RU2410031C1 - Method of controlling effectiveness of correcting intestinal microbiocenosis disturbances - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: to control the effectiveness of correcting intestinal microbiocenosis disturbances, a quantitative assessment of a pre- and post-therapeutic state of intestinal microbiocenosis is required. Prior to and after a prescribed mode and medication, a patient is examined for a complex of intestinal microbiocenosis values: bifidus bacteria, lactic acid bacilli, nonpathogenic Escherichia coli, hemolytic Escherichia coli, lactose-negative Escherichia coli, enterococci, saprophytic staphylococcus, staphylococcus aureus, clostridia, opportunistic bacteria, Candida fungus counts, and a dysbiosis index is calculated also. If the post-therapeutic dysbiosis index is decreased as compared with the primary values by 51-100 % enables to observe an effect of the applied mode and medication, while no therapeutic effect is considered when the dysbiosis index is decreased by 50 % and less.

EFFECT: method enables to control the effectiveness of correcting the intestinal microbiocenosis disturbances with using both integrated modalities, and single-drug therapy without considering clinical objective and subjective data.

3 ex, 2 tbl

Description

The invention relates to medicine, in particular to infectious diseases, in particular to methods for monitoring the effectiveness of treatment of patients with intestinal dysbiosis.

Recent studies have shown that part of the body’s total homeostasis is a microecological system, which includes tens and hundreds of different species, the total number of which reaches 10 ¹⁵ cells, which is almost 2 orders of magnitude more than the own somatic cells of the macroorganism, and about 60% of the entire microflora populates various niches of the digestive tract (N.I. Ursova, 2006).

Today there is no doubt the enormous importance of eubiosis of the gastrointestinal tract in maintaining homeostasis and the normal functioning of the human body. Intestinal microbiocenosis largely determines the composition of the skin flora and mucous membranes, intestinal dysbiosis creates favorable conditions for the formation and development of a number of diseases and pathological conditions (D.D. Zabolotnaya, 2007).

Currently, there is a steady increase in diseases associated with the imbalance between the macroorganism and various populations of the microbial flora of its individual organs and systems, which has developed during evolution (V.R. Gritsenger, 2005). Conditions that may be caused by a violation of the composition and functions of the intestinal microflora include diarrhea, constipation, colitis, malabsorption syndrome, gastritis, duodenitis, peptic ulcer of the stomach and duodenum; atherosclerosis, diabetes; rheumatoid arthritis, other lesions of the joints and connective tissue; malignant neoplasms of the stomach, colon; bronchial asthma, dermatitis and other allergic diseases; superinfection of various localization, chronization of almost all inflammatory diseases in children and adults (A.I. Khavkin, 2004).

From modern positions, intestinal dysbiosis is regarded as a pathogenetic link in the formation and development of chronic inflammatory diseases of various organs and systems, in connection with which the restoration of normal microbial biocenosis of the gastrointestinal tract in the complex treatment of a wide variety of pathologies is given great importance along with traditional directions.

In the correction of intestinal dysbacterioses, the most studied and to some extent practically implemented direction is the use of biological bacterial preparations based on microorganisms - representatives of the normal human microflora, the so-called probiotics (N.I. Ursova, 2006).

The criteria for monitoring the effectiveness of the correction of intestinal microbiocenosis disorders in the industry standard “Patient Management Protocol. Intestinal dysbacteriosis ”(Industry standard 91500.11.0004-2003“ Protocol for the management of patients. Intestinal dysbacteriosis ”, approved by order of the Ministry of Health of Russia dated 09.06.2003 No. 231), taken as a prototype, are clinical signs and indicators of intestinal microflora. The result of treatment is evaluated by categories: restoration of health, improvement, stabilization, progression.

The disadvantages of this method are: 1) the microbiological criterion is taken into account only in the case of a complete restoration of intestinal microbiocenosis and can only be used to evaluate the effectiveness of treatment with a complex of drugs, since with monotherapy with any one means such a result is almost impossible to achieve; 2) restoration of the content of certain types of intestinal microflora and its incomplete recovery are not taken into account; 3) improvement and stabilization are established according to clinical subjective and objective signs without taking into account changes in the state of the microbial flora of the intestine, which is impossible with latent forms of intestinal dysbiosis; 4) there is no standardization for assessing the effectiveness of the correction of intestinal dysbiosis and its quantitative expression.

The novelty of the proposed method is to determine the effectiveness of the correction of intestinal dysbiosis based on a comparison of the quantitative assessment of the intestinal microbiocenosis state before and after the course of treatment.

, где ИД - индекс дисбиоза, П₁, П₂…П_n - определяемые показатели микробиоценоза кишечника, n - число определяемых показателей, и при достоверном уменьшении в результате проведенного лечения индекса дисбиоза по сравнению с исходными данными на 51-100% судят о наличии эффекта от используемого метода и средства, а при уменьшении индекса дисбиоза на 50% и менее делают вывод об отсутствии эффекта.A significant difference from the previously used methods is that a patient before and after the course of treatment with the prescribed method and means determines a set of indicators of intestinal microbiocenosis: the content of bifidobacteria, lactobacilli, E. coli with normal properties, E. coli with hemolytic properties, E. coli with lactose-negative properties , enterococci, saprophytic staphylococci, Staphylococcus aureus, Clostridia, opportunistic bacteria, fungi of the genus Candida, and the index d isbiosis according to the formula:

where ID is the index of dysbiosis, P ₁ , P ₂ ... P _n are the determined indices of intestinal microbiocenosis, n is the number of determined indices, and if there is a significant decrease in the index of dysbiosis as a result of treatment compared to the initial data by 51-100%, the presence of the effect of the used method and means, and with a decrease in the index of dysbiosis by 50% or less, they conclude that there is no effect.

The method of controlling the effectiveness of correction of intestinal microbiocenosis disorders was studied using the complex BION ^® 3 supplement. The dietary supplement BION ^® 3 is produced by the manufacturer Merck Zelbstmedikatsion GmbH, Germany, state registration number 77.99.23.3.U.1020.2.06 dated 09.02 .2006, contains 12 vitamins: A, E, C, B1, B2, B6, B12, D3, biotin, folic acid, nicotinamide, pantothenic acid, 7 minerals and trace elements: calcium (phosphate), iron, magnesium , iodine, manganese, chromium, molybdenum, selenium, zinc, 3 probiotic s cultures: Lactobacillus gasseri, Bifidobacterium bifidum and Bifidobacterium longum. BION ^® 3 is used to maintain and restore healthy intestinal microflora, compensate for electrolyte loss, and strengthen the immune system.

The study was conducted on 60 male soldiers aged 18 to 25 years who are in the same living conditions, nutrition and physical activity, not exposed to professional immunosuppressive factors, from among inpatients of the infectious ward of the garrison military hospital (military unit 52236) of Orenburg . Subjective and objective signs of impaired intestinal microbiocenosis were present in all patients with varying degrees of severity: stool instability, diarrhea, constipation, discomfort or abdominal pain, nausea, an unpleasant aftertaste in the mouth, and flatulence. Microbiological examination of feces in all patients selected for the study revealed intestinal dysbiosis of 1-2 degrees. All examined were randomly divided into 2 groups of 30 people, each of which included the same number of patients with 1st and 2nd degree of intestinal microbiocenosis disorders.

During the 1st month, patients of the control group (first group) received diet, taking into account available indications, diet, generally accepted courses of enterosorbents, digestive enzymes, antidiarrheals, vitamins, antispasmodics and did not receive preparations containing probiotics or prebiotics.

Patients of the main group (second group) received BION ^® 3 diet and monotherapy, 1 tablet once a day by mouth for 1 month.

At the end of the course of treatment, the patients of both groups again underwent bacteriological examination of feces to determine the state of intestinal microflora.

Intestinal microbiocenosis was studied by the method of Epstein-Litvak R.V. and Vilshansky F.L. The collection of material was carried out as follows: immediately after the morning stool (the subject defecated on a disinfected vessel), the patient's feces in an amount of about 1 g were taken into sterile standard disposable dishes (backstamp) and delivered to the laboratory within 1-2 hours after defecation. Dishes with the delivered material were weighed on a pharmacy scale, the weight of the backprint was taken away from the obtained value, the stool mass was calculated, then it was calculated how much sterile saline should be added to obtain a 1:10 dilution. Sterile glass laboratory tubes were used to prepare stool dilutions. In the process of preparing feces dilution, the biomaterial was carefully rubbed against the walls of the vessel to obtain a uniform suspension. 1 ml of suspension from a fecal dilution 1:10 (10 ^-1 ) sterile pipette with a wide end was transferred to a test tube with 9 ml of sterile saline, resulting in a 10 ^-2 dilution. After thoroughly mixing the contents in a test tube, 1 ml of the suspension was transferred to the next test tube with 9 ml of sterile saline solution, respectively, a 10 ^-3 dilution was obtained in it. In the same way, feces were continued in a series of tubes with 9 ml of sterile saline until a 10 ^-10 dilution was obtained, using a new sterile pipette for each subsequent dilution. From the obtained dilutions, crops were made on nutrient media.

Sowing for bifidobacteria was carried out from stool dilutions 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ . Lowering the pipette to the bottom of the tube, 1 ml of the suspension of the appropriate dilution was sown in a high column (at least 10 ml). Before sowing, Blaurock's medium was preliminarily regenerated by heating, for which it was kept in a boiling water bath for 35–40 minutes, and then cooled in cold water. After seeding, the tubes were placed in a thermostat at a temperature of 37 °. The first smears from Blaurock's semi-liquid medium were stained according to Gram and microscopic in a day, the final scan was done in three days. Gram-positive fairly large bacilli with bifurcation or club-shaped ends were identified as bifidobacteria. In the presence of such microorganisms in smears, the highest dilution of feces from which they grew was determined.

Inoculation for accounting for lactobacilli was carried out from dilutions of 10 ⁵ , 10 ⁶ , 10 ⁷ on lactobar in Petri dishes. The seeded medium was placed in a thermostat at + 37 ° C for 24 hours. Grown colonies of lactobacilli In Gram stained smears, lactobacilli were determined as gram-positive, non-spore, small-sized sticks forming chains. The highest feces were recorded, from which lactobacilli grew.

Inoculation for the isolation of gram-negative bacilli was performed on Endo, Ploskirev, Levin and 3% blood agar. Sowing was carried out from dilutions of 10 ³ , 10 ⁵ , 10 ⁸ 0.1 ml per Petri dish, the material was ground with a sterile spatula.

After a day, lactose-positive and lactose-negative colonies were counted. After setting up an oxidase test, 1-3 oxidase-negative colonies were removed from each group of morphologically homogeneous microorganisms onto Simmons and Kliger media. Oxidase-negative, lactose-positive and negative by Simmons microorganisms were weeded out in a motley row for further identification according to the generally accepted method for the detection of Escherichia.

The remaining (oxide-positive and oxidase-negative) microorganisms isolated on these media were identified by their belonging to a specific family.

On blood agar, morphologically different colonies with hemolytic properties were counted, calculating their percentage of the total number of isolated microorganisms of this family.

Staphylococci were isolated on yolk-salt agar in Petri dishes, on which 0.1 ml of suspension from 10 ³ , 10 ⁴ , 10 ⁵ dilutions was seeded.

After a day of incubation, colonies with a golden pigment and a characteristic iris around the colony were counted (due to the production of the lecithinase enzyme). These staphylococci showed the ability to ferment beckons under anaerobic conditions. By the presence of all these characters, the isolated cocci were assigned to the species S. aureus. The highest stool dilution was determined, from which Staphylococcus aureus was isolated.

Enterococci were isolated in Petri dishes on enterococcagar selective medium, onto which 0.1 ml of suspension was diluted from dilutions of 10 ⁴ , 10 ⁵ , 10 ⁶ . The grown colonies were microscopic to determine the characteristic morphology of the cells in Gram stained preparations. In doubtful cases, Sherman's signs characteristic of enterococci were determined (growth on media with 40% bile and 6.5% sodium chloride, resistance to heat for 30 minutes at + 60 ° C and reduction of methylene blue), and a catalase test was performed.

Yeast-like fungi were isolated from dilutions of feces 10 ² , 10 ³ , 10 ⁴ by inoculation of 0.1 ml of material per Candida agar medium.

After 48 hours of incubation at a temperature of + 37 ° C, the grown colonies were microscopic, staining the smears with methylene blue first, noting the morphology of the cells typical of yeast-like fungi, thus establishing their presence in the corresponding dilutions of the feces of the patient.

Evaluation of the results of bacteriological examination of feces was carried out by comparing the obtained data of the highest dilution of feces, in which the corresponding microorganisms were isolated, with indicators of the relative norm.

To quantify the results of the study, we used a ball scale for determining the degree of deviation from the norm of each studied indicator of the state of intestinal microbiocenosis according to the following table No. 1.

Table 1 Distribution of the values of the main indicators of intestinal microbiocenosis in patients with XT on a deviation scale. Intestinal microbiocenosis status indicator The degree of deviation, points 0 0.5 one 2 3 Bifidobacteria CFU / g, log 9-10 8 7 6 5 and below Lactobacilli CFU / g, log 7-8 6 5 four 3 and below E. coli with normal CFU / g properties, log 7-8 6 or 9 5 or 10 4 or 11 3 and lower or 12 and higher Escherichia coli hemolyzing CFU / g, log 0 1-2 3-4 5 6 and above E. coli lactose-negative CFU / g, log 0-4 5 6 7 8 and above Enterococci CFU / g, log 5-8 9 10 eleven 12 and above Staphylococci (saprophytic, epidermal) CFU / g, log 3-4 5 6 7 7 and above Staphylococcus aureus CFU / g, log 0 one 2 3-4 5 and above Clostridia CFU / g, log 0-3 four 5 6 7 and above Conditionally pathogenic bacteria CFU / g, log 0-3 four 5 6 7 and above Candida fungi CFU / g, log 0-3 four 5 6 7 and above

, где ИД - индекс дисбиоза, П₁, П₂…П_n - определяемые показатели микробиоценоза кишечника, n - число определяемых показателей. Для удобства и упрощения расчета ИД можно принимать во внимание только те показатели микрофлоры кишечника, которые имеют отклонения от нормы.Having calculated the sum of all indicators of the microbiocenosis of this patient, expressed in points, and dividing it by the number of indicators, we obtain the dysbiosis index (ID) according to the following formula:

where ID is the index of dysbiosis, P ₁ , P ₂ ... P _n are the determined indicators of intestinal microbiocenosis, n is the number of determined indicators. For convenience and simplification of the calculation of ID, only those indicators of intestinal microflora that have deviations from the norm can be taken into account.

In each group of patients examined, average IDs were determined before and after the course of treatment; the obtained ID values are presented in table No. 2

table 2 The average values of ID in the groups examined before and after treatment. Intestinal microbiocenosis assessment indicator The first group (control) M ± m (n = 30) The second (main group) M ± m (n = 30) Before treatment After treatment Before treatment After treatment ID 0.97 ± 0.04 0.92 ± 0.04 0.98 ± 0.04 0.37 ± 0.02 * Note: * p <0.05 compared with the control group

As can be seen from table No. 2, both groups were comparable in ID before treatment. After the course of therapy with BION ^® 3, ID significantly decreased in the second (main group) compared with the control group and with the initial data. The degree of improvement in ID was 62%, which indicates a good therapeutic effect of BION ^® 3. In the control group, the decrease in ID was unreliable, amounted to 5% of the initial level, which corresponds to an unsatisfactory therapeutic effect.

Example 1. Patient K., 20 years old, from the first group, was admitted for inpatient treatment in the infectious ward of military unit 52236 with a diagnosis of intestinal dysbiosis of 2 degrees, medical history No. 3535. Complaints of loose stools up to 4-5 times a day, a feeling of discomfort in the abdomen. A history of frequent (2 times a year or more) sore throats, including those complicated by a paratonsillar abscess, for which he received antibiotics repeatedly for a long time. Microbiological analysis of feces: bifidobacteria - 10 ⁶ CFU / g, lactobacilli - 10 ⁵ CFU / g, E. coli with normal properties - 10 ⁵ CFU / g, E. coli with hemolytic properties - 10 ⁵ CFU / g, E. coli with lactose-negative properties - 10 ⁴ CFU / g, enterococci - 10 ⁹ CFU / g, saprophytic staphylococci - 10 ⁵ CFU / g, staphylococcus aureus - 0, opportunistic bacteria - 0, clostridia - 0, Candida fungi - 10 ⁵ CFU / g. ID = 0.72. Within a month he received a diet, enterosorbents, digestive enzymes, antidiarrheals, vitamins. After a month, the state of health is the same, loose stools and abdominal discomfort remain. Bacteriological analysis of feces after treatment: bifidobacteria - 10 ⁶ CFU / g, lactobacilli - 10 ⁵ CFU / g, E. coli with normal properties - 10 ⁵ CFU / g, E. coli with hemolytic properties - 10 ⁵ CFU / g, E. coli with lactose-negative properties - 10 ⁴ CFU / g, enterococci - 10 ⁸ CFU / g, saprophytic staphylococci - 10 ⁴ CFU / g, staphylococcus aureus - 0, opportunistic bacteria - 0, clostridia - 0, Candida fungi - 10 ⁴ CFU / g . ID = 0.59. Compared to the initial data, it decreased by 18%, which indicates an unsatisfactory therapeutic effect and the need to change treatment. When evaluating the treatment of the prototype - stabilization.

Example 2. Patient Z., 21 years old, from the second group. Directed to the infectious diseases department with suspected intestinal infection, medical history No. 3447. Prior to this, he was repeatedly hospitalized in the skin and venereology department for recurrent furunculosis, and received antibiotics for a long time. Complaints of periodic paroxysmal abdominal pain, bloating, frequent loose stools up to 6 times a day, sometimes with mucus, low-grade fever. Microbiological analysis of feces: bifidobacteria - 10 ⁷ CFU / g, lactobacilli - 10 ⁵ CFU / g, E. coli with normal properties - 10 ⁵ CFU / g, E. coli with hemolytic properties - 0, E. coli with lactose-negative properties - 0, enterococci - 10 ⁷ CFU / g, saprophytic staphylococci - 10 ³ CFU / g, Staphylococcus aureus - 10 ³ CFU / g, Proteus vulgaris - 10 ⁵ CFU / g, Clostridia - 0, Candida mushrooms - 10 ⁴ CFU / g. Diagnosis: intestinal dysbiosis 2 degrees. ID = 0.69. Within 1 month I received BION ^® 3. As a result of treatment, stool and temperature returned to normal, abdominal pain and flatulence stopped. The study of intestinal microflora after the treatment: bifidobacteria - 10 ⁹ CFU / g, lactobacilli - 10 ⁸ CFU / g, E. coli with normal properties - 10 ⁵ CFU / g, E. coli with hemolytic properties - 0, E. coli with lactose-negative properties - 0 , enterococci - 10 ⁷ CFU / g, saprophytic staphylococci - 10 ³ CFU / g, staphylococcus aureus - 0, vulgar proteus - 10 ⁴ CFU / g, clostridia - 0, fungi of the genus Candida - 10 ⁴ CFU / g. ID = 0.18. A significant decrease in ID was 74%, which corresponds to a good therapeutic effect. When evaluating the treatment of the prototype is an improvement.

Example 3. Patient T., 19 years old, from the second group. I was hospitalized in the infectious diseases department with a diagnosis of Food toxicoinfection, gastroenteric variant, moderate severity, medical history No. 3171. At the end of the course of treatment, stool instability, alternating stools with liquid up to watery up to 5-6 times a day, weakness, intermittent spastic pain in the lower abdomen persisted. In crops for the disgroup of pathogenic microflora is not allocated. Bacteriological analysis of feces: bifidobacteria - 10 ⁸ CFU / g, lactobacilli - 10 ⁶ CFU / g, E. coli with normal properties - 10 ⁵ CFU / g, E. coli with hemolytic properties - 0, E. coli with lactose-negative properties - 0, enterococci - 10 ⁹ CFU / g, saprophytic staphylococci - 10 ³ CFU / g, hemolytic streptococcus - 10 ⁵ CFU / g, cytrobacter - 10 ⁴ CFU / g, clostridia - 0, fungi of the genus Candida - 10 ⁴ CFU / g. Diagnosed with intestinal dysbiosis of the 2nd degree, due to opportunistic flora and hemolytic streptococcus. ID = 0.5. A monthly course of BION ^® 3 treatment was carried out, after which he felt normal, abdominal pain stopped, and stool became stably formed. Bacteriological analysis of feces after treatment: bifidobacteria - 10 ¹⁰ CFU / g, lactobacilli - 10 ⁸ CFU / g, E. coli with normal properties - 10 ⁵ CFU / g, E. coli with hemolytic properties - 0, E. coli with lactose-negative properties - 0, enterococci - 10 ⁷ CFU / g, saprophytic staphylococci - 10 ³ CFU / g, hemolytic streptococcus - 0, cytrobacter - 0, clostridia - 0, fungi of the genus Candida - 10 ⁴ CFU / g. ID = 0.14. A significant decrease of 72%, the therapeutic effect is good. When evaluating the treatment of the prototype is an improvement.

The advantages of the proposed method are as follows:

1) the microbiological criterion is taken into account for all variants of the effect of correction of intestinal dysbiosis, including in case of incomplete restoration of intestinal microflora, and can be used in assessing the effectiveness of treatment with both a complex of agents and monotherapy; 2) the restoration of the content of certain types of intestinal microflora and its incomplete recovery are taken into account; 3) it is possible to evaluate the improvement of the intestinal microbiocenosis without taking into account clinical objective and subjective data, especially with latent forms of intestinal dysbiosis; 4) it is proposed to standardize the assessment of the effectiveness of the correction of intestinal dysbiosis and its quantitative expression; 5) simplicity in execution.

The proposed method can find practical application in assessing the effectiveness of probiotics and other drugs in various forms and severity of intestinal dysbiosis in order to determine the optimal indications for their purpose and duration of treatment, to select the most effective combination of drugs of different directions of action, and also to be included as a criterion evaluation of the effectiveness of treatment of disorders of intestinal microbiocenosis in the industry standard "Protocol for the management of patients. Intestinal dysbacteriosis. "

Literature

1. Gitsenger V.R. Dysbiosis and their correction: Textbook / V.R. Gitsenger, T.V. Khaibekova, A.A. Poslavskaya et al. - Saratov, 2005. - 80 p.

2. Zabolotnaya D. D. The state of intestinal microbiocenosis in patients with chronic tonsillitis / D.D. Zabolotnaya, K.G. Seleznev, E.S. Lavrentieva // Journal of Vushny, nasal and throat twigs. - Kyiv: Enlightenment, 2007. - No. 3-s. - S.101.

3. Industry standard 91500.11.0004-2003 "Protocol for the management of patients. Intestinal dysbacteriosis ”, approved by order of the Ministry of Health of the Russian Federation dated 09.06.2003 No. 231.

4. Ursova N.I. Intestinal dysbacteriosis in children / N.I. Ursova. - M .: Borghez Company, 2006. - 239 p.

5. Khavkin A.I. Violations of the microecology of the intestine. Principles of correction / A.I.Khavkin. - M., 2004 .-- 40 p.

Claims (1)

A method for monitoring the effectiveness of correction of intestinal microbiocenosis disorders by comparing a quantitative assessment of the intestinal microbiocenosis state before and after the course of treatment, characterized in that the patient before and after the course of treatment with the prescribed method and means determines a set of indicators of intestinal microbiocenosis: the content of bifidobacteria, lactobacilli, E. coli with normal properties, E. coli with hemolytic properties, E. coli with lactose-negative properties, enter dams, saprophytic staphylococci, Staphylococcus aureus, clostridia, opportunistic bacteria, Candida fungi and dysbiosis index are calculated by the formula:

where ID is the index of dysbiosis, P ₁ , P ₂ ... P _n are the determined indices of intestinal microbiocenosis, n is the number of determined indices, and if there is a significant decrease in the index of dysbiosis as a result of treatment compared to the initial data by 51-100%, the presence of the effect of the used method and means, and with a decrease in the index of dysbiosis by 50% or less, they conclude that there is no effect.