RU2235328C1 - Method of predicting course of viral encephalitis - Google Patents

Abstract

FIELD: neurology.

SUBSTANCE: invention relates to predicting development of autoimmune demyelinization process in the course of infection such as viral encephalitis. For prediction of the course of viral encephalitis in an acute disease period, IgE and IgG antibody levels are investigated with the aid of immunoenzymatic method. When indicated levels are increased, development of autoimmune demyelinization is predicted, whereas with normal IgE and IgG levels favorable course of disease without autoimmune demyelinization is predicted.

EFFECT: enabled prediction of the course of disease in convalescent and later periods.

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Description

The invention relates to medicine, in particular to neurology, and for predicting the development of autoimmune demyelination processes in the process of an infection such as viral encephalitis. The ability to determine demyelination in this infection gives the clinician reason to conduct adequate treatment, to prevent the complications of such a serious neuropathology as viral encephalitis and its consequences.

More than a hundred years ago, a separate group of diseases was identified, characterized by the development of severe progressive organic lesions in the central nervous system (CNS), the pathophysiological basis of which is the destruction of the myelin sheath of the conductors of the brain and spinal cord. It is on this basis that a number of CNS pathologies have been allocated to the group of so-called demyelinating diseases, which include multiple sclerosis, retrobulbar neuritis, multiple encephalomyelopoliradiculoneuritis, subacute sclerosing panencephalitis, leukoencephalitis and others.

Today, the most common point of view regarding etiopathogenesis is that demyelination is a consequence of a viral infection in a genetically predisposed organism, leading to an autoimmune attack on myelin antigens. It is believed that the main autoantigen in this process is the main myelin protein (MBP), therefore, it is associated with the possibility of diagnosis and treatment of this category of patients. It is known that in the process of ontogenesis, it mediates immunological tolerance to the structural components of the central nervous system. Tolerance is maintained by complex regulatory mechanisms, the principle of which is reduced to the fact that non-immunogenic forms of MBP expressing specific immunoregulatory determinants lacking encephalitogenic properties circulate in vivo and activate the T-suppressor clone, which in turn inhibits the proliferation of T-clone autoreactive helpers. Many researchers attribute the development of demyelination to the fact that, under certain conditions, MBP cancels immunological tolerance. As a result, the intensive production of an autoreactive clone of T-helpers begins, which recognize MBP as a “stranger” and, using isolated interleukins, trigger the synthesis of antibodies to MBP. Thus, the determination of the content of antibodies to MBP can serve as a reliable diagnostic marker for the presence, features of the course and prognosis of the demyelinating process, as well as the effectiveness of pathogenetic therapy.

The importance of autoimmune demyelination in viral encephalitis is recognized by various researchers. However, the prediction of this process in the early period of the development of the disease was not carried out.

The study of antibodies to MBP in other demyelinating diseases, for example, for multiple sclerosis, revealed a certain correlation of IgG to MBP and the course of the autoimmune process (1).

In relation to viral encephalitis, no studies were found that describe the possibility of predicting demyelination based on the study of IgE and IgG antibodies to MBP.

Until now, both the diagnosis and prognosis of the course of this infection and its consequences have been carried out clinically, assessing the severity of the course of the disease by the severity of symptoms, including intoxication, by general immunity disorders, etc. (2).

In modern practical neurology, the prognosis of the development of acute viral encephalitis is determined by the etiology of the disease. For example, it is believed that in the prognostic plan, the most difficult variant of the course of viral encephalitis is herpetic encephalitis, as well as encephalitis complicated by cytomegalovirus infection. The prognosis of the development of acute viral encephalitis is also determined by the clinical picture of the disease, in particular, a suspicion of the possible development of demyelination is a violation of visual, auditory and motor functions in the dynamics of the disease. Unfortunately, such changes are detected in patients sometimes quite late, sometimes after 6-12 months from the onset of the disease and are already a consequence of the development of persistent and irreversible neurological disorders, often not amenable to reverse development. In some cases, computed tomography (CT) or magnetic resonance imaging (MRI) is prognostically effective. However, in the early stages of the disease, demyelination on CT and MRI is rarely detected and, as a rule, this kind of diagnosis is carried out in patients who already have obvious clinical signs of autoimmune demyelination. Any specific markers of the demyelinating process in practical neurology are not used to assess the prognosis of the course of acute viral encephalitis, while the use of markers such as the determination of IgE and IgG antibodies to MBP in the acute period of viral encephalitis allows a high degree of confidence to reveal the debut of autoimmune demyelination in nervous tissue.

The purpose of the invention is the early prediction and increase the accuracy of this prediction due to the quantitative assessment of markers of autoimmune demyelination specific for viral encephalitis.

For this, in the acute period of the disease, IgE and IgG antibodies to MBP were determined in the patient.

The content of antibodies to MBP was determined using the NIIVS named after I.I. Mechnikov RAMS enzyme-linked immunosorbent assay for the quantitative determination of IgE and IgG antibodies to MBP in serum (3).

The method is based on enzyme-linked immunosorbent assay (ELISA) using immunoglobulin reagents in the calibration system. Thus, a calibration system for the quantitative determination of IgG antibodies (AT) against MBP includes a solid phase immobilized with murine anti-IgG MKAT, a set of calibrants from human IgG and an anti-IgG MKAT conjugate with horseradish peroxidase. The calibration system for determining IgE AT for MBP consists of a solid phase immobilized with mouse anti-IgE MKAT, a set of calibrants prepared from human IgE and an anti-IgE MKAT conjugate with horseradish peroxidase.

To determine IgG and IgE AT specific for MBP in the patient's blood serum, the solid phase, immobilized pig MBP, patient serum in working dilution and the conjugate corresponding to the desired antibody isotype are used.

Quantitative accounting of antibodies is carried out according to the corresponding calibration curve of the dependence of OD on the concentration of IgG and IgE in calibrants, which quantitatively correlate the content of IgG AT to MBP with the concentration of IgG and IgE in calibrants. The concentration of IgG AT to MBP is measured in IgG units, where 1 unit of IgG is equal to 1 μg / ml. The concentration of IgE AT to MBP is measured in units of IgE, where 1 unit of IgE is equal to 1 kE / L.

The proposed method was evaluated by the indicators of convergence, reproducibility, specificity, the minimum definition boundary and the region of linearity.

When determining the convergence, measured in 10 replicates in blood serum with different levels of AT to MBP, the average deviation (m) was in the range of 0.58-3.45, the standard deviation (δ) was in the range of 1.78-10.91 and coefficient of variation did not exceed 6.67%.

When determining reproducibility, measured in 3 replicates in 3 different dimensions: on different days, by different laboratories and using different series, the average deviation (m) was between 1.45-23.15, the standard deviation (δ) was within 2 , 52-40.10 and the coefficient of variation did not exceed 9.87%.

The minimum border for determining the content of IgG AT to MBP was 0.31 μg / ml.

The minimum limit of determining the content of IgE AT to MBP was 0.84 kE / L.

The area of linearity, that is, the deviation between the maximum and minimum concentrations did not exceed 10%.

The specificity of the method with respect to its ability to detect antibodies of only IgG or IgE isotype was confirmed in the ELISA inhibition, where after incubation of IgG samples with multiple dilutions of MKAT against human IgA, IgM and IgE, the so-called “backcasting” effect was obtained only in case of incubation of IgG with anti-IgG MKAT. Accordingly, after incubation of IgE with multiple dilutions of MKat against anti-human IgG, IgA and IgM, the effect of “reverse propagation” was obtained only in the case of incubation of IgE with anti-IgE MKAT.

The specificity of the method with respect to its ability to detect antibodies to MBP was confirmed in the MBP serum depletion test, where after preliminary incubation of blood serum samples with MBP during subsequent ELISA, there was a significant decrease in OD in depleted samples compared to native serums.

The determination of IgG AT for MBP was carried out in 450 samples of serum from healthy individuals aged 6 months to 50 years. The selection criteria for the control group were: the absence of neurological, autoimmune, allergic and chronic diseases in the acute stage, as well as immunodeficiency states and acute viral infections.

As a result of the study, the following average IgG AT content for MBP was obtained in practically healthy people:

- for children aged 6 months to 11 years - 22 mcg / ml;

- for children aged 12 to 16 years and for adults - 40 mcg / ml.

These values are taken based on the average IgG content in these groups, taking into account the size of the upper boundary of the confidence interval.

Determination of IgE AT for MBP was carried out in the same 450 blood serum samples of healthy individuals aged 6 months to 50 years. The selection criteria for the control group were: the absence of neurological, autoimmune, allergic and chronic diseases in the acute stage, as well as immunodeficiency states and acute viral infections.

The average content of IgE AT for MBP in a group of practically healthy people, determined taking into account the average values and the upper limit of the confidence interval for all age groups, was 8.0 kE / L.

In 194 patients with viral encephalitis in various periods of the disease, an increase in the level of antibodies to MBP was found.

Thus, the average level of IgG AT to MBP was 325 ± 8.5 μg / ml, and IgE AT to MBP was 31 ± 4.9 kE / L.

A legitimate question may arise, why determine the antibodies of both isotypes, if the levels of these antibodies are closely related. In this regard, it should be clarified that it is not always the same patient that we reveal a significant difference in the content of IgG AT to MBP at different stages of the disease, especially this kind of pattern occurs in the very early period of an exacerbation of the disease. As a rule, the first signal of the onset of exacerbation is an increase in the level of IgE antibodies to MBP.

It should be noted that a similar kind of prognostic character of changes in the content of IgE-specific IgE antibodies was detected in patients with acute viral encephalitis (Table 1).

As can be seen from table 1, in patients with viral encephalitis, in the pathogenesis of which autoimmune demyelination predominated already in the acute period of the disease, the level of IgE antibodies to MBP predominates compared with that of viral encephalitis without the predominance of demyelination in pathogenesis. Moreover, the level of IgG antibodies to MBP was quite high in the acute period in all cases of viral encephalitis. Only the dynamics revealed a significant difference in the content of IgG antibodies to MBP in patients with different variants of the pathogenesis of viral encephalitis. From this we can conclude the importance of determining IgE antibodies to MBP for the timely determination of the prognosis of viral encephalitis.

Equally important is the study in the dynamics of humoral markers of demyelination to assess the effectiveness of certain therapeutic measures.

Thus, the proposed method for the quantitative determination of the content of IgG and IgE AT for MBP has diagnostic and prognostic significance. It allows you to differentiate different options for the course of the demyelinating process, the effectiveness of therapeutic measures, and also allows you to timely assess the risk of autoimmune disorders in the central nervous system. All this allows us to characterize the method of quantitative determination of IgG and IgE AT for MBP as a fairly reliable diagnostic tool, the use of which in wide neurological practice can significantly increase the efficiency and accuracy of diagnosis of demyelinating CNS pathology.

Example 1. Patient I., 1 year. Ill acutely. The temperature rose to 37.5 ° C, slight catarrhal phenomena, lethargy were noted. On the 4th day of the disease, twitching in the left hand appeared. Hospitalized in the CIB number 1. Upon admission, drowsy, adynamic. Moderately pronounced meningial syndrome, muscle hypotension and constant cramps in the left limbs, periodically choking. In CSF - cytosis 100/3, lymphocytes 71%, protein 0.264 g / l. Analysis of AT to MBP revealed an increase in the level of IgG AT to MBP to 570 μg / ml and IgE AT to MBP to 38 kE / L. According to a virological blood test, herpes encephalitis caused by HSV-1 was diagnosed. On the 10th day, the condition worsened sharply and the child was transferred to intensive care. The condition at admission to intensive care is serious. The child is inconsistent, adynamic, lethargic, drowsy, choking, hypersalivation is noted. A limitation of the mobility of the soft palate and a sharp decrease in the pharyngeal reflex were revealed. The reaction of the pupils to the light is saved. Rough descending strabismus due to the left eye. The left nasolabial fold is distinctly smoothed. He does not hold his head. The range of motion in the left hand is reduced. Muscle tone in the arms on the left above the pyramidal type. Tendon reflexes in the hands on the left are enlivened. An intermittent symptom of Babinsky on the left. During therapy with acyclovir, the condition improved. On the 22nd day of the disease, the temperature rose again and the child's condition worsened sharply, a gross hyperkinetic syndrome and hemibolism appeared. A CT scan performed on day 24 of the disease revealed a total darkening of the right hemisphere. The level of IgG AT to MBP on day 22 was 920 μg / ml, and IgE AT to MBP was 51 kE / L. In the dynamics on CT, diffuse changes in the right hemisphere, regarded as demyelination, were revealed. The level of IgG AT to MBP in the dynamics of the period was 600 μg / ml, and IgE AT to MBP - 36 kE / L.

Example 2. Patient D., 3 years. He became acutely ill with an increase in temperature to 39 ° C, slight catarrhal phenomena were noted, on the second day of the disease, vomiting up to 2 times a day joined. Within 5 days, the condition worsened, vomiting increased, high temperature persisted. The child was hospitalized in hospital No 1. According to a virological blood test, herpes encephalitis caused by HSV-1 was diagnosed. In CSF - cytosis 504/3, lymphocytes 55%, protein 0.230 g / l. The level of IgG AT to MBP on the day of admission was 340 μg / ml and IgE AT to MBP was 27 kE / L. On the 7th day from the onset of the disease, convulsive status developed, coma 2 on the Glasgow scale for 3 days. Upon admission to intensive care, the child’s condition is severe, lethargic, and dynamic. In neurological status, a light peripheral paresis of the VII pair, left-sided paresis with a low tone, which was transformed into spastic. On CT on the right revealed a vast area with a reduced density. The level of IgG AT to MBP on the 20th day from the onset of the disease was 560 μg / ml, and the IgE AT to MBP was 41 kE / L.

During therapy with acyclovir, the condition improved slightly. In dynamics on CT, foci of demyelination in the right hemisphere were revealed. The level of IgG AT to MBP in the dynamics of the period was 580 μg / ml, and IgE AT to MBP - 45 kE / L.

Example 3. Patient E., 11 years old. The disease began with the appearance of a maculopapular rash. Rubella was diagnosed. On the 3rd day from the beginning of the rashes, the temperature rose again to 37.5 ° C, small clonic twitches of the arms and legs appeared, symmetrical on both sides. The girl was hospitalized in the CIB number 1. Upon admission, the condition is serious, coma of the 1st degree, frequent tonic tension of the entire trunk and limbs. Mydriasis, scleritis, evenly reduced muscle tone, lack of tendon reflexes. In CSF - cytosis 160/3, lymphocytes 76%, protein 0.495 g / l. The level of IgG AT to MBP on the day of admission was 280 μg / ml, and the IgE AT to MBP was 6 kE / L. After symptomatic therapy on day 7, consciousness recovered, tendon reflexes and muscle tone returned to normal, mydriasis was not detected. A bright rash persisted. On day 18, a CT scan was performed on which no focal pathology of the brain was found. The level of IgG AT to MBP was 143 μg / ml, and the IgE AT to MBP was 4 kE / L. In the separated dynamics on CT, no deviations from the age norm were revealed. The level of IgG AT to MBP was 51 μg / ml, and the IgE AT to MBP was 4 kE / L.

As can be seen from tables 1 and 2 in patients with viral encephalitis, in the pathogenesis of which autoimmune demyelination predominated already in the acute period of the disease, the level of IgG AT to MBP predominates significantly compared to that with viral encephalitis without the predominance of demyelination in pathogenesis. Moreover, the level of IgG AT to MBP was quite high in the acute period in all cases of viral encephalitis. Only the dynamics revealed a significant difference in the content of IgG AT to MBP in patients with different variants of the pathogenesis of viral encephalitis. From this we can conclude the importance of determining IgG AT to MBP for the timely determination of the prognosis of the development of viral encephalitis.

Literature

1. Leonovich A.L. Neurology, Minsk, 1996, pp. 103-116.

2. Diseases of the nervous system. Edited by Yakhno N.N., vol. 1, Moscow, Medicine, 1995, p. 313-325.

3. Gervazieva V. B., Vorobeva N. L. “Enzyme-linked immunosorbent assay for the quantitative determination of IgG antibodies to myelin basic protein.” Immunology, No. 2, 2000, pp. 52-56.

Claims (1)

A method for predicting the course of viral encephalitis, characterized in that in the acute period of the disease, IgE and IgG antibodies to the main myelin protein are quantified in the blood serum of the patient by the enzyme immunoassay and, with an increase in the level of IgE and IgG antibodies, the development of autoimmune demyelination is predicted, and with normal IgE antibodies and an increase in the level of IgG antibodies predict a favorable course of the process without the development of autoimmune demyelination in the convalescence and long-term periods of the disease.