RU2424768C2 - Method of predicting disease course in children in case of acute respiratory viral infections - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to immunology and clinic laboratory diagnostics, and can be used to predict course of acute respiratory viral infections (ARVI) in children in the first days of disease and timely administration of immunomodulating medications. For this purpose by means of ELISA immunologic indices of spontaneous and induced interferon-γ in vitro (IFH-γ) are determined. Index of interferon-γ stimulation (IS IFH-γ) is calculated by division of index of induced level by index of spontaneous level of interferon-γ. Also carried out is calculation of lymphocyte activation index (LAI IFH-γ) per 1000 lymphocytes by division of index of induced interferon-γ by absolute number of patient's lymphocytes. Additionally in blood plasma determined is content of interleukin-10 (IL-10). If values of IS IFH-γ are higher than 3, LAI IFH-γ equals or is higher than 40, IL-10 is from 30 to 60 pg/ml favourable outcome of disease is predicted with therapeutic treatment which does not include immunomodulators. If IS IFH-γ is lower than 3, LAI IFH-γ is lower than 40, IL-10 is from 60 to 100 pg/ml, predicted are severe course of disease and development of complications, which requires urgent treatment by immunomodelling therapy. If IS IFH-γ is lower than 3, LAI IFH-γ is lower than 30, IL-10 is higher than 100 pg/ml, predicted are severe course of disease with development of bronchopulmonary complications and possible chronisation of pathologic process and recurrent ARVD, which requires additional introduction of immunomodelling medications.

EFFECT: increase of accuracy of early prediction of disease course severity and development of complications in children with ARVI, including those from 1 month old, which makes it possible to carry out necessary anti-viral and immunomodelling therapy, aimed at strengthening immunity cell responses, in due time.

3 tbl, 3 ex

Description

The invention relates to medicine, namely to immunology and clinical laboratory diagnostics. It can be used to predict the course of a disease, such as acute respiratory viral infection (ARVI), in children aged 1 month to 18 years. The invention allows to predict the development of complications in children and at the early stage of the disease to prescribe therapeutic drugs that prevent the adverse course of the disease, in particular, to choose the optimal immunomodulating therapy.

The invention is based on a method for assessing the overall immunoreactivity of a child’s body according to individual indicators of interferon (IFN) status and the content of interleukin-10 (IL-10) in the patient’s blood plasma in the first days (1st, 2nd day) of acute respiratory viral infections.

Factors that determine the severity of the course, the peculiarity of clinical manifestations and the outcome of acute respiratory viral infections, in addition to the properties of the pathogen, are characteristics of the ability of the immune system to adequately respond to an infectious antigen. An acute inflammatory process, as a rule, is accompanied by disorders in the immune system, which in most cases are reversible. However, the development of complications may be associated with the initial defect in the immune system or with insufficient functions of macrophage and immunocompetent cells.

In the diagnosis and prognosis of the course of diseases, changes in the production of cytokines are used quite widely.

Known work:

Pitkäranta A., Nokso-Koivisto J., Jäntti V., Takala A., Kilpi T., Hovi T. Lowered yields of virus-induced interferon production in leukocyte cultures and risk of recurrent respiratory infections in children. Journal of Clinical Virology. - Volume 14, Issue 3, December 1999, P. - 199-205.

It describes how a decrease in the level of production of interferons by leukocytes in vitro increases the risk of repeated respiratory infections in children. The disadvantage of this forecasting method is the complexity of the method using cell cultures and test virus.

Known patent of the Russian Federation 2159936, G01N 33/68, Arinenko R.Yu., Anikin V.B., Iovlev V.I., Malinovskaya V.V. A method for choosing the type of immunomodulating therapy and determining the prognosis of the disease outcome based on the analysis of the patient's interferon status.

This invention relates to a method for selecting immunomodulating therapy and determining a prognosis of a disease based on the interferon status of the patient. The method includes determining the activity of total serum interferons (S-IFN), leukocyte production in vitro of interferon-α and interferon-γ.

The determination is carried out in whole blood samples incubated in RPMI-1640 medium, 100 μl, with the addition of Newcastle disease virus (NDV) and phytohemagglutinin (PHA) for the induction of IFN-α and IFN-γ, respectively. It is carried out by titration of the test samples in cell culture, followed by incubation with the virus of vesicular stomatitis. In this case, transplantable cultures sensitive to the action of IFN are used. This method allows you to predict the course of the disease, however, it is time-consuming, time-consuming and requires special equipment and skills of the contractor, as it involves working with tissue culture and viruses.

Known RF patent No. 2169923, G01N 33/53, Senko O.V. A method for predicting the recurrence rate of infectious diseases of the respiratory tract and the persistence of the healing effect in preschool children.

The authors propose a method for predicting the possible recurrence rate of infectious respiratory diseases using a number of biochemical parameters. The method includes assessing the quality and quantity of correlation between the data of laboratory parameters.

Known RF patent No. 2008686, G01N 33/53, Sizyakina L.P., Chernyshov V.N., Andreeva I.I., Bulgarianov N.K. A method for predicting the development of complications in children with acute respiratory viral infections.

This method is based on the determination in the early period of the disease of the number of peripheral blood neutrophils expressing receptors for the Fcj fragment of immunoglobulin G (Fc jP) in the reaction of rosette formation with sheep red blood cells. If the percentage of neutrophils with FcjP increases above 58%, complications are predicted.

Known patent of the Russian Federation No. 2297003 G01N 33/68, Useynova N.N., Shovkun V.A., Misernitsky Yu.L. A method for predicting the incidence of recurrent diseases in young children suffering from acute respiratory diseases.

The method was carried out as follows: in a patient in the morning on an empty stomach, venous blood was collected in an amount of 5 ml, mononuclear cells were isolated and cultured for 24-48 hours at 37 ° C in the presence of 5% CO2 in RPMI1640 culture medium with 10% inactivated calf serum, 5 × 10.5 M2-mercaptoethanol, 2 mmol of glutamine, 10 mmol of HEPES buffer, 50 μg / ml of gentamicin with the addition of mitogens (lipopolysaccharide-c for inducing tumor necrosis factor-alpha and phorbolmyristiate acetate for inducing interferon-gamma). In parallel, spontaneous production was determined by adding an equal volume of culture medium. The level of cytokines in supernatants was determined by the enzyme immunoassay using Protein Contour assay kits. The ratio of induced and spontaneous production of these cytokines was calculated, and when the ratio index for tumor necrosis factor alpha was lower than 0.85, and for interferon gamma below 0.45, the possibility of repeated respiratory diseases was determined. Here we are only talking about a predisposition to repeated acute respiratory infections.

The prototype of the claimed invention is the method described in the dissertation Obraztsova E.V. “Interferon preparations and its inducers in the treatment of influenza and acute respiratory viral infections in children” (Author. Diss. Cand. Sc. Medical Sciences. - St. Petersburg, 2007. - 23 p.).

The method includes the determination of spontaneous and induced production of leukocytes in vitro IFN-α and IFN-γ according to the method of S. S. Grigoryan. Based on the results obtained, the ratio of the induced and spontaneous production of these cytokines is calculated - the stimulation index (IS) of the IFN-α or -γ, the lymphocyte activation index (IAL) - IFN-α or -γ.

These indicators give an idea of the activity of immunocompetent cells (ICC), which characterizes the reserve capabilities of interferon-producing cells. This allowed the author to develop criteria for the appointment of drugs of interferon and its inducers in children with various acute respiratory viral infections.

The interferon system (IFN) provides non-specific antiviral resistance of the body and affects the whole complex of its specific and non-specific defense reactions.

A reflection of the functional state of the IFN system is IFN status, the assessment of which is carried out according to several well-known indicators [General Virology Issues, ed. O. Kiseleva, 2007, St. Petersburg], the main of which are:

1. The amount of IFN-α and -γ circulating in the blood (serum IFN).

2. The level of production of IFN-α by leukocytes (or lymphocytes) in vitro upon its induction by viral inducers.

3. The level of production of IFN-γ by leukocytes (or lymphocytes) - when it is induced by mitogens in vitro.

The task of the invention is to develop a method for predicting the course of acute respiratory viral infections in children at the earliest stages of the disease by determining the immunoreactivity of the patient’s body, which is practically applicable and economically viable.

According to the diagnostic results, the use of immunotherapy methods aimed at enhancing cellular immunity reactions, in addition to complex therapeutic treatment, is becoming a promising direction in the treatment of complicated and protracted ARVI.

The proposed forecasting method is easily reproducible, does not require special conditions (for example, sterile boxes) associated with working with tissue culture and test viruses for research. It is less time-consuming in terms of the number of actions taken during the analysis. The advantage is that the study does not require blood serum, which allows you to take blood in a smaller volume, and this is an important condition when examining young children: from 1 month.

The goal is early prediction of the severity of the disease and the development of complications in children with acute respiratory viral infections, and simplification of the diagnostic method.

The most informative indicators were selected from a sufficiently large amount of clinical material, which made it possible to adequately assess the functional state of the sick child’s immune system, namely interferon-gamma and interleukin-10. Earlier in this combination, these indicators were not used.

When determining changes in selected indicators, one can evaluate the severity of the pathological process and the possibility of developing complications of acute respiratory viral infections, and the percentage of prognosis accuracy is increased compared with known combinations of other indicators of a child’s immunoreactivity.

The essence of the invention is the establishment of a digital pattern in the in vitro values determined by spontaneous and phytohemagglutinin-induced production of IFN-γ leukocytes with calculation of the stimulation index (IS), lymphocyte activation index (IAL), as well as the introduction of the interleukin-10 level in the blood plasma into the accuracy of the forecast. Taking into account the interdependence of all these indicators allows us to prescribe an adequate therapeutic treatment that prevents possible complications.

The authors have shown the possibility of using only data from SP IFN-γ and IP IFN-γ in vitro. An additional determination of the level of IL-10 in blood plasma allows us to assess the direction and intensity of the immune response of the body of children with acute respiratory viral infections.

IL-10 is produced by type 2 T-helpers, responsible for switching to a humoral antigen-specific immune response, and plays the role of an antagonist of a number of cytokines, including interferon-gamma. This fact may be an unfavorable sign, as it is associated with the manifestations of immunosuppression in the form of suppression of the proliferative response of T-lymphocytes and, accordingly, less effective protection against viral infection [Children's infections. 2003. No3. p. 58-61].

The interferon system is known to belong to early cytokine reactions in the development of infection. INF-γ, produced primarily by type I T-helpers and cytotoxic lymphocytes, is a powerful immunostimulant and inducer of nonspecific protection, stimulates phagocytosis, as well as the activity of neutrophils and natural killers, promotes adhesion of granulocytes to endothelial cells, and also regulates the strength of the immune response [Immunology . 2002. No2. - p.77-80].

An increase in spontaneous production of interferon-gamma occurs with the development of inflammatory processes in the upper respiratory tract (Acta Paediatrica. - 2008. - Volume 74. - Issue 1, P. - 118-121).

For diagnosis and prognosis, in contrast to the prototype method, only a study of phytohemagglutinin-induced (PHA) and spontaneous production (SP) of IFN-γ in vitro was carried out. For this analysis, a patient in the morning on an empty stomach took blood from a vein or finger in a volume of 1.0 ml into a test tube with heparin 10-15 units / ml. The blood selected in this way can be stored for 5-6 hours at + 4 ° C without losing all the necessary properties.

The remaining blood was centrifuged and the level of interleukin-10 was determined in plasma. The determination was carried out by ELISA standard kits manufactured by LLC "Cytokine", St. Petersburg.

Further analysis of the results was carried out automatically using an enzyme immunoassay reader at a wavelength of 490 nm. The level of IFN in the analyzed sample is determined using a calibration curve depending on the optical density. This makes it possible to obtain reproducible results.

The authors used an indicator called the IFN-γ production stimulation index (IFN-γ IS), which gives an idea of the degree of ICC activity, their ability to respond by IFN-γ synthesis to the introduction of an inducer, which characterizes the reserve capabilities of interferon-producing cells. IP IFN-γ was determined by dividing the level of induced production of IP IFN-γ by the value of the index of spontaneous production of SP IFN-γ:

Due to the fact that there are age-related features in the content of interferon-producing cells, the main of which are lymphocytes, the following indicator was introduced - the lymphocyte activation index (IAL IFN) - an indicator that gives an idea of the individual IFN producing activity of lymphocytes (in terms of a thousand lymphocytes), which is the quotient of dividing IP IFN-γ by the absolute number of patient lymphocytes:

Statistical processing of the results showed a certain pattern. In 36 healthy children, SP IFN-γ levels of 10-50 pg / ml and IFN-γ were obtained at 150-450 pg / ml. IS IFN-γ = 9-45. IAL IFN-γ> 40. The level of IL-10 = 2-50 pg / ml.

In acute respiratory viral infections with no complications, the most common patients at the onset of the disease were patients with a high level of SP IFN (in 61.6% of cases). In children with initially low levels of IFN-γ and IP IFN-γ, with IL-10> 60 pg / ml more often than in other groups, patients with pneumonia and bronchitis met more often, while with high levels of IFN-γ ( > 50 pg / ml) and IP IFN-γ (> 4.0), the level of IL-10 <50 pg / ml in 90.9% of cases, there were children with acute respiratory viral infections without bronchopulmonary lesions, and only 9.1% were detected bronchitis.

The highest levels of IFN-γ SP in blood (58.3 ± 4.3 pg / ml) and IFN-γ IS> 4, with IL-10 <50 pg / ml, were detected in children with acute respiratory viral infections without complications, and the lowest - with pneumonia (39.0 ± 5.4 pg / ml, IP IFN-γ <3 at the level of IL-10> 60 pg / ml), the differences in the indicators are statistically significant (p <0.05).

IP IFN-γ, as well as IAL IFN-γ, were significantly lower in children undergoing acute respiratory viral infections with damage to the lower parts of the respiratory tract, which indicates a reduced ability of the body to counteract the pathogen, that is, the level of damage to the respiratory tract to a certain extent depends on the activity of the patient's ICC . The content of IL-10 in blood plasma in patients with severe ARVI disease and with the presence of complications was statistically significantly higher than in children with moderate severity and without complications.

With a low activity of ICC (low IF IF and low IS), a prolonged course of the disease is more common than with a higher activity of ICC. The presence of a high interferon-producing activity of cells even in individuals with a low level of IFN-associated SP helps to reduce the duration of both intoxication syndrome and the disease as a whole (Table 1).

Table 1 The prognostic value of two methods for predicting the level of respiratory tract damage in children with SARS Indicator IP IFN-γ IL-10 The claimed method of IP IFN-γ + IL-10 (abs. /%) The method is a prototype of IF IF-α + IF IF-γ (abs. /%) ≤2.0; > 50 > 2.0; ≤50 ≤2.0; ≤2.0 > 2.0; > 2.0 ARI, n = 298 4,5 ± 0,4 30.5 ± 1.3 56 / 19.0 242 / 81.0 71 / 24.0 227 / 76.0 ARI, bronchitis, + complications, n = 93 2.6 ± 0.7 * 55.9 ± 2.1 * 81 / 87.0 # 12 / 24.0 # 32 / 34.0 61 / 66.0 ARI, pneumonia, prolonged course, n = 42 1.2 ± 0.4 * 76.4 ± 1.9 * 37 / 88.0 # 5 / 12.0 # 27 / 64.0 15 / 36.0 * - p <0.05 - in relation to ARI without bronchopulmonary lesions
# - p <0.05 - in relation to the prototype method

The sensitivity of the claimed method is 87%, which is significantly higher than that of the prototype method - 66.0%.

The specificity of the claimed method is 81.0%, and the prototype method is 76.0%.

Thus, the claimed method significantly more accurately predicts the development of complications and a protracted course of acute respiratory viral infections in children, which will allow for timely administration of immunomodulating therapy taking into account the patient's immune response.

The dependence of IFN producing activity of ICC patients on the content of the absolute number of lymphocytes in them and their ability to produce IFN-γ was revealed (Table 2).

table 2 The characteristic of IFN-γ IAL in children with acute respiratory viral infections, depending on the level of damage to the respiratory tract Indicator IAL IFN-γ (abs.%) ≤30.0, n = 118 > 30.0, n = 294 ARI, n = 317 69 / 21.8 248 / 78.2 # ARI, bronchitis, n = 73 35 / 47.9 38 / 52.1 ARI, pneumonia, n = 22 14 / 63.6 * 8 / 36.4 * Abs. the number of lymphocytes, 10 ⁹ / l 4.6 ± 0.5 2.5 ± 0.3 # IAL IFN-γ (10 ^-3 pg) 21.1 ± 1.2 51.1 ± 2.9 # * p <0.05 - in relation to ARI without bronchopulmonary lesions
# - for children with IAL IFN-γ≤30,0 · 10 ^-3 pg

When the absolute number of lymphocytes in the blood is> 2.0 × 10 ⁹ / L in children, IFN-γ IAL> 30.0, the disease proceeds more easily with a significant decrease in the duration of the febrile period and intoxication syndrome, complications less often develop.

The nature of the course of the disease also depends on the individual interferon-producing activity of the ICC. So, the protracted course of the disease is most often found in patients with low IFN-γ IAL (≤30 pg 10 ^-3 ).

By the method claimed in the invention, 578 children from 1 month to 18 years were examined. The reliability of the results and conclusions are given in table 3.

Findings:

The revealed pattern in the combination of immunoreactivity indices for different nature of the course of the disease is statistically significant.

Thus, when determining SP IFN-γ and IF IF-γ in the first days of ARVI disease, calculating IF IF-γ and IFN-γ IAL, determining the level of interleukin 10 in the blood plasma, one can correctly assess the state of immunoreactivity of the sick child’s body and with sufficient the degree of confidence to predict the nature of the course of the disease.

The authors first established what a minimum of immunity characteristics is necessary and sufficient for an adequate assessment of the immune response of children with acute respiratory viral infections.

Variants of the immune response are inextricably linked with the clinical features of the course of the disease. The complex of immunity indicators used by the authors made it possible, with a high degree of accuracy - up to 87%, to carry out short-term forecasting of complicated and protracted ARVI.

Determination of the content in the blood plasma of IL-10 contributes to a more accurate and early assessment of the severity of the disease, the development of complications, as well as possible recurrent SARS.

If in healthy children the levels of IFN-γ SP are 10-50 pg / ml, IFN-γ -150-450 pg / ml, IP IFN-γ = 9-45, IAL IFN-γ> 40, IL-10 = 2- 50 pg / ml, then in case of acute respiratory viral infection when the following indicators are established - SP IFN-γ = 50-75 pg / ml, IP IFN-γ≥150 pg / ml, IP IFN-γ> 3, as well as IAL IFN-γ≥ 40, the level of IL-10 = 30-60 pg / ml - predict a favorable outcome of the disease.

When SP IFN-γ = 50-75 pg / ml, IP IFN-γ = 90-100 pg / ml, IP IFN-γ≤3, as well as IAL IFN-γ≤40, level IL-10 = 60-100 pg / ml - predict a severe course and the development of complications requiring immuno-replacement therapy.

When SP IFN-γ = 20-35 pg / ml, IP IFN-γ = 60-100 pg / ml and IP IFN-γ <3, as well as IAL IFN-γ≤30, the level of IL-10> 100 pg / ml - they predict a prolonged course with the development of bronchopulmonary complications and a possible chronicization of the pathological process and repeated acute respiratory viral infections.

This method of assessing the immune response of patients with a diagnosis of acute respiratory viral infections allows you to timely predict the development of a protracted and complicated course of the disease and timely carry out the necessary antiviral and immunomodulating therapy aimed at enhancing cellular immune responses. The method can be used in the work of infectious departments of children's hospitals.

Clinical example.

1. Girl M., 5.7 g., A formalized examination card No. 152, was examined on admission on the first day of an infectious disease of the respiratory tract.

SP IFN-γ - 55 pg / ml; IP IFN-γ - 190 pg / ml; white blood cells (absolute amount) 5.8 × 10 ⁹ / l; lymphocytes - 45%. IS IFN-γ = 3.5, IAL IFN-γ = 72.9; IL-10 = 32.4 pg / ml. These indicators suggest a smooth course of the disease, which was further confirmed by clinical and laboratory data. Diagnosed with SARS. The duration of the disease is 6 days. Moderate changes in immunological parameters corresponded to rapid clinical recovery from acute respiratory viral infections and confirmed the correct prognosis and choice of treatment methods.

2. Boy I., 4.6 g., A formalized examination card No. 120, examined on admission on the first day of an infectious disease of the respiratory tract.

SP IFN-γ - 50 pg / ml; IP IFN-γ - 90 pg / ml; white blood cells (absolute amount) 7.4 × 10 ⁹ / l; lymphocytes - 36%. IS IFN-γ = 1.8, IAL IFN-γ = 33.8; IL-10 = 73.6 pg / ml. The result obtained suggests a protracted course with the development of complications, which was confirmed by clinical and laboratory data. Diagnosed with acute bronchitis. In addition to the symptomatic, substitution therapy with interferon preparations (viferon or analogues) was prescribed. The duration of the disease is 10 days.

3. Boy V., 6.5 g., Formalized examination card No. 128, examined on admission on the first day of an infectious disease of the respiratory tract. A history of frequent acute respiratory infections.

SP IFN-γ - 35 pg / ml; IP IFN-γ - 70 pg / ml; white blood cells (absolute amount) 5.2 × 10 ⁹ / l; lymphocytes - 36%. IS IFN-γ = 2.0, IAL IFN-γ = 37.4; IL-10 = 138.5 pg / ml. These indicators suggest a protracted course with the development of complications, which was confirmed by clinical and laboratory data. Diagnosed with pneumonia, right-sided catarrhal otitis media. Conclusion: low immune reactivity, poor prognosis of the course of the disease. Additionally, immunomodulating therapy was prescribed. The duration of the disease is 16 days. Thus, the preliminary forecast after the laboratory examination of the proposed method was correct.

Claims (1)

A method for predicting the course of the disease in children with acute respiratory viral infections (SARS) in the early days of the disease, including the determination of spontaneous and induced interferon-γ in vitro (IFN-γ) by immunoenzymatic analysis and calculating its stimulation index (IFN-γ) by dividing the indices of the induced level by spontaneous, calculating the index of activation of lymphocytes (IAL IFN-γ) by 1000 lymphocytes, by dividing the indicator of induced interferon γ by the absolute number of lymphocytes of the patient (IAL IFN-γ), characterized in that the blood plasma additionally determines the content of interleukin 10 (IL-10), and then conclude: with IP IFN-γ greater than 3, IFA-γ IAL equal to or greater than 40, IL-10 from 30 up to 60 pg / ml - a favorable outcome of the disease is predicted with therapeutic treatment without the inclusion of immunomodulators; with IP IFN-γ less than 3, IFN-γ IAL less than 40, IL-10 from 60 to 100 pg / ml - a severe course of the disease and the development of complications that require urgent treatment by immuno-replacement therapy are predicted; moreover, with IP IFN-γ less than 3, IFN-γ IAL less than 30, IL-10 more than 100 pg / ml, a prolonged course is predicted with the development of bronchopulmonary complications and a possible chronicity of the pathological process and repeated acute respiratory viral infections, which requires additional inclusion of immunomodulating drugs.