RU2817993C1 - Method for selective decontamination of staphylococcus aureus, klebsiella pneumoniae in children using bacteriophages in treating atopic dermatitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to paediatrics, allergology – immunology and microbiology, and concerns a method for selective decontamination of Staphylococcus aureus, Klebsiella pneumoniae in children. Bacteriophages are used in the presence of Micrococcaceae – Staphylococcus aureus and Enterobacteriaceae – Klebsiella pneumonieae families or their associations in the analysed material when analysing the microflora composition. Bacteriophages used are Bacteriophagum Staphylococcum in dose of 1–3 ml per os 2 times day and 1–3 ml per rectum before bedtime for no more than 8 days and Klebsiella polyvalent bacteriophage purified in dose of 1–3 ml per os 2 times day and 1–3 ml per rectum before bedtime for no more than 8 days. Introduction of bacteriophages is course. Frequency rate of the course is no more than 5 courses with an intercourse break of not more than 14 days.

EFFECT: invention provides preservation and acceleration of formation of a unique and individual composition of the intestinal microbiota of a child, formation of an immunoregulatory balance, as well as allows to achieve complete absence of clinical manifestations and aggravations of AD in catamnesis.

1 cl, 1 tbl, 1 ex

Description

The invention relates to medicine, in particular to pediatrics, allergology - immunology and microbiology, and can be used for the treatment of food allergy (FA), atopic dermatitis (AD).

A significant increase in the number of predominantly young patients with allergic pathology today reflects all the signs of a pandemic and, unfortunately, the reasons for this phenomenon remain unclear. Currently, due to the lack of a general doctrine and modern schemes for both the treatment and prevention of allergic diseases (hereinafter referred to as AD), conceptually new approaches to the treatment and prevention of this pathology are being developed.

The consistently steady increase in the prevalence of AD dictates the need to search for qualitatively new effective treatment and preventive strategies. For a long time, various types of research have been carried out on hypotheses that make it possible to effectively treat and prevent allergies, but, unfortunately, to no avail.

The hypothesis of eliminating contact with possible allergens to prevent allergic manifestations, analysis of the results of multicenter, cohort European studies (GINI, LISA, COALA), confirmed the lack of convincing evidence of the effectiveness of limiting highly allergenic foods in the diet of pregnant and lactating mothers, children with a hereditary predisposition to allergies (Smirnova G.I. Intestinal microbiota and the use of probiotics in the prevention and treatment of atopic dermatitis in children // Attending physician. - 2016. - No. 1 - P. 6-10; Belyaeva I.I., Bombardirova E.P., Turti T.V., Mitish M.D., Potekhina T.V., N.A. Kharitonova Ontogenesis and dysontogenesis of intestinal microbiota in young children: trigger mechanism of child health disorders // Issues of modern pediatrics. 2017. Vol. 1. S - 29-38; Schwiertz A, Rusch V. Microbiota of the Human Body Implications in Health and Disease. Advances in Experimental Medicine and Biology 2016. https://doi.org/10.1007/978-3. -319-31248-4).

One of the latest hypotheses, proposed by researchers from Australia and the USA, involves the use of vitamin D for favorable colonization of intestinal microflora, which helps reduce the permeability of the intestinal barrier and excessive exposure of the immune system to food antigens (Kuznetsova, M.V. Monitoring the colonization of opportunistic microflora in newborns during stay in medical institutions / M.V. Kuznetsova, T.I. Avdeeva // Med. almanac. - No. 6. - P. 156-159).

The most well-known and actively studied hypothesis of the possible role of probiotics in the prevention of AD causes concern on the part of the scientific community and requires additional evidence. The recently announced conclusion of the EAACI working group states that there is no convincing evidence for the use of probiotics for the prevention of food allergies and anaphylaxis (Nikiforova, E.M. Features of the development of dermatorespiratory syndrome in children with atopic dermatitis in the first year of life / E.M. Nikiforova, M.A. . Chueva // Volgograd. scientific journal. - 2011. - P. 33-35; Intestinal microbiota and brain: mutual influence and interaction / O.K. Pediatrics. - 2015. - No. 6. - P. 134-140).

It has now been reliably established that the triggering mechanism for allergies is the processes of competitive interaction between the lacto and bifid flora of an infant with aggressive strains of opportunistic pathogenic flora (hereinafter referred to as OPM) of hospital origin. Intestinal dysbiosis caused by excessive growth of the UPM against the background of slow growth of bifid flora in children with a hereditary predisposition to allergies indicates a correlation of the risk of developing diseases not only of diseases of the digestive system, but also of dermatorespiratory lesions of the central nervous system.

A very relevant and one of the promising, actively studied areas for correcting intestinal biocenosis today is the use of bacteriophages, which are an alternative to treatment with antibiotics and probiotic drugs. Selective phage decontamination (hereinafter referred to as SPD) of excessive growth of Staphylococcus aureus and Klebsiella pneumonieae and their association as the main factor in the implementation of the atopic phenotype, makes it possible to preserve and accelerate the formation of a unique and individual composition of the child’s intestinal microbiota, capable of forming an immunoregulatory balance. Methods of influencing the microbial communities of a child’s intestine should be as delicate as possible, taking into account the individual genetic characteristics of the intestinal microecosystem and the possibility of developing anaphylaxis.

The prior art knows a method for treating atopic dermatitis in children with probiotic preparations, taking into account the results of microbiological examination of stool (patent RU 2702229, published 10/07/2019 Bulletin No. 28). To do this, a microbiological study of feces is carried out, performing species identification of all isolated cultures of microorganisms using MALDI-TOF mass spectrometry. After that, a probiotic is selected in accordance with the patient’s deficiency or excess of representatives of specific types of microorganisms from representatives of the normal microflora.

The closest in technical essence is a method for complex treatment of intestinal dysbiosis (patent RU 2337697, published on November 10, 2008 Bulletin No. 31). To do this, selective decontamination with anti-Klebsiella bacteriophage is carried out as antibacterial therapy for 10 days. After this, probiotics are administered against the background of prebiotic therapy, including Hilak-Forte, an enzyme preparation and Linex in therapeutic doses. At stage I dysbacteriosis of bififorms, 1 capsule is administered 2 times a day, for dysbiosis of II and III degrees. - 2 capsules 2 times a day, as well as Lactobacterin - 5 doses 1 time a day. At the same time, an infusion from a collection of medicinal plants is introduced, including cinquefoil rhizome, sage leaves, yarrow herb, St. John's wort herb, elecampane rhizome with roots, string herb, mint leaves, fennel fruits and buckthorn bark at a component ratio of 2:2:2:2, respectively: 2:2:2:2:1 1/3 cup 3 times a day, 30 minutes before meals, for 4 weeks.

The above methods are not effective enough in the treatment of AD due to the occurrence of subsequent relapses of the disease and the impossibility of stopping the atopic march in follow-up, have a limited number of patients, and are quite labor-intensive.

The technical result consists in preserving and accelerating the formation of a unique and individual composition of the child’s intestinal microbiota, in the formation of an immunoregulatory balance. The proposed method makes it possible to achieve a complete absence of clinical manifestations and exacerbations of AD in follow-up.

This technical result is achieved by the fact that the method of selective decontamination of Staphylococcus aureus, Klebsiella pneumoniae in children includes the use of a bacteriophage in the presence of the families Micrococcaceae - Staphylococcus aureus and Enterobacteriaceae - Klebsiella pneumonieae or their associations in the test material when analyzing the composition of the microflora, while Bacteriophagum are used as bacteriophages Staphylococcum in a dose of 1-3 ml per os 2 times / day and 1-3 ml per rectum before bedtime for no more than 8 days and Klebsiella polyvalent bacteriophage purified in a dose of 1-3 ml per os 2 times / day and 1-3 ml per rectum before bedtime for no more than 8 days, the introduction of bacteriophages is a course, and the frequency of the course is no more than 5 courses with an intercourse break of no more than 14 days.

The proposed method of SFD of Staphylococcus aureus and Klebsiella pneumonieae and their association is carried out in children with a hereditary predisposition to AD (the fact of the presence of the disease in the family among relatives of the 1st and 2nd degrees of kinship) no later than 5-6 weeks of life. To do this, initially, the cultural composition of the intestinal contents is determined on Baird Parker Agar and Congo Red Agar (CRA) nutrient media. For objectivity, it is advisable to conduct the study twice. The method of bacteriological examination of feces for dysbiosis is carried out in accordance with OST 91500.11.0004-2003 “Protocol for the management of patients. Intestinal dysbiosis." The collection is carried out in a sterile container for biomaterial. After delivery to the bacteriological laboratory, the microbiologist prepares, analyzes the qualitative and quantitative composition of the microflora and, after the study period, issues a conclusion on the state of the microflora of the distal intestine. If the studied material contains the families Micrococcaceae - Staphylococcus aureus and Enterobacteriaceae - Klebsiella pneumonieae or their association, after determining the sensitivity, SFD is carried out using production phage strains.

The optimal recommended dose of bacteriophage to achieve a significant clinical effect depends on lg CFU/g. So in the presence of Staphylococcus aureus and Klebsiella pneumonieae :

- in an amount of up to 2 lg CFU/g, the dose of the bacteriophage is no more than 1 ml 2 times a day per os and a single dose of 1 ml per rectum for a course duration of no more than 8 days,

- in an amount of 3-4 lg CFU/g, the dose of the bacteriophage is no more than 2 ml 2 times a day per os and a single dose of 2 ml per rectum for a course duration of no more than 8 days,

- in an amount of 4-6 or more lg CFU/g, the recommended dose of the bacteriophage is 3 ml 2 times a day per os and a single dose of 3 ml per rectum for a course duration of no more than 8 days.

SFD of Staphylococcus aureus is carried out using Bacteriophagum Staphylococcum in a dose of 1-3 ml per os 2 times / day. and 1-3 ml per rectum before bedtime for 8 days. SFD of Klebsiella pneumonieae is carried out using Klebsiella polyvalent bacteriophage purified in a dose of 1-3 ml per os 2 times a day and 1-3 ml per rectum before bedtime for 8 days.

The inter-course break for SFD is no more than 14 days. The most preferable number of courses to achieve a significant clinical effect in case of laboratory-confirmed sensitivity to bacteriophage is no more than 3. In the case of laboratory-confirmed lack of sensitivity to bacteriophage, the multiplicity is no more than 5 courses.

Advantages of the method:

1. The proposed method of SFD of Staphylococcus aureus and Klebsiella pneumonieae and their association in children in the first weeks of life with a hereditary predisposition to AD using bacteriophages makes it possible to preserve and accelerate the formation of a unique and individual composition of the child’s intestinal microbiota, capable of forming an immunoregulatory balance in the Th1/Th2 system.

2. The method of phage correction of intestinal microbiota is financially low-cost and does not require complex invasive laboratory and instrumental research methods.

3. Carrying out SFD in the form of monotherapy makes it possible to achieve clinical manifestations: a decrease in the intensity and complete disappearance of intestinal colic already on the 2-3rd day of use. Reduction of pathological inclusions in the stool and normalization of the frequency of defecation on days 3-4, objective dynamics of skin syndrome on days 5-6 of phage therapy (decrease in the SCORAD index). The presence of a positive clinical picture will be determined by changes in the microbiological composition of the intestinal biotope.

4. The use of SFD makes it possible to achieve complete decontamination of Staphylococcus aureus and Klebsiella pneumonieae and subsequently, due to competitive growth, an increase in the dynamics of lg CFU/g of lacto and bifid flora.

The results of the SFD are presented as an example.Staphylococcus aureus and Klebsiella pneumonieae and their associations in 60 children with AD. Follow-up for 7 years did not reveal any child from the atopic march (development of allergic rhinitis and bronchial asthma) (Table 1).

Table 1. Dynamics of decontamination of Staphylococcus aureus and Klebsiella pneumonieae and their associations in children with AD during SFD (3 courses)

Microflora Norm in children, CFU/g Before treatment After treatment R indicator/CFU/g number of children/abs
(%) indicator/CFU/g number of children/abs
(%) Staphylococcus aureus ≤0 10 ⁷ 21(35) 10³ 5(8.33) 0.002 Klebsiella pneumoniae ≤10 ⁴ 10 ⁸ 11(18.33) 10 ⁴ 2(3.33) 0.020 Staphylococcus aureus and Klebsiella pneumonieae ≤10 ^0-4 10 ⁷ 10 ⁶ 10 (16.66) - -

SFD of Klebsiella pneumoniae and Staphylococcus aureus and their associations allows us not to disturb the genetically determined diversity of the intestinal microbiota of a child in the first weeks of life and makes it possible to achieve a rapid clinical effect.

Below is an example of the implementation of the claimed method of treatment.

Patient M., born April 25, 2020, according to the mother, complaints about the baby’s general restlessness, mainly in the evening, which, in her opinion, is associated with abdominal pain and itching of the skin, absence of stool for up to 2 days with the presence of mucus in the stool, greenery, streaks of blood.

Allergic anamnesis: aggravated on the maternal and paternal lines: father - seasonal allergic rhinitis, maternal grandmother - food allergy, bronchial asthma.

Anamnesis morbi briefly: Child from 2 pregnancies, 2 births, which occurred against the background of mild anemia, chronic pyelonephritis, bronchitis, carriage of CMV and toxoplasmosis. Urgent birth at 38.5 weeks in an anterior-occipital presentation. Birth weight 3.2 kg, height 52 cm, o. heads 33cm, about. chest 33 cm, Apgar score 8/9 points, attached to the breast on the first day, sucked actively. Breastfed from birth, the mother is very careful about the diet and care of the baby, strictly following the recommendations of the local pediatrician. At the 3rd week of life, intestinal colic appeared, which was relieved by the use of drugs from the semethicone group. At the age of 4 weeks, skin syndrome debuted with the appearance of erythematous elements on the face with subsequent generalization. In accordance with the Federal Clinical Guidelines, a diagnosis of Atopic dermatitis of infancy, erythematous-squamous form, widespread (L20.8) was made. Against the background of the therapy, Bak-set baby, sub simplex, D-panthenol 5% ointment for external use, there was no positive dynamics, and therefore, he was referred to a consultation with an allergist-immunologist.

Status praesens objectivus: The general condition at the time of the first visit is satisfactory, the reaction to examination is adequate, the child is active. Visually, there is an erythematous-squamous rash with a predominant localization on the upper half of the body, areas of bright painful hyperemia on the elbows, knees, and groin area; active desquamation of the epithelium on the scalp. SCORAD index 46.1 points. Visible mucous membranes are pale pink, clean. Breathing through the nose is difficult due to moderate swelling of the mucous membrane; with anxiety, “grunting” nasal breathing. Pharynx without signs of pathomorphological changes. There is puerile breathing in the lungs. Percussion pulmonary sound over the entire surface. The heart sounds are clear, rhythmic, and the tone ratio is correct. The abdomen is of the correct configuration, somewhat swollen, palpable in all parts, the liver and spleen are within the age norm. The lymphatic system is without pathological changes. Stool: periodic delays of up to 2 days, the presence of mucus and greenery, the presence of blood streaks twice in the last week. Diuresis is not impaired. There are no meningeal signs. There is no pathology on the part of the cranial nerve. Reflexes are alive and symmetrical.

RESULTS OF CLINICAL AND LABORATORY STUDIES

Hemogram at the time of treatment: RBC - 3.95 - 1012 /l, HGB - 116 g/l, WBC - 6.19 /l, EOS - 6%, NEUT: p.o. - 1, p.o. - 41, LYM - 40, MON - 12.

Blood biochemistry: ALT - 12.4 U/L, AST 17.2 U/L, total bilirubin 14 µmol/L, urea - 3.2 mmol/L, createnine - 43.0 mmol/L, glucose 2.69 mmol/L .

Coprogram: consistency - viscous, color - yellow-green, smell - sour, pH - 5.7, mucus - in small quantities, no soluble protein, stercobilin, fecal detritus, neutral fat, fatty acids, soaps - in small quantities, ammonia , muscle, connective tissue fibers, starch, plant fiber - absent, erythrocytes - 4-6 in the p/z, leukocytes - 6-8 in the p/z.

Fecal calprotectin - 547 mcg/g.

General urine analysis is the age norm.

Stool culture on UPM: ( Staphylococcus aureus 10 ⁶ CFU/g, Klebsiella pneumonieae 10 ⁵ CFU/g).

DIAGNOSIS: (ICD 10) Atopic dermatitis, infantile, erythematous-squamous form, widespread (L20.8). Proctocolitis induced by dietary proteins (K52.2).

TREATMENT: First course: Staphylococcal bacteriophage (Bacteriophagum Staphylococcum) series P30, Klebsiella polyvalent bacteriophage purified series U50.

Second and third courses: Staphylococcal bacteriophage (Bacteriophagum Staphylococcum) series P26F5, Klebsiella bacteriophage (Klebsiella polyvalent bacteriophage purified) series U51/0120F. The recommended dose of bacteriophages in this clinical example was 3 ml per os 2 times a day and 3 ml per rectum before bedtime Bacteriophagum Staphylococcum for 8 days, then Klebsiella polyvalent bacteriophage purified for 8 days with an inter-course break of 14 days.

CLINICAL EFFECT:

During the therapy, on the second day there was a decrease in the intensity of intestinal colic with complete disappearance of the pain syndrome by the fifth day. The dynamics of a decrease in the amount of mucus and greens was noted on the fifth day of phage therapy, complete disappearance on the ninth day. According to my mother, my general condition and well-being have significantly improved, the frequency of daytime sleep and the duration of sleep at night have increased. The extinction of the skin syndrome was noted on the third day, complete relief on the fourteenth day. The full therapeutic course in a particular case was three episodes of SFD. Carrying out phage therapy in the form of monotherapy made it possible to quite successfully decontaminate excessive growth of Staphylococcus aureus from 10 ⁶ lg CFU/g to “no growth” of Klebsiella pneumonieae from 10 ⁵ lg CFU/g to 10 ³ lg CFU/g.

The optimal clinically effective dose of bacteriophage for SFD depends on lg CFU/g. and is 1 ml 2 times a day per os and a single dose of 1 ml per rectum at 2 lg CFU/g, 2 ml 2 times a day per os and a single dose of 2 ml per rectum at 3-4 lg CFU/g and 3 ml 2 times a day per os and a single dose of 3 ml per rectum at 4-6 or more lg CFU/g.

Recommended dosages of bacteriophages, frequency of courses, no more than 3, intercourse interval have shown their effectiveness in more than 30 thousand patients with AD and are justified by the following reasons; treatment is carried out for children in the first weeks of life with a high predisposition to AD; the recommended doses of the drug minimize the development of acute anaphylaxis reactions, primarily to the preservative; minimal doses of the drug have a very significant effect on parental compliance, as shown by many years of experience in using the technique; In addition, an important point in the treatment of AD is a significant reduction in the burden on the financial component of the family budget; the use of bacteriophage dosages recommended by the manufacturer in the context of AD therapy is inappropriate and entails a high probability of developing side effects.

The given examples of treatment using the SFD method show a high therapeutic effect, do not cause side effects, and have no contraindications.

Thus, the claimed method is simple, universal, with a personalized approach and focused on use in primary healthcare.

Claims (1)

A method for selective decontamination of Staphylococcus aureus, Klebsiella pneumoniae in children, including the use of a bacteriophage in the presence of the families Micrococcaceae - Staphylococcus aureus and Enterobacteriaceae - Klebsiella pneumonieae or their associations in the test material when analyzing the composition of the microflora, while Bacteriophagum Staphylococcum is used as bacteriophages in a dose of 1-3 ml per os 2 times a day and 1-3 ml per rectum before bedtime for no more than 8 days and Klebsiella polyvalent bacteriophage purified at a dose of 1-3 ml per os 2 times a day and 1-3 ml per rectum before bedtime for no more than 8 days , the introduction of bacteriophages is a course, and the frequency of the course is no more than 5 courses with a break between courses of no more than 14 days.