RU2402775C1 - Method of diagnosing early manifest congenital syphilis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for diagnostics of early manifest congenital syphilis values of cellular, humoral link of immunity and phagocytosis in acute period of disease are determined. If values of relative number of CD₈ ⁺ - subpopulation of T-lymphocytes are over 20.0% and phagocytic number of neutrophils is over 4.2 units with respect to control group, and if values of IRI (CD₄ ⁺/CD₈ ⁺) is lower than 2.9 and relative content of CD₉₅ ⁺ - lymphocytes is lower than 19.6%, than in control group, early manifest congenital syphilis is diagnosed.

EFFECT: acceleration and increase of accuracy of manifest form of ECS diagnostics.

3 tbl, 2 ex

Description

The invention relates to medicine, namely to infectious immunology, and can be used in pediatric infectology for the diagnosis of early congenital syphilis (PBC) manifest.

Syphilis is one of the classic sexually transmitted diseases, the causative agent of which can affect various organs and cause a cyclic, chronic infection [1]. In recent years, a steady increase in the incidence of syphilis has been recorded in Russia, which is accompanied by a regular increase in the incidence of congenital syphilis (BC) [2]. In turn, the peculiarity of syphilis transmission and the general low resistance of the fetus to infections explain the peculiarities of the course of the sun [3]. Cases of early congenital syphilis (PBC) with active manifestations and fatal outcomes have become more frequent [4].

Syphilitic infection still has not lost its scientific relevance, both in terms of epidemiological and diagnostic. However, despite the significance of this problem, many issues remain open and require further study. If several decades ago it was considered important to improve the specific treatment of syphilis, today interest has shifted to the study of serodiagnosis, the laws of the epidemiology of syphilis and the study of the immunopathogenesis of this infection [5].

The diagnosis of RVS is usually carried out taking into account the following main criteria [6]:

- medical history: lack of treatment or inadequate treatment of the mother;

- X-ray examination of long tubular bones;

- a higher positivity of serological reactions in the venous blood of a child in comparison with cord blood;

- a higher positivity of serological reactions in the venous blood of the child taken on the 7-8th day of treatment, in comparison with the blood of the mother;

- pathology in the cerebrospinal fluid;

- temperature reaction of exacerbation after the start of treatment;

- A positive IgM-ELISA with venous blood of the child (as an additional criterion).

The clinical manifestations of RVS depend on the timing of fetal infection and the premorbid state of the mother. The complexity of the interpretation of the diagnosis of PBC manifest is associated with multiple organ failure, and therefore with a variety of clinical manifestations, most of which are not pathognomonic and require differential diagnosis with other intrauterine infections (IUI) [7].

Currently, it is noted that PBC more often began to proceed in a latent form or with erased clinical manifestations. The latter are characterized by low-symptomness and scarcity of rashes, sometimes negative serological reactions and even negative immunofluorescence (RIF) reactions and the reaction of immobilization of pale treponemas (RIT), which complicates the diagnosis of the disease, especially in infants [8]. In case of monosymptomatic PBC, the only specific symptom of the disease is more often a lesion of the skeletal system (60%), or asymptomatic meningitis (neurosyphilis) with positive serological reactions in the cerebrospinal fluid. These clinical manifestations require x-ray examination and lumbar puncture, which may cause certain difficulties [7], while the diagnosis of PBC can be “missed” or regarded as “hidden”.

There is also a pathomorphism of the clinical manifestations of PBC manifest, which, according to many authors, could be due to the uncontrolled intake of readily available antibacterial drugs by the mother and early massive antibacterial therapy, which begins when a newborn is suspected of IUI in the maternity hospital [9].

Most researchers associate the possibility of verification of PBC with the need to detect specific class M immunoglobulins (JgM) by enzyme-linked immunosorbent assay (ELISA) or RIF-abs, which due to the selective permeability of the placenta are not detected in children in the absence of syphilis (with transplacental transmission of maternal antibodies). However, the quality of test systems for detecting IgM, currently used in Russia, does not allow us to fully rely on the result. Also, in practice, their identification may cause difficulties associated with the concentration of Jg data in serological reactions with class G antibodies. All of the methods proposed today for the removal of JgG cannot be adapted for practical use due to the extreme methodological complexity, complexity and high cost [ 8, 10, 11].

At the same time, despite the difficulties, an early diagnosis of PBC is necessary for the timely conduct of appropriate treatment - prophylactic or specific [6].

There is no doubt the key role of the immune system in the development of any pathological processes. The reactivity of the immune system largely determines the severity of any infection process. The various parts of the immune system are closely interconnected, and therefore the failure of one of them leads to the failure of the entire mechanism of immunoreactivity [12, 13].

It is known that with syphilitic infection in the human body, a specific inflammatory process occurs, non-sterile or infectious immunity is formed, which persists all the time the pathogen is in the patient's body [14, 15]. To date, the mechanism of immunity in syphilis has not been finally established, although it is obvious that all parts of the immune system participate in its formation [14, 16]. Their study in syphilis is relevant and necessary: to create a model of the interaction of the pathogen and the immune system, to study the characteristics of immune reactions in various clinical forms of infection in order to improve diagnosis and treatment, to determine the “immunological criteria” for cure for syphilis patients and the rationality of immunocorrective therapy [ 16].

Cellular immunity is one of the earliest responses of a human macroorganism to the introduction of pale treponema and plays a significant role in the formation of immune defense [14, 15]. At the same time, these roles of the humoral immunity in the pathogenesis of syphilis are contradictory [16, 17]. In recent years, studies on the immunological characteristics of patients in various periods of syphilitic infection [18], in pregnant women [14], HIV-positive individuals, and also with seroresistance after treatment of syphilis have begun to appear more and more often in the literature. At the same time, we did not come across studies on the immunology of PBC.

The objective of the invention: accelerating the diagnosis of PBC manifest.

The problem is solved due to the fact that when the values of the relative number of CD ₈ ⁺ - subpopulations of T-lymphocytes more than 20.0% and the phagocytic number of neutrophils more than 4.2 units. in relation to the control group, as well as when the IRI value (CD ₄ ⁺ / CD ₈ ⁺ ) is less than 2.9 and the relative content of CD ₉₅ ⁺ lymphocytes is less than 19.6% than in the control group, manifestative PBC is diagnosed.

The method is as follows.

A study of the cellular and humoral immunity, as well as phagocytosis.

The population and subpopulation composition of blood lymphocytes was evaluated using flow cytometry using FACS Calibur (Becton Dickinson, USA) and Simul Test IMK-Lymphocyte Kit reagents (USA). The analysis was performed using the Simul Set program.

The contents of CD ₃ ⁺ -, CD ₄ ⁺ -, CD ₈ ⁺ -, CD ₁₉ ⁺ -, CD ₁₆ ⁺ -, CD ₉₅ ⁺ - cells in the peripheral blood were studied. The ratio of CD ₄ ⁺ / CD ₈ ⁺ cells (immunoregulatory index (IRI)) was determined, which is used to assess the immune status of patients with autoimmune disorders, immunodeficiency or immune reactions. It is shown that the relative percentage of CD ₄ ⁺ subpopulation is reduced, and the relative percentage of CD ₈ ⁺ subpopulation increased in many patients with congenital or acquired immunodeficiency such as severe combined immunodeficiency and acquired immunodeficiency syndrome (AIDS). IRI, equal to 1.5-2.5, corresponds to a normal state, more than 2.5 - hyperactivity, less than 1.0 - immunodeficiency. In severe inflammatory processes, IRI can be less than 1.0. An increase in IRI to 3.0 is often observed in the acute phase of various inflammatory diseases due to an increase in T-helpers and a decrease in the level of T-suppressors. When the inflammatory process subsides, these indicators and their ratio normalize [19].

The concentration of immunoglobelins of classes A, M, G in the blood serum was determined by the method of radial immunodiffusion in a gel according to G. Mancini [20].

The content of circulating immune complexes (CIC) in blood serum was determined in a reaction with polyethylene glycol according to the method of V. Haskova et al. [21].

The phagocytic activity of peripheral blood neutrophils was studied in reaction with latex particles. The percentage of phagocytic neutrophils and the phagocytic number, i.e., the average number of particles absorbed by one neutrophil, were calculated.

Phagocytosis is the absorption by a cell of large particles visible through a microscope. The study of phagocytosis is important in the comprehensive analysis and diagnosis of immunodeficiency states. The most informative for assessing the activity of phagocytosis is considered the phagocytic number and the index of completion of phagocytosis [19].

The selection was made from 364 children aged 0 to 3 months who constituted a risk group for PBC. In this group, we identified 64 children with the highest probability of developing RVS. In 34 of them, RVS was diagnosed (in 16 children - manifest, and in 18 - hidden). Prevention treatment according to the PBC scheme was received by 30 children (with perinatal contact for syphilis), who made up the control group.

Statistical processing of the results of studies [22] revealed in children with RVS a manifest change in the functional activity of the T-cell immunity.

Table 1 Indicators of cellular immunity in children aged 0 to 3 months with manifestative PBC Control group (n = 30) PBC manifest (n = 16) CD ₃ ⁺ - cells (%) 71,567 ± 1,144 72.125 ± 1.712 CD ₃ ⁺ - cells, (10 ³ / μl) 4331.6 ± 295.724 3725.5 ± 145.942 CD ₃ ⁺ CD ₈ ⁺ , (%) 20, ± 1,026 23.625 ± 1.179
p <0.05 CD ₃ ⁺ / CD ₈ ⁺ 2.938 ± 0.213 2.174 ± 0.122
p <0.05 CD ₁₉ ⁺ - cells (%) 12,133 ± 1,136 13.813 ± 1.84 HLA-DR ⁺ - cells (%) 1.474 ± 0.14 2,313 ± 0,514 HLA-DR ⁺ - cells (10 ³ / μl) 80.579 ± 11.811 117.938 ± 25.705 CD ₉₅ ⁺ CD ₈ ⁺ - cells (%) 19.632 ± 2.928 11.875 ± 1.879
p <0.05 Note: p - significance of differences in the indicators of patients with RVS manifest relative to the control group

The results of immunological studies are presented in table. 1, show that in the group of children with manifestative PBC in the period before the treatment of the disease revealed a statistically significant increase, relative to the control group, of the percentage of CD ₃ ⁺ cells, expressed by an increase of more than 20.0% (1.2 times (p <0 , 05)) the relative content of CD ₈ ⁺ - a subpopulation of T-lymphocytes. A change in the ratio of CD ₄ ⁺ / CD ₈ ⁺ cells less than 2.9, compared with the parameters of the control group, led to a drop in IRI, which indicates a decrease in the functional activity of the T-cell immunity. Also revealed a significantly significant decrease in the percentage of CD ₈ ⁺ cells carrying the apoptosis induction receptor (CD ₉₅ ⁺ ), less than 19.6% (1.7 times (p <0.05)) than in the control group.

The CD95 antigen is also called Fas or APO-1, a transmembrane glycoprotein that is a member of the group of receptors for tumor necrosis factor. It is expressed in significant quantities on peripheral blood CD ₄ ⁺ and CD ₈ ⁺ T lymphocytes and, to a lesser extent, on B lymphocytes and NK cells. This antigen is also expressed on granulocytes, monocytes, tissue cells and neoplastic cells. Binding of CD95 to a Fas ligand (CD95L) induces apoptosis in cells [19].

A study of the phagocytic activity of peripheral blood neutrophils in children of this group revealed an increase of more than 4.2 units. the number of actively "working" cells against the background of their increased absorption capacity (Table 2).

table 2 Indicators of phagocytic activity in children aged 0 to 3 months with manifestative PBC Control group (n = 30) PBC manifest (n = 16) FI (%) 43,462 ± 1,182 46.933 ± 0.753 PS (units) 4.219 ± 0.087 4.847 ± 0.119
p <0.001 Note: p - significance of differences in the indicators of patients with RVS manifest relative to the control group

Indicators of humoral immunity, in particular JgM, confirm our data.

Table 3 Indicators of humoral immunity in children aged 0 to 3 months with manifestative PBC Control group (n = 30) PBC manifest (n = 16) JgM (g / l) 0.707 ± 0.079 1,053 ± 0,198
p <0.05 CEC (units) 22.05 ± 1.364 29.75 ± 6.556 Note: p - significance of differences in the indicators of patients with RVS manifest relative to the control group

From table 3 it is seen that the reaction of the humoral immunity was characterized by an increase in the level of JgM in blood serum in the group of children with PBC manifest compared with the parameters of the control group. The content of circulating immune complexes (CECs) in the blood was not significantly different from the control values for manifestative PBC.

Thus, the results of the studies established in children with RVS manifest in the period before treatment of the disease changes in the phenotypic composition of the circulating pool of lymphocytes. A marked increase in the percentage of CD ₈ ⁺ subpopulations of T-lymphocytes is noted against the background of a decrease in the relative content of CD ₈ ⁺ cells carrying the apoptosis induction receptor (CD ₉₅ ⁺ ) and a drop in IRI (CD ₄ ⁺ / CD ₈ ⁺ ). In addition, there was an increase in the phagocytic number of neutrophils relative to normal values.

The results obtained made it possible to determine statistically significant changes in indicators of the immune status in children with manifestative PBC and to identify quantitative and qualitative indicators characterizing this infectious process.

The proposed method can be used for early diagnosis of PBC manifest, which will facilitate the timely conduct of appropriate treatment - prophylactic or specific.

Example 1

Patient K., 10 days old, was transferred to an infectious diseases hospital from a maternity hospital with a diagnosis of congenital syphilis? From the medical history of the disease, it is known that the mother is 4 weeks old. During pregnancy, secondary syphilis was detected and she received a course of specific treatment with retarpenom. Preventive treatment during pregnancy was not carried out.

A girl was born from an urgent delivery by cesarean section. The mass at birth, 3850, screamed immediately, was not applied to the chest according to the severity of the condition. In a child’s maternity ward, blood for syphilis in the Wassermann (RV) 2+ reaction.

When translating, the child’s condition is severe, lethargic, squeaky cry, nutrition through a probe. An examination revealed severe hepatosplenomegaly, rhinitis, osteochondritis of the second century, anemia, signs of hypertension-hydrocephalic syndrome. Moreover, similar clinical manifestations can be observed with other IUIs. Examination of blood serum revealed sharply positive serological reactions to syphilis, but in the enzyme-linked immunosorbent assay ELISA-JgM neg. Given the severity of the condition of the child, lumbar puncture was not performed.

A survey of blood serum. Blood was drawn from a vein in the morning on an empty stomach in sterile hermetically sealed polymer tubes (vakuteynerov) containing anticoagulant:

- For the study of the cellular component of immunity and immunohematological studies - tubes with K ₃ EDTA (1.5 + 0.15 mg per ml of blood) - at least 2 ml of blood.

- For research of phagocytosis - test tubes with heparin - not less than 5 ml.

- For the study of the humoral link - “dry test tube” (without anticoagulant) - at least 2 ml.

Blood tubes gently swayed so that the anticoagulant on the walls of the tube dissolved in the sample. Blood tubes were placed in a container and delivered to the AIDS center, trying to avoid shaking along the way.

For each patient sample, 6 tubes A to F were labeled, 4 μl of antibodies were added to each of them: CD ₄₅ / CD ₁₄ ; negative control; CD ₃ / CD ₁₉ ; CD ₃ / CD ₄ ; CD ₃ / CD ₈ ; CD ₃ / CD ₁₆ + CD ₅₆ . At the bottom of each tube, 50 μl of blood is carefully added and mixed, then incubated for 20 min at room temperature in the dark. The lyse solution was diluted 1:10 with distilled water at room temperature and 0.5 ml was added to the test tubes, thoroughly mixed. The tubes were incubated for 10 min at room temperature in the dark, then centrifuged for 5 min at 1000 rpm. The supernatant was drained, 2 ml of Cell Wash (cell washer) was added and thoroughly mixed, then centrifuged again for 5 min at 1000 rpm. The supernatant was again drained, 0.35 ml of Cell Wash was added and mixed thoroughly. The prepared samples were analyzed on a FACS Calibur flow cytometer using the SimulSET program using FACS Calibur (Becton Dickinson, USA) and Simul Test IMK-Lymphocyte Kit reagents (USA). We studied the content of CD ₃ ⁺ -, CD ₄ ⁺ -, CD ₈ ⁺ -, CD ₁₉ ⁺ -, CD ₁₆ ⁺ -, CD ₉₅ ⁺ cells in peripheral blood. The ratio of CD ₄ ⁺ / CD ₈ ⁺ - cells was determined (immunoregulatory index (IRI)).

The concentration of immunoglobelins of classes A, M, G in blood serum was determined by G. Mancini method of radial immunodiffusion in a gel.

The content of circulating immune complexes (CIC) in serum was determined in a reaction with polyethylene glycol according to the method of V. Haskova et al.

The phagocytic activity of peripheral blood neutrophils was studied in reaction with latex particles. The percentage of phagocytic neutrophils and the phagocytic number, i.e., the average number of particles absorbed by one neutrophil, were calculated.

Examination by this method showed a pronounced increase in the percentage of CD ₈ ⁺ - subpopulations of T-lymphocytes to 25% and, accordingly, a decrease in IRI to 1.39. A decrease in the relative number of CD ₉₅ ⁺ lymphocytes to 13% and an increase in the number of actively "working" blood neutrophils with an increase in their absorption capacity to 5.3 units were also found.

Based on the medical history, clinical data and a comprehensive examination, the child was diagnosed with Early congenital syphilis with clinical manifestations (hepatosplenomegaly, osteochondritis of the II degree, rhinitis, anemia).

Example 2

Patient D., 17 days old, transferred from a somatic hospital with a diagnosis of congenital syphilis? From the medical history of the disease, it is known that the mother revealed latent syphilis 1.5 years before pregnancy and she received a course of specific treatment, preventive treatment was not carried out.

A boy was born from premature (at 31 weeks), independent birth. The mass at birth in 1650, screamed after resuscitation, was not applied to the chest. In the first day of life, according to the severity of the condition, due to prematurity and immaturity, he was transferred to the intensive care unit of the somatic hospital. When examined for IUI, congenital syphilis is suspected. After stabilization, the child was transferred to an infectious diseases hospital for further examination and treatment.

When translating, the condition of the child is closer to severe, lethargic, sucking is reduced. The examination revealed severe hepatosplenomegaly, rhinitis, osteochondritis of the second century, periostitis, rhinitis, anemia, signs of hypertension syndrome and depression syndrome. Positive serological reactions to syphilis were detected in the blood serum, but the reaction of Wasserman (RW) and ELISA-JgM neg. Moreover, the child has positive ELISA-JgG to herpes simplex virus and cytomegalovirus infection. From a lumbar puncture, the mother categorically refused.

The study of immunological parameters by this method showed a pronounced increase in the percentage of CD ₈ ⁺ - subpopulations of T-lymphocytes to 23.3% and, accordingly, a decrease in IRI to 1.42. Also found a decrease in the relative number of CD ₉₅ ⁺ lymphocytes to 13.9% and an increase in the number of actively "working" blood neutrophils with an increase in their absorption capacity to 4.8 units.

Based on the medical history, clinical data and a comprehensive examination, the child was diagnosed with Early congenital syphilis with clinical manifestations (hepatosplenomegaly, grade II osteochondritis, periostitis, rhinitis, anemia).

Thus, the results of a study of immunological parameters established in children with RVS manifest in the period before treatment of the disease changes in the phenotypic composition of the circulating pool of lymphocytes. A marked increase in the percentage of CD ₈ ⁺ subpopulations of T-lymphocytes is noted against the background of a decrease in the relative content of CD ₈ ⁺ cells carrying the apoptosis induction receptor (CD ₉₅ ⁺ ). An increase in CD ₈ ⁺ cells leads to a violation of the ratio

CD ₄ ⁺ / CD ₈ ⁺ - cells in the blood, which leads to a drop in IRI. This indicator indicates a decrease in the functional activity of the T-cell immunity. In addition, there was an increase in the number of actively "working" blood neutrophils and an increase in their absorption capacity.

Using the method for assessing immunological parameters in children at risk for PBC with sufficient accuracy will allow diagnosing PBC manifest in the early stages, which means that the necessary treatment of the disease can be timely and adequately carried out with the inclusion of appropriate immunocorrecting drugs in the complex of therapeutic measures.

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Claims (1)

A method for the diagnosis of early congenital syphilis manifest, including determining the values of the cellular, humoral immunity and phagocytosis in the acute period of the disease, characterized in that when the values of the relative number of CD ₈ ⁺ subpopulations of T-lymphocytes are higher than 20.0% and the phagocytic number of neutrophils is more than 4, 2 units in relation to the control group, as well as when the IRI value (CD ₄ ⁺ / CD ₈ ⁺ ) is less than 2.9 and the relative content of CD ₉₅ ⁺ lymphocytes is less than 19.6% than in the control group, they are diagnosed with PBC manifests.