RU2319152C2 - Method for predicting toxoplasma infection in children - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: after blood sampling one should simultaneously detect the subclasses of immunoglobulins IgG-3 and IgG-4 due to fulfilling modified complement binding reaction (m-CBR) and detect the level of antibodies IgG due to immunoenzymatic assay (IEA). At the presence of antibodies in m-CBR in subclass IgG-3 in titers being 1:10-1:80 and the level of antibodies IgG in IEA in titer being >200 IU/ml one should establish acute form of the disease mentioned. At the presence of antibodies in m-CBR in subclass IgG-4 in titers being 1:20-1:50 and the level of antibodies IgG in IEA being <200 IU/ml one should state upon chronic form of the disease.

EFFECT: higher accuracy of diagnostics.

3 ex, 2 tbl

Description

The invention relates to medicine, in particular to laboratory diagnosis of toxoplasma infection, and can be used in healthcare practice to identify acute and chronic course of the disease.

The urgency of the problem of toxoplasmosis is due to the wide distribution of T.gondii on all continents and in all climatogeographic zones. According to serological tests in different countries of the world, from 4 to 68% of the population are infected with toxoplasma infection. In Russia, infection is 25%. Toxoplasmosis infection increases with age:

1 year - 2%, 2 years - 10%, 4 years - 22%, in 15-19 years - 38.2%, 40-44 years - 65.8%. However, despite a small percentage of infections in children, toxoplasmosis is one of the main places among the causes of perinatal pathology. For every 1000 newborns in Germany and France, there are 1 to 8 children with congenital toxoplasmosis (BT). According to Swedish researchers, toxoplasmosis is the cause of severe hydrocephalus in 98% of cases.

Toxoplasmosis is the cause of every third case of chorioretinitis with impaired vision and every fifth case of lymphadenitis.

Despite a fairly detailed study of toxoplasma infection (TI), there are a number of factors, such as the prevalence of latent forms of infection, the complexity of the diagnosis of manifest forms, the lack of effectiveness of antiprotozoal therapy, which indicate the need to develop algorithms for the diagnosis, treatment and follow-up of this category of patients aimed at to determine the acute or chronic phase of the disease.

A known method of parasitological diagnosis of TI, when the pathogen is detected by direct microscopy of the material, in histological sections of internal organs or by isolating the pathogen in susceptible laboratory animals using bioassays. For this purpose, use blood, cerebrospinal fluid, pleural fluid, puncture of the lymph node and bone marrow, etc. For direct microscopy, imprint smears on slides are prepared from the test material, dried, fixed with methyl alcohol, then stained according to Romanovsky and microscopic. The direct microscopy method is simple and affordable, but it is effective in a small percentage and requires additional material costs, which does not ensure the accuracy of diagnosis (Gracheva LI Review of diagnostic methods in 1999)

A known method of serological research (reaction with the Sabin-Feldman-RK dye, complement-RSK complement binding reaction, indirect immunofluorescence-RNIF reaction, enzyme-linked immunosorbent assay-ELISA), which detects the presence of specific antibodies in the blood serum of the person being examined. The essence of the method is to identify specific antibodies in the test serum using a toxoplasma antigen and labeled antiserum. The disadvantage of the RK method is the complexity and complexity of execution and requires live toxoplasmas, which is not available for most laboratories. Antibodies detected in RNIF appear at the end of the first week of infection and persist for a long time. The ELISA method is based on indirect enzyme-linked immunosorbent assay on the solid phase using horseradish peroxidase as a marker enzyme. The disadvantages of the RNIF and ELISA methods are the obligatory re-examination after 2-4 weeks, and by increasing the titers of JgG antibodies, it will be possible to judge the developing infection, but if the infection is longer than 12 months, the determination of the dynamics of IgG-toxo does not carry positive information, since the levels these antibodies are not associated with clinical manifestations, while the detection of IgM antibodies, which are markers of acute infection, presents great difficulties due to the possibility of obtaining a false-positive result in RNIF, due to the presence of m antinuclear antibodies and rheumatoid factor. In ELISA, the detection of IgM antibodies can give both positive and negative results. Thus, each of the proposed methods does not fully reflect the diagnosis of toxoplasma infection due to the fact that during the initial examination of the patient it is impossible to establish the phase of the process, and therefore, does not ensure the accuracy of diagnosis and cannot serve as a criterion for the appointment of antiprotozoal therapy.

To eliminate the above drawbacks, the authors propose a fundamentally new method of diagnosing toxoplasma infection by using enzyme-linked immunosorbent assay (ELISA) and a modified complement binding reaction (m-CSC), characterized in that after blood sampling, the ELISA and m-CSC reactions are set up simultaneously, differentiate toxoplasma low-avidity antibodies in subclasses of immunoglobulins IgG-3, IgG-4 and highly avid IgG antibodies and in terms of the presence of antibodies in m-CSC in the JgG-3 subclass in titers of 1: 10-1: 80 and the level of highly avid IgG antibodies in ELISA in the titers> 200 IU / ml, the acute form of the disease is established, and in the presence of antibodies in subclasses of IgG-4 in titers 1: 20-1: 50 and the level of IgG antibodies in ELISA <200 IU / ml establish the chronic form of the disease.

The technical result of this invention is to improve the accuracy of diagnosis and expressivity.

The authors first proposed a method for the diagnosis of TI, based on the simultaneous formulation of ELISA and m-RSK, which allows to determine both low-avid and highly avid AT. In m-CSCs, we identify weakly ATs, which need to enhance the avidity of sorption of complement proteins, which not only enhances AT's affinity for antigenic epitopes of pathogen proteins, but also is included in the body's immune defense process. Our studies have proved that the addition of the C3b compliment system protein to the AT-AG complex creates a pronounced possibility of capture by phagocytes (macrophages) that have a receptor for this complement protein in their structure. In case of primary infection or exacerbation, the process of combining AT-AG and complement quickly enough leads to the formation of high molecular weight immune complexes. During the course of the infection, the degree of AT avidity increases and an immune complex (IR) is formed, which energetically binds to the Fc-receptor of phagocytes.

Thus, after numerous analyzes, the authors established the fact that for the full functioning of the pathogen elimination system in the IR structure, two types of AT are necessary - at the initial stage of infection, the presence of low-type antibodies, when sorption on a macrophage is associated with the receptor for the C3-b complement fraction, and highly avid, when sorption on a macrophage involves the macrophage receptor for the Fc fragment of AT.

According to the invention, in contrast to the above methods, the authors, on the basis of clinical studies in a small group of patients, having received an unexpectedly positive result, propose to diagnose an acute form of toxoplasma infection based on the detection of low-grade antibodies of the IgG-3 subclass in the blood in titers 1: 10-1: 80 by the method m-CSCs and highly similar IgG antibodies in titers> 200 IU / ml in ELISA. The authors also found that in chronic TI, antibodies are detected in m-CSC in the IgG-4 subclass at titers of 1: 20-1: 50 and the level of IgG antibodies in ELISA is <200 IU / ml.

After conducting 925 studies on the diagnosis of TI in order to establish the forms of the infectious process, the authors empirically determined that for the acute form of the infectious process, antibody titers in the IgG-3 subclass within 1: 10-1: 80 are necessary, and if the process is chronic, antibody titers in IgG-4 subclass within 1: 20-1: 50.

Table 1 Characterization of forms of toxoplasma infection in children with simultaneous formulation of ELISA and m-RSK Methods IFA m-RSK Acute form IgG> 200 IU / ml IgG-3 1: 10-1: 80 Chronic form IgG <200 IU / ml IgG-4 1: 20-1: 50

The method is as follows: the blood delivered to the laboratory, taken by venipuncture in a volume of 2.0 ml, is centrifuged to obtain serum, which is involved in both the m-RSK and ELISA reactions. The m-CSC method is based on the standard complement binding reaction with a significant difference, which is a quantitative determination of the lamb erythrocyte lysis level in the hemolytic system. This level of lysis is tested with the presence of a digital indicator, determined by the concentration of oxidase enzymes released from these lysed red blood cells.

The enzyme-linked immunosorbent assay is carried out with the Vector-best toxo test system with the detection of antibodies of the IgM and IgG class in standard execution.

The results obtained after ELISA and m-RSK are analyzed and when receiving an AT IgG titer of> 200 IU / ml in ELISA and antibodies in m-RSK in the JgG-3 subclass in titers of 1: 10-1: 80, an acute form of TI is diagnosed. And when receiving IgG antibodies in ELISA <200 IU / ml and in the IgG-4 subclass in titers of 1: 20-1: 50, a chronic form of infection is diagnosed in m-CSC.

An analysis of the known methods at the filing date of the application shows that there are no such methods that allow, based on the initial examination, to determine the phase of infection and to accurately diagnose. Based on the proposed method, etiological therapy is carried out in the acute phase of TI, which significantly improves the prognosis of the disease.

As evidence, we give the following examples:

1. J. Artem, 3 months, amb. card No. 522, doctor's note: congenital toxoplasmosis, central chorioretinitis, convalescence of meningoencephalitis: in ELISA IgG antibodies in titer 400 IU / ml, in m-CSC IgG-3 in titer 1:60 was detected. An acute form of the infectious process was established, antiprotozoal therapy was prescribed, which led to an improvement in the condition of the child (the syndrome of vegetative-visceral disorders disappeared, weight growth rates improved).

2. L. Anya, 10 months, amb. card No. 144, dz: chronic toxoplasmosis, central chorioretinitis: IgA antibodies at 180 IU / ml were detected in ELISA, in m-CSC antibodies in the IgG-4 subclass in titer 1:20. The diagnosis of chronic toxoplasmosis has been confirmed.

3. O. Arthur, 13 years old, amb. card No. 105, dz: acquired toxoplasmosis, acute course, myocardial dystrophy. No IgM was detected in ELISA, IgG antibodies were detected in a titer of 300 IU / ml, IgG-3 in a titer of 1:80 were detected in m-CSC. The acute phase of acquired toxoplasmosis was diagnosed. Got a course of antiprotozoal therapy. After 3 months, antibodies were not detected in m-CSC, IgG antibodies were detected in ELISA at a titer of 800 IU / ml - they do not need antiprotozoal therapy.

The given examples indicate the high information content of the developed method for the diagnosis of acute and chronic phases of infection.

table 2 Children examined Including, the diagnosis is confirmed Abs % Total 94 one hundred BT, manifest fourteen 14.8 BT, latent course and residual manifestations 26 27.6 PT, acute course 21 22.3 PT, latent flow 33 35.1

Testing the method on the clinical material of children with a diagnosis of lymphadenopathy, subfebrile condition of unknown etiology, IUI of unspecified genesis showed its high efficiency. Positive results were obtained both at the initial stage of the development of the disease, and with an exacerbation of chronic infection.

The authors first proposed a method that allows the initial examination to determine the phase of infection and prescribe adequate therapy. The advantage of this method is its high specificity, accuracy, and the ability to use it both to detect primary infection and to exacerbate chronic.

The method developed by the authors can be recommended to clinicians for widespread use in healthcare practice to determine both acute and chronic forms of infection, followed by the appointment of etiological treatment.

Claims (1)

A method for diagnosing a toxoplasma infection in children by examining a patient’s blood, characterized in that immediately after the blood sampling, subclasses of IgG-3 and IgG-4 immunoglobulins are carried out by setting a modified complement binding reaction (m-CSC) and determining the level of IgG antibodies by enzyme-linked immunosorbent assay ( ELISA), and in the presence of antibodies in m-CSC in the IgG-3 subclass in titers 1: 10-1: 80 and the level of IgG antibodies in ELISA in a titer> 200 IU / ml, the acute form of the disease is established, and in the presence of antibodies in m- CSC in the IgG-4 subclass in credits 1: 20-1 : 50 and the level of IgG antibodies in ELISA <200 IU / ml establish a chronic form of the disease.