the test material on artificial nutrient media, cell cultures or chicken embryos in order to isolate and identify a pure culture of the pathogen or pathogens. In bacteriology, the cultural method is called bacteriological, in mycology - mycological, in protozoology - protozoological, in virology - virological.

Biological method (experimental or bioassay) consists of infecting sensitive laboratory animals or other biological objects (chicken embryos, cell cultures) with the test material. It is used to isolate a pure culture of the pathogen, determine the type of toxin, the activity of antimicrobial chemotherapy drugs, etc.

Serological method consists in determining the titer of specific antibodies in the patient’s blood serum, less often - in detecting a microbial antigen in the test material. Immune reactions are used for this purpose.

Allergological method consists of identifying an infectious allergy (HRT) to a diagnostic microbial drug allergen. For this purpose, skin allergy tests are performed with the corresponding allergens.

The diagnostic value of these methods is unequal. The leading method of microbiological diagnostics is bacteriological method, since it allows you to isolate and identify the pathogenic microbe, i.e. the root cause of the disease. Other methods are less informative, since they make it possible to detect changes in the body caused by the presence of a microbe in it. The second place in importance is serological method, since the interaction between antigen and antibody is characterized by a high degree of specificity. The information content of the other three methods is low, and they usually serve as a complement to the bacteriological and serological methods. Thus, microscopy of the material under study does not always allow microbes to be seen and identified under a microscope. They can only be detected when the material is highly contaminated with them. Even if bacteria are detected under a microscope, it is impossible to identify them morphologically to species. As is known, the entire species diversity of bacteria comes down to 4 main morphological forms: cocci, rods, convoluted and branching forms. Therefore, according to micro-

Based on the scopic picture, it is possible to roughly attribute the bacteria seen to a large taxon, for example, gram-positive cocci. Only in isolated cases, when bacteria have a unique morphology, can their genus be determined microscopically. The information content of the microscopic method of fungi and protozoa is higher, since fungi and protozoa, being eukaryotes, have larger sizes and a more characteristic morphology.

The diagnostic capabilities of the biological method are limited by the fact that laboratory animals are immune to most pathogens of anthroponotic human infections, so it is not possible to cause an experimental infection in them.

The capabilities of the allergological method are limited by the fact that most microbes in the human body do not cause HRT.

Since microbiological studies are one of the most expensive types of laboratory research, the microbiologist is faced with the task of making a reliable microbiological diagnosis with the least amount of time, effort and money. Therefore, to make a diagnosis, 1-5 diagnostic methods are used so that the selected set of methods guarantees the correct answer.

Of particular importance are express diagnostic methods, which make it possible to make a microbiological diagnosis within a short period of time (from several minutes to several hours) from the moment the test material is delivered to the laboratory. Express methods include RIF, ELISA, RIA, PCR, the use of biochips, chromatography, etc. Features of the diagnosis of anaerobic infections are described in the materials of the disk.

Along with traditional classical methods of microbiological diagnostics, in recent years molecular biological diagnostic methods (DNA probes, PCR, ligase chain reaction - LCR, chromatography, electrophoresis, immunoblot, biochips, etc.) have become increasingly important.

Molecular biological diagnostic methods are based on the identification of DNA and RNA specific to a given microbial species, and include hybridization based on DNA probes and PCR-based diagnostics.

Post-analytical The stage of microbiological diagnostics consists of the clinical interpretation of laboratory results. In this case, the attending physician must assess the etiological significance of microbes isolated from the patient, adjust the empirical antimicrobial chemotherapy administered to the patient based on microbiological monitoring data, etc.

15.5.1. Methods for microbiological diagnosis of bacterial infections

In bacteriology, bacterioscopic, bacteriological, and biological methods are used to detect pathogens in the material under study.

The advantages of the bacterioscopic method are simplicity, speed, and efficiency. However, it finds limited application, since it can be used only if there are any morphological or tinctorial features of the pathogen and its sufficient content in the material being studied. This method is indicative.

The main and most accurate method for diagnosing bacterial infections is bacteriological, which is used for almost all diseases, despite its disadvantages: the duration of the study (from 4-5 days to 2 months), danger (since a pure culture of the pathogen accumulates), and comparative high cost. If the material under study is expected to contain a pathogen in sufficient quantities, the material is inoculated on solid nutrient media to obtain isolated colonies. If the content of microbes is insignificant, the material under study is first inoculated on liquid nutrient media - enrichment media. Identification of the isolated pure culture is carried out by morphological, tinctorial, cultural, biochemical, antigenic and toxigenic properties (depending on the type of pathogen). Determining the listed properties allows you to determine the type of pathogen. For the purpose of epidemiological marking, intraspecific identification of the isolated culture is carried out: its phagovar, biovar, etc. are determined. In addition, to prescribe rational treatment, as a rule, the sensitivity of the isolated culture to antibiotics is determined.

In the microbiological diagnosis of diseases caused by opportunistic microbes, representatives of normal microflora, it is mandatory to determine the number of pathogens in the material under study.

The biological method is uneconomical and inhumane, and therefore has limited application. White mice, guinea pigs, rabbits, monkeys and other animals are used as experimental animals.

The diagnosis of an infectious disease can also be established using a serological method, which makes it possible to detect either specific antibodies in the patient’s serum or specific antigens directly in the test material. Antibodies to the causative agent of the disease appear, as a rule, by the end of the first week of illness. The inability to detect them in the first days of the disease is a serious drawback of the method, especially in cases where the disease is acute. In addition, many diseases require studying the dynamics of antibody formation and identifying an increase in the number of antibodies, which also does not allow a quick diagnosis. The disadvantage of this method is that it cannot be used to accurately identify the pathogen and determine its antibiogram. But at the same time, it is a completely safe, relatively inexpensive method that allows you to make a diagnosis in a short time. Currently, for a number of diseases, not only the quantity, but also the classes of immunoglobulins are determined.

For some diseases, the serological method is used to identify specific antigens in the test material. Since the specific antigens that make up the pathogen are present in the pathological material from the first minutes of the disease, this version of the serological method is used for accelerated (during the first day of illness) or even rapid diagnosis (within several hours) of infectious diseases.

As an auxiliary method for a small group of infectious diseases, the allergological method is used, which makes it possible to identify increased sensitivity to the specific antigen (allergen) that is the causative agent of the disease.

15.5.2. Methods for microbiological diagnosis of viral infections

In virology, methods for laboratory diagnosis of viral infections have their own specifics, taking into account the peculiarities of the biology of viruses. Virusoscopic, virological and serological laboratory diagnostic methods are used.

The viruscopic method involves detecting the virus in the material under study under a microscope. An electron microscope is used more often, and a fluorescent microscope is used less often. Light microscopy is practically not used due to the negligible size of viruses. Only to detect large viruses using superstaining methods can a light microscope be used. In addition, using a light microscope, you can identify intracellular inclusions that form in affected cells during some infections.

The virological method consists of infecting a sensitive biological model (laboratory animals, chicken embryos or cell cultures) with the test material, indicating the virus and its subsequent identification. When laboratory animals are infected, viruses are usually identified based on the clinical picture of the disease, pathological and anatomical changes, tentatively and finally, for example, using the hemagglutination reaction. The same reaction makes it possible to detect viruses in a chicken embryo, which, as a rule, does not show visible changes upon autopsy. In cell culture, the presence of the virus is determined by its cytopathic effect (including the formation of intracellular inclusions), hemadsorption, the phenomenon of plaque formation, hemagglutination reaction, and the absence of changes in the color of the indicator. Identification of the virus is carried out using serological reactions (RPGA, RTGA, RN, RSK, ELISA, etc.). The virological method allows you to accurately determine the nature of the pathogen, but it requires sufficient time (5-7 days or more), significant material costs and is unsafe.

A special feature of the serological method in virology is the study of paired sera. The first serum is taken from the patient during the acute period at the onset of the disease, stored at a temperature of 4-8? C, and the second serum is taken after 10-14 days. Serums

investigated simultaneously. The disease is indicated by seroconversion, i.e. an increase in antibody titer in the second serum relative to the first. Seroconversion of 4 times or higher is diagnostic. Since many viral diseases are acute, this version of the serological method is usually used for retrospective diagnosis.

The leading method of laboratory diagnosis of viral infections is virological.

Accelerated and express diagnosis of viral diseases is carried out in the same way as for bacterial infections.

15.5.3. Features of microbiological diagnosis of mycoses

To diagnose fungal infections, a microscopic method is usually used. The mycological method consists of inoculating pathological material on special nutrient media, isolating a pure culture of the pathogen and identifying it by morphological, cultural and biochemical properties. The peculiarity of this method is its duration - several weeks due to the slow growth of fungi. Detection of antibodies during serological testing is possible from the 2-4th week of illness. For some diseases, specific antigens are detected in the test material. The allergological method is rarely used. Often, for mycoses, a histological method is used, which consists in detecting fungal elements (spores, conidial heads, etc.) in organs and tissues affected by fungi. For this purpose, histological thin or ultrathin sections of tissue are prepared, stained using special histological and histochemical methods and examined using light and, if necessary, electron microscopy.

15.5.4. Features of microbiological diagnosis of protozoal infections

Microscopic examination of pathological material consists of preparing both native preparations (“thick drop”) and smears stained using the Romanovsky-Giemsa method, and is the main method for diagnosing diseases caused by protozoa. In some cases, serological and allergological diagnostic methods are used.

15.6. Principles of immunological diagnosis of human diseases

Immunodiagnostics is a branch of immunology that studies and develops methods for diagnosing infectious and non-infectious diseases associated with the function of the immune system.

Many infectious diseases have currently undergone significant changes, which is reflected in an increase in the proportion of mild, erased and asymptomatic forms, an increase in the allergic component, and a high frequency of mixed infections. This complicates the traditional diagnosis of diseases, so the importance of immunodiagnostics aimed at searching for pathogen antigens or specific immune changes in the patient’s body is increasing.

Immunoreactivity (immune status, immune profile) refers to the ability of the immune system to mount an immune response at a given time. It is characterized by the concentration of immunoglobulins, the number of lymphocytes and leukocytes, the ratio of T and B cells and functional indicators, in particular the ability of immunocompetent cells to respond to stimulation.

15.7. Quality control of laboratory tests

An important element of the work of a microbiological and immunological laboratory is obtaining accurate and comparable test results, for which it is necessary to monitor the quality of the studies performed. Quality control can be internal and external.

In-lab quality control - a system of control measures that are carried out in a separate laboratory by the personnel of this laboratory and are aimed at ensuring an appropriate quality level of the laboratory’s work.

External quality control - a system of control measures that are carried out within the framework of the unified Federal System of External Quality Assessment (FSVOK) of laboratory research by groups of experts and are aimed at ensuring the correct organization of technological processes for the production of laboratory research.

The federal system of external assessment of the quality of laboratory research consists of sections, within each of which

The quality of a certain type of laboratory research is assessed. The structure of FSVOC includes expert groups for the development and implementation of external quality control in various types of laboratory tests.

Tasks for self-preparation (self-control)

A.Name a microbiological research method that allows you to establish the type of pathogen:

1. Allergic.

2. Microscopic.

3. Cultural.

4. Biological.

B.Name the main task of the bacteriological research method.

B.On the 7th day of the disease, serum was taken from a patient with a suspected viral infection, in which specific antiviral antibodies were detected. Assess the reliability of the research result obtained.

G.Name the type of laboratory to which material from a patient suspected of having a particularly dangerous infection should be sent.

Course work

"Methods of clinical virology"

Introduction

Laboratory diagnosis of viral infections is carried out mainly using electron microscopy, sensitive cell cultures and immunological methods. As a rule, one method is chosen to make a diagnosis depending on the stage of the viral infection. For example, all three approaches may be useful in diagnosing varicella, but the successful use of microscopy and cell culture techniques depends on the ability to collect satisfactory samples relatively early in the disease.

To a large extent, the success of viral diagnostics depends on the quality of the samples obtained. For this reason, laboratory staff themselves must be directly involved in collecting the necessary samples. Characteristics of the samples, as well as methods for their delivery to the laboratory, are described by Lennett, Schmidt, Christ, et al.

Most reagents and instruments used in laboratory diagnostics can be purchased from various companies. In most cases, the same reagent is produced simultaneously by several companies. For this reason, we have not indicated individual companies, unless the reagent is supplied by only one company. In all other cases, you should refer to the general list of suppliers indicated in table. 1.

We did not aim to provide a comprehensive description of all currently available methods for diagnosing human viral infections. First of all, we characterized the main methods. As you gain experience working independently, these basic techniques can be used to solve more complex problems.

1. Electron microscopy

For electron microscopic diagnosis of viral infections, thin sections of affected tissue can be used. The most common material used for electron microscopy is feces or liquid.

Table 1. List of companies supplying reagents and equipment

vesicles that characterize some diseases, such as chickenpox. When analyzing such material, viruses can be detected using negative staining, which results in electron-dense material delineating the components of the virion. The method is effective when the virus concentration is high in the test samples, such as in feces or vesicular fluid. In cases where the content of viral particles in samples is low, the probability of detecting the virus can be increased by concentrating the virus by ultracentrifugation or by aggregating it with specific antibodies. The latter method is also convenient for identifying viruses. Here we will describe the electron microscopic method for diagnosing rotavirus infection and the immunoelectron microscopy method using the example of detecting specific antibodies to parvoviruses. Electron microscopy methods are described in more detail by Field.

2.1 Direct electron microscopic examination of feces

1. Dip the tip of a Pasteur pipette into the feces and draw up enough material to obtain a 1 cm smear.

2. Resuspend the fecal smear in electron microscopic negative contrast dye until a translucent suspension is obtained. Negative contrast dye is a 2% solution of phosphotungstic acid in distilled water.

3. To obtain an electron microscopic specimen, a drop of the suspension is placed on an electron microscopy grid coated with a carbon-formvar film. During this operation, the mesh is held with a pair of thin tweezers.

4. The drug is left in air for 30 s.

5. Excess liquid is removed by touching the edge of the glass with filter paper.

6. The drug is dried in air.

7. If necessary, the viable virus is inactivated by irradiating both sides of the grid with ultraviolet light with an intensity of 440,000 μW-s/cm2. In this case, a short-wave ultraviolet lamp with a filter is used. The lamp should be at a distance of 15 cm from the grid; Irradiation time for each side is 5 minutes.

8. Rotavirus virions can be characterized under a transmission electron microscope with a magnification of 30,000 to 50,000.

2.2 Immunoelectron microscopy

The immunoelectron microscopy method described below is only one of many similar immunological methods. To study virus-specific antibodies, in addition, a method is used that involves binding to the microscopic network of protein A. The working concentration of antiviral antibodies is determined by trial and error in the range from 1/10 to 1/1000. The concentration we indicate is usually used in routine work. To obtain optimal results for the interaction of antibodies with the virus, serum containing parvovirus is titrated in the same way.

1. 10 µl of antiserum to human parvovirus is diluted 100 times with PBS. The solution is heated in a water bath to 56°C.

2. Melt 10 ml of 2% agarose in PBS in the usual manner and cool to 56 °C in a water bath.

3. At 56 °C, mix 1 ml of diluted antiserum with 1 ml of 2% agarose.

4. Transfer 200 µl of the resulting mixture into two wells of a 96-well microtiter plate.

5. The agarose is allowed to set at room temperature. The tablet can be stored at 4°C for several weeks if it is sealed with adhesive tape.

6. Add 10 µl of serum containing parvovirus to a well containing a mixture of agarose and antiserum.

7. An electron microscopy grid with a pre-prepared carbon-formvar coating is placed on the less shiny side on a drop of serum.

8. The mesh is kept for 2 hours at 37 °C in a humid chamber.

9. Using thin tweezers, take out the mesh and apply a drop of 2% phosphotungstic acid to the surface of the mesh that was in contact with the serum.

10. After 30 s, the excess paint is washed off, the preparation is dried and the virus is inactivated.

Aggregated viral particles are examined under a transmission electron microscope at a magnification of 30,000 to 50,000.

3. Identification of viral antigens

Viruses found in tissues or tissue fluids can be identified by virus-specific proteins using the antigen-antibody reaction. The product of the antigen-antibody reaction is tested against a tag that is introduced either directly into antiviral antibodies or into antibodies directed against virus-specific antibodies. Antibodies can be labeled with fluorescein, radioactive iodine, or an enzyme that cleaves the substrate causing a color change. In addition, the hemagglutination reaction is used to identify the virus. In everyday practice, the described methods are used mainly to detect hepatitis B virus antigens in the blood and search for antigens of various viruses that cause various respiratory diseases.

Currently, many companies produce erythrocyte, radioactive and enzymatic diagnostics, including those for detecting the hepatitis B virus. We do not consider it advisable to outline methods for working with these diagnostics: it is enough to follow the attached instructions. Below we will focus on the immunofluorescence method for identifying respiratory syncytial virus in nasopharyngeal secretions.

3.1 Identification of respiratory syncytial virus in nasopharyngeal secretions by immunofluorescence

The method for obtaining preparations of nasopharyngeal secretions is described by Gardner and McQuillin. In laboratory conditions, this operation is performed in two stages. First, a smear of nasopharyngeal mucus is prepared on a glass slide. The resulting smears can be stored fixed at -20°C for many months. At the second stage, smears are stained to detect the respiratory syncytial virus antigen. For this purpose, the method of indirect immunofluorescence is used.

3.1.1 Preparation of preparations of nasopharyngeal secretions

1. Mucus from special forceps is washed off with 1-2 ml of PBS and transferred to a centrifuge tube.

2. Centrifuge for 10 minutes at 1500 rpm in a tabletop centrifuge.

3. The supernatant is drained.

4. The cell pellet is carefully resuspended in 2-3 ml of PBS until a homogeneous suspension is obtained. To do this, use a wide-necked Pasteur pipette.

5. The resulting suspension is transferred to a test tube.

6. Add another 2-4 ml of PBS to the suspension and mix by pipetting. Large clots of mucus are removed.

7. Centrifuge for 10 minutes at 1500 rpm in a tabletop centrifuge.

8. The supernatant is discarded, the sediment is resuspended in such a volume of PBS that the resulting suspension is easily separated from the walls of the tube.

9. The resulting suspension is applied to a marked glass slide.

10. The glass is dried in air.

Fix in acetone for 10 min at 4°C.

12. After fixing, the glass is dried again in air.

13. The resulting preparations are stained immediately or stored at -20 °C.

3.1.2. Staining technique

1. Print and dilute commercial RSV antiserum in PBS to the recommended working concentration.

2. Using a Pasteur pipette, apply one drop of antiserum to the prepared preparation.

3. The drug is placed in a humid chamber.

4. The drug is incubated for 30 minutes at 37 °C.

5. Samples are carefully washed with PBS to remove excess antibodies in a special reservoir.

6. Samples are washed in three shifts of PBS, 10 minutes each.

7. Dry the samples, remove excess PBS with filter paper and air dry.