RU2702229C1 - Method for treating atopic dermatitis in children with probiotic preparations based on results of microbiological examination of feces - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to dermatology, and concerns treating atopic dermatitis in children with probiotic preparations. That is ensured by microbiological examination of faeces, performing type identification of all recovered cultures of microorganisms by MALDI-TOF mass spectrometry. Thereafter, the probiotic is selected in accordance with a deficiency or excess in the patient of representatives of specific microorganisms of normal microflora.

EFFECT: method provides reduced length of treatment and prolonged clinical remission ensured by individual selection of the probiotic preparation.

1 cl, 4 tbl, 3 ex

Description

The invention relates to medicine, namely to dermatology, and can be used to increase the effectiveness of treatment and increase the duration of remission in children with atopic dermatitis.

Atopic dermatitis in children remains an important medical and social problem, the significance of which is determined by the steady increase in the incidence of dermatosis, its chronic, recurrent course and difficulty in conducting therapy. In recent years, children with atopic dermatitis noted torpidity, inefficiency of standard therapeutic approaches. Treatment of atopic dermatitis depends on the clinical manifestations of the disease in each individual child [Albanova, V.I. Atopic dermatitis: a training manual / V.I. Albanova, A.N. Pampura - M .: GEOTAR-Media, 2016. - 128 p.].

For the treatment of atopic dermatitis, many approaches have been proposed based on the systemic and local use of detoxification, desensitizing, antihistamines, anti-inflammatory, immunosuppressive and sedative drugs, as well as physiotherapeutic effects. At the same time, the known methods of treatment are not effective enough, long-lasting, do not reduce the tendency to relapse, due to the lack of a comprehensive effect on the body of the drugs used.

Difficulties in the treatment of atopic dermatitis in children, accompanied by a persistent chronic course and a tendency to relapse, are caused by its etiopathogenesis.

Significant factors in the pathogenesis of atopic dermatitis are impaired digestive function, in particular, impaired intestinal microbiocenosis. It has been proved that the absence of correction of the concomitant pathology of the gastrointestinal tract aggravates the severity of the skin process and shortens the time of remission [Belmer S.V., Korotkiy N.G., Tikhomirov A.A., Narinskaya N.M. Atopic dermatitis in children: from general concepts to the state of the digestive system // Issues of Pediatric Dietetics. - 2016. - T. 14, No. 6. -FROM. 40-46].

To date, in the detection and correction of dysbiotic disorders in patients with atopic dermatitis, the routine method of bacteriological sowing of feces has been used, which allows to establish the generic origin of bacteria, to identify the presence of conditionally pathogenic bacteria, to a first approximation, to evaluate the spectrum of obligate flora (bifidumbacteria, lactobacilli, Escherichia with various enzymatic activity) [Chaplin A.V., Brzhozovsky A.G., Parfenova T.V., Kafarskaya L.I., Volodin N.N., Shkoporov A.N., Ilyina E.N., Efimov B.A. Learning Wee diversity of bacteria of the genus Bifidobacterium intestinal microflora using the method of MALDI-TOF mass spectrometry // Vestnik RAMS. - 2015. - No. 70 (4). - S. 435-440].

However, this approach did not always provide an effective result, since for the correct assessment of the composition of obligate flora, it is necessary to take into account the species affiliation of bacteria. Currently, this approach is technically achievable in wide clinical practice using modern methods, in particular, species identification of all selected cultures of microorganisms using MALDI-TOF mass spectrometry.

The MALDI-TOF mass spectrometry method is based on Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) mass spectrometry with laser desorption-ionization. The isolated protein extracts or directly bacterial stands are mixed with a saturated solution of the matrix substance, which leads to their joint crystallization. The laser pulse causes ionization and explosive evaporation of the matrix together with the studied proteins. The resulting ions are accelerated in an airless environment by an electrostatic field, after which they fly through the site without acceleration and crash into the target of the detector; in this case, the device registers the flight time of the ions, which will reflect their mass-charge ratio. Thus, the MALDI-TOF mass spectrometry method allows you to record the protein spectrum of a pure bacterial culture, which, when compared with the database, makes it possible to carry out identification accurate to the species. This technology allows you to analyze a large number of bacterial strains quickly and with high accuracy [Nomura F. Proteome based bacterial identification using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS): A revolutionary shift in clinical diagnostic microbiology. Biochim Biophys Acta. 2015; 1854 (6): 528-537].

Given the significance of microbiological changes in dysbiosis, the following severity levels of microbiological disorders are distinguished (in children older than 1 year):

1st degree -

- a decrease in the content of bifidobacteria to 10 ⁸ -10 ⁷ CFU / g,

- lactobacilli up to 10 ⁶ -10 ⁵ CFU / g,

- typical Escherichia up to 10 ⁶ -10 ⁵ CFU / g,

- an increase in typical Escherichia (possibly) to 10 ⁹ -10 ¹⁰ CFU / g.

2nd degree -

- a decrease in the content of bifidobacteria to 10 ⁷ CFU / g and below,

- lactobacilli - up to 10 ⁵ CFU / g and below,

- increase the content of hemolytic Escherichia or other opportunistic bacteria to 10 ⁵ -10 ⁷ CFU / g,

- or the detection of associations of opportunistic microorganisms at a concentration of 10 ⁴ -10 ⁵ CFU / g.

3rd degree -

- a decrease in the content of bifidobacteria to 10 ⁷ CFU / g and below,

- lactobacilli up to 10 ⁵ CFU / g and below,

- detection of associations of opportunistic microorganisms at a concentration of 10 ⁷ -10 ⁸ CFU / g [Harchenko O.F. The problem of dysbiosis in children in modern conditions // Medical News. - 2013. - S. 50-56].

Currently, the standard for the diagnosis of atopic dermatitis in children does not include the definition of intestinal dysbiosis. At the same time, the appointment of an adequate diagnostic program allows you to correctly assess the situation and conduct both the etiopathogenetic treatment of the main pathology and the correction of microecological disorders.

The main approach to the correction of dysbiosis is the use of probiotics - living microorganisms that have a positive effect on the microbial balance of the intestine.

Four generations of probiotics are distinguished.

Monocomponent preparations (Colibacterin, Bifidum-bacterin, Lactobacteria) containing 1 strain of bacteria are referred to the 1st.

2nd generation drugs (Bactisubtil, Biosporin, Sporobacterin, etc.) are based on the use of non-specific microorganisms for humans and are self-eliminating antagonists.

3rd generation drugs include multicomponent probiotics containing several symbiotic strains of bacteria of the same species (Acylact, Acipol, etc.) or different species (Linex, Bifiform) with a mutually reinforcing effect. From drugs of the 1st generation, they differ in a more balanced composition.

Bifid-containing preparations immobilized on a sorbent (Bifidumbacterin forte, Probifor) are referred to the 4th generation. Sorbed bifidobacteria effectively colonize the intestinal mucosa, providing a more pronounced protective effect than non-sorbed analogues.

A known method for the prevention and / or treatment of skin diseases, including atopic dermatitis, the essence of which is that an artificial mixture for infants includes a probiotic bacterial strain of Bifidobacteria and / or Lactobacilli. The composition in accordance with the invention contains from 10 ³ to 10 ¹² CFU of the probiotic bacterial strain, more preferably from 10 ⁷ to 10 ¹² CFU, per g of dry weight of the composition [patent RU 2609838, 2017]. The disadvantage of this method is the lack of an initial study of the state of the intestinal microbiota.

A known method of using the therapeutic and prophylactic fermented milk product "Narine-forte" containing strains of Lactobacillus acidophilus [RF Patent No. 2180790, 2002]. The product is used to treat dysbiosis and related diseases of the gastrointestinal tract, normalize the immune status of the body, reduce the severity of allergic reactions, etc. However, this tool is not effective enough.

A known method of using the tool "Bionorm" containing dry biomass of living bacteria Lactobacillus rhamnosusodi 83 and Lactococcus lactis ssp lactis odi 83 [Patent RU No. 2252770, 2005] The disadvantage of this method is the need for prolonged use in atopic dermatitis (6-12 months or more) .

The closest analogue of the invention is a method characterized in that for the treatment of patients with atopic dermatitis used a probiotic composition containing a culture of Lactobacillus salivarius and a culture of Bifidobacterium breve in a concentration of from 1 × 10 ⁷ to 1 × 10 ¹¹ CFU, preferably 1 × 10 ⁹ CFU / g, which caused a decrease in the SCORAD index. In addition, the introduction of the indicated probiotic composition for 12 to 24 weeks, preferably 16 weeks, can reduce / block the secretion of the cytokine IL-4 [patent RU 2605293, 2016]. The disadvantage of this method is its use for adult patients with atopic dermatitis.

The objective of the invention is to develop a method of treating children with atopic dermatitis, taking into account the results of microbiological studies of feces to increase therapeutic efficacy.

The technical result of the invention is to reduce treatment time and increase the duration of clinical remission.

The proposed method for the treatment of atopic dermatitis in children is as follows. Before prescribing a probiotic, feces are collected from patients in accordance with OST 91500.11.0004-2003 "Protocol for the management of patients. Intestinal dysbiosis ”, conduct a qualitative and quantitative study of the composition of the cavity microflora of the intestine with species identification of all selected cultures of microorganisms using the MALDI-TOF mass spectrometry method.

Material for research is collected and delivered in accordance with MU4.2.2039-05 "Technique for collecting and transporting biomaterials to microbiological laboratories." For the study, 1 gram of native feces is selected, which is ground in a sterile mortar with 9 ml of physiological saline, and the first ten-fold dilution is thus obtained. From it, seeding is carried out on solid nutrient media for the isolation of Shigella and Salmonella (Shigella salmonella agar), as well as on liquid nutrient media for the isolation of Salmonella (selenite medium).

The following dilutions are prepared from the first ten-fold dilution: 10 ^-3 and 10 ^-5 . From a 10 ^-5 dilution, 0.1 ml is inoculated onto Endo media (to isolate enterobacteria and other gram-negative bacilli), Levin (to isolate enterobacteria) and Saburo with chloramphenicol (to isolate mushrooms), yolk-salt agar (to isolate staphylococci); 0.01 ml for 5% blood agar (for the isolation of streptococci, enterococci and other microorganisms). Separately, crops are carried out to detect motile microorganisms according to the Shukevich method in test tubes with beveled meat and peptone agar.

In addition to the standard study on bifido and lactobacilli, carried out in the form of ten-fold dilutions on Wednesday of Blaurock and MRS from tubes in which growth was noted, reseed on dense selective media for lactobacilli, meat peptone agar, 5% blood agar, universal chromogenic medium. Crops are incubated at 37 ° C under anaerobic conditions, which are created using gas generating packages of commercial production in a hermetically sealed anaerostat for 2-5 days. This method allows for species identification of bifidobacteria and lactobacilli.

All grown microorganism cultures were identified using MALDI-ToF mass spectrometry on a Bruker Microflex LT® instrument. The selection of a probiotic is carried out in accordance with a deficiency or excess of representatives of specific types of microorganisms from representatives of normal microflora.

When evaluating the clinical effectiveness of the proposed method for the treatment of atopic dermatitis in children, the following criteria are considered:

1) the dynamics of objective clinical manifestations (based on changes in the standardized severity index - SCORAD indicator);

2) the dynamics of indicators of the degree of dysbiosis;

3) comparison of treatment outcome indicators.

The possibility of practical implementation of the proposed method is illustrated by examples of its specific use in patients.

Example 1

Patient C, aged 2.5 years. Was admitted to the hospital with complaints of rashes on the skin of the extremities, accompanied by itching, restless sleep. Mother and father consider themselves healthy. Registered since 1.5 years, against the background of treatment prescribed by a dermatologist, periods of unstable remission were noted. On examination, the skin process is diffuse, represented by foci of hyperemia in the thighs, knee bends and lower legs. When assessing the severity of clinical manifestations, the SCORAD index was 17 points.

Microbiological examination of feces with identification of cultures of microorganisms by MALDI-TOF mass spectrometry revealed the content of Bifidobacterium longum - 10 ⁸ CFU / g, Lactobacillus acidophilus -10 ⁴ CFU / g, Escherichia coli (fermenting lactose) - 10 ⁶ CFU / g, Enterococcus faecium - 10 ⁶ CFU / g.

Diagnosed with atopic dermatitis of mild severity, 1st degree of violation of intestinal microflora.

Due to the fact that the level of Lactobacillus acidophilus is reduced, the drug Acidophilus (Acidophilus probiotic) containing this type of lactobacillus is prescribed.

A month after completing the course of basic treatment with the additional intake of Acidofilus daily, half an hour before meals, 1 capsule of the drug, washed down with half a glass of water 1 time per day for 14 days, the spectrum of microorganisms isolated from feces was: Bifidobacterium longum - 10 ⁹ CFU / g, Lactobacillus acidophilus - 10 ⁸ CFU / g, Escherichia coli (fermenting lactose) - 10 ⁷ CFU / g, Enterococcus faecium - 10 ⁶ CFU / g.

There was a decrease in the intensity of skin itching, a decrease in hyperemia and skin infiltration. After the end of therapy, the SCORAD index was 8 points. The patient was discharged from the hospital in a state of remission of the skin process, which was observed for one year.

Example 2

Patient K., 14 years old, is under observation at the institute’s clinic since 2 years old with a diagnosis of atopic dermatitis. He repeatedly underwent inpatient treatment, including desensitizing, detoxification, systemic and external corticosteroid therapy, and was discharged with improvements. By the age of 8, the skin process has become resistant to the therapy, frequent exacerbations combined with short periods of remission. When assessing the severity of clinical manifestations with this exacerbation, the SCORAD index before treatment was 32.8 points, which corresponds to the moderate severity of atopic dermatitis.

Functional digestive disorders in the form of intestinal colic, sporadically loose stools with an unpleasant odor, loss of appetite, and sometimes nausea are observed.

The indicators of bacteriological analysis of feces are as follows:

Bifidobacterium infantis - 10 ⁵ CFU / g, Lactobacilus acidophilus - 10 ⁵ CFU / g, Escherichia coli (lactose fermenting) - 10 ⁵ CFU / g Enterococcus durans - 10 ⁵ KOE / g.

Due to the fact that the analysis showed a decrease in the content of Bifidobacterium infantis and Lactobacilus acidophilus, LINEX ^® (containing Lactobacillus acidophilus, Bifidobacterium infantis, Enterococcus faecium) in the form of capsules 2 capsules 3 times / day was added to the treatment of atopic dermatitis. within 14 days.

As a result, there was a decrease in the intensity of skin itching and a decrease in hyperemia, infiltration and peeling of the skin. The spectrum of microorganisms was: Bifidobacterium infantis - 10 ⁹ CFU / g, Lactobacilusacidophilus - 10 ⁷ CFU / g, Escherichia coli (fermenting lactose) - 10 ⁶ CFU / g, Enterococcus faecium - 10 ⁷ CFU / g.

The SCORAD index was 19.6 points, which corresponds to a mild severity of atopic dermatitis. There was an improvement in gastrointestinal symptoms (decreased colic, decreased stool frequency). With further observation, the duration of remission was 9 months.

Example 3

Patient T., 9 years old, repeatedly received courses of inpatient treatment. Hereditary burden of allergic diseases in the mother's side (allergic rhinitis). A history of early conversion to artificial feeding. Sick from an early childhood, is registered with a dermatologist. The skin process is common. The patient repeatedly received courses of systemic corticosteroids, antihistamines.

At the time of going to the clinic, the rashes were diffuse, accompanied by excruciating itching. When assessing the severity of clinical manifestations, the SCORAD index before treatment was 55.1 points, which corresponds to a severe severity of atopic dermatitis.

Complaints of a feeling of fullness in the stomach, belching, decreased appetite, intestinal colic, unstable stool.

Determination of the species composition of microflora revealed the content of Bifidobacterium infantis - 10 ⁷ CFU / g, Lactobacillus acidophilus - 10 ⁵ CFU / g, Klebsiella oxytoca - 10 ⁵ CFU / g, Citrobacter braakii - 10 ⁵ CFU / g, Raoultella ornithinolytica - 10 ⁶ CFU / g .

Due to the decrease in the level of Bifidobacterium infantis, Lactobacillus acidophilus, the detection of associations of opportunistic microorganisms, the antimicrobial agent Bactisubtil (containing Bacillus cereus, an antagonist of conditionally pathogenic and pathogenic 3 enterobacteria) was prescribed for the basic course of treatment for severe atopic dermatitis. once a day for 7 days and the probiotic LINEX ^® (containing Lactobacillus acidophilus, Bifidobacterium infantis, Enterococcus faecium) in the form of capsules 2 capsules 3 times / day for 14 days.

As a result of the therapy, a decrease in the intensity of skin itching, a decrease in hyperemia, infiltration and peeling of the skin were noted. After the end of the course of therapy, only foci of moderate lichenification and dry skin were preserved. The spectrum of microorganisms was: Bifidobacterium infantis - 10 ⁹ CFU / g, Lactobacillus acidophilus - 10 ⁷ CFU / g, Escherichia coli (lactose fermenting) - 10 ⁶ CFU / g, Enterococcus faecium - 10 ⁴ CFU / g.

There was an improvement in symptoms from the gastrointestinal tract, an improvement in appetite, and occasionally rare colic persisted. The SCORAD index was 23.3 points, which corresponds to the moderate severity of atopic dermatitis. The duration of remission was 8 months.

By the proposed method, 30 children with atopic dermatitis with concomitant dysbacteriosis from 3 to 12 years old, including 17 girls and 14 boys, were treated at the Republican Dermatovenerologic Dispensary State Health Institution. The duration of the disease ranged from 0.5 to 12 years. A specific characteristic of the intestinal microbiota is given by MALDI-TOF mass spectrometry, and dysbiosis is established. Previously, patients were treated with antihistamines, detoxification therapy, systemic and topical corticosteroids, which did not allow for complete clinical remission.

A correlation was established between the severity of atopic dermatitis and the degree of dysbiosis. With the first degree of dysbiosis, the SCORAD index was 17.0 ± 4.5, with the second degree - 32.9 ± 7.1, with the third degree - 61.3 ± 11.2 (table 1).

Further, the patients were randomly divided into two groups.

1 group of children with atopic dermatitis received standard treatment [Federal clinical guidelines for the management of patients with atopic dermatitis [Electronic resource] / V.V. Chikin, L.F. Znamenskaya, K.N. Monakhov [et al.]: Russian Society of Dermatovenerologists and Cosmetologists. - M., 2013 .-- 40 p. - URL: http://www.cnikvi.ru/docs/clin_recs/bolezni-kozhi-i-pridatkov-kozhi /].

In addition to standard treatment, the 2nd group of patients with atopic dermatitis simultaneously received a drug that regulates the equilibrium of intestinal microflora (probiotic) in accordance with the deficiency or excess of representatives of specific types of microorganisms from normal microflora.

After the end of the course of treatment, the SCORAD index decreased by 10 points or less in 20.0% (n = 3) of the patients of the first group and in 13.3% (n = 2) of the patients of the second group. The SCORAD index decreased by 11-15 points in 20.0% (n = 3) of patients in both groups. A decrease in the index by 16-20 points was observed in 26.7% of patients (n = 4) in the first group and in 46.7% (n = 7) in the second group (table 2).

As a result of the proposed method of treatment, the proportion of children with a severe degree of dysbiosis decreased from 60.0% (n = 9) to 20.0% (n = 3), with an average degree of dysbiosis - from 26.6% (n = 4) to 20.0% (n = 3). At the same time, the number of patients with a mild degree of dysbiosis increased from 13.3% (n = 2) to 46.0% (n = 7), and the diagnosis of dysbiosis was made in two children (table 3).

When comparing the indicators of treatment results, first of all, a reduction in the duration of treatment is noted: with standard treatment, the period is from 21 days, in the declared case - 14 days. Clinical recovery in the first group was observed in 26.7% of children (n = 4), in the second group - in 43.2% of children (n = 6). Significant improvement was noted in the first group in 26.7% of children (n = 4), in the second group - in 53.3% of children (n = 8). Improvement was observed in the first group - in 46.6% of children (n = 7), and in 6.7% (n = 1) in the second group. The duration of the remission period was 2-4 months in the first group of examined children, and from 8 to 12 months in the second group (table 4).

Thus, the use of the proposed method for the treatment of atopic dermatitis in children provides a significant increase in the effectiveness of therapy, lengthening the period of remission and can be recommended for use in clinical practice.

* - differences between groups are significant (p <0.05).

* - significance of differences p <0.05, ** - significance of differences p <0.01 in terms of the beginning and end of treatment.

Note: T0 is the zero period, that is, the starting point of treatment, T14 is the 14-day treatment period.

Claims (1)

A method for treating atopic dermatitis in children, comprising administering a probiotic preparation against the background of basic treatment, characterized in that microbiological examination of feces is preliminarily carried out, specific cultures of microorganisms are identified by MALDI-TOF mass spectrometry, and probiotic is selected in accordance with the deficiency or the patient’s excess of representatives of specific types of microorganisms from representatives of normal microflora.