RU2734718C1 - Method of reducing manifestation of non-motor symptoms in patients with parkinson's disease - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: present invention refers to medicine, namely to neurology, gastroenterology and microbiology, and concerns reducing the manifestations of non-motor symptoms in Parkinson's disease (PD). That is ensured by intestinal microbiocenosis correction by introducing an autoprobiotic with Enterococcus faecium content of not less than 10KOE per 1 ml. Autoprobiotic is orally administered in dose of 50 ml 2 times a day for a course of at least 20 days.EFFECT: it provides effective correction of non-motor PD symptoms, particularly improvement of bowel motility and evacuation capacity without side effects, due to absence of conflict with immune system, as well as with other representatives of patient's own microbiota.1 cl, 2 ex, 3 tbl, 5 dwg

Description

The invention relates to clinical medicine and microbiology, namely to neurology and gastroenterology, and can be used to improve the quality of life of neurological patients, improve the strategy and tactics of increasing the efficiency of medical care to the population.

Currently, Parkinson's disease (PD) is recognized as a multisystem disease: in addition to the central nervous system, the neurodegenerative process involves the Meissner and Auerbach plexuses of the enteric nervous system, the distal sympathetic "terminals" of the adrenal glands, salivary glands, myocardium, pelvic plexus, and other structures of the peripheral autonomic nervous system. PD is one of the most common and severe neurodegenerative diseases in humans with a chronic progressive course, accompanied by degeneration of dopaminergic nigrostriatal neurons and dysfunction of the basal ganglia. Motor and non-motor symptoms of PD are caused by common pathogenetic mechanisms: pathological accumulation of the α-synuclein protein, excitotoxicity, oxidative stress and neuroinflammation [Mosley R.L., Hutter-Saunders J.A., Stone D.K. Inflammation and adaptive immunity in Parkinson's disease. Cold Spring Harb. Perspect. Med. 2012; 2 (1): 81-93.].

H. Braak has data on the detection of α-synuclein aggregates in the cells of the Meissner and Auerbach plexuses in the latent presymptomatic period of PD. Apparently, the involvement of parasympathetic neurons of the intestinal submucosa is the earliest event in the development of the disease, which indicates the role of exogenous alimentary (infectious and toxic) factors. This is how the alimentary concept of PD development was formulated, suggesting that when the intestinal barrier function is disturbed, the pathological agent can penetrate into the central nervous system along the fibers of the vagus nerve, affecting its dorsal nucleus and other neurons of the caudal trunk [Hawkes CH, Del Tredici K, Braak N. Parkinson's disease: a dual-hit hypothesis. Neuropathol Appl Neurobiol. 2007; 33: 599-614].

Primary lesion of the gastrointestinal tract many years before the development of motor symptoms was confirmed both clinically and pathomorphologically. It is known that the most frequent autonomic disorders (constipation, urinary disorders, and erectile dysfunction) may precede the development of PD (within 2-24 years, on average for 18 years), and according to the HAAS study, the risk of developing PD in persons with less than one peristaltic movement intestines per day, 2.7-4.1 times higher than in people with more active peristalsis [Abbott RD, Petrovich H., White LR, Masaki KH, Tanner CM, Curb JD, Grandinetti A, Blanchette PL, Popper JS , Ross GW. Frequency of bowel movements and the future risk of Parkinsons disease // J. neurol. - 2001. - Aug. No. 57 (3). - P. 456-462.].

The most common manifestations of autonomic dysfunction in PD are orthostatic hypotension, gastrointestinal disorders, urinary disorders, hypersalivation, impaired sympathetic innervation of the skin [Kim J.S., Sung H.Y. Gastrointestinal Autonomic Dysfunction in Patients with Parkinson's Disease. Journal of movement disorders, 2015, 8 (2), 76-82]. The incidence of certain autonomic disorders in PD patients ranges from 70.6% to 96%, and according to some authors, up to 100% [Zhang TM, Yu SY, Guo P, Du Y, Hu Y, Piao YS, Zuo LJ, Lian TH, Wang RD, Yu QJ, Jin Z, Zhang W. Nonmotor symptoms in patients with Parkinson disease: A crosssectional observational study. Medicine (Baltimore). 2016 Dec; 95 (50): e5400.]. Dysfunction of the gastrointestinal tract in PD includes constipation, malnutrition, dental problems, dysphagia, impaired gastric emptying, difficulty in defecation, calamification, Helicobacter pylori infections, and intestinal dysbiosis. The most common syndrome is constipation - in 28-61% of patients [Poewe W. Non-motor symptoms in Parkinson's disease. Eur. J, Neurol. 2008; 15 (Suppl. 1): 14-20]. All patients with PD showed a slowdown in intestinal peristalsis, and an increase in the transit time of food through the colon was also observed in PD patients who did not complain of constipation [Kupsky WJ, Grimes MM, Sweeting J., Bertsch R., Cote LJ Parkinson's disease and megacolon: concentric hyaline inclusions (Lewy bodies) in enteric ganglion cells. Neurology. 1987; 37: 1253-1255.].

The quantitative and qualitative composition of the intestinal microbiota in PD is changed, there is an increase in the content of representatives of the Enterobacteriaceae family, a decrease in the number of Faecalibacterium prausnitzii, lactobacilli and prevotella [Scheperjans F., Aho V., Pereira PA, Koskinen K., Paulin L., Pekkonen E. et al ... Gut microbiota are related to Parkinson's disease and clinical phenotype. Mov. Disord. 2015; 30 (3): 350-8.] There is evidence of translocation of Escherichia coli into the mucous membrane of the colon [Niehaus I., Lange J.H. Endotoxin: Is it an environmental factor]. There is evidence of the ability of bacteria belonging to the Lactobacillaceae family to modulate the activity of neurons of the enteric nervous system and afferent signal transmission through the vagus nerve, which can alter the intracellular secretion of α-synuclein [Paillusson S., Clairembault T., Biraud M., Neunlist M., Derkinderen P. Activity-dependent secretion of alpha-synuclein by enteric neurons. J. Neurochem. 2013; 125 (4): 512-7.]. Bacteria of the genus Prevotella not only break down a wide range of plant polysaccharides and mucin glycoproteins in the intestinal wall, but also interact with the immune system [Scher J.U., Sczesnak A., Longman R.S., Segata N., Ubeda C., Bielski C. et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. Elife. 2013: e01202.]. People with elevated serum Prevotella have higher levels of neuroactive short-chain fatty acids, as well as thiamine and folate.

Thus, a decrease in the population of prevotella can lead to a decrease in the amount of these compounds in a patient with PD. A low level of Prevotellaceae is an indicator of a decrease in mucin synthesis, which leads to an increase in intestinal permeability [Wright D.P., Rosendale D.I., Robertson A.M. Prevotella enzymes involved in mucin oligosaccharide degradation and evidence for a small operon of genes expressed during growth on mucin. FEMS Microbiol. Lett. 2000; 190 (1): 73-9.], Which, in turn, can lead to local and systemic effects of bacterial endotoxins, which can cause an increase in the expression of α-synuclein in the intestine. Significant in the pathogenesis of the disease is the indirect systemic effect of the microbiota on neuroinflammation through the immune system and the circulatory system. Inflammation associated with a change in intestinal microbiocenosis can contribute to the formation of α-synuclein inclusions, subsequently peripherally induced inflammation, immunosuppressive and autoimmune processes lead to damage to dopaminergic neurons. Thus, support of microbial balance and microbiota functions in PD patients is an important component of successful PD treatment.

Movement disorders in this disease, the need for frequent medication intake, and restriction of daily activity lead to a deterioration in the quality of life of patients. Parkinson's disease is also characterized by autonomic and mental disorders, which lead to permanent limitation of all aspects of a person's normal life. The presence of motor (postural instability, resting tremor and stiffness) and non-motor symptoms in PD have an extremely unfavorable effect on the quality of life of patients [Moroz MD, Baida A.G. Assessment of the quality of life of patients with Parkinson's disease and secondary parkinsonism // Young Scientist. - 2016, No. 8. - S. 411-415. - URL https://moluch.ru/archive/112/28584/ (date of access: 17.12.2019].

Evaluation of the temporal and causal relationship between changes in the intestinal microbiota and the development of pathology of the nervous and immune systems in PD is an urgent area of research in modern biomedical science. The interrelation of disturbances in the intestinal microbiocenosis and the role of dysbiosis in maintaining the pathological process served as the basis for the use of probiotic therapy in the complex treatment of PD patients, aimed primarily at improving the patient's quality of life with this incurable disease.

So, several clinical studies have shown that regular consumption of a fermented milk drink containing probiotic microorganisms can significantly reduce the severity of constipation in patients with PD [Cassani E., Privitera G., Pezzoli G., Pusani C., Madio C., Iorio L . et al. Use of probiotics for the treatment of constipation in Parkinson's disease patients. Minerva Gastroenterol. Dietol. 2011; 57 (2): 117-21.]. [M. Barichella, C. Pacchetti, C. Bolliri et al. Probiotics and prebiotic fiber for constipation associated with Parkinson disease. Neurology Sep 2016, 87 (12) 1274-1280].

It has been shown that improving bowel motility in patients with PD leads to improved absorption of drugs prescribed to patients, including for the treatment of PD and relieving symptoms [Danique L.M. Radder, Nienke M. de Vries, Niels P. Riksen, Sarah J. Diamond, Ditza Gross, Daniel R. Gold, John Heesakkers, Emily Henderson, Adrianus L.A. J. Hommel, Herma H. Lennaerts, Jane Busch, Ray E. Dorsey, John Andrejack, Bastiaan R. Bloem. (2019) Multidisciplinary care for people with Parkinson's disease: the new kids on the block !. Expert Review of Neurotherapeutics 19: 2, pages 145-157.]

Another study conducted by Tamtaji O. et al showed a significant improvement in intestinal motor function, a decrease in clinical indicators of inflammation and oxidative stress in PD patients who took a multistrain probiotic compared with a placebo group [Tamtaji OR, Taghizadeh M, Daneshvar Kakhaki R, Kouchaki E, Bahmani F, Borzabadi S, Oryan S, Mafi A, Asemi Z. Clinical and metabolic response to probiotic administration in people with Parkinson's disease: A randomized, double-blind, placebo-controlled trial. Clinical Nutrition. -2019. - Vol. 38. - pp. 1031-1035.]

Numerous studies indicate the neuroprotective activity of antibiotic therapy with minocycline in patients with PD, one of the mechanisms of which is the ability to influence the balance of the intestinal microbiota by decreasing the Firmicutes / Bacteroidetes ratio [Parashar A., Udayabanu M. Gut microbiota: Implications in Parkinson's disease. Parkinsonism Relat. Disord. 2017; 38: 1-7. doi: 10.1016 / j.parkreldis.2017.02.002.].

A known method of selection of funds that affect intestinal motility disorders and pain and bacterial strains of Lactobacillus gasseri LG345A (DSM 27123) and Lactobacillus gasseri LG621A (DSM 27126), selected using the proposed method [RF Patent 2701348. Method of selection of funds that affect violations bowel motility and pain / Connolly Eamonn, Kunze Wolfgang, Bienenstock John; applicant and patentee BIOGAYA AB (SE). - No. 2015112102; declared 09/03/2013; publ. 26.09.2019, Bul. No. 27. - 21 S.]

Closest to the claimed method is a method for the treatment of neurodegenerative diseases, which include Parkinson's disease, according to which, along with the basic therapy, a probiotic strain Enterococcus faecium L3 is used [Patent RU 2642246. The use of a probiotic strain of the microorganism Enterococcus faecium L-3 for the treatment of neurodegenerative diseases / Abdurasulova I.N., Tarasova E.A., Ermolenko E.I., Matsulevich A.V., Suvorov A.N. - No. 2014154366; declared 12/30/2014; publ. 24.01.2018, Bul. No. 3.]

The objective of the present invention is to provide a method for improving the quality of life of patients suffering from Parkinson's disease, by correcting the main non-motor symptoms of the disease, namely, defecation disorders, manifested in constipation, calamification and modification of the feces shape.

The essence of the invention lies in the fact that a patient who has not taken antibiotics, probiotic products and preparations for at least 10 days, takes feces in an amount of at least 1 ml, clones of indigenous strains of Enterococcus faecium are isolated from them, using a dense nutrient medium, containing sodium azide and a crystal violet dye, then, according to the nature of the color, the colonies are selected, pure cultures are obtained by reseeding three pink colonies with a burgundy center on three sectors of Petri dishes with the same medium, and then incubated in a thermostat under aerobic conditions at t = 37 ° C, check for the absence of pathogenicity genes using PCR, select clones devoid of pathogenicity factors, which ferment soy hydrolyzate in a volume of at least 10 ml per liter, then choose one of the starter cultures that changed the structure of the hydrolyzate earlier than others and use it for preparation two liters of an individual autoprobiotic product containing at least 10 ⁸ CFU per 1 ml, which is administered to the patient orally, 50 ml 2 times a day for a course of at least 20 days.

A significant advantage of the proposed method is that when using indigenous bacteria isolated from the body individually and reintroduced to correct dysbiotic conditions, there is no conflict with the immune system and with other representatives of the patient's own microbiota. A method for producing an autoprobiotic based on Enterococcus faecium, a representative of the indigenous microflora of the host intestine, was protected by patent RU No. 2460778.

The technical result of this invention is to increase the effectiveness of the treatment of Parkinson's disease by reducing the manifestation of non-motor symptoms, improving intestinal motility, its evacuation capacity and restoring the composition of the intestinal microbiocenosis.

Example 1.

To study the effectiveness of the claimed invention, a blinded, randomized, comparative clinical study was conducted.

40 patients with Parkinson's disease (56-77 years old) were examined. Patients with Parkinson's disease received basic therapy for the underlying disease. They were divided into two groups. One of the Probiotic groups (20 people: 9 women, 11 men, 66.63 ± 10.1 years old) received a probiotic in the form of a starter culture based on E. faecium L3. Patients from the second group "Autoprobiotic" (20 people: 16 women, 4 men 63.84 ± 8.78 years old) received 10 days of autoprobiotic in the form of a starter culture based on indigenous strains of their own microbiota.

All patients underwent a neurological examination assessing the severity of movement disorders using the Hyun and Yara stage assessment scale. In all patients, the severity of non-motor manifestations of the disease was also assessed using diagnostic scales: hospital anxiety scale, hospital scale of depression (GDS), geriatric scale of depression (GDSD). We also used the PDQ-20 Perceived Deficit Questionnaire - a Perceived Deficit Questionnaire consisting of 20 questions aimed at identifying cognitive changes in patients, the Unified PD Rating Scale (UPDRS), part 2, Non-Motor Symptom Scale for PD (PD NMS), Bristol Scale feces, Scales for assessing gastrointestinal dysfunction in patients with PD, in addition, patients kept a well-being diary, in which they indicated the frequency of bowel movements, stool properties and described subjective sensations. A detailed clinical examination was performed before, during (to exclude the occurrence of side effects) and after probiotic / autoprobitic therapy.

In the group of patients receiving autoprobiotic and probiotic, there was a decrease in the severity of vegetative manifestations of the disease: shortening of the interval between bowel movements (in 100%), decrease in general weakness (in 75%), decrease or disappearance of nausea after eating (in 90%), decrease in discomfort in the stomach after eating (85%).

Changes in the non-motor symptom score before and after administration of the probiotic and autoprobiotic are shown in FIG. 1. When comparing the scale of non-motor states, a tendency towards their disappearance was noted (Table 1.).

The average values of the frequency of defecations before and after the administration of the probiotic and autoprobiotic are shown in Figure 2. There was a tendency to an increase in the number of defecations, which was reduced in almost all patients.

р<0,05 (при сравнении группы больных до и после введения аутопробиотика и пробиотика).FIG. 2 shows the number of bowel movements per week before and after administration of the probiotic and autoprobiotic.

p <0.05 (when comparing a group of patients before and after administration of an autoprobiotic and a probiotic).

In the group of patients receiving the autoprobiotic, a decrease in the severity of non-motor symptoms was observed (Fig. 3, Table 3). There was also an improvement in the Bristol scale of patients after a course of probiotic and autoprobiotic therapy (Fig. 3).

р<0,05 (при сравнении группы больных до и после введения аутопробиотика и пробиотика).FIG. 3 shows the Changes in the Bristol Stool Scale before and after administration of probiotic and autoprobiotic.

p <0.05 (when comparing a group of patients before and after administration of an autoprobiotic and a probiotic).

The following are the results of evaluating the number of individual representatives of intestinal microbiocenosis in healthy volunteers and patients with PD (before and after administration of probiotics and autoprobiotics) using the bacteriological method (Fig. 4). The study revealed an increase in the number of enterococci against the background of the introduction of probiotic and indigenous enterococci, a decrease in the amount of Proteus before and after the introduction of autoprobiotic.

р<0,05 (при сравнении группы больных до и после введения аутопробиотика).FIG. 4. shows the content of marker bacteria before and after the introduction of autoprobiotic and probiotic (bacteriological study).

p <0.05 (when comparing the group of patients before and after the administration of autoprobiotic).

The following are the results of assessing the number of individual representatives of intestinal microbiocenosis of healthy volunteers and patients with PD (before and after administration of probiotics and autoprobiotics) using RT-PCR (Fig. 5). The study revealed a statistically significant increase in the number of enterococci against the background of the introduction of probiotic and indigenous enterococci, a tendency to an increase in the amount of proteus against the background of the introduction of a probiotic, and its decrease after exposure to autoprobiotic, a decrease or elimination of Clostridium difficile and C. perfringens in the groups receiving probiotic and autoprobiotic.

р<0,05 (при сравнении группы больных до и после введения аутопробиотика).FIG. 5. shows the content of marker bacteria before and after the introduction of autoprobiotic and probiotic (RT-PCR).

p <0.05 (when comparing the group of patients before and after the administration of autoprobiotic).

Example 2. Correction of non-motor symptoms using autoprobiotic

Patient K., 69 years old. Consultation 1. Complaints about: changes in handwriting, difficulties in initiating movements, stoop, stiffness, slowness, constipation, recurrent aching pains in the cervical, thoracic and lumbar spine, limited mobility in the neck, constipation, calorification. She considers herself ill since the summer of 2018, when she began to notice stiffness, sluggishness, and a significant increase in urinary incontinence. Since March 2019, a change in handwriting and a violation of fine motor skills appeared, I turned to a neurologist at the place of residence, according to the patient, they suggested a diagnosis of Parkinson's disease. 06/24/2019 consulted by a neurologist, diagnosed with CVD, DEP II, Parkinson's disease, consultation with a parkinsonologist was recommended. 06/25/2019 consulted by a neurologist, diagnosed with Parkinson's disease, akinetic-rigid form, stage 2.5 on the Hen and Yar scale, with predominantly right-sided symptoms. In April 2019, she underwent inpatient treatment with the diagnosis "Chronic colitis with constipation, chronic combined hemorrhoids of the II degree." 06/28/2019 hospitalized for emergency indications with anamnestic bleeding, outpatient treatment is recommended.

Neurological status

The condition is satisfactory. Consciousness is clear, contact. Locally preserved. The emotional background is stable. The movement of the eyeballs in full. Eye slits D = S. No nystagmus, no diplopia. Hypomimia is easy. Midline tongue. Oral automatism reflexes: not triggered. There is no dysphagia. There is no dysarthria. Dysphonia is light. The strength of the muscles of the limbs is full, D = S. Muscle tone is increased by extrapyramidal type, D> S. Reflexes from the extremities of medium liveliness, D = S. Samples for bradykinesia are positive, D> S. There is no postural kinetic tremor. KP - satisfactory. She is stable in the Romberg position. Thevenar test is positive (4 steps). Width, step height are reduced. Meningeal symptoms were not identified. Controls the functions of the pelvic organs.

Neuropsychological testing before using an autoprobiotic

Sheehan's Anxiety Scale = 8; hospital scale of anxiety and depression = 1/8; geriatric depression scale = 3; Bristol stool scale = 6; non-motor symptoms = 9; PDQ-20 = 2; frequency of bowel movements per week = 3.

After consultation, the use of an autoprobiotic was prescribed in addition to the main therapy.

To isolate autoprobiotic enterococci from the patient's feces, delivered within two hours after sampling, 9 ml of sterile phosphate buffer (physiological sodium chloride solution with the addition of sodium phosphates of two and mono-substituted sodium phosphates, mixed in a proportion necessary to create a pH of 7, was added to 1 g of material, 4). Some of the faecal samples were frozen and stored at -20 ° C for analysis of the microbiota composition using RT-PCR and metagenomic analysis (16SpRNA).

A suspension of feces at a dilution of 1:10 was inoculated in an amount of 100 μL on a selective agar nutrient medium for enterococci containing sodium azide (NICF, St. Petersburg). Cultivated for 24-48 hours at 37 ° C. Three colonies of E. faecium of pink color with a burgundy center, typical in morphology, were selected on a medium with sodium azide and transferred to a new Petri dish with a dense azide medium in order to accumulate pure cultures, seeded separately in three sectors of the dish. Cultivated for 24 hours at 37 ° C. Pure cultures were sent for genetic analysis. The species was determined by PCR using primers highly specific to certain types of enterococci, and the presence or absence of pathogenicity factors for at least five pathogenicity genes. Detection of PCR results was carried out using horizontal electrophoresis in 1% agarose gel.

To prepare autoprobiotics, we selected one bacterial clone belonging to the microorganism species of interest (E. faecium), which does not contain any of the pathogenicity genes.

Obtaining dairy-free starter culture based on selected E. faecium strains. produced as follows. A separate colony of the selected identified pure culture of the microorganism was plated on 10 ml of 5% broth from soy protein isolates (sterilized by autoclaving at 120 ° C for 20 min) and incubated for 24-48 hours at 37 ° C. Then, the obtained 10 ml of starter culture was used as seed to obtain a larger volume of starter culture. To do this, 10 ml of starter culture was inoculated per liter of sterile 5% broth from soy protein isolates heated at 37 ° C. After receiving the starter, the product was stored until use in a refrigerator at 4 ° C for no more than 3 days, after which the starter was transferred to the patient. To preserve the strains obtained, the finished product was poured into two test tubes and sent to storage at -75 ° C for up to a year, for possible re-production of the autoprobiotic.

Consultation 2.

Neuropsychological testing after using an autoprobiotic

Sheehan's Alert Scale = 6; hospital scale of anxiety and depression = 0/5; geriatric depression scale = 2; Bristol stool scale = 5; non-motor symptoms: 5; PDQ-20 = 4; frequency of bowel movements per week = 5.

After 20 days of taking the drug, the patient's feces were re-sampled. A study of the patient's feces before and after oral administration of an autoprobiotic against the background of basic therapy using RT-PCR and metagenomic analysis showed that the patient had a decrease in the representation of Prevotellus, an increase in the population of beneficial bacteria Lachnospira, Ruminococcus and Streptococcus (Tables 2 and 3). Basically, the number of enterococci increased, which in metagenomic analysis are not recognized as a separate family when comparing the composition of streptococci (Table 3). A decrease in opportunistic bacteria Bacteroides fragilis and an increase in the content of fecalibacteria were of fundamental importance.

Thus, the patient showed positive dynamics on the scales for assessing anxiety and depression, the severity of non-motor manifestations of Parkinson's disease decreased, stool frequency increased, feces became more formed, and the patient also reported stopping caloing. All these changes occurred against the background of restoration of the intestinal microbiocenosis composition.

It is especially important what happened with a decrease in prevotellus, an increase in the content of which is interpreted as a negative link in the pathogenesis of PD, and an increase in fecalibacteria belonging to butyrate-producing bacteria that enhance motor skills and stimulate the production of mucus that protects the mucous membrane.

Conclusion

The studies conducted allow us to consider autoprobiotics based on E. faecium as an effective drug that improves the quality of life of patients by reducing non-motor symptoms (general weakness, nausea and discomfort after eating), improving intestinal motility, its evacuation ability, and restoring the composition of intestinal microbiocenosis.

Claims (1)

A method of reducing the severity of non-motor symptoms in patients with Parkinson's disease by correcting intestinal microbiocenosis, which consists in the fact that a patient who has not taken antibiotics, probiotic products and drugs for at least 10 days, takes feces in an amount of at least 1 ml , from them clones of indigenous strains of Enterococcus faecium are isolated using a dense nutrient medium containing sodium azide and a crystal violet dye, then the colony selection is carried out by the nature of the color, pure cultures are obtained by reseeding three pink colonies with a burgundy center into three sectors of a Petri dish with such the same medium, after which they incubate in a thermostat under aerobic conditions at t = 37 ° C, check for the absence of pathogenicity genes using PCR, select clones devoid of pathogenicity factors, with which soy hydrolyzate is fermented in a volume of at least 10 ml per liter, then choose that from the ferment that changed the ghee structure earlier than others drolysate, and use it to prepare two liters of an individual autoprobiotic product with a content of at least 10 ⁸ CFU per 1 ml, which is administered to the patient orally, 50 ml 2 times a day, for a course of at least 20 days.

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