Corinbacteria diphtheria. Corinbacteria

One of the most dangerous diseases is the difftheria, the pathogen is referred to as Corynebacterium of the form (SPP), bacterium having a rod shape.

The body of a healthy person contains a small number of corenebacteria in the colors. In pathological changes, additional infectiousness, the vital activity of microorganisms leads to the disease.

Bacteria is divided into several types, each of which is unique, has the peculiarities of a specific nature. Based on the variety, microorganisms are affected by the skin, affect the work of the internal organs. People who have a weak immunity are risky. Bacteremia will begin development in the case when bacteria will form abdominal, venous catheters.

If men or women have Corinbacteria, the likelihood of septic arthritis, pneumonia is great.

Mobilongus

There are hidden infectious diseases that include hazardous bacteria, such as Mobiluncus SPP and Corynebacterium SPP, present in DNA. The presence in the urine, in sperm or in a smear of dangerous bacteria, will lead to inflammatory processes. Men develops the pathology of the urogenital tract, leading to orchoepidimitimitis, non-phonococcal and other.

Often, mobile microorganism is found in female vaginal discharges, both having bacterial and healthy. With the accumulations of the Mobilunkus in the area of \u200b\u200bthe rectum, the vagina contamination may occur during anal sex.

To diagnose the presence of bacteria, use several methods:

• Polymerase chain reaction;
• Bacterioscopic examination.
• Serological methods.

You can get infected with a disease solely upon contact with a sick person. People who previously suffered the disease also carry the danger to others, having a bacterium-causative in the body.

Bacteria are transmitted by air-droplet, or settle on household items: these are attributes of dishes, bedding, clothing, things related to personal hygiene category and other things. If an infected person has contact with food, they also become causes of infection.

People contacting patients with acute form of diphtheria increase the risk of their own contamination by air-droplet.

Difteria often proceeds asymptomatic for a long time, without the absence of hospitalization, the patient is able to infect many healthy people around. The recovered patient is a carrier for another 3-8 weeks, and sometimes the term increases to 3-5 months.

Treatment

In order for the Corinbacteria to be the cause of the appearance of diseases of reproductive organs, urination systems, before planning a pregnancy, two partners need to pass tests for the presence of bacteria.

If tests show a positive result, doctors write out the course of antibiotics. It is forbidden to engage in self-treatment, a clearly selected treatment diagram for each patient is necessary.

Men living in hot and dry climate have a predisposition to the emergence of erytress, pathology relating to skin dermatitis. The disease is manifested in the field of corporal folds, signs are similar to dermatitis or thrush (the second name -).

When the presence of Corinbacteria in a woman is diagnosed, an accurate determination of the quantity is important. With a moderate enough course of drugs. If the volume exceeds the norm, additional research is carried out to identify other vaginal infectious pathologies. When discovery, the accompanying diseases are first cured.

When prescribing treatment, a woman needs to postpone conception. When the minimum of 30 days after complete recovery, you can think about pregnancy.

Microbiological research, allowing to identify diphtheria causative agent (C. Diphtheriae) in the studied biomaterial.

Russian synonyms

Sowing Lefler Bacillus, sowing on BL, sowing on the diphtheria stick.

Synonyms English

Corynebacterium Diphtheriae Sulture, Diphtheria Sulture.

Research method

Microbiological method.

What kind of biomaterial can be used for research?

Mazz from the zea and nose.

How to research?

Preparation is not required.

General research information

Corynebacterium Diphtheriae (Lefeler Bacillus) is the gram-positive bacteria of the CoryneBacterium genus, which are causers of diphtheria and capable of developing diphtheria toxin. The disease is transmitted by air-droplet, the source of infection is sick people or bacteria carriers.

The incubation period is an average of 2-5 days. The fibrinous inflammation of the mucous membranes of the rotogling and the respiratory tract with the formation of pseudommbran and with the symptoms of general intoxication is occurring.

With the toxic form of diphtheria, the heart and the nervous system can also be affected. In some cases, asymptomatic carriage is possible.

DIYPTERIY Diagnosis is based on clinical data, sowing on diphtheria is carried out to confirm.

What is the study?

• To confirm the diagnosis of "Difteria".
• For differential diagnosis of diseases occurring with similar symptoms, such as angins of various origin, paratonzillar abscess, infectious mononucleosis, acute laryngotrachite, epiglotte, bronchial asthma.
• To assess the effectiveness of the antibacterial therapy.

When is the study assigned?

• With suspected diphtheria.
• When it is known that the patient contacted with diphtheria patients.
• After conducting antibacterial therapy - not less than 2 weeks after the end of the course of antibiotics.
• In some cases, before hospitalization in the hospital (with a prophylactic target).

What do the results mean?

Reference values: No growth.

The detection of the diphtheria causative agent confirms the diagnosis of "diphteria" or, if there are no symptoms of the disease, testifies to bacterication. With a negative resulting in the patient with a suspicion of diphtheria, the diagnosis can be confirmed in the case when the contact persons have a positive resulting, that is, the diphtheria causative agent is distinguished.

Causes of positive results

• Diphtheria or asymptomatic carriage C. diphtheriae.

Causes of negative results

• No diphtheria. An exception is cases when antibiotics treatment was carried out at the time of study.

What can affect the result?

Previous antibacterial therapy.

Important comments

DIIFTERIY Diagnosis is based on a clinical picture of the disease, so treatment must be started before obtaining a laboratory confirmation of the disease. With a positive result of sowing, it is necessary to explore the allocated strain C. Diphtheriae for toxigencies.

• Sowing on the flora with the definition of sensitivity to antibiotics

Who appoints a study?

Infectiousist, therapist, general practitioner, pediatrician, ENT.

Literature

1. MacGregor R.R. Corynebacterium Diphtheriae. In: Principles and Practice of Infectious Disease / G.L. Mandell, Bennett J.E., Dolin R (EDS); 6th Ed. - Churchill Livingstone, Philadelphia, PA 2005. - 2701 p.
2. Efstratiou A. LaboTory Guidelines Caused by Corynebacterium Diphtheriae and C. Ulceran / A. Efstratiou, R.C. Georg // Communicable Disease and Public Health. - 1999. - Vol. 2, No. 4. - P. 250-257.
3. Current Approaches to the Laboratory Diagnosis of Diphtheria / A. Efstratiou // J. Infect. DIS. - 2000. - Vol. 181 (Suppl 1). - P. S138-S145.

Corynebacterium diphtheriae was discovered and then highlighted in a clean culture 100 years ago. Its ultimate etiological significance in the occurrence of diphtheria was confirmed several years later, when a specific toxin was obtained, causing the death of animals in phenomena, similar to the diphtheria observed in patients. CoryNebacterium Diphtheriae refers to the genus Corynebacterium, a group of corinform bacteria. Corynebacterium Diphtheriae is straight or slightly curved sticks with extensions or pointers at the ends. The division on the break and splitting ensures the characteristic location in the form of Roman figures V or spread fingers, but often in the smears there are single sticks. The large clusters that are in the smears prepared from the population mucus, the nose, the wound discharge, have a foil-shaped character. The average length of the sticks of them is 1-8 microns, width - 0.3 / 0.8 microns. They are stationary, disputes and capsules do not form. CoryNebacterium Diphtheriae is an optional anaerobom. Diffex sticks are resistant to dry. At a temperature of 60 ° C in pure cultures are destroyed for 45-60 minutes. In pathological products, i.e., if there are protein protection, they can maintain viability over an hour at a temperature of 90 ° C. Low temperatures do not have a destructive effect on diphtheria sticks. In disinfectants of the usual concentration, they quickly die.

It is necessary to note the extremely large polymorphism of diphtheria sticks, manifested in the change in their thickness and shape (swollen, flavoid, segmented, filamentous, branching), in terminal thickening, and sometimes in the central part after 12 hours of cultural growth, the grains of the Babes are detected during a special color Ernsta, representing the clusters of volutin. It is evidence that volutin is a long-chain inorganic polyphosphate. M. A. Peshkov involves their metaphosphate nature. A. A. Imshnetsky believes that Voliutin is a by-product of metabolic processes. It is known that phosphorus is needed to form grains. There are assumptions about the need for manganese and zinc for this process.

Volyutin grains are found in daily cultures, and then the number of bacteria with the presence of grains decreases, in the cytoplasm there are also nucleotides, intracitoplasmic membranes - lysosomes, vacuol.

Bacteria is painted by all aniline paints. When painting according to the method of gram - positive. For coloring volutin grains, the method of the neosser is used. When painting with this method of grain Volyututin, having a big affinity for methylene blue, steadfastly stained in blue, and from the body of the bacterium with additional coloring chrysoid or bismarkbrush, methylene blue is supplanted.

The causative agent of diphtheria is heterotrof, that is, refers to the group of bacteria requiring organic substances for its growth. The cultivation used for growing medium should contain as a source of carbon and nitrogen amino acids - alanine, cystin, methionine, valine, etc. in connection with this cultivation environmental media are mediums containing animal protein: blood, serum, ascitic fluid. Based on this, a classical leffler medium was created, and then the Clauberga, Tindall medium, the accumulation medium.

On the leffler medium, the diphtheria colony has a brilliant, wet surface, smooth edges, yellowish color. After a few days of growth, the radialochargence of colonies and weakly pronounced concentric lines appear. The diameter of the colonies reaches 4 mm. The first signs of growth appear after 6 hours of stay in the thermostat at 36-38 ° C. The growth is clearly visible 18 hours after sowing. The optimal pH value for the growth of the diphtheria stick is 7.6. Corinbacteria diphtheria is very often distinguishable from other types of Corinbacteria. To determine the species, a complex of culture and biochemical signs is used.

The view of the Corinbacteria of diphtheria is heterogeneous, it is divided into 3 cultural and biochemical types of Gravis, Mitis, Intermediate, for two varieties - toxygenic and non-operational, a number of serological types and phageotypes.

Currently, in most territories, two cultural and biochemical types are circulated - GRAVIS and MITIS. The intermediate type, which previously highlighted also quite widely, is rarely found lately. The most clearly differentiation of types can be carried out in the form of colonies when growing culture on blood agar with the addition of television. Colonies of GRAVIS type 48-72 h are reached in diameter 1-2 mm, there are wavy edges, radial aperture and a flat center. They are considered to compare with a flower of daisies. Colonies are matte due to the ability of the bacterium to restore televurt, which is then connected to the generated hydrogen sulfide, gray-black. When growing on the crop broth, GRAVIS type form on the surface of the crumbling film. When sowing on the GISS medium with the addition of serum, they split polysaccharides - starch, dextrin, glycogen with an acid formation.

Mitis cultures on blood agar with televisers are growing in the form of round, slightly convex, with a smooth edge, black matte colonies. When growing on the broth give a uniform turbidity and precipitate. Starch, dextrin and glycogen they do not break.

In the smears of gravis type wands are more often short, and the type Mitis is thinner and long.

A comparative electron microscopic study of diphtheria sticks of various biochemical types has shown the presence of three-layer cellular shells in the types of GRAVIS and MITIS. The shell at the type intermediate is two-layer and almost 3 times thicker. Between cytoplasma and shell there are spaces filled with grains, which may be related to exotoxin. The oblique is the descriptive of the bacteria, which is created by the separation walls between the daughter cells. The chromosomal apparatus, the types of Gravis and Mitis are represented by conventional grains with vacuoles, at the type of intermediate - distributed over the entire cytoplasm. In the electron microscope, a multilayer shell is visible, the presence of which explains why diphtheria sticks are sometimes gram-negative.

Colonies of diphtheria bacteria are in S-, R- and SR-forms, the latter are considered intermediate. N. Morton believes that the colonies of S-forms are inherent in the type of Mitis, SR-forms - GRAVIS type. In addition to these basic forms, there are colonies of a mukoid type - m-form, dwarf colonies - D-forms and gonidial colonies - L-forms. All of them are considered for the forms of dissociative variability.

Diffex bacteria must be distinguished from difteroids and a false-informitian stick.

A large number of studies are devoted to the variability of the diphtheria stick. The possibility of atypical forms in laboratory conditions was confirmed by the work of the epidemiological profile.

Recognized by the large number of researchers biochemical, morphological, the physico-chemical variability of the diphtheria bacterium makes it difficult to carry out bacteriological diagnostics in some cases, forces a comprehensive study of cultures.

We have distributed all cultures allocated in a variety of epidemiological situation, on 8 groups; They included all possible morphological options of the Corynebacteria representatives of interest:

1st group - short chopsticks, about 2 μm long, without grains;

The 2nd group is short chopsticks, about 2 μm long, but occasionally with grains;

3rd group - sticks of medium size, 3-6 μm long, 0.3-0.8 microns width, without characteristic graininess;

4th group - sticks of medium size, 3-7 μm long, 0.3-0.8 μm width, slightly curved, occasionally with grains;

5th group - sticks of medium size, 3-6 μm long, 0.3-0.8 μm width, slightly curved, grainy;

6th group - long sticks, 6-8 μm long, 0.3-0.6 μm wide, slightly curved, occasionally with grains;

7th group - long sticks, 6-8 μm long, 0.3-0.8 microns width, usually curved, without grains;

The 8th group is short, coarse sticks, about 2 μm long, about 1 μm width, without grains.

The location of the sticks during the distribution in groups was not taken into account, but usually the characteristic location corresponded to morphology.

In 1, 2, 3 and 8 groups, which corresponded on the morphology of the gofman sticks, the location was group, parallel or in the form of single individuals, in 4, 5 and 6 groups, mainly corresponding to true diphtheria bacteria, chopsticks Arranged at an angle or in the form of single individuals. In the 7th group of sticks, more often were randomly intertwining among themselves. In the 8th group of sticks were located in the form of single individuals.

Of the 428 studied crops 111 for the aggregate of the signs were to be attributed to the true diphtheria, 209 were the cultures of the gofman sticks and 108 amounted to a group of atypical cultures. In crops close to diphtheria, the atypical has been manifested in a decrease in biochemical activity, sometimes in the decomposition of urea; In crops, morphologically close to the wands of the corrugation, in the preservation of a positive cysteine \u200b\u200bsample, the ability to decompose one of the sugars.

Of the 111 diphtheria cultures of morphologically typical, 81 culture (73%), 28 crops (27%) had morphology of gofman sticks. Of the 111 diphtheria cultures, there were 20 gravis cultures and only 9 of them are attributed to 1 and 2nd morphological groups.

Cultures that were attributed to the totality of signs to the cultures of the gofman sticks, in 20% of cases had morphology of typical diphtheria cultures.
25% of studied strains were attributed to atypical cultures, their morphology corresponded to both diphtheria sticks and gofman wands.

Thus, the biochemical and morphological properties of crops are far from always coincide, and biochemical atypoticity, as well as morphological, is more often observed in crops allocated during the period of reducing the incidence, and therefore reducing the level of carriage.

It is necessary to note the overall decrease in the biochemical activity of cultures over the past 10-15 years. The index of this is the belated fermentation of sugars, sometimes coming to the 5-6th day, as well as the various biochemical activity of colonies of the same culture.

The biochemical identification of pure crops allocated under conditions of various epidemiological situation shows that although morphology and biochemical properties often do not coincide, the general principle of the distribution of cultures established according to the data of morphology does not change. Both, during the distribution of crops in morphological and biochemical data, and when they are fully identified with the inclusion of serological reactions, the distribution principle remains the same: atypical cultures are more common during the epidemic well-being, the gofman sticks are more often found during epidemic disadvantage and seed longer than true diphtheria.

The study of the toxic properties of isolated, crops on solid nutrient media showed that even in the period of epidemic well-being there is a sufficient number of carriers of toxygenic diphtheria sticks. It should be noted that toxic properties are not always able to catch even in crops isolated from patients. This indicates the need to improve the methods of determining the culture toxigenicity.

The results of the reaction of agglutination of atypical crops, isolated in a variety of epidemiological situation, showed the presence of the same patterns for serological properties that were noted by us when studying morphology and biochemistry of cultures. The atypotic of crops isolated in a safe area, according to Serology, was deeper than in disadvantaged areas. Thus, in a safe area, a positive reaction of agglutination was given by 26% of atypical crops, in disadvantaged - 19%.

One of the main properties of the diphtheria stick is the ability to toxinization. The toxinogenesis of the Corinbacteria diphtheria is determined by the genome contained in the oppage, therefore, the main means of aggression - toxicosis is not associated with chromosome bacteria.

Difteric toxin is a protein with a molecular weight of 6200 dalton. The force of toxin is determined by the formulation of intradermal samples on the presence of necrotic action and on the effect on susceptible animals (death). The toxin strength is measured using a minimum deadly dose, which is the smallest amount of toxin, which can cause the death of a Guinea pig weighing 250 g per 4-5 days with intraperitoneal administration. Toxin has antigenic properties that are preserved when processing formalin, removing its toxic properties. This made it possible to use it to prepare a prophylactic drug.

The toxin molecule consists of two fragments, one of which is thermostable and has enzymatic activity, and the second thermolabile and performs a protective function. Proved intracellular synthesizing toxin with highlighting it through the cell wall tubules. Synthesis of toxin occurs when the microbe is cultivated in a liquid medium - meat-pepton broth with the addition of glucose, maltose and growth factors at pH 7.8-8.0.

According to the latest data, diphtheria toxin is a product of viral origin. As a confirmation, I. V. Chistyakova puts forward the ability of non-toxigenous Corinbacteria to transform into toxic under the influence of the phage. The possibility of the conversion of non-operational crops into toxygenic was confirmed in experiments on single-cell cultures. The described phenomenon is called a leased conversion. With the help of moderate viruses obtained from the toxic strains of GRAVIS, it was possible to convert a non-tariff version of the Corinbacteria of GRAVIS diphtheria into toxic.

E. V. Bakulina, M.D. Krylova suggested that the focal conversion may be significance in the epidemic process. In this regard, the study of its role in the formation of toxic strains of Corinbacterium diphtheria in nature was launched. The possibility of the implementation of the conversion of toxity not only in phage-bacteria systems, but also in natural conditions was shown. But among the local cultures, this process, according to a number of researchers, is far from frequently. The reasons for this are probably the lack of producers of moderate phages, different from the reference strains of the phagezensitivity of local strains, and therefore they cannot be recipients of the converting phages of the well-known spectrum of action.

Only in terms of the microbial population resolved the conversion of toxic properties in diphtheria sticks under the action of staphylococcal and streptococcal phages. In the work of recent years, the issue of phage conversion in the epidemic process receives even more reserved assessment. It is believed that Corinophage tox + in the epidemic process of diphtheria does not play an independent role. Non-toxigenic stick carriers can be infected with TAX + phage only with a toxic strain, and staphylococcal phages are not able to convert non-toxigenous Corinbacteria. To carry out the conversion in the direction of toxiosis in the human body, it is necessary, apparently, the presence of a close communication of the carrier having a converting phage, with a carrier that makes skin-sensitive to this phage of the strain. In addition to the toxinization ability, diphtheria bacterium has such pathogenic factors as hyaluronidase, neuraminidase, deoxyribonucleosis, catalase, eseratsis, peroxidase. The study of extracellular metabolic products has shown the lack of differences between toxigenic and non-toxigenous diphtheria corinbackers.

Currently, serological and phacks can be used for intraslide typing of the Corinbacteria of diphtheria in except the biochemical method described above.

The presence of serological types is due to type-specific, thermal stable, surface and thermolabile antigens.

There are a number of sulfuric typing schemes. We use the scheme proposed by V. S. Suslova and M. V. Pelievina, but it cannot provide the classification of all non-operational strains. The number of serological types is growing. I. Ewing set the presence of 4 serological types - A, B, C and D; D. Robinson and A. Peeney 5 types - I, II, III, IV and V. L. P. Deljagina allocated 2 more serological types. It is believed that the number of serological types is much larger, and mainly due to the type of Mitis. Of the existing few data literature data in the allocation of one or another serotype with various forms of the infectious process and a different epidemiological situation is not established. Along with the data on various aggressiveness of crops belonging to different serological types, there are reports in which serological communication with the pathogenicity of cultures is rejected.

It is characteristic that various serological types are found in various territories. Serological typing can be used for epidemiological analysis.

In the conditions of sporadic morbidity, limitations of the number of media, when much more difficult to search for a source of infection, the value of the phagotype method acquires, allowing to divide the Corinbacterium on serological and culture options. Marking can be carried out according to the properties of the phages isolated from the culture and the sensitivity of culture to specific bacteriophages. The most widely used scheme proposed by R. Saragea and A. MaximeSco. It allows you to label toxic and non-operational strains of all culture options. With the help of 22 typial phages of culture, it can be divided into 3 groups in which 21 Fagiovariants are combined: 1st group - toxygenic and non-operational strains of type Mitis (Fagalovatives I, LA, II, III); 2nd - toxigenous and non-operational strains of type InterMedins and non-target GRAVIS (Fagalovatives IV, V, VI, VII); 13 Fagalovants (from VIII to XIX) entered the 3rd group, which combined GRAVIS toxic strains.

The scheme was tested on a large number of strains isolated in Romania and obtained from museums of 14 countries. The phageotype was positive in 62% of strains, especially successfully were marked with strains of GRAVIS. Among the latest belonging to one of the Fagalovariants was set by 93%. Specific reactions with typical phages in toxic strains of type GRAVIS according to the scheme of these authors are based on the infection of strains of various viruses.

In our country, research in the field of phageotype was conducted by M. D. Krylov. The author has developed a phage marking scheme, which is based on the principle proposed by Williams and Rippon to typing plasmo-adagging staphylococci: FagiovanderAnager was indicated by the title of the Phage, which was lysed in test breeding. Phages and Fagalovants in the scheme of M. D. Krylova are denoted by the letters of the Latin alphabet: in capital - phages, giving drain and semi-loom lysis, listened - lysis in the form of plaques. Based on this, a modified diagram of the phageotype of non-toxigenous Corinbacteria variant of the GRAVIS variant and the phageotype diagram of the toxigenous Corinbacterium variant GRAVIS is developed.

Full title:
Bacteria; Actinobacteria; Actinobacteria (Class); Actinobacteridae; ActinomyCetales; Corynebacterineae; Corynebacteriaceae; Corynebacterium; Corynebacterium Diphtheriae.

Introduction

Diphtheria - acute infectious disease caused by toxigenic Corinbacterium Diphtheria (CoryneBacterium Diphtheriae) transmitted by air-droplet, characterized by local fibrium inflammation of predominantly mucous membranes of rotational and nasopharynx, as well as phenomena of general intoxication and damage internal organs. Significantly less than clinical symptoms The disease can cause toxic strains Corynebacterium ulcerans. The introduction of mass immunization into health care in the 40-50s led to a significant decrease in morbidity and almost complete elimination of diphtheria in the UK and many other countries. However, the recent diphtheria epidemic in Russia and other countries indicates that the epidemic morbidity may appear where the coverage of preventive vaccinations is reduced. In Western Europe, diphteria is rare, however, a dispute has been observed. Moreover, most cases of infection are associated with staying in endemic areas: in Industan, Southeast Asia, South America and in some countries formed from the Republic of USSR.

Etiology

The CoryneBacterium Diphtheriae diphtheria detector was discovered in 1883 in the sections of diphtheria films, and in 1884 Leffler allocated it in clean culture. According to the modern classification of bacteria, the genus CoryNebacterium combines inconsistent species causing the disease in humans. The view of S. Diphtheria is heterogeneous by culture, morphological and enzymatic properties, and therefore there are three biovars: gravis, mitis and intermedius. Biovar received its name thanks to the proposed connection with the severity of clinical flow. Type GRAVIS (short polyphonic sticks, fermented starch) isolated predominantly with severe and complicated forms of diphtheria, accompanied by high mortality. Mitis type (long twisted polymorphic sticks, do not ferment the starch) prevailed with easily underlined forms, and Intermedius occupied an intermediate position. This dependence was confirmed during the period of this diphtheria epidemic in Russia. Diffex sticks are straight or slightly curved, 1 - 8 μm long, 0.3 - 0.8 microns width, polymorphic. Under the microscope it can be seen that they are better painted around the poles, where the presence of granules (inclusions) is noticeable. In the smears of Corinbacteria are located at an angle, taking the appearance of "spreading fingers". Optimal growth temperature for C. Diphtheriae 37.0 ° C. Difteria causative agents are well cultivated on media containing protein. They adversely act straight sun rays, high temperatures. When boiling, they die after 1 min, disinfectants (chlorine lime, 1% solution of chlorine, lysol, etc.) act on them. In the dried state, up to 1 month are stored. The main factor in the pathogenicity of Corinbacteria is exotoxin. Excotoxin-producing pathogens are defined as toxic strains. There are cornebacteria that do not produce exotoxin (non-etoxy strains) that do not cause diseases. Diffexy toxin was opened in 1888 the ability to produce diphtheria toxin products (DT) - the main factor in the virulence of C.diphtheriae - the pathogen of diphtheria. The molecular composition of the toxin was deciphered in the 60s - 70s of our century. Differic toxin is a protein with a mol. Weight 62 - 63 cd. With soft hydrolysis, disintegrates on two fragments - A and V. According to the antigenic composition, a fragment A is non-unugene. With the introduction of its animal, it did not have toxicity, and in the culture of tissue - cytotoxicity. A fragment is a thermal stable protein that determines the toxic effect. Analysis conducted by monoclonal antibodies to fragments A and in toxin showed that people have 80% of the antibodies formed to a fragment A and only 20% - to fragment V. Only 3 representatives of the Corynebacterium genus are potentially toxigenic: C.diphtheriae, C. ulcerans and c.seudotuberculosis. The ability of these types to develop DT depends on the action of two factors: 1) Liezing by B-Fago or other corinophages, which contain a structural gene (tox-gene) toxin molecules; 2) low extracellular iron concentration. It is with the action of DT most of the symptoms of diphtheria and mortality from this infection are associated. Despite the fact that diphteria is a rare disease in the UK and other countries of Western Europe, the frequency of extraction of non-operational strains C.diphtheriae has recently increased significantly. In most cases, they are allocated in patients with pharyngitis. However, there are reports of cases of endocarditis and lesions of other organs and systems in Europe and Australia. As a result, reliable, specific and accurate methods for determining diphtheria toxin are necessary for differentiation of sporadic toxic strains from circulating non-operational strains. Methods for determining diphtheria toxin. The perfect test for determining toxizations should be simple, quick, reliable and sensitive, to correlate well with the biological activity of DT. Recently, a number of genotypic, phenotypic and biological methods for determining DT were investigated. Molecular methods based on the polymerase chain reaction (PCR) to determine the toxin gene, have certain advantages over phenotypic tests. They give a faster and easily interpretable answer. Their use is becoming increasingly common due to the greater availability of equipment for PCR. However, the main disadvantage of PCR-based methods is that they do not provide information on the ability of a micro organism to express biologically active DT. Netoxygenic, but at the same time, tox-gene-generating strains (NTTB), which have part of a complete DT gene, but not capable of expression of the biologically active form of toxin. As a result, the use of only PCR does not give a final result in determining toxiosis. Therefore, PCR is recommended to be used only as an additional method to phenotypic tests. Test of immunoprecipitation ELA - the most commonly used microbiological laboratories of the whole world a phenotypic method for determining toxity. The problem of incorrect interpretation of non-specific precipitation lines, especially where the electric test is not performed routine, led to a decrease in the number of laboratories using it in their work, especially in non-hendemic regions. Different phenotypic methods for determining DT, which, however, or have not found wide use, or have no significant advantages compared to an electric diet for microbiological diagnosis of diphtheria. Immuno-immunimal analysis (ELISA) and tests with immunochromatographic strips (ICS) were widely used to identify microbial antigens and markers. Given the said, we developed, standardized and conducted studies of an amplified ELISA and ICS test to determine DT. In addition to toxin, the wand has a number of cell wall antigens. The main ones are polysaccharides, peptopolysaccharides, proteins and lipids. Surface antigens of the cell wall provoke a typical antihive response, and the deep - species-specific. In the surface layers of the cell wall of the causative agent, a Cord factor was found, which contributes to the adhesion of Corinbacteria. The presence of antitoxic antibodies in the blood serum of ill-faced diphtheria was found at the end of the last century.

Epidemiology

Difteria - anthropotic disease. The source of infection is a patient diphtheria or a bacteria carrier toxic strains of Corinbacteria. From patients with severe diseases, the causative agent is allocated in more quantities than from people who have suffered a disease is easy. However, patients with light and erased formations of diphtheria are more dangerous in epidemiological terms, if the true nature of the disease is not recognized and is not isolated in a timely manner. In the dissemination of the disease, a special role belongs to bacteriars, although they highlight the pathogen quantitatively much less intense than patients with manifestic forms. The most dangerous bacteria carriers that distinguish the microbes for a long time (up to 1 months or more), which is more often observed in patients with concomitant diseases of the upper respiratory tract and rotogling. The mechanism of transmission of the pathogen air-droplege (when conversation, sneezing, cough). The main path of transmission of the pathogen is air-drip, as well as contact-household - through dishes, towel, toys, etc. Known "Milk" Flares of diphtheria associated with infection through infected dairy products. The pathogen of diphtheria is stable in the external environment. In the diphtheritic film, it remains for 3 - 5 months, in dust - up to 2 months, on food products - up to 12 - 18 days, in the droplets of saliva, remaining on the walls of the glass, on the handles of the doors, on children's toys, diphtheria bacteria can be saved 15 days. The survival rate of Corinbacteria on the subjects of the environment in the autumn-spring period can reach 5.5 months, and all this time the pathogenic properties of the pathogen are preserved. The contagiosity index during diphtheria is 15-20%, i.e. When circulating the pathogen among the non-immunized population, diphtheria is dropped 15-20%. The most frequent outcome of infection is bacteridation. During the epidemic of influenza, the circulation of toxigenic Corinbacteria increases at 7 - 15 times. The incidence of diphtheria affects a number of factors, including the state of natural and artificial, i.e. vaccine, immunity. The infection is defeated if 70% of children under 2 years and 70% of adults are covered by vaccination. Social and environmental factors occupy a certain place.

Pathogenesis

The entrance gate is usually the mucous membranes of the octopling (microbes use mucus as a habitat), nose, larynx, less often an eye, genital organs, skin cover. The pathogen is fixed at the injection site, there is a multiplied, highlighting exotoxin. In the process of livelihoods, the Corinbacteria also produce other biologically active substances: hyaluronidase, neuraminidase - necrotic and diffusion factors. Pathological changes in the body of the patient - intoxication, the local inflammatory process, early and late complications are caused by the damaging effect of toxin, which is to block the synthesis of protein cell. Under the action of toxin, the permeability of membranes increases. This is assisted by another bacterial flora, which is available in the Rothoglotka. The gravity of the flow of diphtheria and the outcome of the disease significantly affect the level of antitoxic immunity in the patient, the degree of toxiosis of the strain of Corinbacteria and the dose of the causative agent.
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Mostly childhood, which is manifested by the deep intoxication of the body with diphtheria toxin and characteristic fibrinous inflammation at the location of the pathogen. The name of the disease occurs from the Greek word diphthera - the skin, film, since a dense, grayish-white film is formed at the exposure place of the pathogen.

The causative agent of diphtheria - Corynebacterium Diphtheriae - was found for the first time in 1883. E. Chlebsz in cuts from the film, was obtained in a clean culture in 1884 by F. Lefefler. In 1888, E. Ru and A. Iersen discovered his ability to produce exotoxin, which plays a major role in etiology and the pathogenesis of diphtheria. Obtaining in 1892 Antitoxic serum by E. Bering and the use of it since 1894 for the treatment of diphtheria made it possible to significantly reduce mortality. Successful offensive on this disease began after 1923 due to the development of the city of the method of obtaining diphtheria anatoksin.

The causative agent of diphtheria refers to the genus CoryneBacterium (Actinobacteria class). In the morphological terms, it is characterized by the fact that the cells are milk-shaped thickened at the ends (Greek. Sughu - Bulava), form branching, especially in old cultures, and contain grainy inclusions.

Morphology Corinbacteria

S. Diphtheriae - straight or slightly curved fixed sticks with a length of 1.0-8.0 μm and a diameter of 0.3-0.8 μm, the dispute and capsules do not form. Very often, they have blown at one or both ends, often contain metachesomatic granules - grains of volutin (polymetafosphates), which, when stained with methylene blue, acquire a bluish-purple color. To detect them, a special method of staining in neussa is proposed. At the same time, the sticks are painted in straw-yellow, and the grain of volutive - in a dark brown color, and usually are usually in poles. Corynebacterium Diphtheriae is well colored by aniline dyes, gram-positive, but in old cultures is often discolored and has a negative color in gram. It is characterized by severe polymorphism, especially in old cultures and under the influence of antibiotics. The content of r + c in DNA about 60 mol%.

Biochemical properties Corinbacteria

The diphtheria stick is an aerobic or optional anaerobom, the temperature optimum for growth of 35-37 ° C (growth boundaries 15-40 ° C), the optimal pH of 7.6-7.8. Nutritional media is not very demanding, but it grows better on media containing serum or blood. Election for diphtheria bacteria are rolled whey environments of the RU or Lefeflera, the growth on them appears after 8-12 hours in the form of convex, the amount with the pins of the colonies of a grayish-white or yellowish-cream color. The surface of their smooth or slightly grainy, on the periphery of the colony is somewhat more transparent than in the center. Colonies do not merge, as a result of which the culture acquires the type of shagreen skin. On the broth, the growth is manifested in the form of uniform turbidity, or the broth remains transparent, and a gentle film is formed on its surface, which gradually thickens, crumbs and flakes sedates to the bottom.

A feature of diphtheria bacteria is their good growth on blood and serum media containing such concentrations of potassium television, which suppress the growth of other types of bacteria. This is due to the fact that S. Diphtheriae restore potassium television to a metal tellurium, which, laying out in microbial cells, gives the colonies a characteristic dark gray or black color. The use of such environments increases the percentage of the sample of diphtheria bacteria.

Corynebacterium Diphtheriae is fermented by glucose, maltose, galactose with the formation of acid without gas, but do not ferment (usually) sucrose, have cystinase, do not have urease and do not form indole. According to these features, they differ from those corneformal bacteria (dipteroids) that are more often found on the mucous membrane of the eye (Corynebacterium Xerosus) and nasopharming (corynebacterium pseiidodiphtheriticum) and from other difteroids.

In nature, there are three basic variants (biotypes) of the diphtheria stick: gravis, intermediate and mitis. They differ in morphological, cultural, biochemical and other properties.

The division of diphtheria bacteria on biotypes was made taking into account that at what form of the flow of diphtheria in patients they are highlighted with the highest frequency. The GRAVIS type is more frequent from patients with severe diphtheria and calls group flashes. The type of Mitis causes lighter and sporadic cases of diseases, and the type of intermedius occupies an intermediate position between them. Corynebacterium Belfanti, previously attributed to Mitis biotype, is highlighted in an independent, fourth, biotype. Its main difference from GRAVIS and Mitis biiotypes - the ability to restore nitrates into nitrites. CoryneBacterium Belfanti strains have pronounced adhesive properties, and among them are found both toxygenic and non-target options.

Antigenic structure of Corinbacteria

Corynebacteria is very heterogeneous and mosaic. In the pathogens diphtheria of all three types, several dozen somatic antigens were found, for which they are divided into serotypes. In Russia, serological classification was adopted, which distinguishes 11 difterine bacteria serotypes, of which 7 main (1-7) and 4 additional, rarely encountered serotypes (8-11). Six serotypes (1, 2, 3, 4, 5, 7) refer to the GRAVIS type, and five (6,8,9,10,11) - to the type of Mitis. The disadvantage of the serotype method is that many strains, especially non-target, have spontaneous agglutination or polyaggglutinity.

Phagotyping Corynebacterium Diphtheriae.

Differentiation of diphtheria bacteria proposed various phageotype diagrams. According to the scheme M. D. Krylova using a set of 9 phages (A, B, C, D, F, G, N, I, C), it is possible to typing most of the toxigenous and non-operational strains of GRAVIS. Taking into account the sensitivity to the specified phages, as well as the culture, antigenic properties and the ability to synthesize Corrinis (bactericidal proteins) M. D. Krylov allocated 3 independent Corinbacterius groups of the GRAVIS (I-III) type. In each of them there are subgroups of toxygenic and their non-operational analogues of diphtheria causative agents.

Corinbacterium resistance

Corynebacterium Diphtheriae exhibits a large resistance to low temperatures, but quickly dies at high temperatures: at 60 ° C - for 15-20 minutes, with boiling - after 2-3 minutes. All disinfectants (lysol, phenol, chlorine, etc.) in the usually used concentration destroy it in 5-10 minutes. However, diphtheria causative agent well transfers drying and can remain a long time to maintain viability in dry mucus, saliva, in dust particles. In a fine aerosol, diphtheria bacteria retain viability within 24-48 hours.

Factors of pathogenicity Corinbacteria

The pathogenicity of CoryneBacterium Diphtheriae is determined by the presence of a number of factors.

Factors of adhesion, colonization and invasion

The structures responsible for adhesion are not identified, but without them the diphtheria wand could not colonize cells. Their role is performed by some components of the cell wall of the causative agent. Invasive properties of the pathogen are associated with hyaluronidase, neuraminidase and protease.

Toxic glycolipid contained in the cell wall of the pathogen. It is 6.6 "-dether trehalose containing Coriosolic acid (C32H6403) and Corimisticic acid (SZ2H62AZ) in an equimolar relationship (Tregalozo-6.6" -dicormineikolat). Glycolipid has a destructive effect on the tissue cells at the place of reproduction of the pathogen.

Exotoxin, due to the pathogenicity of the pathogen and the nature of the pathogenesis of the disease. Non-etoxy options S. Diphtheriae diphtheria do not cause.

Exotoxin is synthesized in the form of an inactive precursor - a single polypeptide chain with M.M. 61 cd. Its activation is carried out by its own bacterial protease, which cuts the polypeptide into two associated disulfide bonds of the peptide: A (M.M. 21 KD) and in (M.M. 39 KD). The peptide in performs an acceptor function - it recognizes the receptor, binds to it and forms an intramampled channel through which peptide a cell penetrates and implements the biological activity of toxin. Peptide A is an ADF-RibrosylTransferase enzyme, which ensures the transfer of adenosine diaphosphate from above by one of the amino acid residues (histidine) of the protein factor of EF-2 elongation. As a result of Modification, EF-2 loses its activity, and this leads to the suppression of the synthesis of protein ribosomes at the translocation stage. Toxin is synthesized only by such S. Diphtheriae, which carry the genes of moderate converting proof in their chromosome. Opero, encoding the synthesis of toxin, is a monocystron, it consists of 1.9 thousand pairs of nucleotides and has a TOXP promoter and 3 sections: toxs, toxa and toxb. The toxs section encodes 25 amino acid residues of the signal peptide (it provides toxin yield through the membrane into the periplasmic space of the bacterial cell), toxa - 193 amino acid residues of peptide A, and TOXB - 342 amino acid residues of the peptide in toxin. The loss of the profhag cell or mutation in the tox-operon makes a cell lowoxygenic. In contrast, the lysogenization of non-target S. Diphtheriae converting phage turns them into toxygenic bacteria. This is definitely proven: the toxigenicity of diphtheria bacteria depends on the lysogenation by their converting tox-corinophages. Corimophages are integrated into the chromosome of corinbacteria using the site-specific recombination mechanism, and the strains of diphtheria bacteria may contain in their chromosomes of 2 recombination sites (ATTB), and corinophages are integrated into each of them with the same frequency.

Genetic analysis of a number of non-toxigenic strains of diphtheria bacteria, carried out with the help of labeled DNA probes, carrying fragments of tox-opera corinophag, showed that in their chromosomes there are sequences of DNA, the homologous tox-opera corinophagus, but they either encode inactive polypeptides or are in " silent "condition, i.e. inactive. In this regard, there is a very important issue in epidemiological terms: can non-target diphtheria bacteria turn into toxic in natural conditions (in the human body), just as in vitro? The possibility of such conversion of non -oxigenic cultures of Corinbacteria into toxic, with the help of phage conversion was shown in experiments on guinea pigs, chicken embryos and white mice. However, whether it happens during the natural epidemic process (and if it happens, then as often), while it was not possible to establish.

Due to the fact that diphtheria toxin in the body of patients has selective and specific impact on certain systems (mostly sympathetic-adrenal system, heart, vessels and peripheral nerves) are affected, it is obvious, it not only oppresses the biosynthesis of protein in cells, but also Causes other violations of their metabolism.

To detect the toxiosis of diphtheria bacteria, you can use the following methods:

• Biological samples on animals. The intradermal infection of the guinea pigs by the filtrate of the broth culture of diphtheria bacteria causes them necrosis at the injection site. One minimum deadly toxin dose (20-30 ng) kills a guinea pig weighing 250 g at subcutaneous introduction to the 4-5th day. The most characteristic manifestation of the toxin action is the defeat of the adrenal glands, they are increased and sharply hyperemic.
• Infection of chicken embryos. Differic toxin causes their death.
• Infection of cell cultures. Diffex toxin causes a distinct cytopathic effect.
• The method of solid-phase immunoassay analysis using antitoxins labeled peroxidase.
• Using a DNA probe for direct detection of tox operon in chromosome of diphtheria bacteria.

However, the most simple and widespread way to determine the toxiosis of diphtheria bacteria is serological - method of precipitation in the gel. Its essence is as follows. The strip of sterile filter paper with a size of 1.5 x 8 cm is wetted by antitoxic contamination serum containing 500 AE per 1 ml, and applied to the surface of the nutrient medium in Petri dish. The cup is dried in a thermostat for 15-20 minutes. Test cultures are float on both sides of the paper. There are several strains on one cup, one of which is obviously toxyne, serves as a control. Cups with crops are incubated at 37 ° C, the results are taken into account after 24-48 hours. Owing to the counter diffusion in the gel of antitoxine and toxin, a clear line of precipitation is formed in the place of their interaction, which merges with the line of precipitation of the control toxic strain. Strips of non-specific precipitation (they are formed if other antimicrobial antibodies are present in serum except antitoxin) appear late, weakly expressed and never merge with a strip of precipitation of the control strain.

Post-infectious immunity

Durable, persistent, actually lifelong, repeated cases of the disease are rarely observed - in 5-7% of the breakdowns. Immunity is mainly antitoxic in nature, the antimicrobial antibodies have fewer importance.

To estimate the level of anti-informous immunity, a package of chic was previously widely used. To this end, 1/40 DIM toxin for boils in a volume of 0.2 ml was introduced for intradermal children. In the absence of antitoxic immunity after 24-48 hours at the injection site, a red and swelling of a diameter of more than 1 cm appears. Such a positive chic reaction indicates either the complete absence of antitoxine, or that its content is less than 0.001 AE / ml of blood. Negative chic reaction is observed when the content of antitoxine in the blood is higher than 0.03 AE / ml. With the content of antitoxine below 0.03 AE / ml, but above 0.001 AE / ml, the chic reaction can be either positive, or, sometimes negative. In addition, toxin itself has a pronounced allergenic property. Therefore, to determine the level of anti-informy immunity (quantitative content of antitoxine), it is better to use RPGA with erythrocyte diagnosis, sensitized diphtheria anatoxin.

Epidemiology diphtheria

The only source of infection is a person - a patient, a recovering or healthy bacteria carrier. Infection occurs by air-drip, air-dust, as well as through various items used in patients or healthy bacteria carriers: dishes, books, linen, toys, etc. in case of infection of food (milk, creams, etc. p.) It is possible to infect an alimentary way. The most massive selection of the pathogen takes place in the acute form of the disease. However, the greatest epidemiological importance is face with erased, atypical forms of the disease, as they are often not hospitalized and not immediately detected. The patient diphtheria is contagious during the entire period of the disease and part of the period of recovery. The average limit of bacterianesis in recovering varying from 2 to 7 weeks, but can continue until 3 months.

Healthy bacteria carriers play a special role in diphtheria epidemiology. Under the conditions of sporadic morbidity, they are the main distributors of diphtheria, contributing to and preserving the pathogen in nature. The average duration of the carriage of toxic strains is somewhat less (about 2 months) than non-operational (about 2-3 months).

The reason for the formation of healthy carriage of toxygenic and non-operational diphtheria bacteria is uncovered, since even a high level of antitoxic immunity does not always ensure the full liberation of the body from the causative agent. Perhaps a certain meaning is the level of antibacterial immunity. Priority epidemiological importance is the carriage of toxygenic strains of diphtheria bacteria.

Symptoms of diphtheria

To the diphtheria are susceptible people of any age. The causative agent can penetrate the human body through the mucous membranes of various organs or through damaged skin. Depending on the localization of the process, the diphtheria of the oz, nose, larynx, ear, eyes, genitals and skin is distinguished. Mixed forms are possible, such as diftere and skin, etc., the incubation period is 2-10 days. In the clinically pronounced form of diphtheria, the characteristic fibrinous inflammation of the mucous membrane is developing at the location of the pathogen. Toxin, produced by the pathogen, first affects epithelial cells, and then nearby blood vessels, increasing their permeability. In the exudent exudate, the fibrinogen is contained, the coagulation of which leads to the formation of a grayish-white-white mucous membrane on the surface of the film raids, which are tightly fed to the fabric and when it causes bleeding. The consequence of damage to blood vessels can be the development of local edema. Particularly dangerous is the difterey of the language, since it can cause diphtheria cereals due to edema of the mucous membrane of the larynx and voice ligaments, from which 50-60% of patients with diphtheria of children had died as a result of asphyxia. Diffex toxin, entering blood, causes overall deep intoxication. It affects mainly cardiovascular, sympathetic-adrenal system and peripheral nerves. Thus, the symptoms of diphtheria are made up of a combination of local signs that depend on the localization of the entrance gate, and the general symptoms caused by the poisoning of toxin and manifest themselves in the form of adamisia, the lethargy, pallhenia of the skin, lowering blood pressure, myocarditis, paralysis of peripheral nerves and other disorders. Difteria in graft children, if observed, proceeds, as a rule, in a light form and without complications. Mortality in the period before the use of serotherapy and antibiotics was 50-60%, now 3-6%.

The material for the study serves mucus from the zea and nose, film with almonds or other mucous membranes, which are the place of the entrance gate of the pathogen. Crops are produced on televised serum or blood media and at the same time on crushed whey environments RU (crushed horse serum) or lefflera (3 parts of bovine serum + 1 part of sugar broth), on which the growth of Corinbacteria appears after 8-12 hours. The dedicated culture is identified by The aggregate of morphological, cultural and biochemical properties, whenever possible use the methods of serm and phageotypes. In all cases, checking on toxyness is obligatory to one of the above methods. The morphological features of Corinbacteria are better to study, using three methods of dyeing drug-smear: in gram, neussser, and methylene blue (or toloidin blue).

Specific diphtheria prevention

The main method of combating diphtheria is the mass planned vaccination of the population. For this purpose, various vaccine versions are used, including combined, i.e., aimed at simultaneous creation of immunity against several pathogens. The highest distribution in Russia received the vaccine DCD. It is an adsorbed aluminum hydroxide suspension of couch bacteria killed by formalin or america (20 billion in 1 ml), and contains diphtheria anatoxine at a dose of 30 flocculating units and 10 units of binding of tetanus anatoxine in 1 ml. Children vaccinate from 3 months of age, and then conduct revaccinations: the first after 1.5-2 years, subsequent aged 9 and 16 years, and then every 10 years.

Thanks to the mass vaccination initiated in the USSR in 1959, the incidence of diphtheria in the country by 1966 compared to 1958 was reduced 45 times, and its figure in 1969 was 0.7 per 100,000 population. Fistened in the 80s. XX century Reducing the volume of vaccinations led to serious consequences. In 1993-1996 Russia covered the diphtheria epidemic. Adults were hurt, mostly not received vaccinations, and children. In 1994, almost 40 thousand patients were registered. In this regard, mass vaccination was resumed. During this period, 132 million people were vaccinated, including 92 million adults. In 2000-2001 The coverage of children by vaccinations within the prescribed period was 96%, and a revaccination is 94%. Due to this, the incidence rate of diphtheria in 2001 decreased compared to 1996 at 15 times. However, in order to bring the level of morbidity to isolated cases, it is necessary to cover the vaccination of at least 97-98% of children of the first year of life and to ensure the mass revaccination in subsequent years. To achieve complete elimination of diphtheria in the coming years, it is unlikely possible due to the common carriage of toxic and non-operational diphtheria bacteria. To solve this problem, a certain time will also be required.