RU2743453C1 - Method of predicting infectious complications of critical condition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine. The method for predicting infectious complications of critical condition is proposed. The method presupposes that one should determine the concentration of extracellular DNA in the plasma of peripheral blood during the first 24 hours after a patient is admitted to the intensive care unit. After that one should determine the intensity of exDNA oxidation in terms of the amount of oxidized deoxyguanosine (8-oxodG) in exDNA per 1 mln of nucleotides and calculate the protection index. Based on the values ​​of the protection index, infectious complications of critical condition are predicted.

EFFECT: invention provides increase in the predictive value of the concentration of extracellular DNA (exDNA) as a predictor of critical condition.

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Description

Technology area

The invention relates to medicine, in particular to anesthesiology, resuscitation and is intended to predict the development of an infectious process (pneumonia or sepsis) and the likelihood of death within 28 days in resuscitation patients. The result of using the invention is an increase in the predictive value of the concentration of extracellular DNA (cfDNA) as a predictor of a critical state (CS) by calculating the protection index (IZ) - the ratio of the number of oxidation-modified guanosine residues - 8-oxodG per million nucleotides in cfDNA to cfDNA concentration in blood plasma , determined once upon admission to the intensive care unit (ICU).

Infectious complications of CS, in particular sepsis, including septic shock (SEPSIS-3, 2016) remain the leading cause of death in the ICU [1]. Preventing the lethal outcome of sepsis requires the development of new strategies for personalizing treatment, requiring reliable and highly informative predictive biomarkers. The early use of such biomarkers in CS medicine allows for timely stratification of patients by risk groups for an unfavorable course and death. The latter contributes to the adoption of the optimal decision on the patient's treatment, including the expediency of using high-tech methods and the individualization of drug doses [2]. Promising markers for the prognosis of the course and predicting the outcome of diseases and pathological conditions, including CS, include the amount of cfDNA in blood plasma [3-7]. To date, the main criterion under consideration for the amount of cfDNA for predicting the outcomes of pathological conditions is its concentration in blood plasma [8-14]. However, heterogeneity of nosological forms and concomitant comorbidity may be a source of variability in the cfDNA content, which limits its informative value as a predictor of CS outcome, and, in particular, sepsis [14].

State of the art

Currently, a number of methods are known for predicting the development of nosocomial infection and the outcomes of infectious complications in intensive care patients.

1. Methods for determining the patient's susceptibility to nosocomial infection and making a prognosis for the development of septic syndrome. Authors: Casali Marie-Angelique, Lepal Alain, Monneret Guillaume, Mougins Bruno, Pashot Alexander (RU 2663724). The invention relates to biotechnology, specifically to determining the prognosis of the development of sepsis in patients with an infectious process, and can be used in medicine. The known method allows for overexpression of the target gene S100A9 in relation to a certain threshold value to determine the prognosis of the development of sepsis in a patient. To determine gene expression, the authors of the known method propose to study whole blood in resuscitation patients with already developed septic shock, or in patients who have undergone this condition. Thus, this method makes it possible to predict the outcomes of infection and sepsis in resuscitation patients.

However, the use of this method does not allow predicting the development of the infectious process, in contrast to the proposed method, which allows already on the first day of admission of a patient to the ICU to predict the development of infection, in particular pneumonia or sepsis, and the outcomes of these conditions, in particular the probability of death within 28 days ...

2. Early diagnosis and prediction of systemic inflammatory response syndrome and sepsis in asymptomatic patients based on the determination of IL-6. Authors: Grueb Susanne; Smolle-Juttner Freyja-Maria; Neuboeck Nicole; Weinberg Annelie-Martina (WO 2011116872). The known method relates to the field of medicine and is intended for the early diagnosis and prediction of the systemic inflammatory response syndrome (SIRS) and sepsis in asymptomatic surgical patients. The invention is based on determining the concentration of interleukin 6 (IL-6) in the blood of patients over time: before and after surgery. This indicator makes it possible to identify these conditions a few days before the development of clinical manifestations of the systemic inflammatory reaction and sepsis. However, this method makes it possible to predict the development of infection, systemic inflammatory response and sepsis only in surgical patients by repeated determination of IL-6.

At the same time, the claimed method makes it possible to predict the development of infection, the development of pneumonia or sepsis, and the outcomes of these conditions in intensive care patients who have suffered a stroke on the first day of admission to the ICU by a single determination of the IZ ratio of the intensity of cfDNA oxidation and the concentration of total cfDNA.

(WO 2019110706). Изобретение относится к области медицины и может быть использовано для прогнозирования риска развития сепсиса или SIRS. Известный способ основан на использовании высокопроизводительного транскрипционного и биоинформатического анализа. В исследование вошел 21 пациент с диагнозом множественная миелома или лимфома, поступившие в отделение для трансплантации костного мозга. Образцы крови забирались до химиотерапии, по ее окончанию и на стадии нейтропении. Авторы известного способа показали, что экспрессия одиннадцати генов в периферических мононуклеарах была различна (CHAT, CNN3, ANKRD42, LOC100505725, EDAR, GPAT2, ENST00000390425, MTRM8, C6orfl92, LOC 10289230, XLOC-005643). Ha основании чего предсказали развитие SIRS или сепсиса за 48-162 часа до возникновения таких состояний. Несмотря на полученные результаты, данный способ применим только для пациентов, подготовленных к трансплантации костного мозга. Интерпретация и использование обнаруженных генов на всех пациентов реаниматологического профиля сомнительна в виду специфического воздействия на иммунные клетки (химиотерапия с комбинацией лекарственных средств), приводящая к подавлению их образования и гибели костного мозга.3. Methods for predicting the risk of developing sepsis or SIRS. Authors: Nguyen Catherine, Labiad Yasmine, Costello

(WO 2019110706). The invention relates to medicine and can be used to predict the risk of developing sepsis or SIRS. The known method is based on the use of high-performance transcriptional and bioinformatic analysis. The study included 21 patients with a diagnosis of multiple myeloma or lymphoma who were admitted to the department for bone marrow transplantation. Blood samples were taken before chemotherapy, after chemotherapy, and at the stage of neutropenia. The authors of the known method showed that the expression of eleven genes in peripheral mononuclear cells was different (CHAT, CNN3, ANKRD42, LOC100505725, EDAR, GPAT2, ENST00000390425, MTRM8, C6orfl92, LOC 10289230, XLOC-005643). Based on this, the development of SIRS or sepsis was predicted 48-162 hours before the onset of such conditions. Despite the results obtained, this method is applicable only to patients prepared for bone marrow transplantation. The interpretation and use of the detected genes in all resuscitation patients is questionable due to the specific effect on immune cells (chemotherapy with a combination of drugs), leading to the suppression of their formation and bone marrow death.

The inventive method makes it possible to predict the development of infection, the development of pneumonia or sepsis and the outcomes of these conditions in intensive care patients who have suffered a stroke on the first day of admission to the ICU by a single determination of the IZ ratio of the intensity of cfDNA oxidation to the concentration of total cfDNA.

4. Risk assessment tool for patients with sepsis. Authors: Liaw Patricia, Fox-Robichaud Alison, Selvaganapathy Ponnambalam Ravi, Liaw Kao-lee, Dwivedi Dhruva, Mcdonald Ellen (WO 2019006561). The invention relates to medicine and can be used to predict 28-day mortality in patients with sepsis. To predict 28-day mortality, the authors of the known method use the following combination of indicators: the concentration of cfDNA, C-reactive protein, lactate, creatinine, platelet count, and also take into account the Glasgow Coma Scale in resuscitation patients on the first day with manifestation of sepsis. The known method for predicting 28-day mortality in patients with sepsis does not allow assessing the risk of developing severe pneumonia or sepsis, while the prognosis in septic patients is made on the basis of dynamic changes in the studied parameters. Adopted as a prototype.

The inventive method allows for a single determination of IZ on the first day of admission to the ICU to predict not only 28-day mortality in intensive care patients who have suffered a stroke, but also the development of infection, including the development of pneumonia or sepsis.

In the studied scientific, medical and available patent literature, no method was found for predicting the development of the infectious process and 28-day mortality in intensive care patients with stroke on the basis of a single determination of the ratio of the amount of 8-oxodG per million nucleotides in cfDNA to cfDNA concentration in blood plasma.

The claimed invention is aimed at solving the problem of predicting the onset and outcome of infectious complications, in particular pneumonia or sepsis, as well as the likelihood of death within 28 days in resuscitation patients.

The use of the proposed method in clinical practice makes it possible to personalize the approach to the treatment of patients with life-threatening conditions by increasing the predictive value of determining the concentration of cfDNA as a predictor of CS by determining the IZ ratio of the amount of 8-oxodG per million nucleotides in cfDNA to the concentration of cfDNA in blood plasma, with a single dose. determination of IZ to the patient during the first day of admission to the ICU.

The specified technical (diagnostic) results in the implementation of the invention are achieved due to the fact that, as in the known method, the concentration of cfDNA in the plasma of the peripheral blood (C) of patients of the intensive care profile is determined.

The peculiarity of the proposed method is that the intensity (I) of cfDNA oxidation is additionally determined in the form of the amount of oxidized deoxyguanosines 8-oxodG in the cfDNA composition per 1 million nucleotides, and the protection index (IZ) is calculated according to the formula IZ = I / C × 100, where IZ - protection index, I - intensity of cfDNA oxidation - amount of 8-oxodG per 10 ⁶ nucleotides, C - cfDNA concentration in plasma, ng / ml.

These tests are performed once during the first day after the patient is admitted to the ICU.

Disclosure of the essence of the invention

To improve the outcomes of life-threatening conditions - sepsis, severe injuries, vascular catastrophes, a search is underway for biomarkers that would allow the patient to choose optimal therapy methods at an early stage in the ICU, personalizing treatment if possible. One of the promising biomarkers in this regard is the cfDNA content in blood plasma.

The level of content and changes in the dynamics of the concentration of circulating DNA fragments, including the organ-specific fraction of cfDNA, today can be important for assessing the degree of damage to an organ of interest, the likelihood of a complicated course, and the prognosis of outcomes of an emergency / critical condition in ICU patients. The sources of cfDNA circulating in the bloodstream can be the nuclei of dying cells of organs and tissues, damaged mitochondria, the pool of which is renewed as a result of mitophagy, and microorganisms.

BcDNA is a biomarker of cellular damage in various conditions. An increase in the concentration of total cfDNA is observed in CS, such as severe trauma, sepsis, vascular accidents, as well as in physiological and borderline conditions, including physical exertion and acute and chronic psycho-emotional stress [5-15].

DNA is susceptible to oxidative damage leading to the formation of various oxidized nucleosides. One of the most widespread and studied structural damages of cfDNA is the formation of 8-oxo-2'-deoxyguanosine (8-oxodG), the quantitative content of which in the blood circulation is detected in the form of individual molecules or as part of cfDNA using immunological methods and anti-8-oxodG -antibodies, or when using high pressure liquid chromatography associated with mass spectrometry [16]. However, despite the fact that CS are accompanied by oxidative stress and the intensity of cfDNA oxidation may increase, there are currently no data on the relationship of oxidized cfDNA with CS outcomes, the development of infectious complications, including a life-threatening condition - sepsis. The authors of the invention found that fragments of cfDNA modified by oxidation intensively penetrate cells, causing a more pronounced stimulation of the expression of genes of the antioxidant system and autophagy (Hmox1 and Becn1) in comparison with the non-oxidized form of cfDNA. The authors of the proposed method suggested that the ratio of the content of oxidized DNA fragments with a pronounced cytoprotective effect to the total content of cfDNA, which has a damaging effect on cells, can be informative for predicting the clinical course of CS: the higher the ratio (IZ), the more favorable the outcome of CS and the less life-threatening complications characteristic of these patients (infections, including severe pneumonia or sepsis). This position was verified in the ICU neuroresuscitation profile using biomaterial (blood plasma) of patients with ischemic stroke.

The essence of the invention lies in the fact that on the basis of a single joint determination of the concentration of cfDNA, the intensity of its oxidation in the peripheral blood plasma of resuscitation patients during the first days after admission to the ICU and calculation of the quantitative ratio of the content of 8-oxodG molecules in the circulating cfDNA to the total content of cfDNA in the plasma of patients with CS, referred to as IZ, it becomes possible on the first day of hospitalization to identify patients with a favorable and unfavorable course of CS - ischemic stroke. The authors of the proposed method found a high information content of the calculated IZ for predicting the development of an infectious process, including pneumonia or sepsis, as well as for predicting the outcome of treatment of hospitalized patients - the probability of 28-day mortality.

The authors of the proposed method empirically found that with a value of IZ less than 450, the likelihood of an infectious process increases, and with a lower value of IZ - less than 223 - a lethal outcome. The invention provides an effective prediction of the development of the infectious process and 28-day mortality in stroke patients by jointly determining the concentration of cfDNA and the intensity of its oxidation in peripheral blood plasma.

The statistically processed research results presented below confirm the predictive value of these criteria.

Predicting the development of pneumonia

Determination of IZ reliably predicted the development of pneumonia in intensive care patients after stroke.

The chances of developing pneumonia in ICU patients with IZ at admission less than 450 are 11 times higher (p = 0.0007, odds ratio (OR) = 11.053 (95% CI: 2.261-54.018) sensitivity 59.6%, specificity 88.2% , with a cutoff value of IZ = 450).

Predicting the development of an infectious process (pneumonia or urological infection)

Determination of IZ reliably predicted the development of infection in intensive care patients.

The chances of developing an infectious process in ICU patients with IZ on admission less than 450 are 11.4 times higher (p = 0.0002, odds ratio (OR) = 11.4 (95% CI: 2.891-44.946) sensitivity 64.3%, specificity 86.4%, with a cutoff of 450).

Predicting the development of sepsis

Determination of IZ reliably predicted the development of sepsis in patients with intensive care.

The chances of developing an infectious process in ICU patients with IZ at admission less than 450 are 7.1 times higher (p <0.005, odds ratio (OR) = 7.105 (95% CI: 1.8-28.013) sensitivity 58.7%, specificity 83 , 3%, at 450 cutoff)

Prediction of 28-day mortality in patients with an infectious process after a stroke.

The determination of IZ reliably predicted 28 day mortality in intensive care patients with the course of the infectious process after stroke.

The risk of death in intensive care patients who have suffered a stroke with the development of an infectious process is significantly higher with an IR value of more than 223: odds ratio (OR) = 9.1 (95% CI: 2.72-30.269), area under the AUC curve = 0.801, sensitivity 77.8%, specificity 80.4%, at a cutoff of 223; P = 0.0001, log-rank method.

Implementation of the invention

A method for predicting the development of infectious complications, including the course of the infectious process, the development of pneumonia, the development of sepsis, and predicting outcomes in patients with sepsis includes:

- venous blood sampling upon admission to the ICU;

- determination of cfDNA concentration in peripheral blood plasma;

- determination of the intensity of cfDNA oxidation (the amount of oxidized deoxyguanosine per 1 million bases (8-oxodG / 10 ⁶ nucleotides) circulating in the peripheral blood plasma;

- determination of the likelihood of the risk of developing infectious complications, in particular sepsis or pneumonia, for CS according to the formula: IZ = intensity of oxidation / total concentration of cfDNA × 100, where IZ is the protection index.

Isolation of cfDNA from plasma and determination of its concentration is carried out according to the methodological techniques described earlier [16].

Venous blood is taken within 24 hours after the patient is admitted to the ICU (according to the SEPSIS-3 criteria) into VACUETTE® TUBE K3EDTA tubes, Greiner bioone, Austria). To obtain plasma from whole blood, the samples are centrifuged at 22 ° C, 400 g, for 10 minutes. The supernatant is removed and transferred to a clean tube. In order to determine the quantitative content in plasma, DNA is isolated by extraction with organic solvents. The DNA pellet is dissolved in 40 μl of deionized water. The cfDNA concentration (ng / ml) in plasma is measured by fluorescence with an intercalating dye PicoGreen (Invitrogen, USA) using an EnSpire plate spectrofluorometer (Perkin Elmer, Finland) at an excitation wavelength of 480 and an emission wavelength of 520 nm.

Method for determining the intensity of cfDNA oxidation (Dot immunoblotting for 8-hydroxy-deoxyguanosine).

Determination of the intensity of cfDNA oxidation is carried out according to the methodological techniques described earlier [16]. Quantitative determination of 8-hydroxy-deoxyguanosine is carried out by dot-immunoblotting using an OPTITRAN BA-S 85 nitrocellulose filter (0.45 μm) (Sigma-Aldrich, USA, catalog Z750697) and a solution of antibodies to 8-hydroxy-deoxyguanosine (SantaCruz, USA, catalog No. 66036) at a dilution of 1:50.

Clinical examples

Example # 1

Patient K., 66 years old, was in the ICU with the main diagnosis: ischemic stroke in the basin of the right middle cerebral artery. Concomitant diseases: type 2 diabetes mellitus, atherosclerotic cardiosclerosis. Upon admission, the following were determined: I = 1289, C = 227; FROM = 1289/227 × 100 = 569. During the observation period, the infectious process was not detected. On the third day, the patient was transferred to a specialized department for further observation and rehabilitation. The lethal outcome within 28 days from the moment of hospitalization was not registered.

Example No. 2

Patient D., 67 years old, was in the ICU with the main diagnosis: ischemic stroke in the basin of the right middle cerebral artery. Concomitant diseases: atherosclerotic cardiosclerosis, a permanent form of atrial fibrillation. Upon admission determined: I = 1069, C = 308; FROM = 1069/308 × 100 = 347. During the observation period, the patient was diagnosed with sepsis against the background of the development of a urinary tract infection. The lethal outcome within 28 days from the moment of hospitalization was not registered.

Example No. 3.

Patient A., 59 years old, was in the ICU with the main diagnosis: ischemic stroke in the basin of the left middle cerebral artery. Concomitant diseases: atherosclerotic cardiosclerosis, chronic kidney disease. Upon admission, the following were determined: I = 509, C = 213; FROM = 509/213 × 100 = 239. During the observation period, the patient was diagnosed with sepsis against the background of pneumonia and urinary tract infection. After 7 days, he was transferred to a specialized department for further observation and rehabilitation. The lethal outcome within 28 days from the moment of hospitalization was not registered.

Example No. 4.

Patient S. 87 years old was in the ICU with the main diagnosis: ischemic stroke in the basin of the left middle cerebral artery. Concomitant diseases: atherosclerotic cardiosclerosis, a permanent form of atrial fibrillation. Upon admission, the following were determined: I = 1175, C = 835; FROM = 1175/835 × 100 = 141. During the observation period, the patient was diagnosed with sepsis against the background of pneumonia. On the 21st day of being in the ICU, death occurred.

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Claims (1)

A method for predicting infectious complications of a critical state, including determining the concentration (C) of extracellular DNA (cfDNA) in the peripheral blood plasma of resuscitation patients, characterized in that the intensity (I) of cfDNA oxidation is additionally determined in the form of the amount of oxidized deoxyguanosines 8-oxodG in cfDNA on 1 million nucleotides, the protection index (IZ) is calculated by the formula IZ = I / C × 100, where IZ is the protection index, I is the intensity of cfDNA oxidation per 10 ⁶ nucleotides, C is the concentration of cfDNA in plasma (ng / ml); if the IC value is less than 450, a high risk of an infectious process in the form of pneumonia or sepsis is predicted; if the IC value is less than 223, a high risk of death is predicted within 28 days after hospitalization; the determination of the concentration of cfDNA and the intensity of oxidation of cfDNA in the peripheral blood plasma is carried out once during the first days after the patient is admitted to the intensive care unit.