RU2522678C2 - Method for prediction of severity of chronic obstructive pulmonary disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and aims at treating chronic obstructive pulmonary disease (COPD). When taking into account a quantity of courses of antibiotic administration for aggravated COPD during previous 12 months, a standard 6-minute walking test involves estimating a 6-minute distance, the heart rate prior to the walking test and oxygen saturation after the walking test; a biomaterial is sampled from the posterior wall of the pharynx by oropharyngeal smears to recover DNA and to sequence; observing proteobacteria involves recoding of the derived data into a qualitative variable; genome DNA is recovered from the COPD patients' blood that is followed by genetic typing by CD14 rs2569190 and IL18 rs1946518 genes; the genetic typing data are pre-recorded into digital values to calculate a discriminant function and to make a forecast.

EFFECT: presented method enables a higher accuracy, information value and sensitivity and enables predicting a severity of COPD effectively, as well as differentiating a severe and very severe clinical course of a moderate and mild COPD.

1 dwg, 4 tbl, 2 ex

Description

The invention relates to medicine, namely to pulmonology, and can be used to predict the severity of chronic obstructive pulmonary disease (COPD).

Despite the fact that many clinical studies are devoted to the study of COPD, the problem of rational pharmacotherapy of chronic obstructive pulmonary disease remains unresolved. The need to create a new method for predicting the severity of the disease in patients with COPD is related to the heterogeneity of the rate of progression of disease severity in various patients, the difference in the effectiveness of their existing standard pharmacotherapeutic approaches and high rates of health care resources for managing patients with COPD. In addition, COPD, associated with frequent exacerbations and rapid progression, is characterized by early disability of patients and a high risk of death, which makes it possible to attribute the problem of personalized COPD therapy to one of the most urgent problems of modern medicine. It must be emphasized that, in view of the lack of fundamental knowledge, the classification of COPD is based on clinical signs, and not on certain pathophysiological or genetic mechanisms.

Currently, it is relevant to study the composition of microorganisms of oropharyngeal smears and determine the genetic polymorphism in patients with COPD for an integrated approach to determining and predicting the severity of the disease with the prospect of personifying treatment of chronic obstructive pulmonary disease [1].

Currently, several methods for predicting the severity of COPD are known.

Thus, a team of authors from the State Far Eastern Scientific Center for Physiology and Respiratory Pathology SB RAMS proposed a method for predicting a stable course of chronic obstructive pulmonary disease based on measuring the volume of forced expiration in the first second (FEV1) in l, determining the change in the volume of forced expiration in the first second (FEV 1 ) in% of the initial value after a bronchial provocative pharmacological test with a 0.1% solution of acetylcholine chloride and the solution of the discriminant equation: D = -2.94 · OFV1 + 1.34 · OFV1. When the value of D is greater than -19.34, a stable course of chronic obstructive pulmonary disease is predicted (RU No. 2262889 publ. 10/27/2005) [2]. In the light of recent studies, it is obvious that this method is not sufficiently informative, since it does not take into account the severity of the patient’s clinical condition, the frequency of exacerbations of the disease, genetic factors and the contribution of the microbiome to the progression of the disease.

A team of authors from Surgut State University proposed a method for assessing the severity of bronchial asthma by comparing bronchial patency and reversibility of bronchial obstruction during monitoring. Assessment of patency of the bronchial tree was carried out according to the peak flow curve; assessment of bronchial obstruction - by measuring the peak expiratory flow rate (PSV) before using a bronchodilator and 30 minutes after consuming a bronchodilator. Then, the obtained values of the values were recorded in the formula by which the index was calculated. If the index reached a threshold value, severe bronchial asthma was diagnosed (RU No. 2348352, publ. 05.20.2008) [3]. The method proposed by the authors has a limited scope and is not informative regarding the prognosis of the severity of chronic obstructive pulmonary disease.

A team of authors from the Military Medical Institute of the Federal Border Service of the Russian Federation at the Nizhny Novgorod State Medical Academy proposed a method

for predicting bronchial obstruction in chronic bronchitis, based on a comprehensive study of the concentration of interleukin-1 (IL-1) in blood, saliva, bronchoelveolar fluid. The distribution coefficient (CR) for (IL-1) is determined as the ratio of the amount of IL-1 of blood to the amount of IL-1 of saliva and it is determined with an increase in the content of IL-1 by more than 10 times in blood, 2 times in saliva, unchanged level bronchoalveolar interleukin-1. When CR for IL-1 more than 1.0 predict bronchial obstruction (RU No. 2245550 publ. 01/27/2005) [4]. This method is more suitable for detecting bronchial obstruction in the early stages of COPD, but is not informative for assessing and predicting the severity of the disease.

Closest to the proposed one is a method for predicting the progression of COPD, proposed by a team of authors from GU Far Eastern Scientific Center for Physiology and Respiratory Pathology SB RAMS. The method consists in conducting an immunological study to determine the ability of immunocompetent cells to produce transforming growth factor ^β 1 (TGF- ^β 1) and fibroblast growth factor (bFGF). At a spontaneous level of TGF- ^β 1 ^≥ 1122.71 ± 20.6 pg / ml and bFGF ^≤ 7.2 ± 0.08 pg / ml, a favorable course of COPD with a decrease in FEV _{1 of} no more than 50 ml per year is predicted, and at a spontaneous level TGF- ^β 1≤1025.5 ± 11.7 pg / ml and bFGF 40.1 ± 0.9 pg / ml predict an unfavorable course of COPD with a decrease in FEV1 by more than 50 ml per year (RU No. 2370773 publ. 23.06.2008 g.) [5]. Taking into account modern scientific data, it should be concluded that the method is not sufficiently informative, since it does not take into account the role of microbiotic communities inhabiting the respiratory tract of a patient with COPD and the peculiarities of genetic polymorphism in the development of exacerbations of COPD and its progression.

A new technical problem is improving the accuracy, information content and sensitivity of the method.

To solve the problem in a method for predicting the severity of chronic obstructive pulmonary disease, including a study of the patient’s venous blood, the number of antibiotic courses for exacerbating COPD over the previous 12 months is additionally taken into account, a standard test with 6-minute walking is performed with an estimate of the distance traveled by the patient for 6 minutes, the heart rate before the test with walking and the level of oxygen saturation after performing the test with walking, determine the quality and if ety composition posterior pharyngeal wall mikrobioticheskogo community method oropharyngeal swabs with subsequent isolation of the DNA by conducting sequencing and transcode the received data into qualitative variable on the principle of assigning a numerical value 1 if there is more than 50 colony-forming units of microorganisms from the group of Proteobacteria, per 1 cm ² of the posterior pharyngeal wall, and otherwise, assigning a digital value of 0, genomic DNA is isolated from the venous blood of patients with COPD by phenol-chloroform extraction with after By carrying out genotyping by polymorphisms of the CD14 rs2569190, IL18 rs1946518 genes, the genotyping data are pre-transformed into digital values by the CD14 rs2569190 gene, assigning 0 to the TT genotype, 1 to the TC genotype and 2 to the CC genotype, and assigning 0 to the IL18 rs1946518 gene, assigning 0 1 - ACi genotype 2 - SS genotype, and determine the probability of an individual being assigned to a group of severe and very severe COPD patients or to groups of mild to moderate COPD patients, calculating the discriminant function of F1 and F2:

F1 = -0.0349 * Path + 1.970188 * Micro-0.20922 * Heart rate + 0.531974 * TLR4-0.49016 * Oxygen saturation post + 0.257794 * IL18B-0.14965 * Antibiotics;

Where

-0.0349; 1.970188; 0.20922; 0.531974; 0.49016; 0.257794; 0.14965 - numerical values are coefficients;

Path - the distance traveled by the patient (m) in 6 minutes in the standard test with a 6-minute walk;

Micro - numerical value of colony forming units of microorganisms from the group of proteobacteria (mainly Haemophilus) per 1 cm ^{2 of the} posterior pharyngeal wall;

ChSSpre - heart rate before the test with a 6-minute walk;

TLR4 and IL18B - genotypes;

Post oxygen saturation - oxygen saturation after the test with a 6-minute walk;

Antibiotics - the number of antibiotic courses for exacerbation of COPD in the previous 12 months;

further, to assign the patient to a specific prognostic group, the F1 value is evaluated, and if the function value is> -67.826, the patient is predicted to have severe and very severe COPD;

and with a value of ≤-67.826, in order to clarify the distribution, use the function Ф2, which is calculated by the formula:

Ф2 = 0.001983 * Path-0.574 * Micro-0.10173 * HRspre + 0.748163 * TLR4-0.2893 * Oxygen saturation post + 1.16181 * IL18B + 1.039555 * Antibiotics;

Where

0.001983; 0.574; 0.10173; 0.748163; 0.2893; 1.16181; 1.039555 - numerical values are coefficients;

Path - the distance traveled by the patient (m) in 6 minutes in the standard test with a 6-minute walk;

Micro - numerical value of colony forming units of microorganisms from the group of proteobacteria (mainly Haemophilus) per 1 cm ^{2 of the} posterior pharyngeal wall;

ChSSpre - heart rate before the test with a 6-minute walk;

TLR4 and IL18B - genotypes;

Post oxygen saturation - oxygen saturation after the test with a 6-minute walk;

Antibiotics - the number of antibiotic courses for exacerbation of COPD in the previous 12 months;

and with a function value of ≤-31.5079, moderate COPD is predicted;

- with a function value> -31.5079, mild COPD is predicted.

The method is as follows

A study of the patient’s venous blood is carried out, the number of antibiotic courses taken for exacerbation of COPD over the previous 12 months is also taken into account, a standard test with 6 minutes of walking is carried out with an estimate of the distance traveled by the patient in 6 minutes, the heart rate before the walking test and the level of saturation oxygen after performing the walking test, determine the qualitative and quantitative composition of the microbiotic community of the posterior pharyngeal wall by the method of oropharyngeal smears with subsequent isolation of D NK, by sequencing and transcoding the obtained data into a qualitative variable according to the principle of assigning a digital value of 1 in the presence of more than 50 colony forming units of microorganisms from the group of proteobacteria per 1 cm ^{2 of the} posterior pharyngeal wall, otherwise, assigning a digital value of 0, genomic DNA is isolated from venous blood patients with COPD by phenol-chloroform extraction followed by genotyping by polymorphisms of the genes CD 14 rs2569190, IL18 rs1946518, pre-transcode the genotyping data in digital values for the CD14 rs2569190 gene, assigning 0 to the TT genotype, 1 to the TS genotype and 2 to the CC genotype, and according to IL18 rs1946518 gene, assigning 0 to the AA genotype, 1 to the AC genotype and 2 to the CC genotype, and determine the probability of assigning the individual to a group of patients with severe and very severe COPD or groups of patients with mild to moderate COPD, calculating the discriminant function of F1 and F2:

F1 = -0.0349 * Path + 1.970188 * Micro-0.20922 * Heart rate + 0.531974 * CD14-0.49016 * Oxygen saturation post + 0.257794 * IL18B-0.14965 * Antibiotics;

Where

-0.0349; 1.970188; 0.20922; 0.531974; 0.49016; 0.257794; 0.14965 - numerical values are coefficients;

Path - the distance traveled by the patient (m) in 6 minutes in the standard test with a 6-minute walk;

Micro - numerical value of colony forming units of microorganisms from the group of proteobacteria (mainly Haemophilus) per 1 cm ^{2 of the} posterior pharyngeal wall;

ChSSpre - heart rate before the test with a 6-minute walk;

CD14 and IL18B are genotypes;

Post oxygen saturation - oxygen saturation after the test with a 6-minute walk;

Antibiotics - the number of antibiotic courses for exacerbation of COPD in the previous 12 months;

further, to assign the patient to a specific prognostic group, the value of F1 is evaluated and,

- if the value of the function is ≤-70.4332, the patient is assigned to the group of patients with mild COPD;

- if the value of the function is> -70.4332 and ≤-67.826, the patient is assigned to the group of patients with moderate COPD;

- if the value of the function is> -67.826, the patient is assigned to the group of patients with severe and very severe COPD;

further, when the patient is assigned to the COPD group of severe and very severe course after calculating the first discriminant function, further analysis is stopped, and if the patient is distributed into the COPD group of mild or moderate severity in order to clarify the distribution, the calculation procedure for the second function F2 is repeated according to the formula

Ф2 = 0.001983 * Path-0.574 * Micro-0.10173 * HRspr + 0.748163 * CD14-0.2893 * Oxygen saturation post + 1.16181 * IL18B + 1.039555 * Antibiotics;

Where

0.001983; 0.574; 0.10173; 0.748163; 0.2893; 1.16181; 1.039555 - numerical values are coefficients;

Path - the distance traveled by the patient (m) in 6 minutes in the standard test with a 6-minute walk;

Micro - numerical value of colony forming units of microorganisms from the group of proteobacteria per 1 cm ^{2 of the} posterior pharyngeal wall;

ChSSpre - heart rate before the test with a 6-minute walk;

CD14 and IL18B are genotypes;

Post oxygen saturation - oxygen saturation after the test with a 6-minute walk;

Antibiotics - the number of antibiotic courses for exacerbation of COPD in the previous 12 months;

And with a function value of ≤-31.5079, moderate COPD is predicted;

- with a function value> -31.5079, mild COPD is predicted.

There is no research data in the scientific and technical literature that would establish relationships between the severity of COPD and the characteristics of the microbiome of the posterior pharyngeal wall in combination with patient genotyping data for polymorphic variants of the CD14 and IL18B genes. Also, the proposed combination of features for the differential diagnosis of COPD was not found. Thus, the invention meets the criterion of "novelty" and "significant differences".

The method has been tested in clinical conditions, thus meeting the criterion of "industrial applicability".

Bacterial contamination, as a factor in provoking exacerbations of COPD, has been considered for a long time. Under normal conditions, mucociliary clearance and an active pool of alveolar macrophages, together with secreted IgA, mucin and other antimicrobial defense factors, are an effective defensive system of the lower respiratory tract. Against the background of chronic diseases with persistent inflammation (COPD), some of these lines of defense do not function, contributing to the development and maintenance of bacterial contamination in the respiratory tract. Mucostasis and violation of the drainage function of the bronchi against the background of local and systemic immunodeficiency create favorable conditions for the constant colonization of the bronchial tree with microorganisms [6]. The respiratory tract in patients with COPD (especially severe and very severe) is often colonized by bacteria. The most common seeding is Haemophilus influenzae, Haemophilus spp., Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas spp., Klebsiella spp. In the late stages of the disease [7].

An exacerbation of the infectious process in the respiratory system is an important factor exacerbating bronchial obstruction, leading to an increase in all signs of the disease and its further progression. It is the transformation of bacterial contamination into an infectious process that is the most common cause of the development of exacerbations of COPD [8].

Of course, every infectious dependent exacerbation of COPD worsens the prognosis of the disease. From a clinical point of view, all exacerbations should be considered as a factor in the progression of the disease. Typically, over the course of a year, a patient with COPD suffers from one to four exacerbations or more. Their frequency varies markedly depending on the severity of the disease and the adequacy of the chosen treatment regimen. So, with OOB ₁ 50-55% of the due, the average frequency of exacerbations is 1.9-2.1 exacerbations per year, and in patients with OOB ₁ less than 40%, 2-3 exacerbations per year. Therefore, one of the main criteria for the effective treatment of stable COPD is to reduce the frequency of exacerbations and increase the duration of the patient's life without episodes of exacerbation of the disease [1].

Given the above, it becomes necessary to take into account the severity of bacterial contamination and the species characteristics of the microbiome of the respiratory tract in predicting the severity of COPD. In the presented method, the assessment of the microbiome in smears from the posterior pharyngeal wall is used, since a correlation is established between oropharyngeal microbiotic communities and communities obtained from brush biopsy specimens of bronchi and lavage fluid [9].

As a result of the studies, information was obtained on the composition of microbiotic communities in oropharyngeal smears in patients with COPD. It was decided to use discriminant analysis calculated on the basis of more than one factor. In order to improve the accuracy and informativeness of the method, it was also decided to use the patient genotyping data for polymorphic variants of the immune response and inflammation regulatory genes IL18 rs5744247, IL18 rs1946518, CD14 rs2569190, IFNG rs2069705, MCP1 rs1024611. Known sources of information do not have data on studies that would establish relationships between the severity of COPD and the characteristics of the microbiome of the posterior pharyngeal wall in combination with patient genotyping data for polymorphic variants of the immune response and inflammation regulatory genes.

The coefficients of the discriminant models are selected based on the analysis of clinical and laboratory research data of patients conducted on the basis of SBEI HPE SibGMU of the Ministry of Health and Social Development of Russia, using logistic regression analysis.

The development of the proposed method for predicting the severity of COPD using a classification function based on a combination of clinical and functional factors, identifying the quantitative and species composition of the microbiome of oropharyngeal smears by molecular genetic methods (isolation of bacterial DNA followed by sequencing), as well as determining the genetic polymorphism of the immune response genes and regulation of inflammation (genes of the TLR family, genes of cytokines and their receptors) by DNA extraction from whole blood for a gene The following experimental work and clinical studies preceded the isolation with RFLP analysis.

The basis for the development of the proposed method was an in-depth clinical, medical history, physical and functional examination of 45 patients with COPD (15 patients with mild COPD, 15 patients with moderate COPD and 15 patients with severe and very severe COPD).

Patients were included in the study, subject to the following criteria:

Inclusion criteria for patients with COPD:

1. Men aged 40 to 70 years old with a previously documented diagnosis of COPD;

2. Patients currently smokers or former smokers with a smoking index of more than 10 packs / year;

3. Patients having, according to the results of the study of the function of external respiration, postbronchodilation values of FEV1 <80% of the due and FEV1 / FVC <70%. Patients with stage II COPD - FEV1 = <80-50% of due, patients with stage III COPD - FEV1 = <50-30% of due, patients with stage IV COPD - FEV1 = <30% [10].

3. Patients who signed written informed consent prior to inclusion in the study;

4. A documented clinical diagnosis of COPD at least 12 months before the start of the study (a photocopy of an outpatient card dated and signed by the researcher must be attached to the patient's KFM);

5. Stable course of the disease (no exacerbations for at least 4 weeks before inclusion in the study).

Exclusion Criteria:

Lack of informed consent;

Smoking> 10 pack / year (for patients with AD);

Signs of a respiratory infection at the time the patient was included in the study;

Antibacterial therapy courses of any duration during the last 4 weeks prior to inclusion;

Women in pregnancy and lactation;

The presence of any cancer;

Patients with other diseases of the broncho-pulmonary system: α1-antitrypsin deficiency, bronchiectatic disease, pulmonary pulmonary emphysema, lung cysts, asbestosis, resection of a part of the lung or transplantation (according to history);

Patients with severe concomitant pathology in the stage of decompensation, which, according to the researcher, can affect the results of the study;

Helminthic invasion (according to the anamnesis);

Alcoholism and drug addiction;

Mental disorders, severe damage to the central nervous system in the stage of decompensation (according to the anamnesis);

Patients with hypersensitivity to therapy;

Potential danger from conducting functional tests and instrumental examination (according to the researcher’s doctor);

Refusal of the patient to participate in the study.

In the framework of this study, a comparative analysis of the clinical characteristics of patients depending on the severity of the disease was performed. All the studied indicators are given according to the results of a survey of patients and the study of available medical documentation, as well as according to the survey.

Patients with severe and very severe COPD according to the GOLD classification were characterized by greater severity of dyspnea and cough, a more significant history of previous exacerbations of COPD, hospitalizations associated with them, the frequency of antibiotic and systemic glucocorticosteroid courses.

The extraction of biomaterial from the oropharynx for microbiota identification and venous blood sampling for molecular genetic studies was performed in all patients included in the study [11].

Next, DNA was extracted from smears using FastDNA® SPIN Kit for Soil kits (MPBIO, USA), followed by sequencing, and a qualitative and quantitative analysis of the composition of the microbiome.

Genomic DNA was isolated from the venous blood of patients with COPD by phenol-chloroform extraction. Then, genotyping was performed on the selected genes of the immune response and regulation of inflammation IL18 rs5744247, IL18 rs1946518, CD14 rs2569190, IFNG rs2069705, MCP1 rs1024611.

Also, the following indicators were identified that were included in the construction of the discriminant model: demographic (gender, age, height, weight), anamnestic (smoking history, number of exacerbations of COPD and hospitalizations for the previous 12 months, frequency of antibiotic and systemic glucocorticosteroid therapy over the past 12 months ), clinical and functional (the severity of cough and sputum production, the severity of shortness of breath on the mMRC scale, exercise tolerance in the test with 6 minutes of walking (distance traveled), taking into account the acid saturation ode, counting heart rate and respiratory rate before and after the test), laboratory (microbiome colonization according to the results of the study of oropharyngeal smears, patient genotyping data for polymorphic variants of the immune response and inflammation regulating genes: IL18 rs5744247, IL18 rs1946518, CD14 rs2569190, TLR2 rs4696480, IL12RB1 rs11575934, IL12RB1 rs401502, IL17F rs1889570, IFNG rs2069705, IFNGR2 rs17880053, MCP1 rs1024611).

For the purposes of the discriminant analysis genotyping data were converted to a digital value in accordance with Table 1. The data on the determination microbiota were converted to a variable quality in the following way: in the presence of more than 50 colony-forming units of microorganisms proteobacteria groups per 1 cm ² oropharynx feature is set to 1, otherwise, 0.

The construction of discriminant function was carried out in two stages. First, prioritization of predictor variables was performed using univariate multinomial regression. Further analysis included variables for which the achieved significance level in the regression model did not exceed 5%. These variables were then used for stepwise discriminant analysis using the forward stepwise algorithm. The result of the analysis is discriminant functions that allow you to determine the constants of discrimination and to classify dependent variables (severity). Due to the lack of a test sample, the cross-validation procedure using the leave-one-out algorithm was used to assess the validity of discriminant models. The calculations were performed in the R language environment using the MASS and nnet packages.

The calculation results of the univariate multinomial are presented in table 2.

In accordance with the data of the regression analysis, the following variables were used to determine the severity of COPD in order to construct a discriminant function: microbial, hospitalizations, mMRC, IL18 rs5744247, IL18 rs1946518, CD14 rs2569190, IFNG rs2069705, MCP1 rs1024611, exacerbations, antibiotic pathway, oxygen post - before and after the test with walking, respiratory rate before and post - before and after the test with walking.

The step-by-step discriminant analysis established a significant contribution of the variables presented in Table 3. The coefficients for the discriminant models are presented in Table 4. The stratification of patients in accordance with the discriminant functions is shown in Figure 1. It is obvious that the most effective is the difference in the most severe course of COPD (COPD severe (Stage 3 according to GOLD) and very severe course (stage 4 according to GOLD)) from two other groups (COPD 1: COPD mild course (stage 1 according to GOLD); COPD 2 (moderate COPD (stage 2 according to GOLD), which can be separated using an additional function.The value of ¹ 'Wilks lambda statistics for the whole model was 0.068, which indicates a high classifying ability of the calculated discriminant functions.

The average values of the first discriminant function in the available sample are as follows: COPD1, -70.9993; COPD 2, -69.8672; COPD, -65.7848. In accordance with this, the value of the discrimination constant for the COPD1 and COPD2 groups is (-70.9993-69.8672) /2=-70.4332; and for the COPD2 and COPD groups - (- 69.8672-65.7848) /2=-67.826. Thus, the patient classification rules for the first discriminant function will be as follows:

- if the value of the function is <-70.4332, the patient is classified as COPD1;

- if the function value is> -70.4332 and <-67.826, the patient is assigned to the COPD class 2;

- if the value of the function is> -67.826, we assign the patient to the class of COPD. The share of classification achieved for the first function

is 79.83%, and for the second - 20.17. That is, the use of an additional classification function significantly increases the classification efficiency. In accordance with this and the data presented in FIG. 1, the second function can be used to further reclassify patients assigned to the COPD1 and COPD2 groups.

The average values of the second classification function in the available sample are as follows: COPD1, -30.159; COPD2, -32.8568. Accordingly, the discrimination constant is (-30.159-32.8568) /2=-31.5079.

The rules for patient discrimination for the second function will be as follows:

- if the value of the function is <-31.5079, the patient is assigned to the COPD2 group;

- if the value of the function is> -31.5079, refer the patient to the COPD1 group. The second function should only be applied to patients,

classified using the first function in the COPD1 and COPD2 groups.

The use of these functions in relation to the existing training sample (initial data set) made it possible to correctly classify COPD patients according to severity in 86.7% of cases. Three patients from the COPD group were erroneously assigned to the COPD group and one patient from the COPD group was mistakenly assigned to the COPD group. Using cross-validation analysis, the correctness of the prediction was 80%.

Thus, based on the results of the study, discriminant models were proposed for classifying COPD patients by severity using criteria not directly used for such an analysis. This suggests that the resulting models can be used to predict and not diagnose patients.

Examples of the method

Example 1

I.O.F. Valentin Nikolaevich A.

Date of birth: 12.31.1941.

Complaints: persistent cough during the day with profuse separation of whitish sputum, pronounced shortness of breath with minimal physical exertion, episodes of respiratory discomfort in the morning, high need for berodual N (up to 12 doses per day).

Medical history: The diagnosis of “chronic obstructive pulmonary disease, very severe course (according to the GOLD classification)” was made in October 2002 by a pulmonologist in Tomsk. Disease experience of 10 years. From March 2007 to the present, the patient receives Seretide Multidisk 50/500 μg for 1 inhalation 2 times a day (1000/100 μg / day for AF). From the moment of diagnosis, Berodual N is used as a symptomatic therapy in the “on demand” mode. Need up to 12 doses per day. The above complaints are recorded against the background of current therapy. Over the past year, the patient has registered 3 exacerbations of COPD of moderate and severe severity, 2 hospitalizations in a specialized medical hospital, the patient received 2 courses of antibacterial therapy over the next 12 months for exacerbation of COPD.

Anamnesis of life: The patient smokes for 40 years at 20-40 cigarettes per day, the smoking index is 40 pack / year. Since 2007, the patient has been diagnosed with Stage II hypertension, risk 2, HK0. From the moment of diagnosis to the present, the patient receives Enap 5 mg 2 times a day and Indapamide 2.5 mg in the morning.

Objective status: Height: 174 cm., Weight 72 kg., HELL: 125/85 mm. mercury, heart rate: 70 beats. min

The condition is satisfactory. On palpation of the chest, pain points were not found. During auscultation, breathing is tough, scattered dry rales are heard with calm breathing. For other organs and systems without features.

Spirometry: FEV 1 = 26.1%, FVC = 60.1%. Negative AOFV test for reversibility 1 = 4.5%. Conclusion: violations of bronchial patency III degree obstructive type.

Forecast: very severe COPD.

To clarify, an additional study was conducted according to the proposed method

According to the proposed method, the patient specified the number of antibiotic courses due to exacerbation of COPD in the previous 12 months - 2 courses, a standard test with 6-minute walk was performed with an estimate of the distance traveled by the patient in 6 minutes - 120 meters, heart rate before the test with walking (70 in 1 minute) and the level of oxygen saturation after performing the walking test (92%), the qualitative and quantitative composition of the microbiotic community of the posterior pharyngeal wall was determined by the method of oropharyngeal swabs more than 50 colony forming units of microorganisms from the group of proteobacteria (mainly Haemophilus) per 1 cm of the posterior pharyngeal wall were identified - a numerical value of 1 was assigned, genomic DNA was isolated from venous blood by phenol-chloroform extraction followed by genotyping of the selected immune response genes and regulation of inflammation, in particular, TLR4 rs2569190 - the GG genotype was identified, assigned the value 2, IL18 rs1946518 - the GT genotype was identified, the numerical value 1 was assigned, the discriminant function F1 was calculated:

F1 = -0.0349 * Path + 1.970188 * Micro-0.20922 * Heart rate + 0.531974 * TLR4-0.49016 * Oxygen saturation post + 0.257794 * IL18B-0.14965 * Antibiotics =; -0.0349 * 120 + 1.970188 * 1-0.20922 * 70 + 0.531974 * 2-0.49016 * 92 + 0.257794 * 1-0.14965 * 2 = -60.93;

The result obtained by the function F1 = -60.93, which is> -67.826, therefore, the patient was predicted to have a severe and very severe course of chronic obstructive pulmonary disease

According to the observations, the forecast turned out to be correct, and corresponds to 4 stages according to the spirometric classification GOLD, 2011.

Example 2

I.O.F. Ivan Anatolyevich X.

Date of birth: 02.23.1952.

Complaints: periodic cough in the morning with a slight separation of light sputum, moderate shortness of breath with accelerated walking, climbing uphill, episodes of respiratory discomfort in the morning.

Medical history: The diagnosis at the clinic "chronic obstructive pulmonary disease, mild severity" was made in November 2007 by a pulmonologist in Tomsk. Disease experience 5 years. From January 2010 to the present, the patient receives Salbutamol on-demand, over the past 6 months, the need for the drug has increased to 2-4 doses per day, shortness of breath has intensified, and exercise tolerance has decreased. The above complaints are recorded against the background of current therapy. Over the past year, the patient did not have exacerbations of COPD, did not seek medical help, the patient did not receive antibacterial therapy for exacerbations of COPD over the previous 12 months.

Given that according to the GOLD spirometric classification, a patient with stage 1 COPD (mild course) is registered in the patient, regular basic therapy was not prescribed to the patient.

Anamnesis of life: The patient smokes for 20 years at 20 cigarettes per day, the smoking index is 10 packs / year. Since 2008, the patient has been diagnosed with coronary heart disease, angina pectoris FCII, HK0. IHD does not receive regular therapy. Objective status: Height: 184 cm., Weight 76 kg., Blood pressure: 120/80 mm. mercury, heart rate: 68 beats. min

The condition is satisfactory. On palpation of the chest, pain points were not found. During auscultation, breathing is tough, wheezing is not heard. For other organs and systems without features.

Spirometry: FEV1 = 81.1%, FVC = 106.1%. The test for reversibility is negative ΔОФВ1 = 3%. Conclusion: violations of bronchial patency I degree obstructive type.

Forecast: mild COPD.

A study was performed on the patient to clarify the prognosis of the course of the disease according to the proposed method in order to justify the need for the appointment of basic therapy with prolonged drugs.

According to the proposed method, the patient specified the number of antibiotic courses due to exacerbation of COPD for the previous 12 months - 0, a standard test with 6-minute walk was performed with an estimate of the distance traveled by the patient in 6 minutes - 480 meters, heart rate before the test with walking (68 per 1 minute) and the level of oxygen saturation after performing the walking test (97%), the qualitative and quantitative composition of the microbiotic community of the posterior pharyngeal wall was determined by the method of oropharyngeal swabs - m less than 50 colony forming units of microorganisms from the group of proteobacteria per 1 cm ^{2 of the} posterior pharyngeal wall - assigned a numerical value 0, genomic DNA was isolated from venous blood by phenol-chloroform extraction followed by genotyping by CD14 rs2569190 polymorphisms - TT genotype identified, assigned value 0, IL18 rs19465 - the AS genotype was identified, the numerical value 1 was assigned, the discriminant function F1 was calculated:

F1 = -0.0349 * Path + 1.970188 * Micro-0.20922 * Heart rate + 0.531974 * CD14-0.49016 * Oxygen saturation post + 0.257794 * IL18B-0.14965 * Antibiotics =; -0.0349 * 480 + 1.970188 * 0-0.20922 * 68 + 0.531974 * 0-0.49016 * 97 + 0.257794 * 1-0.14965 * 0 = -78.26;

The result obtained according to the function F1 = -78.26, which is ≤-70.4332, thus, the patient had a mild course of chronic obstructive pulmonary disease.

In order to clarify the distribution, an additional calculation was performed for the second function Ф2, using the appropriate rules of discrimination:

Ф2 = 0.001983 * Path-0.574 * Micro-0.10173 * HRpr + 0.748163 * CD14-0.2893 * Oxygen saturation post + 1.16181 * IL18B + 1.039555 * Antibiotics = 0.001983 * 480-0.574 * 0-0.10173 * 68 + 0.748163 * 0-0.2893 * 97 + 1.16181 * 1 + 1.039555 * 0 = -34.76583;

The result on the function Ф2 = -34.76583, which is ≤-31.5079, indicated that the patient is predicted to have a course of chronic obstructive pulmonary disease of moderate severity.

After clarifying the severity of COPD by the proposed method, the patient was prescribed basic therapy with Spiriva, 18 mcg per day daily, for 3 months. During the second examination, the patient practically did not complain, the condition improved significantly: physical exercise tolerance increased, the severity of shortness of breath and cough decreased.

Thus, the proposed method, based on the use of a combination of clinical and functional factors, identification of the quantitative and species composition of the microbiome of oropharyngeal smears by molecular genetic methods (isolation of bacterial DNA with subsequent sequencing), as well as determination of genetic polymorphism by the genes of the immune response and regulation of inflammation (genes of the TLR family, genes of cytokines and their receptors) by the method of DNA extraction from whole blood for genotyping with the use of RFLP analysis, allows to predict the severity of the course of chronic obstructive pulmonary disease, to differentiate the severe and very severe course of the disease from COPD of mild and moderate course. The method is characterized by high sensitivity (correct

classification of patients with COPD by severity in 86.7% of cases), which makes the method promising for widespread use in clinical practice, since it allows the timely appointment of more appropriate therapeutic and preventive measures.

Sources of information used in compiling

descriptions:

1. Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the Diagnosis, Management and Prevention of Chronic Obstructive Pulmonary Disease. Updated 2010 ().

2. Patent RU No. 2262889 "Method for predicting a stable course of chronic obstructive pulmonary disease" authors Kolosova VP, Kolosova AV

3. Patent RU No. 2348352 "Method for the diagnosis of the severity of bronchial asthma" authors Meshcheryakova VV, Titova EL

4. Patent RU No. 2245550 "A method for predicting bronchial obstruction in chronic bronchitis" by the authors Postnikova LB, Kubysheva NI

5. Patent RU No. 2370773 "A method for predicting the progression of chronic obstructive pulmonary disease of a stable course of chronic obstructive pulmonary disease" authors Kalinina EP, Lobanova EG, Ivanova EM

6. Sehti S., Murphy T.F. Infection in the pathogenesis and course of chronic obstructive Pulmonary Disease. N. Engl. J. Med. 2008; 359: 2355-2365.

7. Bilde L., Rud Svenning A., Dollerup J., Baekke Borgeskov H., Lange P. The cost of treating patients with COPD in Denmark - A population study of COPD patients compared with non-COPD controls. Respir. Med. 2007; 101: 539-546.

8. Monso E., Rosell A., Bonet G. et al. Risk factors for lower airway colonization in chronic bronchitis. Eur. Respir. J. 1999; 13: 338-342.

9. Porcher C, Malinge M.C., Picat C. et al / A simplified method for determination of specific DNA or RNA copy number using quantitative PCR and an automatic DNA sequencer // Biotechniques. - 1992. - No. 1. - P.106-114.

10. Standardization of Spirometry, 1994 update. American Thoracic Society. Am. J. Respir. Crit. Care Med. - 1995. - Vol. 152, No. 3. - P.1107-1136.

11. Barnes, P.J. Chronic Obstructive Pulmonary Disease: molecular and cellularmechanisms / P.J. Barnes, S.D. Shapiro, R.A. Pauwels // Eur. Respir. J. - 2003. - No. 22. - P.627-688.

application

Table 1. Transcoding of genotypic data into digital values

Table 2. Results of multinomial regression calculations for the severity of COPD and predictor variables

Note: LR is likelihood ratio statistics; R ² is the coefficient of determination; AIC - Akaike information criterion; P-value - achieved significance level of the regression model

Table 3. Statistics for discriminant analysis for variables remaining in the model after a step-by-step algorithm.

Table 4. Odds of discriminant models for the severity of COPD

Fig 1. Distributions of patients with COPD in the axes of canonical variables calculated on the basis of discriminant analysis data. Four patients with incorrect classification were selected (see text).

Table 1. Genes Genotypes IL18 rs5744247 CC-0 Cg-1 Gg-2 IL18 rs1946518 AA-0 AC-1 CC-2 CD14 rs2569190 AA-0 AG-1 Gg-2 TLR2 rs4696480 TT-0 AT-1 AA-2 IL12RB1 rs11575934 Gg-0 Cg-1 CC-2 IL12RB1 rs401502 Gg-0 AG-1 AA-2 IL17F rs1889570 Gg-0 AG-1 AA-2 IFNG rs2069705 CC-0 TC-1 TT-2 IFNGR2 rs17880053 DD-0 DG-1 Gg-2 MCP1 rs1024611 TT-0 TC-1 CC-2 Table 2. Variables Lr R ² Aic P-value Microbiome 18.2 0.434 56.7 0.00011 Floor 2.15 0.065 72.8 0.341 Hospitalizations 18.7 0.442 56.3 8.85E-05 Cough 5.46 0.157 73.5 0.243 Sputum 8.59 0.235 70.3 0.0723 mMRC, points 25.1 0.544 57.8 0.000323 IL18 rs5744247 13 0.353 65.9 0.0111 IL18 rs1946518 17.6 0.443 61.4 0.0015

CD14 rs2569190 17.6 0.443 61.4 0.0015 TLR2 rs4696480 4.8 0.148 74.1 0.309 IL12RB1 rs11575934 5.84 0.177 69.1 0.0539 IL12RB1 rs401502 7.97 0.233 71 0.0927 IL17F rs1889570 6.7 0.2 72.2 0.153 IFNG rs2069705 9.92 0.282 69 0.0418 IFNGR2 rs17880053 6.57 0.197 72.4 0.16 MCP1 rs1024611 12.6 0.342 66.4 0.0137 Age 1.62 0.0492 73.3 0.446 Exacerbations 15.2 0.379 59.7 0.00049 Antibiotics 12.7 0.328 62.2 0.00174 Smoking 1.27 0.0388 73.7 0.531 The weight 3.64 0.108 71.3 0.162 Height 0.145 0.00454 74.8 0.93 6-minute walk test 30.7 0.617 44.2 2.13E-07 Oxygen Saturation - Pre 13.6 0.346 61.4 0.00113 Respiratory rate - pre 11.8 0.307 63.2 0.00281 Heart Rate - Pre 7.18 0.201 67.7 0.0276 Oxygen Saturation - Fast 16.7 0.407 58.2 0.000235 Respiratory Rate - Fast 8.56 0.235 66.4 0.0138 Heart rate 3.79 0.112 71.1 0.15

abbreviations - post 0.15 Table 3. Variables Wilks'lambda Partial lambda F-remove (2.21) p-level Way 0.156543 0.435806 13.59332 0.000163 Micro 0.110151 0.619350 6.45325 0.006536 Heart Rate - Pre 0.126356 0.539922 8.94725 0.001547 CD14 rs2569190 0.088066 0.774675 3.05408 0.068510 Oxygen Saturation - Fast 0.079541 0.857698 1.74207 0.199531 IL18 rs1946518 0.084464 0.807708 2.49976 0.106211 Antibiotics 0.080745 0.844914 1.92731 0.170424

Table 4. Variables Function 1 Function 2 Way -0.0349 0.001983 Micro 1.970188 -0.574 Heart Rate - Pre -0.20922 -0.10173 CD14 rs2569190 0.531974 0.748163 Oxygen Saturation - Fast -0.49016 -0.2893 IL18 rs 1946518 0.257794 1.16181 Antibiotics -0.14965 1.039555

Claims (1)

A method for predicting the severity of chronic obstructive pulmonary disease (COPD), including a study of the patient's venous blood, characterized in that it additionally takes into account the number of antibiotic courses for exacerbation of COPD in the previous 12 months, conduct a standard test with 6-minute walk with an estimate of the distance traveled the patient for 6 minutes, the heart rate before the test with walking and the level of oxygen saturation after performing the test with walking, carry out the sampling of biomaterial from the back the pharyngeal wall by the method of oropharyngeal smears followed by DNA extraction, sequencing and, when proteobacteria are detected, the data are transcoded into a qualitative variable to determine the qualitative and quantitative composition of the microbiotic community of the posterior pharyngeal wall according to the principle of assigning a digital value of 1 in the presence of more than 50 colony forming units of microorganisms from the group of proteobacteria on 1 cm ² of the posterior pharyngeal wall, otherwise assigning a digital value 0, vyd extract genomic DNA from venous blood of patients with COPD using phenol-chloroform extraction followed by genotyping according to the immune response genes and regulating inflammation CD14 rs2569190 and IL18 rs1946518, pre-transcoding the genotyping data into digital values by the CD14 rs2569190 gene, assigning 0 to the TT genotype 0, TS genotype and 2 - SS genotype, and according to IL18 rs1946518 gene, assigning 0 AA genotype, 1 - AS genotype and 2 - SS genotype, and determine the probability of an individual being assigned to a group of patients with severe and very severe COPD or to groups who are sick x COPD of mild to moderate severity, calculating the discriminant function of F1 and F2:
F1 = -0.0349 * Path + 1.970188 * Micro-0.20922 * Heart rate + 0.531974 * CD14-0.49016 * Oxygen saturation post + 0.257794 * IL18-0.14965 * Antibiotics;
Ф2 = 0.001983 * Path-0.574 * Micro-0.10173 * HRspr + 0.748163 * CD14-0.2893 * Oxygen saturation post + 1.16181 * IL18 + 1.039555 * Antibiotics,
Where
-0.0349; 1.970188; 0.20922; 0.531974; 0.49016; 0.257794; 0.14965; 0.001983; 0.574; 0.10173; 0.748163; 0.2893; 1.16181; 1.039555 - numerical values are coefficients;
Path - the distance traveled by the patient (m) in 6 minutes in the standard test with a 6-minute walk;
Micro - numerical value of colony forming units of microorganisms from the group of proteobacteria per 1 cm ^{2 of the} posterior pharyngeal wall;
ChSSpre - heart rate before the test with a 6-minute walk;
CD14 and IL18 are genotypes;
Post oxygen saturation - oxygen saturation after the test with a 6-minute walk;
Antibiotics - the number of antibiotic courses for exacerbation of COPD in the previous 12 months;
moreover, to assign the patient to a specific prognostic group, the F1 value is evaluated and, with a function value> -67.826, the patient is predicted to have COPD of a severe and very severe course; and with a value of <-67.826, in order to clarify the distribution, the function Ф2 is used, and with a value of function Ф2≤-31.5079, moderate COPD is predicted, and with a value of Ф2> -31.5079, moderate COPD is predicted.

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