RU2324936C1 - Method of indication of shigella epidemic strains - Google Patents

Abstract

FIELD: medicine; infectious diseases.

SUBSTANCE: suspension of Shigella, identified till species level, is added to the beef-extract broth at concentration of 100 million of CFUs/ml and the electric resistance of solution is determined in limits up to 2000 kOhm, in 1 and 6 hours of bacteria fermentation at 37°C. If the electric resistance of solution is increased 4.0-6.0 times, the isolated Shigella strain is considered as epidemic.

EFFECT: enables high reliability of indicating epidemic Shigella strains, reduces study time and accelerates interpretation of epidemic episodes.

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Description

The invention relates to medicine, namely to the microbiological laboratory diagnosis of infectious diseases.

In the late 80s, an increase in acute intestinal infections in the Russian Federation was noted. In the Tyumen region, the incidence of acute intestinal infections in the period from the beginning of the 90s to the present has exceeded the republic-wide indicators by 2-3 times. In 2003 and 2004 the incidence rates of dysentery were 78.7 and 106.5 per 100 thousand people, respectively, which was 2 times higher than the incidence rate in the Russian Federation (Report on the health status of the population of the Tyumen Region in 2004, Tyumen, 2005, p. 93). The increase in the incidence of dysentery is due to the circulation among the susceptible population of epidemic strains of dysentery. Therefore, the identification of epidemic Shigella strains is an urgent problem in the modern period.

Identification of Shigella species is currently carried out to determine the mobility of bacteria, the formation of hydrogen sulfide, the breakdown of glucose, lactose, indole, xylose, adonite, the ability to utilize Na citrate, destroyed by a dysenteric bacteriophage, and serological identification (A.S. Labinskaya. Microbiology. M. Moscow. - 1978. - S.228-238). This method is selected as an analog.

A known method for the identification of epidemic strains of shigella (V.I. Pokrovsky. Enterobacteria. A guide for doctors. M., Medicine. - 1985. - P.87-121), selected as a prototype. Shigella isolated from the patient are identified by type, and then epidemiological marking of the isolated shigella is carried out: the pathogen biovar is determined (by the ability to break down various carbohydrates), colicinogenicity using indicator strains, make up the antibioticogram of the isolated pathogen. If Shigella strains isolated from different patients during an epidemic outbreak are identical in biovar, colicinogenicity and antibiotic profile, then these strains are classified as epidemic. In Shigella sonnei strains, 7 biovars are distinguished, in Shigella flexneri - 13, in Shigella boydii - 8 biovars.

One of the significant disadvantages of the prototype is the duration of laboratory tests (epidemiological labeling of Shigella strains is carried out within 3 days), and expensive reagents are used for laboratory diagnostics (at the rates of 01.01.2006, the cost of identifying 2 Shigella strains is 200 rubles), 5 hours of working time of the average medical worker are spent on laboratory tests. In total, the costs of identifying 2 epidemic Shigella strains are 500 rubles.

The objective of the present invention is to develop a new, objective, economical express method for indicating epidemic Shigella strains.

The technical result is a simple, low-cost express method for indicating epidemic Shigella strains, based on the fact that during the cultivation of Shigella in a nutrient medium, epidemic strains cause a more intense increase in electrical resistance compared to sporadic strains of dysentery pathogens. A well-known method was used to determine the electrical resistance of solutions for a new purpose, namely to indicate epidemic Shigella strains with more intense reproductive activity compared to sporadic Shigella strains.

The technical result of the method for indicating epidemic Shigella strains is achieved by the fact that the identified Shigella culture, to the type according to the invention, is introduced into the meat and peptone broth at a concentration of 100 million CFU / ml and the electrical resistance of the solution is determined within 2000 kOhm after 1 hour and 6 hours of bacterial cultivation at a temperature of 37 ° C, with an increase in the electric resistance of the solution by 4.0-6.0 times, the isolated shigella culture is classified as epidemic.

The essence of the method lies in the fact that in accordance with the invention, epidemic strains have higher reproductive activity compared to sporadic strains and as a result, the resistance of suspension of bacteria in the nutrient medium after 6 hours of cultivation of bacteria increases 4.0-6.0 times, and when cultivating sporadic strains with less reproductive activity, the resistance of suspension of bacteria increases by 2.0-3.0 times. An objective method of detecting the electrical resistance of a suspension of bacteria was used, which allows one to detect an increase in the concentration of microbial suspension in a nutrient medium.

The proposed method is as follows:

Shigella isolated from the patient is grown at a temperature of 37 ° C on standardized culture media for the cultivation of enterobacteria and identified to form, then an 18-hour Shigella culture is brought to a working concentration of microbial suspension equal to 1 billion CFU / ml. The resulting suspension of bacteria in an amount of 0.5 ml is introduced into a test tube with 4.5 ml of meat-peptone broth, then steel electrodes are introduced into a test tube with a microbial suspension and after 1 and 6 hours of bacterial cultivation at a temperature of 37 ° C, the electrical resistance of the solution is in the range of 2000 kOhm using a digital multimeter M-832. With an increase in the electrical resistance of Shigella suspension by 4.0-6.0 times after 6 hours of cultivation of bacteria, the studied Shigella suspension is classified as an epidemic strain.

The method of indicating epidemic Shigella strains allows the investigation of epidemic outbreaks to identify Shigella epidemic strains and to develop anti-epidemic measures in foci of infection, given the etiology of the pathogen. The proposed method gives objective results and reduces the time of laboratory studies.

Examples of specific performance of the proposed method

Example 1

Patient V., 6 years old, was on an outpatient basis in a children's clinic number 3 in Tyumen. S.sonnei culture was isolated from the patient’s feces. The identification of the pathogen was carried out to a species in the Department of General Microbiology of the Federal State Health Institution “Center for Hygiene and Epidemiology in the Tyumen Region” according to generally accepted methods for enterobacteria, in particular according to morphological, cultural, biochemical and serological properties. Using microbiological techniques, the isolated shigella were assigned to the sporadic strain S.sonnei (biotype 3d, colicinotype 1).

At the same time, after identification of the isolated Shigella from the patient to the species, the belonging of the S.sonnei isolated to the epidemic strain was determined using the proposed method for indicating epidemic Shigella strains. An 18-hour culture of identified shigella was taken for the study, the bacterial suspension was brought to a working concentration of 1 billion CFU / ml, then the resulting bacterial suspension in an amount of 0.5 ml was introduced into a test tube with 4.5 ml of meat-peptone broth and steel electrodes were installed in the solution . After 1 and 6 hours of cultivation of bacteria in a nutrient medium at a temperature of 37 ° C, the electrical resistance of the solution was determined in the limit of 2000 kOhm using an M-832 digital multimeter. After 1 hour of cultivation of bacteria, the instrument readings were 25 kOhm, and after 6 hours - 51 kOhm. The resistance of the solution increased by 2.0 times. The isolated S.sonnei strain is assigned to the sporadic strain.

Example 2

Patient M., 3 years old, was hospitalized in the infectious ward of the regional clinical hospital No. 1 of Tyumen. S.sonnei culture was isolated from the patient’s feces. The identification of the pathogen was carried out to the species in the Department of General Microbiology of the Federal State Health Institution “Center for Hygiene and Epidemiology in the Tyumen Region” according to generally accepted methods for enterobacteria. Using microbiological techniques, the isolated shigella were assigned to the epidemic strain S.sonnei (biotype 3c, colicinotype 8).

At the same time, after identification of the isolated Shigella from the patient to the species, the belonging of the S.sonnei isolated to the epidemic strain was determined using the proposed method for indicating epidemic Shigella strains. For the study, an identified 18-hour Shigella culture was taken, a suspension of bacteria was brought to a working concentration of 1 billion CFU / ml, then the resulting suspension of bacteria in an amount of 0.5 ml was introduced into a test tube with 4.5 ml of meat-peptone broth and steel electrodes were installed in the solution . After 1 and 6 hours of cultivation of bacteria in a nutrient medium at a temperature of 37 ° C, the electrical resistance of the solution was determined in the limit of 2000 kOhm using an M-832 digital multimeter. After 1 hour of cultivation of bacteria, the instrument readings were 18 kOhm, and after 6 hours - 87 kOhm. The resistance of the solution increased 4.8 times. The isolated S.sonnei culture is assigned to an epidemic strain.

Example 3

Patient T., 23 years old, was on an outpatient basis in the clinic number 17 of Tyumen. The S.flexneri culture was isolated from the stool of the patient. The identification of the pathogen was carried out to a species in the Department of General Microbiology of the Federal State Health Institution “Center for Hygiene and Eridemiology in the Tyumen Region” according to generally accepted methods for enterobacteria. Using microbiological techniques, the isolated shigella suspension was assigned to the epidemic strain S.flexneri (biotype 2a).

At the same time, after identification of the isolated shigella from the patient to the species, the S.sonnei belonging to the epidemic strain was determined using the proposed method for indicating epidemic shigella strains. For the study, an identified 18-hour Shigella culture was taken, a suspension of bacteria was brought to a working concentration of 1 billion CFU / ml, then the resulting suspension of bacteria in an amount of 0.5 ml was introduced into a test tube with 4.5 ml of meat-peptone broth and steel electrodes were installed in the solution . After 1 and 6 hours of cultivation of bacteria in a nutrient medium at a temperature of 37 ° C, the electrical resistance of the solution was determined in the limit of 2000 kOhm using an M-832 digital multimeter. After 1 hour of cultivation of bacteria, the instrument readings were 12 kOhm, and after 6 hours - 70 kOhm. The resistance of the solution increased by 5.8 times. The isolated S.flexneri culture is assigned to an epidemic strain.

The given examples illustrate the possibility of using the proposed method for indicating epidemic and sporadic Shigella strains. The method was tested on the basis of the Department of General Microbiology FGUZ "Center for Hygiene and Epidemiology in the Tyumen Region." A study of 23 Shigella cultures isolated from patients during epidemic outbreaks and sporadic diseases was carried out. The research results are presented in tables 1 and 2.

The proposed method allows to save the expense of material resources for laboratory research and reduces the time required for laboratory research (table 3).

The method of indicating epidemic Shigella strains does not require large material costs, is easily reproducible, objective, allows you to accelerate the decoding of epidemic outbreaks, and the reliability of the indication of epidemic Shigella strains is 99%.

Table 1 Identification of epidemic Shigella strains post №№ Type, biotype and colicinotype of Shigella strain established using the prototype Indication of epidemic Shigella strains using the present invention Resistance readings in kΩ through Multiplicity of resistance increase 1 hour 6 o'clock one S.flexneri, biotype 2a 12 70 5.8 2 S.flexneri, biotype 2a fifteen 76 5,0 3 S.flexneri biotype 6 eighteen 81 4,5 four S.flexneri biotype 6 16 88 5.5 5 S.flexneri, biotype 1c fifteen 78 5.2 6 S.sonnei, biotype 3d, colicinotype 1 19 79 4.2 7 S.sonnei, biotype 3c, colicinotype 8 twenty 82 4.1 8 S.sonnei, biotype 3c, colicinotype 8 eighteen 87 4.8 9 S.sonnei, biotype 3d, colicinotype 6 19 90 4.7 10 S.flexneri, biotype 2a 17 73 4.3 eleven S.sonnei, biotype 3d, colicinotype 6 19 93 4.9 12 S.flexneri biotype 6 16 83 5.2

Claims (1)

A method for indicating epidemic Shigella strains, including isolating the pathogen and identifying it to a species, characterized in that the identified Shigella suspension is introduced into the meat and peptone broth at a concentration of 100 million CFU / ml and the solution's electrical resistance is determined within 2000 kOhm after 1 and 6 hours of bacterial culture at at a temperature of 37 ° C, with an increase in the electric resistance of the solution by 4.0-6.0 times, the isolated shigella culture is classified as epidemic.