RU2629335C1 - Method for diagnostics of infectious disease period caused by varicella zoster virus in children - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: test serum is simultaneously added to the wells of two strips immobilized with a varicella zoster virus antigen, incubated for 50-55 minutes, then one strip is treated once with an aqueous solution of 6 M of urea for 15 minutes, after which both strips are washed and conjugated with specific monoclonal antibodies to human IgG antibodies, incubated for 30 minutes, stained after washing with an aqueous chromogen solution for 10-15 minutes at room temperature, the reaction is stopped with a solution of 0.5 M of sulfuric acid, optical density is determined with a spectrophotometer, the avidity index is calculated using the formula: avidity index (AI) = (optical density of the sample treated with a solution of 6 M of urea/optical density of the sample without treatment with a solution of 6 M of Urea)X100%, and with an AI level less than 45% and up to 50% an acute period is diagnosed, with AI more than 50% and less than 65% - an early past infection, with AI of 65% and more than 70% - the late disease period is diagnosed.

EFFECT: method is effective and informative for diagnostics of the period of infectious disease caused by the varicella zoster virus in children, used to establish acute infection, activation of chronic persistent infection, and high protective immunity, which is decisive for determination of disease etiology and choice of adequate therapeutic tactics.

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Description

The invention relates to medicine, namely to virology, and can be used to diagnose the period of the infectious process caused by chickenpox virus.

Chicken pox is an infectious disease caused by the varicella zoster virus, which belongs to the alpha herpes virus family and is transmitted by airborne droplets. The disease is manifested mainly by papular-vesicular rash on the skin, fever, intoxication. Being neurotropic, chickenpox virus can also affect the nervous system and cause the development of severe pathologies. The importance of chickenpox is important in connection with the possible development of a chronic recurring form of infection - herpes zoster, the frequency of which can reach 60-70 people per 100,000 who have had chickenpox. Particularly at risk of developing severe forms and the complicated course of chickenpox are individuals with a T-cell immunodeficiency state, adults, as well as children of the first year of life and children of high school age. The current epidemic process of chickenpox is characterized by a tendency to "mature" infection. The likelihood of becoming pregnant and, therefore, the risk of intrauterine infection of the newborn increases. The immunological prerequisites for the described age-related features of the chickenpox clinic are not well understood. These modern observations dictate the need to expand laboratory methods for in-depth diagnosis of the period of infectious diseases caused by chickenpox virus, with the aim of predicting the severity and outcome of the disease, as well as establishing the period of the infectious process for timely targeted therapy.

Currently, there is a known method for the diagnosis of chickenpox virus, based on the detection of VZV DNA in clinical samples by PCR using synthetic oligonucleotides primers complementary to regions of genes encoding VZV proteins (Invention RU No. 2385872 Synthetic oligonucleotides are primers used to detect Varicella virus DNA -Zoster Human Herpes). This invention allows you to directly detect the virus itself in the biological material of the patient, but does not give an idea of the response of the body and the timing of infection, as well as the possibility of predicting the severity of the pathological process.

The famous "Method for determining the avidity of immunoglobulin G to varicella-zoster virus in serum and cerebrospinal fluid", Kneitz R.-H. et al., 2004. The method is aimed at comparing the avidity of IgG antibodies to varicella-zoster virus (VZV) in paired blood serum and cerebrospinal fluid (CSF) samples. This technique allows you to simultaneously compare the avidity of immunoglobulin G in blood serum and CSF, is used to diagnose acute infection or its exclusion and does not depend on the concentration of specific IgG antibodies. The method was proposed as a diagnostic tool for detecting intrathecal synthesis of antibodies in case of viral encephalitis, multiple sclerosis and is used to evaluate concomitant chickenpox disease with the progression of HIV infection.

However, this method requires mandatory parallel studies of blood serum and cerebrospinal fluid, which is not always advisable to use in routine practice for the diagnosis of diseases caused by varicella zoster virus. In addition, the disadvantages of this method include the need to dilute the test material (serum) in a ratio of 1: 231, which is almost impossible to do in routine laboratory practice due to the need to use additional consumables and perform manipulations that increase the time spent on this study, leading to rise in price of analysis. Additional dilution of the conjugate is also required, which increases the reaction time. Note that when evaluating the results of the described method, only one borderline value of IA (<37%) is used, indicating acute infection, however, when examining the children contingent, it is necessary to take into account the characteristics of the immune system and assume various variants of the body's immune response in accordance with individual and age characteristics a child.

The closest method for diagnosing atypical forms of VZV infection resulting from reactivation of chickenpox virus and shingles is a method based on the analysis of spontaneous production of specific antibodies by peripheral blood mononuclear cells (MPC) arising from endogenous reactivation of the virus (Casanova A.S. et al. A new method for diagnosing an infection resulting from reactivation of chickenpox virus and shingles // Epidemiology and Vaccine Prevention. - 2012. - No. 4. - P. 57-60). The method is based on the analysis of spontaneous production of anti-VZV-IgG in a culture of peripheral blood mononuclear cells (MIC). To this end, mononuclear cells were initially isolated from venous heparinized blood, then cultured in sterile tubes in a culture medium for 48 hours. After determining the viability of the cells, protein synthesis was inhibited in the prototype IPC by incubating the cells in a nutrient medium for 30 minutes, followed by washing and an additional viability test, and then they were again cultured for 48 hours simultaneously with the control sample, which was continued to be studied without treatment with cycloheximide . The described technique is rather laborious due to the costs associated with the manipulations for the isolation of blood cells and a long cultivation process (4 days), which is not consistent with the concept of expressiveness of the diagnostic approach. In addition, given the constant checks on the viability of isolated and cultured MICs, errors are possible related to the accuracy and information content of the results obtained with final accounting. This technique is not possible to use for routine diagnostics in laboratories not intended for conducting cell cultures, due to the lack of special conditions and equipment intended for this. We also note that the method does make it possible to diagnose VZV infection, however, it is difficult to judge the severity of the process in view of obtaining results showing only the intensity of antibody synthesis in MVP, which may depend on both individual characteristics of the humoral immune response and virologesis in the patient’s body. . Thus, this method does not provide accuracy and expressness of diagnosis.

In order to eliminate the above disadvantages, the authors suggest a fundamentally new method for diagnosing the period of an infectious disease caused by the varicella zoster virus in children, the technical result achieved in this method is to increase the accuracy and expressness of diagnosis by detecting the avidity of IgG antibodies with the calculation of digital indicators characterizing periods of diseases, caused by varicella zoster virus.

This is achieved by the fact that in the enzyme immunoassay the test serum is simultaneously introduced into the wells of two strips immobilized with the varicella zoster virus antigen, incubated for 50-55 minutes, then one strip is treated once with an aqueous solution of 6M urea for 15 minutes, after which both strips are washed and add a conjugate of specific monoclonal antibodies to human IgG antibodies to them, incubate for 30 minutes, after washing, stain with an aqueous chromogen solution for 10-15 minutes at room temperature, stop the reaction a solution of 0.5 M sulfuric acid, determine the optical density using a spectrophotometer and calculate the avidity index by the formula:

and with an IA level of less than 45% and up to 50%, an acute period is diagnosed, with more than 50% of IA and less than 65%, an early past infection, and with an IA of 65% and more than 70%, a late period of the disease.

The authors found that increasing the incubation time to 50-55 minutes, in contrast to 30 minutes in the standard immuno-enzyme test, at the stage of formation of antibody antibody complexes with specific VZV proteins adsorbed on the surface of the polystyrene well, led to the formation of a stronger bond and allowed to affect the immune complex with a detergent in a higher concentration. The authors first used a 6M urea solution as a detergent, finding that it is at this concentration and a single exposure of 15 minutes that the antigen-antibody bond dissociates in the well. The use of a less concentrated solution did not allow complete breakdown of weakly-shaped immune complexes, however, an increase in the concentration of detergent led to a complete rupture of the complexes between antibodies and proteins, and the total antibodies were removed during subsequent washes. Thus, a lower concentration of urea did not lead to a complete cleavage of low-avidity bonds, and the use of a higher concentration turned out to be ineffective. The authors, using an aqueous solution of 6 M urea for the first time and washing once, found that the likelihood of delamination of adsorbed proteins from the surface of the wells is significantly reduced, and the effect of the detergent on immune complexes is enhanced.

The authors, having significantly reduced the duration of the test, reduced the necessary contact time of the serum with VZV antigens adsorbed on the surface, shortened the stages of detergent treatment and the exposure time of the bound immune complexes with the conjugate solution for 30 minutes, unexpectedly obtained the optimal result, increasing the expressivity and accuracy of the analysis.

The exposure time with a chromogen solution for 10-15 at room temperature was obtained experimentally and for this modification of the immuno-enzyme method is sufficient and most optimal to obtain the final result. In the formula for calculating the avidity index, the authors used indicators of the optical density of the samples after processing the studied serum samples with a 6M urea solution. An interpretation of the estimated avidity index with an estimate of the period of an infectious disease, including both the acute period, past infection, and high specific protective immunity, and the activation of chronic persistent infection with an increase in IA in dynamics after 2 weeks, is also proposed.

The proposed method is as follows

To ensure studies to establish the avidity of IgG antibodies to VZV, standard equipment can be used for virology laboratories and enzyme immunoassay laboratories (ELISA).

The avidity of antibodies of the IgG class to VZV was determined using reagent kits for enzyme immunoassay for the detection of IgG immunoglobulins based on the solid-phase indirect enzyme-linked immunosorbent assay using purified recombinant varicella zoster antigen.

The principle of determining the avidity of antibodies was to detect antibodies that could break the bond with the VZV antigen when the resulting complex was treated with a detergent - 6M urea solution. When comparing the optical density of the solution exposed to the detergent, and with that without the detergent, the extinction value was calculated as an indicator of the maturity (avidity) of the antibodies.

ELISA was carried out according to the following scheme:

1. Binding of antibodies. The first wells of the two strips were left empty and were considered as a control of the substrate, 90 μl of serum dilution solution (PPC) and 10 μl of test serum pre-diluted with pre-dilution solution (MPS) to obtain a final dilution of 1: 100 were added to all the rest . The same test sera were introduced simultaneously in two rows following the control wells in parallel. The plate was incubated at a temperature of 37 ° C for 50-55 min in a humid chamber. After incubation, the solution was removed and one strip was washed once with a 6 M urea solution with an exposure of 15 minutes, then 3 times with a solution of phosphate-saline buffer with tween (FSB-T), and the second strip was treated with FSB-T, then it was also washed 3 times, as well as experienced strip.

2. The introduction of the conjugate. 100 μl of the working solution of the conjugate — specific monoclonal antibodies to human IgG with horseradish peroxidase — was added to all wells, except for the substrate control. The plate was incubated at 37 ° C for 30 minutes. After incubation, the solution from the wells was removed and washed 5 times with FSB-T solution.

3. The reaction of interaction with the substrate. 100 μl of a 0.05% TMB aqueous solution was added to all wells. The tablet was kept in the dark at room temperature for 15 minutes. After the indicated time, the reaction was stopped by adding 100 μl of a 0.5 M sulfuric acid solution.

4. Accounting for the results of ELISA and calculation of the avidity index (IA) of antibodies. The optical density (OD) was measured using a spectrophotometer in the following mode: main filter - 450 nm, reference filter - 620 nm, no later than 10 minutes after the reaction was stopped.

Calculation of IA antibodies was carried out according to the formula:

and with an IA level of less than 45% and up to 50%, an acute period is diagnosed, with more than 50% of IA and less than 65% - an early past infection, with IA of 65% and more than 70% - a late period of the disease, characterized as a high specific protective immunity, and the possible activation of chronic persistent infection with an increase in IA in dynamics after 2 weeks.

A method for diagnosing a period of an infectious disease caused by varicella zoster virus in children can be confirmed by the following examples:

Example 1

To identify the period of infection, the avidity index of IgG class antibodies to VZV virus was determined in 15 children aged 1 year to 14 years, at the time from 1 to 5 days after chickenpox virus infection and undergoing treatment at the clinic of the Federal State Budget Scientific Research Institute of Biomedical Medicine of Russia. IgG antibodies to VZV were detected in the blood serum of children by the standard method of enzyme-linked immunosorbent assay (ELISA) using commercial test systems. Table 1 presents the definition of the period of the infectious process caused by VZV in children.

* The digital level of antibodies is indicated in units of optical density

In 14 children (86.7%), specific IgG antibodies with low avidity were detected in serum (avidity did not exceed 50, which noted the early phase of the disease with chicken pox. In one patient (No. 3 - the fifth day of the disease), a late period of infection was determined , and the patient (No. 8 — the fourth day of illness) was diagnosed with an early paste infection.

Example 2

At the NIIIDI Clinic, a patient S.S., 12 years old, was hospitalized with the diagnosis of chickenpox encephalitis on the 10th day after the rash to the department of neuroinfections and organic pathology of the nervous system. The diagnosis was confirmed by clinical and liquorological data. When examining biological material (blood, cerebrospinal fluid, oropharyngeal swab), the following results are obtained, presented in Table 2.

GSR of CSF on VZV DNA - positive; PCR of blood for CMV DNA - negative; PCR of blood for HHV DNA 6 - negative; PCR of urine for CMV DNA - negative; ICC studies of smear of the oropharynx on CMV - negative.

* Antibodies were determined by ELISA. The digital level of antibodies is indicated in units of optical density

Laboratory diagnosis: chickenpox encephalitis, early past infection.

Example 3

A patient, F.Yu., 5 years old, was diagnosed with a diagnosis of a viral infection of unknown etiology and suspected herpesvirus infection in the NIIDI clinic at the neuroinfection department. When virological examination of the patient's blood serum for a group of herpes viruses by ELISA, the following results were obtained, are presented in table 3.

* The digital level of antibodies is indicated in units of optical density

Laboratory diagnosis: Herpetic infection etiologically related to HSV1, absence of disease caused by VZV virus

Thus, the authors proposed an effective and informative method for diagnosing the period of an infectious disease caused by the varicella zoster virus in children, with the help of which it is possible to establish acute infection, activation of chronic persistent infection, as well as high protective immunity, which turns out to be a decisive factor in establishing the etiology of the disease and choosing adequate therapeutic tactics.

Claims (3)

A method for diagnosing a period of an infectious disease caused by varicella zoster virus in children by determining antibodies in an enzyme immunoassay, characterized in that the test serum is simultaneously introduced into the wells of two strips immobilized with a varicella zoster virus antigen, incubated for 50-55 minutes, then one strip treated once with an aqueous solution of 6 M urea for 15 minutes, after which both strips are washed and a conjugate of specific monoclonal antibodies to human IgG antibodies is introduced into them, incubated for 30 minutes, after washing, stain with an aqueous chromogen solution for 10-15 minutes at room temperature, stop the reaction with a solution of 0.5 M sulfuric acid, determine the optical density using a spectrophotometer, calculate the avidity index by the formula:

and with an IA level of less than 45% and up to 50%, an acute period is diagnosed, with more than 50% of IA and less than 65%, an early past infection, and with an IA of 65% and more than 70%, a late period of the disease.