Diagnostic technique for early congenital latent syphilis

FIELD: medicine.

SUBSTANCE: diagnosing of early congenital latent syphilis is enabled by determining the values of acute cell-mediated, humoral components of the immune system and phagocytosis. If the CD19+-lymphocyte percentage values exceed 12.1 as related to the reference group, the absolute and relative HLA-DR-antigen number values: more than 80.6×103/mcl and 1.5 % respectively, than in the reference group, and also a phagocytic neutrophil index is more than 48.2 %, latent ECS is diagnosed.

EFFECT: use of the method faster and more accurate diagnosing of latent ECS.

2 tbl, 2 ex

 

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

Syphilis is one of the classic venereal diseases, the pathogen which can infect various organs and cause cyclic, chronic infection [1]. In recent years Russia has registered a steady growth in the incidence of syphilis, which is naturally accompanied by increased incidence of congenital syphilis (CS) [2]. The cases of early congenital syphilis (RVS) with active manifestations and fatal outcome [3].

Syphilitic infection still has not lost its scientific relevance, both in terms of epidemiological and diagnostic. A new rise in the incidence in the 90-ies of XX century he proved himself a high proportion of hidden forms and their predominance among women and children, were once again forced to come back to this problem. However, despite its importance, many questions are still open and require further study. If a few decades ago recognized the important improvement of the specific treatment of syphilis, today the interest has shifted towards studying serodiagnosis, the patterns in the epidemiology of syphilis and study of the immunopathogenesis of this infection have been the AI [3, 4, 5].

The diagnosis of PBC decided to hold in accordance with the following criteria [6]:

- anamnesis data: no treatment or inadequate treatment of the mother;

- x-ray of the long bones;

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

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

- pathology in the liquor;

- temperature reaction exacerbation after the start of treatment;

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

Currently, it is noted that the PBC began to leak in a latent form or erased clinical manifestations. For the last characteristic malosimptomnoe and the scarcity of rash, sometimes negative serological reactions and even negative reactions immunofluorescence (RIF) and the reaction immobilization pale treponemes (RIT), which complicates the diagnosis of the disease, especially in infants [7]. When malosimptomna RVS manifest only specific symptom of the disease often defeat the skeletal system (60%)or asymptomatic meningitis (neurosyphilis) with positive serological reactions in the cerebrospinal fluid. the data of clinical manifestations require radiographic examination and lumbar puncture, what you may experience certain difficulties [8], the diagnosis of PBC can be "skipped" or regarded as "hidden".

Difficulties arise in the diagnosis of PBC hidden, which must be differentiated from transplacental transfer of antibodies from mother to fetus[1, 2, 9, 10]. Diagnosis "hidden RVS requires great responsibility and valid in either case, the slew of titles serological reactions during treatment or at higher titers of serum reactions in the neonate compared to the parent. Many serological reactions are not strictly specific and can give false positive results. Reliable diagnostic criteria domestic has come apart has not yet been.[1, 2, 8, 9, 10].

The possibility of verification RVS by most researchers associated with the need for detection of specific immunoglobulin M (JgM) enzyme-linked immunosorbent assay (ELISA) or REEF-abs, which is due to the selective permeability of the placenta are not detected in children in the absence of syphilis (transplacental transfer of maternal antibodies). However, the quality of test systems for the detection of IgM, currently used in Russia, it is not possible to rely on the result. Also in practice, the identification can occur SL is gnosti, related to concentration in the serological reactions of data Jg with antibodies of class g All of the currently proposed removal methods JgG may not be adapted for practical use due to the extreme methodological complexity, difficulty and high cost[7, 11, 12].

At the same time, despite the difficulties, early diagnosis RVS for timely appropriate treatment - and-prophylactic 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. Different parts of the immune system are closely interrelated, and therefore failure of one of them leads to the disruption of the whole mechanism of immunoreactivity [13, 14].

It is known that syphilis infection in the human body there is a specific inflammatory process, is formed of non-sterile or infectious immunity, which continues all the time the pathogen in the body of the patient [10, 15]. To date, the mechanism of immunity in syphilis definitively not installed, although it is obvious that in its formation involves all parts of the immune system [10, 16]. Their research in syphilis what is relevant and necessary: to create a model of the interaction of the pathogen and the immune system, for studying immune responses in different clinical forms of infection with the aim of improving diagnosis and treatment, to determine the "immunological criteria of cure of syphilis and rationality of holding immunocorrective therapy [16].

Cellular immunity is one of the earliest responses of the organism of a person on the introduction of pale Treponema and plays a significant role in the formation of immune protection [10, 15]. However, details of the role of humoral immunity in the pathogenesis of syphilis controversial [16, 17]. In recent years, the literature increasingly began to work on the study of the immunological characteristics of patients in different periods of syphilis infection [18], in infected pregnant women [14], HIV-positive individuals, as well as seroresistant after treatment of syphilis. While we have not met the study of immunology RVS.

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

The task to solve due to the fact that when the values of the percentage oflymphocytes over 12.1% compared to the control group, the absolute and relative number of HLA-DR antigen: more 80,6×103/μl and 1.5%, respectively, than in the control group, as well as the phagocytic index of neutrophils more or 48.2%, diagnose R. Is With the hidden.

The method is as follows.

Conducted a study of cellular and humoral immunity and phagocytosis.

Population and subpopulation structure of blood lymphocytes was assessed using the method of flow cytofluorometry using FACS Calibur (Becton Dickinson, USA) and reagents Simul Test IMK-Lymphocyte Kit (USA). Analysis was performed using the program Simul Set.

Examined the contents,,,,,-cells in the peripheral blood. To determine the ratiocells (immunoregulatory index (IRI), which is used for assessing the immune status of patients with autoimmune disorders, immunodeficiency or immune reactions. It is shown that the relative percentagesubpopulations are reduced, and the relative percentagesubpopulation is increased in many patients with congenital or acquired immunodeficiency States, such as severe combined immunodeficiency and acquired immunodeficiency syndrome (AIDS). IRI is equal to 1.5 to 2.5, corresponds normalizename state, more than 2.5 - hyperactivity, less than 1.0 - immunodeficiency. In severe inflammatory process IRI can b shall be less than 1.0. The increase IRI to 3.0 is often observed in the acute phase of various inflammatory diseases by enhancing T-helper cells and reduced T-suppressors. When the subsidence of the inflammatory process, these indicators and their normalized ratio [19].

The concentration of immunoglobulines classes A, M, G in the serum was determined by radial immunodiffusion in gel on G.Mancini [20].

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

Phagocytic activity of neutrophils in the peripheral blood was studied in reactions with latex particles. Expected percentage faguoqitirute neutrophils and phagocytic number, i.e. the average number of absorbed single neutrophil particles.

Phagocytosis - the absorption cell of large particles that are visible in the microscope. The study of phagocytosis indices of importance in complex analysis and diagnosis of immunodeficiency. The most informative for assessing the activity of phagocytosis believe phagocytic number and the index of completeness of phagocytosis [19].

The selection was made from 364 children aged 0 to 3 months, constituting a risk for PBC. In this group we identified 64 children with the greatest likelihood of RSU 34 of them are diagnosed with PBC (16 children - manifest, and 18 - hidden). Professor who completed the treatment according to the scheme RVS received 30 children (perinatal contact with the syphilis), who formed the control group.

Statistical processing of the results [22] found in children with RVS hidden changes in the functional activity of T-cell immunity.

Table 1.
Indicators of cellular immunity in children aged 0 to 3 months with RVS hidden
Control group(n=30)RVS hidden (n=18)
CD3+cells, %71,567±1,14468,353±1,531
CD3+cells, 103/µl4331,6±295,7243658,529±255,912
CD3+CD8+, %20,±1,02619,667±1,561
CD4+/CD8+2,938±0,2132,94±0,3
CD19+cells, %12,133±1,13616,882±1,695
p<0.05
HLA-DR+cells, %1,474±0,142,471±0,286
p<0.01
HLA-DR+cells, 103/µl80,579±11,811131,529±16,965
p<0.05
CD95+CD8+cells, %19,632±2,92824,941±3,178
Note: p - significance of differences in characteristics of patients with PBC hidden relative to the control group.

The results of immunological studies presented in table 1 show that in the group of children with RVS hidden in the period before the treatment of the disease is determined by the statistically significant increase of the percentage oflymphocytes over 12.1% relative to the control group. In addition, the increase in the absolute number and the percentage of lymphocytes expressing the activation marker HLA-DR is more of 80.6×103/μl and 1.5% respectively. Also, according to table 2, there is a growing percentage of neutrophils in the peripheral blood of more than 48.2% of relative values of the control group.

CD19 antigen is a glycoprotein, risotti on all peripheral b-lymphocytes, and all predecessors of b-cells. He is absent on plasma cells. Is the earliest marker of b-cells and plays an important role in regulating the activation and proliferation of b-lymphocytes [19].

HLA-DR - antigen is a monomorphic determinant of molecules of class II HLA. The marker is expressed on Langerhans cells, dendritic cells of lymphoid organs, certain types of macrophages, b-lymphocytes, activated T-cells and epithelial cells of the thymus. In the clinic the definition of token use for the quantitative determination of activated T-lymphocytes with the phenotypeHLA-DR+[19].

Table 2.
Indicators of phagocytic activity in children aged 0 to 3 months with RVS hidden
Control group (n=30)RVS hidden (n=18)
PHI (%)43,462±1,18248,222±1,059
p<0.05
FC (%)4,219±0,0874,444±0,105
Note the tion: p the significance of differences of characteristics of patients with PBC hidden relative to the control group.

Thus, the results of these studies have found that children with hidden RVS in the period before the treatment of the disease, an increase in the content in peripheral blood lymphocytes bearing a late marker of activation (HLA-DR) and increased percentage of cells expressing-antigen. In addition, increased phagocytic number of neutrophils relative to normal values.

The results obtained allowed to identify statistically significant changes of immune status in children with hidden RVS and identify quantitative and qualitative indicators of this infectious process.

The proposed method can be used for early diagnosis of PBC hidden that will facilitate timely implementation of appropriate treatment - and-prophylactic specific.

Example 1.

Patient A., 7 day, transferred to the infectious diseases hospital from the hospital with a diagnosis of congenital syphilis? From the anamnesis of the disease it is known that the mother revealed latent syphilis in 32 weeks. pregnancy and she received a course of specific treatment and prophylactic treatment is not finished, as entered in childbirth.

Begot whom I am a boy from premature (36 weeks), independent childbirth. Weight at birth 2485,, cried at once, to his chest was applied on the second day.

When translating the child's condition is moderate. When examining clinical manifestations of syphilis are not identified. In the study of blood sera found positive serological reaction for syphilis, but negative ELISA JgM and increased titers of serum reactions in the dynamics. From lumbar puncture mother flatly refused. These results can also be regarded as a transplacental transfer of maternal antibodies to the fetus.

A survey of serum. The blood from the veins carried out in the morning on an empty stomach in sterile hermetically sealable polymer tubes (vacutainer)containing anticoagulant:

For studies of cellular immunity and holding immunohematological research - tubes with K3EDTA (1,5+0.15 mg per ml of blood) - at least 2 ml of blood.

- To study the phagocytosis - heparinized tubes is not less than 5 ml

To investigate the humoral - dried test tube" (without anticoagulants) - not less than 2 ml

Test tubes with blood gently rocking to the anticoagulant in the tube wall dissolved in the sample. Tubes of blood were placed in a container and transported to the AIDS centre, trying to avoid shaking in the path.

D. the I of each patient sample was labeled with 6 tubes from A to F, in each of which wesli 4 μl of antibody: CD45/CD14; negative control; CD3/CD19; CD3/CD4; CD3/CD8; CD3/CD16+CD56. At the bottom of each tube is carefully added to 50 µl of blood and mixed, then incubated for 20 min at room temperature in the dark. Bred lyse solution 1:10 with distilled water at room temperature and was added to 0.5 ml in the test tubes were thoroughly stirred. The tubes were incubated 10 min at room temperature in the dark, and then centrifuged 5 min at 1000 rpm was Decanted supernatant was added to 2 ml of Cell Wash (wash cells) and thoroughly mixed, then centrifuged 5 min at 1000 rpm was Again decanted supernatant was added 0.35 ml Cell Wash and thoroughly mixed. Prepared samples were analyzed on a flow cytometer FACS Calibur using SimulSET using FACS Calibur (Becton Dickinson, USA) and reagents Simul Test IMK-Lymphocyte Kit (USA). Examined the contents,,,,,-cells in the peripheral blood. To determine the ratiocells (immunoregulatory index (IRI)).

The concentration of immunoglobulines classes A, M, G in serum identify elali by radial immunodiffusion in gel on G.Mancini.

The content of circulating immune complexes (CIC) in the serum was determined in the reaction with ethylene glycol by the method of V. Haskova et al.

Phagocytic activity of neutrophils in the peripheral blood was studied in reactions with latex particles. Expected percentage faguoqitirute neutrophils and phagocytic number, i.e. the average number of absorbed single neutrophil particles.

Examination of blood serum on the proposed method showed higher percentagelymphocytes up to 15%, and the absolute and relative number of HLA-DR antigen to 242×103/µl and 3%, respectively. It is also noted the increased percentage of neutrophils (54%)involved in phagocytosis.

On the basis of medical history, clinical data and the comprehensive examination the child was diagnosed with Early congenital syphilis hidden.

Example 2.

Patient M, 5 d, transferred to the infectious diseases hospital from the hospital with a diagnosis of congenital syphilis? From the anamnesis of the disease it is known that the mother revealed latent syphilis for 2 years before pregnancy and she received a course of specific treatment, prophylactic treatment of a woman was not conducted.

A girl was born from term (37 weeks), independent childbirth. Weight at birth 2655, cried out at once, applied to the chest on the second day.

<> When translating the child's condition is moderate. When examining clinical manifestations of syphilis are not identified. In the study of blood sera found positive serological reaction for syphilis, but negative ELISA JgM, against the background of a specific therapy was positive.

Examination of blood serum on the proposed method showed higher percentage-lymphocytes to 14.5%, and the absolute and relative number of HLA-DR antigen 214×103/ál and 2.5% respectively. It is also noted the increased percentage of neutrophils (52%)involved in phagocytosis.

On the basis of medical history, clinical data and the comprehensive examination the child was diagnosed with Early congenital syphilis hidden.

Thus, the results of the immunological parameters established in children with hidden RVS in the period before the treatment of the disease increased concentration of peripheral blood lymphocytes bearing a late marker of activation (HLA-DR) and increased percentage of cells expressing-antigen. In addition, increased phagocytic number of neutrophils relative to normal values.

Use of the method of assessment of immunological parameters in children at risk for RVS with sufficient the th accuracy will allow for early diagnosis of PBC hidden, so timely and adequately perform the necessary treatment of the disease with the inclusion in the complex of therapeutic measures relevant immunocorrective drugs.

Sources of information

1. Senchuk YA Perinatal infections: Pratic. the textbook / Under the editorship Ayacanora, Simgurusarah. - M.: OOO "MIA", 2005. - Pp.28-47.

2. Volodin N.N. Actual problems of Pediatrics / edited Way. - Moscow: GEOTAR Medicine, 2004. - S-298.

3. Tikhonov LI General overview of the situation of sexually transmitted infections. Analysis of the incidence of congenital syphilis in the Russian Federation / Liikunta // Vestnik St.Petersburg University. dermatol. and venerol. - 1999. No. 2. - P.4-8.

4. Hilippe A. Guide to childhood illnesses: TRANS. with it. / Grancini, Amalgram. - M.: Medgiz, 1960. - S-476.

5. Novoselov V. S. serodiagnosis of syphilis: the doctor's choice / Voskopoulos, Awholesale // Almanac the clinically. the honey. - 2007. No. 15. - P.75-78.

6. Order of the Ministry of health of the Russian Federation of 30 July 2001, No. 291 "on measures to prevent the spread of sexually transmitted infections" (in edition of Order of Ministry of health of the Russian Federation dated 15.11.2001 No. 411).

7. Milevska I.R. OF early congenital syphilis in newborns / Yermilovsky, Iagrlinw, Lamlein // Ukr. obstetrics and female diseases. - 2004. No. S4. - P.59-60.

8. Tereshchenko NR. To the question about the current course of early congenital syphilis and TB diagnosis / Vnte the Eschenko, Mpereira, Lyuberitskaya etc. // Pacific honey. Journe. - 2003. No. 4. - P.45-46.

9. Tereshchenko NR. Pathology of the nervous system in children born to women infected with pale Treponema / Vinereanu, NAV, Won etc. // Pacific honey. Journe. - 2003. No. 4. - P.61-63.

10. Ivanov A. M. Serological diagnosis of syphilis: the possibility of increasing the informativity / Amiano, Aphrodisiac, Intelecto etc. // Ukr. obstetrics and female diseases. - 2004. No. S4. - P.63-64.

11. Prokhorenkov VI Syphilis. Illustrated guide / Under. editor Prof. Viera.holecova. - M: Medical book, 2002. - S-98.

12. Zheleznyakov GF Value of immunological research in infectious diseases in children / Grazalema, VNV // Baby infections. - 2007. - vol. 6. No. 3. - P.7-16.

13. Zhukovskaya WAS the Use of such drugs in the treatment of chronic cervicitis: author. dis.... Kida. the honey. Sciences / Igusa. - Izhevsk, 2007. - 24 S.

14. O.V. Pankratov Dynamics of some indicators of the immune system in pregnant women, patients with syphilis / Overate // Honey. news. - 2006. No. 6. - P.114-119.

15. Rodionov, AN. Syphilis // QuickStart. - SPb: Peter, 2007. - 320 S.

16. Prokhorenkov V.I. Modern ideas about infection when syphilis / Vierkorn, Uphoria // Clinically. dermatol. and venerol. - 2005. No. 4. P.4-8.

17. Borisenko K. the Role of some factors of humoral and cellular immunity in the pathogenesis of syphilis / Kit, Emissionsa, Wavering etc. // Vestnik St.Petersburg University. dermatol. and venerol. - 1984. No. 6. - P.30-34.

18. Borisenko K. Clinical and immunological characteristics of patients with syphilis / Kit, Nambassa, Naihai // Vestnik St.Petersburg University. dermatol. General. - 1985. No. 7. - P.24-27.

19. Kiskun A.A. Immunological and serological investigations in clinical practice / ACO. - M.: OOO "MIA", 2006. - 536 S.

20. Manchini G. Immunochemical quantitation of antigens by single radial diffusion / G.Manchini, A.O.Carbonaro, J.F.Haremans // Immunochemistry. - 1965. - Vol.2, No. 3. - P.235-254.

21. Haskova V. Simple method of circulating immune complex detection in human sera by polyethylene glycol precipitation / V.Haskova, J.Kaslik, J.Riha // J.Immunol. - 1978. Vol.154, N 8. - P.399-406.

22. Glanz S. biomedical statistics: Per. from English. - M.: Practice, 1998. - 495 S.

A method for the diagnosis of early congenital syphilis latent including the determination of the values of the cellular, humoral immunity and phagocytosis in the acute period of the disease, characterized in that, when values of percentagelymphocytes over 12.1% compared to the control group, the absolute and relative number of HLA-DR antigen: more 80,6·103/μl and 1.5%, respectively, than in the control group, as well as the phagocytic index of neutrophils more or 48.2%, diagnose hidden RVS.



 

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1 ex

FIELD: medicine.

SUBSTANCE: differentiated detection large and small circulating immune complexes in blood serum is ensured by a follow-on examination of blood serum clarified by short centrifugation with a method of circulating immune complexes precipitation PEG -6000 of the end concentration 4 % and 6 %. Thereafter, the results are recorded by a turbidimetric method with using a microplate spectrophotometer in a two-wave mode: basic - 340 nm, reference filter - 620 nm. Duration of an incubation step at temperature +18-25°C is 15 minutes. Using the method enables higher effectiveness and reliability of determining the level of large circulating immune complexes, results reproducibility, as well as differential measurement of the level of small CIC which are a diagnostically significant indicator of human body immune responsiveness in many types of a pathology, decreased volume of analysed serums to 0.06 ml, and incubation duration to 15 minutes.

EFFECT: method is suitable for clinical screenings.

1 dwg, 1 tbl, 1 ex

FIELD: measurement equipment.

SUBSTANCE: invention refers to biophysics. In order to determine calcium concentration on the basis of discharged photoproteins, bioluminescent reaction of photoproteins and calcium ions is performed, intensity of the solution fluorescence is measured, calibration dependence of fluorescence intensity on calcium concentration in double logarithmic coordinates is built and logarithm value of calcium concentration is determined as per the logarithm of the measured fluorescence intensity. Fluorescence is initiated with the light source after bioluminescent reaction is completed. Fluorescence intensity is measured at the specified length of excitation and recording wave.

EFFECT: method allows determining calcium concentration in various media as per fluorescence intensity of discharged photoproteins, which allows performing continuous measurements of calcium concentration in "in vivo" system.

2 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: when admitted, an acuity patient is analysed for total protein concentration and creatine phosphokinase activity in blood serum. If the analysed values are within normal limits, the absence of skeletal muscles injures is diagnosed; total protein concentration being within normal limits and creatine phosphokinase hyperactivity indicate the presence of an accompanying injury of the skeletal muscles, while lowered total protein concentration with underlying creatine phosphokinase hyperactivity shows progressing hypoproteinemia.

EFFECT: method provides pre-clinical detection of the presence of the accompanying injures of the muscles and progressing hypoproteinemia.

3 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular toxicology and resuscitation science and can be used for early prediction of pneumonia development in patients. On the first day of staying in hospital functional state of albumin in blood serum is analysed in patients by fluorescent method. Intensity of K-35 probe fluorescence in blood serum albumin in medium with high ionic power is determined. If value of K-35 probe fluorescence intensity in albumin is lower than or equals 36 conv.units, development of pneumonia in patient is predicted.

EFFECT: method allows to increase efficiency of performed treatment in said category of patients.

2 tbl, 4 ex

FIELD: medicine, analytical biochemistry.

SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.

EFFECT: improved assay method.

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