Methods for microbiological diagnosis of dysentery. Microbiological diagnosis of dysentery

Guidelines for students for practical lesson No. 28.

Lesson topic:

Target: Studying methods of microbiological diagnosis, etiotropic therapy and prevention of shigellosis.

Module 2 . Special, clinical and environmental microbiology.

Topic 5: Methods for microbiological diagnosis of dysentery.

Relevance of the topic:Shigellosis is widespread and poses a serious problem in countries with a low sanitary cultural level and a high incidence of insufficient and poor-quality nutrition. In developing countries, the spread of infection is facilitated by poor sanitation, poor personal hygiene, overcrowding and a large proportion of children among the population. In Ukraine, outbreaks of shigellosis are more common in closed groups against the background low level sanitation and hygiene, for example in nurseries and kindergartens, on tourist boats, in psychiatric clinics or shelters for the disabled. Shigella has been the cause of diarrhea in travelers and tourists.

The cause of group diseases can be considered the consumption of food products contaminated due to the negligence of sales workers who are carriers of Shigella. Outbreaks associated with the use of drinking water, swimming in polluted waters also led to infection. However, food and waterborne transmission routes appear to play a lesser role in the spread of shigellosis compared to cholera and typhoid fever, which usually require large doses of pathogens to infect humans. In developing countries, where disease spread is predominantly person-to-person, carriers may be an important reservoir of the infectious agent. In patients who did not take anti bacterial preparations, the excretion of Shigella in feces usually continues for 1–4 weeks, but in a small proportion of cases it continues significantly longer.

Shigellosis is an acute bacterial infection of the intestines caused by one of four types of Shigella. Range clinical forms infections include mild, watery diarrhea and severe dysentery, which is characterized by cramping pain in the abdomen, tenesmus, fever and signs of general intoxication.

Etiology.

The genus Shigella (named after K. Shiga, who in 1898 studied and described in detail the isolated causative agent of bacterial dysentery by A.V. Grigoriev) of the family Enterobacteriaceae consists of a group of closely related species of bacteria with the following properties:

I. Morphological: Shigella are small rods with rounded ends. They differ from other representatives of the family Enterobacteriaceae in the absence of flagella (immobile), do not have spores or capsules, and are gram-negative.

II. Cultural: Shigella are aerobes or facultative anaerobes; optimal cultivation conditions: temperature 37°C, pH 7.27.4. They grow on simple nutrient media (MPA, MPB) in the form of small, shiny, translucent, grayish, round colonies, 1.5 x 2 mm in size. S form. The exception is Shigella Sonne, which often dissociates, forming large, flat, cloudy colonies with jagged edges R shapes (colonies have the appearance of a “grape leaf”). In liquid nutrient media, Shigella produces uniform turbidity, R forms form a precipitate. Liquid medium enrichment is selenite broth.

III. Enzymatic: the main biochemical characteristics necessary for identifying Shigella when isolating a pure culture are the following:

1. absence of gas formation during glucose fermentation;
2. lack of hydrogen sulfide production;
3. no lactose fermentation for 48 hours.

Overall, the four species are further divided into approximately 40 serotypes. According to the characteristics of the main somatic (O) antigens and biochemical properties, the following four species or groups are distinguished: S. dysenteriae (group A, includes: Grigoriev-Shigi, Stutzer-Schmitz, Large-Sachs), S. flexneri (group B), S. boydii (group C) and S. sonnei (group D).

In relation to mannitol, all Shigella are divided into splitting (Shigella Flexner, Boyd, Sonne) and non-splitting (Shigella Grigoriev-Shiga, Stutzer-Schmitz, Large-Sachs) mannitol.

IV. Pathogenicity factors:

1. Invasion plasmidprovides the ability of Shigella to cause invasion with subsequent intercellular spread and reproduction in the epithelium of the colon mucosa;
2. Toxin formation: Shigella has a lipopolysaccharide endotoxin, which is chemically and biochemically similar to the endotoxins of other members of the family Enterobacteriaceae. In addition, S. dysenteriae type I (Shiga bacillus) produces an exotoxin. Since the discovery of the latter, it has been found that it has enterotoxin activity and can cause intestinal secretion, as well as have a cytotoxic effect directed against intestinal epithelial cells; has a neurotoxic effect, which is observed in children with shigellosis. Shiga toxin, entering the blood, along with damage to the submucosal endothelium, also affects the glomeruli of the kidney, as a result of which, in addition to bloody diarrhea, hemolytic uremic syndrome develops with the development of renal failure.

V. Antigenic structure:All Shigella have a somatic O-antigen, depending on the structure of which they are divided into serovars.

VI. Resistance: Temperature 100 0 C kills Shigella instantly. TO low temperatures Shigella is resistant: in river water they persist for up to 3 months, on vegetables and fruits for up to 15 months.Under favorable conditions, Shigella is capable of reproducing in food products(salads, vinaigrettes, boiled meat, minced meat, boiled fish, milk and dairy products, compotes and jelly), especially Shigella Sonne.

Epidemiology.

1. Source of infection:A person suffering from acute and chronic forms of shigellosis; bacteria carrier.

2. Transmission routes:

• Food grade (mainly for S. sonnei)
• Aquatic (mainly for S. flexneri)
• Contact household (mainly for S. dysenteriae)

3. Entrance gateinfections are caused by the gastrointestinal tract.

Pathogenesis and pathological changes.

Once ingested, Shigella colonizes the upper small intestine and multiplies there, possibly causing increased secretion early in the infection. Shigella then penetrates through M cells into the submucosa, where it is absorbed by macrophages. This leads to the death of some Shigella, resulting in the release of inflammatory mediators, which initiate inflammation in the submucosa. Apoptosis of phagocytes allows the other part of Shigella to survive and penetrate the epithelial cells of the mucosa through the basement membrane. Shigella multiply and spread intercellularly within enterocytes, resulting in the development of erosions. When Shigella dies, Shiga and Shiga-like toxins are released, the action of which leads to intoxication. Damage to the mucous membrane is accompanied by swelling, necrosis and hemorrhage, which causes the appearance of blood in the stool. In addition, the toxin affects the central nervous system, which leads to trophic disorders.

Clinical manifestations.

The range of clinical manifestations of shigellosis is very wide, from mild diarrhea to severe dysentery with cramping pain in the abdomen, tenesmus, fever and general intoxication.

Incubation periodranges from several hours to 7 days, most often 2-3 days.Initially, patients experience watery stools, fever (up to 41°C), diffuse abdominal pain, nausea and vomiting. Along with this, patients complain of myalgia, chills, lower back pain and headache. In the coming days from the onset of the disease, signs of dysentery appear: tenesmus, frequent, scanty, bloody-mucous stools. Body temperature gradually decreases, pain can be localized in the lower quadrants of the abdomen. The intensity of diarrhea reaches its maximum around the end of the 1st week of illness. Dysentery with bloody stools is more common and appears earlier in the disease caused by S. dysenteriae type I than in other forms of shigellosis.

For shigellosis Sonne A milder course of the disease is characteristic (gastroenteric or gastroenterocolitic variant). The febrile period is shorter, the symptoms of intoxication are short-lived, and destructive changes in the intestinal mucosa are not typical.

Flexner's shigellosisThere are basically two options clinical course- gastroenterocolitic and colitis.

Extraintestinal complications in shigellosisrare:

1. A complication of shigellosis can be the development of intestinal dysbiosis.
2. Along with headaches, signs of meningitis and seizures may occur.
3. In infections caused by S. dysenteriae type I, cases have been described peripheral neuropathy, and during an outbreak of gastroenteritis caused by S. boydii, cases of Guillain-Barré syndrome (polyneuritis) were encountered.
4. With the exception of children suffering from dystrophy, hematogenous dissemination of the pathogen is relatively rare; cases of shigellosis abscesses and meningitis have also been described.
5. With shigellosis, the development of Reiter's syndrome with arthritis, sterile conjunctivitis and urethritis is possible; this usually occurs 1-4 weeks after the onset of diarrhea in patients.
6. In children, shigellosis is accompanied by hemolytic-uremic syndrome, often in combination with leukemia-like reactions, severe colitis and endotoxin circulation, but bacteremia is usually not detected.
7. Quite rarely, purulent keratoconjunctivitis is caused by Shigella, which has entered the eyes as a result of self-infection with contaminated fingers.
8. Hypovolemic shock and disseminated intravascular coagulation syndrome.
9. Peritonitis, intestinal gangrene, intestinal bleeding.

Immunity: Humans have natural resistance to shigella infection. After past illness immunity is not stable, and after shigellosis Sonne is practically absent. In case of a disease caused by Shigella Grigoriev Shiga, a more stable antitoxic immunity is developed. In protection against infection, the main role belongs to secretory IgA , preventing adhesion, and cytotoxic antibody-dependent activity of intraepithelial lymphocytes, which, together with secretory IgA destroy Shigella.

Diagnostics and laboratory tests.

Purpose of the study: detection and identification of Shigella for diagnosis; identification of bacteria carriers; detection of Shigella in food products.

Material for research: feces, sectional material, food products.

Diagnostic methods:microbiological (bacteriological, microscopic (luminescent); serological; biological; allergy test.

Progress of the study:

1 day of study:Cultures should be done from freshly excreted feces or using rectal swabs (rectal tube); If suitable conditions are not available, the material must be placed in a transport environment. To do this, use intestinal agar (MacConkey or Shigella-Salmonella medium), moderately selective xylose-lysine deoxycholate agar, KLD) and nutrient broth (selenite broth). If the time between collection and inoculation exceeds 2 hours, then preservative solutions should be used: 20% bile broth, combined Kauffmann's medium.

• The feces in the glycerin mixture are emulsified, a drop of the emulsion is applied to the medium and rubbed in with a spatula. Differential media for Shigella are Ploskirev, Endo and EMS (eosin methylene blue agar). Ploskirev's medium (the medium includes: MPA, lactose, bile salts and the indicator brilliant green) is also an selective medium for Shigella, because inhibits the growth of Escherichia coli.
• In parallel with direct sowing, the collected material is inoculated on an enrichment medium - selenite broth.
• All crops are placed in a thermostat.

Day 2 of the study:

• The dishes are removed from the thermostat, suspicious colonies are screened out on Ressel's medium (nutrient medium which includes: agar-agar, Andrede indicator, 1% lactose, 0.1% glucose) and mannitol. Inoculation is carried out with strokes on a beveled surface and an injection into an agar column. The inoculated Ressel medium is placed in a thermostat for 18-24 hours (at the same time, reseeding from the selenite medium is done into differential diagnostic media).
• Smears are made (Gram stain) and examined under a microscope.
• Preparations are prepared as a “hanging” or “crushed” drop.
• Establishment of tentative RA with polyvalent diagnostic shigella sera.
• Sowing suspicious colonies onto agar slants.

Day 3 of the study:

• Microscopy of material from agar slants.
• Cultures that have not fermented lactose on Ressel's medium are subjected to further study: smears are made (Gram staining), and the purity of the culture is checked. In the presence of gram-negative rods, inoculation is carried out on Hiss media, broth with indicator papers (to detect indole and hydrogen sulfide) and litmus milk.
• The inoculated media are placed in a thermostat for 18-24 hours.

Day 4 of the study:

• Accounting for a short “variegated row”.
• Cultures suspicious for their enzymatic and cultural properties in relation to Shigella are subjected to serological identification. Statement of RA on glass (typical and group diagnostic sera). Setting up a deployed RA.

As accelerated methods for shigellosis, they are usedfluorescence microscopy And biological sample(introduction of virulent strains of Shigella into the conjunctival sac (under the lower eyelid) of guinea pigs; conjunctivitis develops by the end of the 1st day).

Tsuverkalov allergy testintradermal allergy test with dysenterine (injection of 0.1 ml of dysenterine into the forearm positive reaction in case of infiltration and hyperemia). Allergy diagnostics are currently practically not used. Tsurvekalov's test is not specific; positive reactions are recorded not only for shigellosis, but also for salmonellosis, escherichiosis, yersiniosis, etc. OKI, and sometimes in healthy individuals.

Treatment and prevention.For treatment and prevention according to epidemiological indications, bacteriophage is used oral administration, antibiotics after determining the antibiogram; in case of dysbacteriosis probiotic preparations to correct microflora. To replenish the loss of fluids and electrolytes, administer a glucose-electrolyte solution inside.

Specific goals:

Interpret the biological properties of shigellosis pathogens.

Familiarize yourself with the classification of Shigella.

Learn to interpret the pathogenetic patterns of the infectious process caused by Shigella.

Determine methods of microbiological diagnosis, etiotropic therapy and prevention of shigellosis.

Be able to:

• Inoculate the test material on nutrient media.
  • Prepare smears and Gram stain.
  • Conduct microscopy of preparations using an immersion microscope.
  • Analyze the morphological, cultural, enzymatic characteristics of Shigella.

Theoretical questions:

1. Characteristics of shigellosis pathogens. Biological properties.

2. Classification of Shigella. The principles underlying it.

3. Epidemiology, pathogenesis and clinical features Shigellosis

4. Laboratory diagnostics.

5. Principles of treatment and prevention of shigellosis.

Practical tasks performed in class:

1. Microscopy of demonstration preparations from pure cultures of shigellosis pathogens.

2. Work on the bacteriological diagnosis of shigellosis: study of fecal cultures on Ploskirev’s medium.

3. Subculture of suspicious colonies on Ressel’s medium and MPB to determine indole formation and H 2 S.

4. Sketching demonstration preparations and microbiological diagnostic diagrams of shigellosis into the lesson protocol.

5. Drawing up the protocol.

Literature:

1. Korotyaev A.I., Babichev S.A., Medical microbiology, immunology and virology / Textbook for medical universities, St. Petersburg " Special literature", 1998. - 592 p.

2. Timakov V.D., Levashev V.S., Borisov L.B. Microbiology / Textbook.-2nd ed., revised. And additional - M.: Medicine, 1983, - 512 p.

3. Pyatkin K.D. Krivoshein Yu.S. Microbiology with virology and immunology.- Kyiv: V i scha school, 1992. - 431 p.

4. Medical microbiology / Edited by V.I. Pokrovsky.-M.:GEOTAR-MED, 2001.-768p.

5. Guide to practical classes in microbiology, immunology and virology. Ed. M.P. Zykova. M. "Medicine". 1977. 288 p.

6. Cherkes F.K., Bogoyavlenskaya L.B., Belskan N.A. Microbiology. /Ed. F.K. Circassian. M.: Medicine, 1986. 512 p.

7. Lecture notes.

Additional literature:

1. Makiyarov K.A. Microbiology, virology and immunology. Alma-Ata, “Kazakhstan”, 1974. 372 p.

2. Titov M.V. Infectious illnesses. - K., 1995. 321 p.

3. Shuvalova E.P. Infectious diseases. -M.: Medicine, 1990. - 559 p.

4. BME, T. 1, 2, 7.

5. Pavlovich S.A. Medical microbiology in graphs: Textbook. allowance for medical Inst. Mn.: Higher. school, 1986. 255 p.

Brief guidelines to work in a practical lesson.

At the beginning of the lesson, the students' level of preparation for the lesson is checked.

Independent work consists of studying the classification of Shigella, analyzing the pathogenetic and clinical signs Shigellosis Study of methods for laboratory diagnosis of shigellosis. Students inoculate biomaterial on nutrient media. Then microslides are prepared, stained with Gram, microscopy is performed, microslides are sketched and the necessary explanations are given. Independent work also includes microscopy of demonstration preparations and their sketching in the lesson protocol.

At the end of the lesson, a test control and analysis of the final results of each student’s independent work are carried out.

Technological map for conducting a practical lesson.

Target training tasks:

1. Feces containing mucus and pus were obtained from a child with ACI (collection of stool was carried out using a rectal tube). What express diagnostic method should be used?

A. ELISA.

B. REEF.

C. RA.

D. RSK.

E. RIA.

1. The causative agent of dysentery was isolated from a sick child with an acute intestinal infection. What morphological features are characteristic of the pathogen?

A . Gram-negative non-motile rod.

B . Gram-positive motile rod.

C . Forms a capsule on a nutrient medium.

D . Forms spores in the external environment.

E . Gram-positive streptobacilli.

3. In a patient who fell ill three days ago and complained of a temperature of 38°C, abdominal pain, frequent loose stools, and the presence of blood in the stool, the doctor clinically diagnosed bacterial dysentery. What microbiological diagnostic method is advisable to use in this case and what material should be taken from the patient to confirm the diagnosis?

A. Bacterioscopic cal.

B. Bacteriological cal.

C. Bacterioscopic blood.

D. Bacteriological urine.

E. Serological blood.

4. Shigella Sonne was isolated from the patient’s feces. What additional research needs to be done to determine the source of infection?

A . Carry out phage typing of the isolated pure culture.

B . Determine the antibiogram.

C . Set up a precipitation reaction.

D . Perform a complement fixation reaction.

E . Set up a neutralization reaction.

5. Among a group of tourists (27 people) who used water from the lake for drinking, after two days, 7 people developed symptoms of acute diarrhea. What material is needed to establish the etiology? of this disease need to be sent to a bacteriological laboratory?

A. Water, feces of patients.

B. Water, the blood of patients.

C. Food products.

D. I'm peeing.

E. Sputum.

6. A significant drawback of the microscopic diagnostic method for acute intestinal infections is its lack of information content due to the morphological identity of bacteria of the family Enterobacteriaceae . What makes this method more informative?

A . Radioimmunoassay.

B . Coombs reaction.

C . Linked immunosorbent assay.

D . Opsonization reaction.

E . Immunofluorescence reaction.

7. A 29-year-old patient was hospitalized with attacks of vomiting, diarrhea, and tenesmus. Feces with pieces of mucus and some blood. A bacteriological study of bacteria from colonies on Ploskirev's medium revealed immobile, gram-negative rods that do not ferment lactose. Name the causative agent of the infectious process.

A. Shigella flexneri.

B. Vibrio eltor.

C. E. Coli.

D. Proteus mirabilis.

E. Salmonella enteritidis.

8. Lettuce, which is believed to be the cause of an acute intestinal infection, was delivered to the microbiological laboratory. What nutrient media are used for primary sowing?

A . Yolk salt agar, MPB.

B. MPA, MPB.

C . Selenite broth, Endo, Ploskireva.

D . Liver broth, Roux medium.

E . Blood agar, alkaline agar.

9. When microbiological research minced meat bacteria belonging to the genus Shigella were isolated. The study of what properties of microbes led to this conclusion?

A . Cultural, tinctorial.

B . Antigenic, cultural.

C . Saccharolytic, proteolytic.

D . Antigenic, immunogenic.

E . Morphological, antigenic.

10. During a microscopic examination of vomit taken from a patient with symptoms of acute intestinal infection, immobile rods were found. In what smear or preparation could the mobility of bacteria be studied?

A . In a Gram-stained smear.

B . In a smear stained according to Ziehl-Neelsen.

C . The preparation contains a “thick drop”.

D . In a smear stained according to Neisser.

E . The preparation contains a “crushed drop”.

Algorithm laboratory work:

1. Study of the biological properties of Shigella.

2. Familiarization with the classification of Shigella.

3. Analysis of the scheme of pathogenetic and clinical manifestations of shigellosis.

4. Study of methods for laboratory diagnosis of shigellosis.

5. Study of the basic principles of therapy and prevention of shigellosis.

1. Preparation of fixed preparations from bacterial culture.
2. Coloring microslides according to Gram.
3. Microscopy of microslides With using an immersion microscope, their analysis and recording in the lesson protocol.
4. Mi croscopy and analysis of demonstration preparations from pure cultures of Shigella.
5. Drawing of demonstration preparations and laboratory diagnostic diagrams of shigellosis into the protocol.
6. Drawing up the protocol.

Dysentery is a severe intestinal infection characterized by an acute onset. Microbiological diagnostics dysentery involves isolating the pathogen from the patient’s feces by inoculating it in a special nutrient medium. The disease must be differentiated from other intestinal diseases and poisonings. Early diagnosis and timely treatment will help avoid complications.

The importance of timely diagnosis

Recognizing dysentery in practice is not so easy because there are infectious and non-infectious diseases with similar clinical manifestations. A characteristic feature of dysentery pathogens (Shigella) is the ability to change resistance to antibacterial drugs. An untimely diagnosis of the disease will lead to infection of a large number of people. Improper use of antibiotics is the cause of bacterial resistance, leading to mass infections and epidemics with fatal outcomes. The source of infection is patients and carriers of bacteria who secrete pathogenic microorganisms with fecal matter. The incubation period of dysentery is 2-3 days.

Clinical symptoms of the disease

• Sudden fever with a body temperature of 40 degrees or higher.
• Diarrhea more than 10 times a day.
• The appearance of blood, mucus, and, in rare cases, pus in the stool.
• Loss of appetite up to complete absence.
• Nausea and vomiting.
• Cutting in the abdomen and right hypochondrium.
• Pain in the rectum.
• Dehydration.
• Dry tongue with a white coating.
• Arrhythmia.
• Reduced blood pressure.
• Disorders of consciousness.

Diagnostic procedures

Diagnosis of the disease includes generally accepted and special methods that establish not only the final diagnosis, but also assess the level of dysfunction of the digestive organs. With dysentery, the diagnosis is made on the basis of the epidemiological picture of the disease, clinical symptoms and research. The main laboratory diagnostics is stool microbiology analysis, which identifies up to 80% of pathogens. The serological method is carried out no earlier than the 5th day of the disease; this type of research complements, but does not replace microbiological analysis. Other methods:

• Coprological examination is a simple and accessible clinical method that detects mucus, blood streaks, red blood cells, neutrophils (up to 50 in the field of view) and altered epithelial cells.
• Sigmoidoscopy - allows you to monitor the healing process. Not for use in children.
• The allergy test method is an auxiliary method based on taking a skin allergy test with dysenterine (Tsuverkalov method).

General blood analysis

Immune cells destroy dysentery pathogens in the intestines, and severe cases of the disease occur when bacteria invade the lymph nodes and subsequently enter the bloodstream. A blood test for dysentery evaluates the patient’s condition and allows timely response to possible complications. Increased erythrocyte sedimentation rate - laboratory value, characterizing the degree of inflammation. Dysentery also causes an increase in the concentration of band neutrophils and monocytes.

How to donate feces for coprogram?

To confirm the disease, a stool test is performed. A coprogram is a detailed laboratory study that evaluates the functioning of the gastrointestinal tract, the speed and efficiency of digestion and intestinal function. Laboratory methods for examining feces reveal the physical and chemical properties of feces, composition, and the presence of foreign organisms and inclusions. Requirements for stool collection:

• The material is taken after the natural act of defecation.
• The collection is carried out in a special container.
• It is prohibited to take biological material obtained as a result of an enema to test stool for dysentery.
• Before the study, it is prohibited to use iron supplements, use rectal suppositories, take laxatives, or drink alcoholic beverages.

Microbiological diagnostics

Bacteriological diagnostics - collection of fecal matter and subsequent sowing of feces in a special nutrient medium. The appearance of colonies of pathogenic bacteria (Shigella) after culture confirms the suspected diagnosis. Bacteriological analysis for dysentery accurately determines the pathogen, its type, subspecies and susceptibility to antibacterial agents, which allows you to choose the right drug for treatment.

The material being tested is feces with foreign impurities, obtained naturally or with a special tube for sigmoidoscopy. A smear is taken from children with a special swab (VD smear or intestinal smear). Drug sensitivity is established by placing Shigella colonies together with various antibiotics. If the vital activity of microorganisms continues near the tablet with an antibiotic, then the drug is not used for treatment; if the microorganisms die, treatment with such an antibiotic is prescribed.

Serological tests for dysentery

In case of negative or questionable results of bacteriological examination, the serological method is used. In the patient's stool it is detected bacterial antigen, and in plasma - specific antibodies. The antibody titer can be determined using the RIGA method, sometimes using the RPGA or RA method. A suspension of a daytime Shegella colony is used as antigens. The disadvantage of the method is that reliable results are obtained only 5 days after the onset of the disease, when the concentration of antibodies reaches the desired level.

Sigmoidoscopy

Due to the fact that the causative agent of dysentery affects the large intestine, sigmoidoscopy - significant method diagnostic, but not definitive. Diagnosis consists of inserting a rectoscope into the anus, equipped with a device that supplies air. By swelling, the intestinal cavity becomes accessible for examination. This method helps to assess the degree of damage to the intestinal epithelium. With dysentery, the intestinal walls become hyperemic as a result of vasodilation. Erosion and hemorrhage occur in some sections. Sigmoidoscopy does not require preparation, but the procedure is not performed if anal fissures or pathologies of the anus are present.

The content of the article

Historical data of dysentery

Etiology of dysentery

Epidemiology of dysentery

Pathogenesis and pathomorphology of dysentery

Dysentery Clinic

Chronic dysentery

Diagnosis of dysentery

Specific diagnosis of dysentery

Differential diagnosis of dysentery

Treatment of dysentery

Prevention of dysentery

Test for dysentery is a collective concept that includes general clinical and specific methods studies that help establish not only the final diagnosis of shigellosis (a more modern name for dysentery), but also assess the degree of disturbances in various organ systems in the body.

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Laboratory diagnosis of dysentery includes:

• general clinical methods (traditional blood and urine tests);
• coprogram;
• biochemical tests;
• bacteriological method;
• serological reactions;
• allergic skin test (rare);
• instrumental studies.

The expediency of a particular diagnostic study is determined by the current medical documentation, namely the protocols for the provision of medical care. Not only the components of the diagnosis of shigellosis are regulated, but also the frequency of their implementation. This detail is important for the so-called decreed group, that is, people working in the food industry and children's groups - a certain number of negative tests is the basis for admission to work.

General clinical methods

Study of the cellular composition of blood

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This routine research method can be quite informative specifically for dysentery, as it reflects the severity of the disease. With a mild course, a general blood test may not reveal any changes or they will be insignificant. On the contrary, in severe forms of the disease, typical for bacterial infection the accents are expressed very violently.

In severe forms of shigellosis, the following can be detected:

• a significant increase in the absolute number of leukocytes (that is, hyperleukocytosis);
• a significant shift of the formula to the left, that is, an increase in the absolute and relative number of band lymphocytes, up to the appearance of young forms;
• toxic granularity of neutrophils;
• increased erythrocyte sedimentation rate;
• a decrease in the level of color index and hemoglobin concentration, the number of red blood cells (erythrocytes), that is, classic signs of anemia.

Pronounced changes in the general blood test indicate not only a severe course of the disease, but also possible development complications such as intestinal bleeding.

On the other hand, even pronounced changes in the general blood test are nonspecific, that is, they can be observed in many other diseases, and therefore are not the basis for making a final diagnosis.

Urine examination

Only in the case of a very severe course of the disease are changes observed in the general urine analysis, which are a consequence of severe intoxication. Such signs include the appearance of red blood cells, a large number of white blood cells and casts, as well as an increase in protein concentration. In order to exclude possible pathology of the urinary tract, a general urine test should be repeated after the clinical symptoms of dysentery have subsided.

Coprogram

Stool analysis reflects all changes typical of dysenteric intestinal damage. In addition, the nature and extent of the identified changes is directly related to the severity of the clinical course of the disease.

When examining stool, the following changes are observed:

• increased number of leukocytes (normally there can be only a few);
• the appearance of red blood cells (normally absent);
• mucus in varying quantities (not normally detected);
• undigested food particles and epithelium as a result of disruption of food digestion processes, as well as damage epithelial tissue intestines.

It is necessary to understand that the obtained results of the coprogram, as well as general clinical tests of urine and blood, cannot be considered as a final diagnosis of shigellosis. To establish the specific type of Shigella that caused the development of clinical symptoms of the disease in a given patient, as well as to assess its sensitivity to certain antibiotics, a full microbiological diagnosis of dysentery is necessary.

Specific diagnostics

If dysentery is suspected, diagnosis involves isolating the pathogen from the patient's biological fluids (primarily from feces, less commonly gastric lavage and vomit) or determining the titer of protective antibodies that are produced in response to the introduction of a microbial agent.

Bacteriological method

It is the most common, informative and accessible in most cases and in the majority of clinics. Stool collection for examination should be carried out in the first days of illness. Feces obtained from in a natural way, as well as taken with a sigmoidoscopy tube or a cotton swab (a kind of smear). Collect biological material in a clean container that has not been treated with disinfectant solutions.

The largest amount of the causative agent of dysentery is detected in those areas of stool where mucus and pus are present. Sowing is done on conventional nutrient media - Levin, Ploskireva, Endo. The result, containing not only comprehensive information about the type of Shigella, but also parameters of sensitivity to antimicrobial drugs, the doctor receives it 3-5 days after collecting the material.

Serological method

Less informative in comparison with the bacteriological method. This is due to the fact that clinical picture uncomplicated and mild dysentery does not last more than 5-7 days, and the serological method for dysentery takes much longer. It is rare for a patient to spend 2-2.5 weeks in a hospital bed awaiting serological results. Serological tests can be useful and informative as a method of retrospective diagnosis or in scientific research.

Most often, an agglutination reaction is performed: the presence of protective antibodies in a certain concentration is detected in the patient’s blood serum using known antigens. It is advisable to evaluate the information content of the agglutination reaction in dynamics.

The reaction of indirect and/or direct agglutination is less specific. Blood serum is taken no earlier than 4-5 days of illness, and again on days 12-14 from the onset of clinical manifestations. Allergy diagnostics

In the middle of the twentieth century, one of the mandatory tests for shigellosis of any severity was a skin allergy test with dysenterin (Tsuverkalov test). In modern medical practice Due to the allergenicity of the population and the non-specificity of this test, most clinics refused to perform it.

Instrumental diagnostics

The most common method is sigmoidoscopy. To carry it out, you only need a competent trained specialist and a portable device. There is no need to allocate a separate room, its special equipment and other technical details.

The sigmoidoscope tube is inserted into the anus to a certain depth. The condition of the mucous membrane is visually assessed lower sections rectum and sphincter. When dysentery is detected:

• ulcerative defects of various sizes;
• diffuse swelling and hyperemia of the mucous membrane;
• areas of hemorrhage.

Sigmoidoscopy is intended to evaluate the effectiveness of the prescribed therapy (whether there are positive dynamics or not, that is, healing of ulcers), as well as to exclude other diseases with similar clinical symptoms (nonspecific ulcerative colitis, tumor formation). In the initial period of shigellosis, this study is not indicated, since the patient’s discomfort from the procedure exceeds its diagnostic value.

Fibercolonoscopy (penetration into higher parts of the intestine) is indicated only if it is necessary to exclude other intestinal diseases (neoplastic tumors).

Thus, only a comprehensive diagnosis of dysentery allows one to correctly assess the severity of the patient’s condition and the effectiveness of the prescribed therapy.

Microbiology of dysentery

Dysentery is an infectious disease characterized by general intoxication of the body, diarrhea and a peculiar lesion of the mucous membrane of the large intestine. It is one of the most common acute intestinal diseases in the world. The disease has been known since ancient times under the name “bloody diarrhea”, but its nature turned out to be different. In 1875, the Russian scientist F.A. Lesh isolated an amoeba from a patient with bloody diarrhea Entamoeba histolytica, in the next 15 years, the independence of this disease was established, for which the name amoebiasis was retained.

The causative agents of dysentery proper are a large group of biologically similar bacteria, united in the genus Shigella. The pathogen was first discovered in 1888 by A. Chantemes and F. Vidal; in 1891 it was described by A.V. Grigoriev, and in 1898 K. Shiga, using serum obtained from a patient, identified the pathogen in 34 patients with dysentery, finally proving the etiological role of this bacterium. However, in subsequent years, other causative agents of dysentery were discovered: in 1900 - by S. Flexner, in 1915 - by K. Sonne, in 1917 - by K. Stutzer and K. Schmitz, in 1932 - by J. Boyd , in 1934 - D. Large, in 1943 - A. Sax. Currently genus Shigella includes more than 40 serotypes. All of them are short, non-motile gram-negative rods that do not form spores or capsules, which grow well on regular nutrient media and do not grow on starvation media with citrate or malonate as the sole carbon source; do not form H 2 S, do not have urease; the Voges–Proskauer reaction is negative; glucose and some other carbohydrates are fermented to form acid without gas (except for some biotypes Shigella flexneri: S. manchester And S. newcastle); As a rule, they do not ferment lactose (with the exception of Shigella Sonne), adonitol, salicin and inositol, do not liquefy gelatin, usually form catalase, and do not have lysine decarboxylase and phenylalanine deaminase. The content of G + C in DNA is 49 – 53 mol%. Shigella are facultative anaerobes, the optimum temperature for growth is 37 °C, they do not grow at temperatures above 45 °C, the optimal pH of the environment is 6.7 - 7.2. Colonies on dense media are round, convex, translucent; in case of dissociation, rough R-form colonies are formed. Growth on MPB in the form of uniform turbidity, rough forms form a sediment. Freshly isolated cultures of Shigella Sonne usually form colonies of two types: small round convex (phase I), large flat (phase II). The nature of the colony depends on the presence (phase I) or absence (phase II) of a plasmid with a molecular weight of 120 MD, which also determines the virulence of Shigella Sonne.

The international classification of Shigella is based on their biochemical characteristics (mannitol-non-fermenting, mannitol-fermenting, slowly lactose-fermenting Shigella) and features of the antigenic structure (Table 37).

In Shigella, O-antigens of different specificity were found: common to the family Enterobacteriaceae, generic, species, group and type-specific, as well as K-antigens; They do not have N-antigens.

Table 37

Classification of bacteria genus Shigella

The classification takes into account only group and type-specific O-antigens. In accordance with these characteristics, the genus Shigella is divided into 4 subgroups, or 4 species, and includes 44 serotypes. In subgroup A (type Shigella dysenteriae) included Shigella, which does not ferment mannitol. The species includes 12 serotypes (1 – 12). Each serotype has its own specific type antigen; antigenic connections between serotypes, as well as with other Shigella species, are weakly expressed. To subgroup B (type Shigella flexneri) include Shigella, which usually ferments mannitol. Shigella of this species are serologically related to each other: they contain type-specific antigens (I – VI), by which they are divided into serotypes (1 – 6), and group antigens, which are found in different compositions in each serotype and by which the serotypes are divided into subserotypes. In addition, this species includes two antigenic variants - X and Y, which do not have typical antigens; they differ in sets of group antigens. Serotype S. flexneri 6 has no subserotypes, but it is divided into 3 biochemical types according to the characteristics of the fermentation of glucose, mannitol and dulcitol (Table 38).

Table 38

Biotypes S. flexneri 6

Note. K – fermentation with the formation of only acid; CG – fermentation with the formation of acid and gas; (–) – no fermentation.

Lipopolysaccharide antigen O in all Shigella Flexner contains group antigen 3, 4 as the main primary structure, its synthesis is controlled by a chromosomal gene localized near the his-locus. Type-specific antigens I, II, IV, V and group antigens 6, 7, 8 are the result of modification of antigens 3, 4 (glycosylation or acetylation) and are determined by the genes of the corresponding converting prophages, the site of integration of which is located in the lac - pro region of the Shigella chromosome.

Appeared in the country in the 80s. XX century and a new subserotype that has become widespread S. flexneri 4(IV:7, 8) differs from subserotype 4a (IV:3, 4) and 4b (IV:3, 4, 6), arose from a variant S. flexneri Y(IV:3, 4) due to lysogenization by its converting prophages IV and 7, 8.

To subgroup C (type Shigella boydii) include Shigella, which usually ferments mannitol. Members of the group are serologically different from each other. Antigenic connections within the species are weakly expressed. The species includes 18 serotypes (1 – 18), each of which has its own main type antigen.

In subgroup D (type Shigella sonnei) included Shigella, which usually ferment mannitol and are capable of slowly (after 24 hours of incubation and later) fermenting lactose and sucrose. View S. sonnei includes one serotype, but colonies of phases I and II have their own type-specific antigens. For the intraspecific classification of Shigella Sonne, two methods have been proposed:

1) dividing them into 14 biochemical types and subtypes according to their ability to ferment maltose, rhamnose and xylose; 2) division into phage types according to sensitivity to a set of corresponding phages.

These typing methods have mainly epidemiological significance. In addition, Shigella Sonne and Shigella Flexner are typed for the same purpose based on their ability to synthesize specific colicins (colicinogenotyping) and sensitivity to known colicins (colicinotyping). To determine the type of colicins produced by Shigella, J. Abbott and R. Chenon proposed sets of standard and indicator strains of Shigella, and to determine the sensitivity of Shigella to known types of colicins, a set of standard colicinogenic strains of P. Frederick is used.

Resistance. Shigella has a fairly high resistance to environmental factors. They survive on cotton fabric and paper for up to 30 - 36 days, in dried feces - up to 4 - 5 months, in soil - up to 3 - 4 months, in water - from 0.5 to 3 months, on fruits and in vegetables – up to 2 weeks, in milk and dairy products – up to several weeks; at a temperature of 60 °C they die in 15 – 20 minutes. Sensitive to chloramine solutions, active chlorine and other disinfectants.

Pathogenicity factors. The most important biological property of Shigella, which determines their pathogenicity, is the ability to invade epithelial cells, multiply in them and cause their death. This effect can be detected using a keratoconjunctival test (injection under the lower eyelid guinea pig one loop of Shigella culture (2 - 3 billion bacteria) causes the development of serous purulent keratoconjunctivitis), as well as by infecting cell cultures (cytotoxic effect) or chicken embryos (their death), or intranasally in white mice (development of pneumonia). The main pathogenicity factors of Shigella can be divided into three groups:

1) factors determining interaction with the epithelium of the mucous membrane;

2) factors that ensure resistance to humoral and cellular defense mechanisms of the macroorganism and the ability of Shigella to reproduce in its cells;

3) the ability to produce toxins and toxic products that determine the development of the pathological process itself.

The first group includes adhesion and colonization factors: their role is played by pili, proteins outer membrane and LPS. Adhesion and colonization are promoted by enzymes that destroy mucus - neuraminidase, hyaluronidase, mucinase. The second group includes invasion factors that promote the penetration of Shigella into enterocytes and their reproduction in them and in macrophages with the simultaneous manifestation of a cytotoxic and (or) enterotoxic effect. These properties are controlled by the genes of a plasmid with a molecular weight of 140 MD (it encodes the synthesis of outer membrane proteins that cause invasion) and the chromosomal genes of Shigella: kcp A (causes keratoconjunctivitis), cyt (responsible for cell destruction), as well as other genes not yet identified. Protection of Shigella from phagocytosis is provided surface K antigen, antigens 3, 4 and lipopolysaccharide. In addition, lipid A of Shigella endotoxin has an immunosuppressive effect: it suppresses the activity of immune memory cells.

The third group of pathogenicity factors includes endotoxin and two types of exotoxins found in Shigella - Shiga and Shiga-like exotoxins (SLT-I and SLT-II), the cytotoxic properties of which are most pronounced in S. dysenteriae 1. Shiga and Shiga-like toxins have also been found in other serotypes S. dysenteriae, they are also formed S. flexneri, S. sonnei, S. boydii, EHEC and some salmonella. The synthesis of these toxins is controlled by the tox genes of converting phages. Type LT enterotoxins are found in Shigella Flexner, Sonne and Boyd. Their LT synthesis is controlled by plasmid genes. Enterotoxin stimulates the activity of adenylate cyclase and is responsible for the development of diarrhea. Shiga toxin, or neurotoxin, does not react with the adenylate cyclase system, but has a direct cytotoxic effect. Shiga and Shiga-like toxins (SLT-I and SLT-II) have a molecular weight of 70 kDa and consist of subunits A and B (the latter of 5 identical small subunits). The receptor for toxins is a glycolipid of the cell membrane.

The virulence of Shigella Sonne also depends on a plasmid with a molecular weight of 120 MD. It controls the synthesis of about 40 outer membrane polypeptides, seven of them are associated with virulence. Shigella Sonne, having this plasmid, form phase I colonies and are virulent. Cultures that have lost the plasmid form phase II colonies and lack virulence. Plasmids with a molecular weight of 120–140 MD were found in Shigella Flexner and Boyd. Shigella lipopolysaccharide is a strong endotoxin.

Features of epidemiology. The source of infection is only humans. No animals in nature suffer from dysentery. Under experimental conditions, dysentery can only be reproduced in monkeys. The method of infection is fecal-oral. Routes of transmission: water (predominant for Shigella Flexner), food, especially milk and dairy products (predominant route of infection for Shigella Sonne), and household contact, especially for the species S. dysenteriae.

A feature of the epidemiology of dysentery is a change in the species composition of pathogens, as well as Sonne biotypes and Flexner serotypes in certain regions. For example, until the end of the 30s. XX century to a share S. dysenteriae 1 accounted for up to 30–40% of all cases of dysentery, and then this serotype began to occur less and less often and almost disappeared. However, in the 1960s - 1980s. S. dysenteriae reappeared on the historical arena and caused a series of epidemics that led to the formation of three hyperendemic foci of it - in Central America, Central Africa and South Asia (India, Pakistan, Bangladesh and other countries). The reasons for the change in the species composition of dysentery pathogens are probably associated with changes in collective immunity and changes in the properties of dysentery bacteria. In particular, the return S. dysenteriae 1 and its widespread distribution, which caused the formation of hyperendemic foci of dysentery, is associated with its acquisition of plasmids that caused multidrug resistance and increased virulence.

Features of pathogenesis and clinic. The incubation period for dysentery is 2–5 days, sometimes less than a day. The formation of an infectious focus in the mucous membrane of the descending part of the large intestine (sigmoid and rectum), where the dysentery pathogen penetrates, is cyclical in nature: adhesion, colonization, introduction of Shigella into the cytoplasm of enterocytes, their intracellular reproduction, destruction and rejection of epithelial cells, release of pathogens into the lumen intestines; after this, the next cycle begins - adhesion, colonization, etc. The intensity of the cycles depends on the concentration of pathogens in the parietal layer of the mucous membrane. As a result of repeated cycles, the inflammatory focus grows, the resulting ulcers, connecting, increase the exposure of the intestinal wall, as a result of which blood, mucopurulent lumps, and polymorphonuclear leukocytes appear in the feces. Cytotoxins (SLT-I and SLT-II) cause cell destruction, enterotoxin – diarrhea, endotoxins – general intoxication. The clinical picture of dysentery is largely determined by the type of exotoxins in to a greater extent produced by the pathogen, the degree of its allergenic effect and the immune status of the body. However, many questions of the pathogenesis of dysentery remain unclear, in particular: the features of the course of dysentery in children of the first two years of life, the reasons for the transition of acute dysentery to chronic, the significance of sensitization, the mechanism of local immunity of the intestinal mucosa, etc. The most typical clinical manifestations of dysentery are diarrhea, frequent urge: in severe cases, up to 50 or more times a day, tenesmus (painful spasms of the rectum) and general intoxication. The nature of the stool is determined by the degree of damage to the large intestine. The most severe dysentery is caused by S. dysenteriae 1, most easily - Sonne dysentery.

Post-infectious immunity. As observations of monkeys have shown, after suffering from dysentery, strong and fairly long-lasting immunity remains. It is caused by antimicrobial antibodies, antitoxins, increased activity of macrophages and T-lymphocytes. Plays a significant role local immunity intestinal mucosa, mediated by IgAs. However, immunity is type-specific; strong cross-immunity does not occur.

Laboratory diagnostics. The main method is bacteriological. The material for research is feces. Pathogen isolation scheme: inoculation on differential diagnostic media Endo and Ploskirev (in parallel on enrichment medium followed by inoculation on Endo and Ploskirev media) to isolate isolated colonies, obtaining a pure culture, studying its biochemical properties and, taking into account the latter, identification using polyvalent and monovalent diagnostic agglutinating sera. The following commercial serums are produced.

1. To Shigella, which does not ferment mannitol:

To S. dysenteriae 1 And 2

To S. dysenteriae 3 – 7(polyvalent and monovalent),

To S. dysenteriae 8 – 12(polyvalent and monovalent).

2. To Shigella fermenting mannitol:

to typical antigens S. flexneri I, II, III, IV, V, VI,

to group antigens S. flexneri 3, 4, 6, 7, 8– polyvalent,

to antigens S. boydii 1 – 18(polyvalent and monovalent), to antigens S. sonnei I phase, II phase,

to antigens S. flexneri I–VI+ S. sonnei– polyvalent.

To quickly identify Shigella, the following method is recommended: a suspicious colony (lactose-negative on Endo medium) is subcultured on TSI medium (English. triple sugar iron) – three-sugar agar (glucose, lactose, sucrose) with iron to determine H2S production; or to a medium containing glucose, lactose, sucrose, iron and urea. Any organism that breaks down urea after 4 to 6 hours of incubation is most likely a member of the genus Proteus and may be excluded. A microorganism that produces H2S or has a urease or acid-forming joint (fermentes lactose or sucrose) can be excluded, although H2S-producing strains should be investigated as possible members of the genus Salmonella. In all other cases, the culture grown on these media should be examined and, if it ferments glucose (change in the color of the column), isolated in its pure form. At the same time, it can be studied in a glass agglutination reaction with appropriate antisera to the genus Shigella. If necessary, other biochemical tests are performed to check genus membership. Shigella, and also study mobility.

To detect antigens in blood (including as part of the CEC), urine and feces can be used following methods: RPGA, RSK, coagglutination reaction (in urine and feces), IFM, RAGA (in blood serum). These methods are highly effective, specific and suitable for early diagnosis.

For serological diagnostics the following can be used: RPHA with the corresponding erythrocyte diagnostic kits, immunofluorescent method (indirect modification), Coombs method (determining titer incomplete antibodies). An allergy test with dysenterine (a solution of protein fractions of Shigella Flexner and Sonne) is also of diagnostic value. The reaction is taken into account after 24 hours. It is considered positive in the presence of hyperemia and infiltrate with a diameter of 10–20 mm.

Treatment. The main attention is paid to restoring normal water-salt metabolism, rational nutrition, detoxification, rational antibiotic therapy (taking into account the sensitivity of the pathogen to antibiotics). Gives a good effect early application polyvalent dysentery bacteriophage, especially tablets with a pectin coating, which protects the phage from the action of HCl gastric juice; V small intestine pectin dissolves, phages are released and exert their effect. For preventive purposes, the phage should be given at least once every three days (the period of its survival in the intestine).

The problem of specific prevention. To create artificial immunity against dysentery, various vaccines were used: from killed bacteria, chemical, alcohol, but all of them turned out to be ineffective and were discontinued. Vaccines against Flexner's dysentery have been created from live (mutant, streptomycin-dependent) Shigella Flexner; ribosomal vaccines, but they also did not find wide application. Therefore, the problem of specific prevention of dysentery remains unresolved. The main way to combat dysentery is to improve the water supply and sewerage system, ensure strict sanitary and hygienic regimes in food enterprises, especially the dairy industry, in child care institutions, public places and in maintaining personal hygiene.