RU2231073C1 - Method for predicting severity degree in the flow of infectious faucial diseases in children - Google Patents

Abstract

FIELD: medicine, pediatrics.

SUBSTANCE: due to the method of radial immunodiffusion in gel one should determine the content of salivary immunoglobin A and its values being below o,4±0,05 mg/ml indicate severe flow of the disease mentioned.

EFFECT: more simplified, accelerated method at increased accuracy of prediction.

5 ex, 2 tbl

Description

The invention relates to medicine, namely to pediatrics, and may find application in medical treatment institutions for predicting the severity of infections of the oropharynx in children.

It is known that the entrance gate of the oropharynx infectious diseases is the mucosa of the oropharynx. Microorganisms (bacteria, viruses) are fixed on the cells of tissues, multiply and in the process of life produce toxic substances that have both local and general effects, which determine all the phenomena of the pathological process.

The initial stage of the infectious process in infectious diseases of the oropharynx is characterized by a specific interaction of the pathogen with the host cells. Secretory immunoglobulin A (sIgA) is the main mucosal secretion immunoglobulin that blocks the adhesion of microorganisms to epithelial cells, thereby providing local immune defense. The quantitative characteristic of sIgA allows us to assess the state of the barrier functions of the mucous membranes along vegetation, the persistence of the microbe and establish prognostic criteria for the course of the infectious process.

A search in the medical, scientific and patent literature revealed various methods for the diagnosis of diphtheria. The well-known “Method for the rapid diagnosis of diphtheria in children” - patent 2129276 Russia, IPC G 01 N 33/53 / Melnikova AV, Zheleznikova GI, Ivanova VV / publ. 04/20/99 bull. No. 11 by detecting diphtheria toxin in the composition of circulating immune complexes (CEC). The disadvantage of this method is the use of blood serum for research, which requires intravenous manipulations.

The well-known “Method for determining the course of toxic diphtheria” - patent 2135998 Russia, IPC G 01 N 33/52 / Ambalov Yu.M., Popolitova CB, Usatkin AV /, publ. 08/27/99 bull. No. 24 by evaluating the biochemical parameters of blood. The disadvantages of this method are that the material for the study is blood plasma, sIgA. A method based on the determination of enzyme activity requires a special study mode, on which the quality of the results depends.

The closest method for predicting the severity of diphtheria is the “Method for predicting the course of diphtheria infection in children” - patent 2146055 Russia, IPC G 01 N 33/569 / Ivanova VV, Bytsenko DS, Kvetnaya AS /, publ. . 02/27/2000 BIPM No. 6. This method is taken as a prototype. The method consists in determining the microbial resistance of the mucosa of the pharyngeal epithelium by detecting secretory immunoglobulin A based on the immunofluorescence reaction and determining the sensitivity of epithelial cells to adhesion of microbial cells. The disadvantage of this method is its complexity, due to the fact that the determination is carried out on epithelial cells in brashbioptata of the oral mucosa. For research use test strains of C. diphtheriae, the cultivation of which takes time. The method is expensive, because equipment requires: a brush from the endoscope, a set of reagents for chemiluminescence and enzyme immunoassay for the diagnosis of viral and bacterial infections.

The aim of the invention is to simplify, accelerate, improve the accuracy of predicting the severity of the course of infectious diseases of the oropharynx in children.

The goal is achieved by identifying and quantitatively characterizing sIgA in saliva by the Mancini method of radial immunodiffusion in a gel, based on measuring the diameter of the precipitation ring formed when the test drug was introduced into wells cut out in an agar layer in which monospecific antiserum was previously dispersed.

The method is as follows: At the initial examination, the child rinses the oral cavity with 50 ml of sterile distilled water, after which saliva is collected for 3 minutes using an automatic pipette with a tip. Saliva is collected in Eppendorf type microtubes. A brass U-shaped frame with a thickness of 1 mm is placed on 7 × 10 cm plates, and a second glass plate lubricated with a hydrophobic liquid is placed on top. The plates are fastened with clamps and 6 ml of the prepared mixture of agar with antiserum are poured into the space between the plates. After the mixture has hardened, the clamps are removed, the frame and the glass plate are removed, and in the agar layer, holes with a diameter of 2 mm are punched out at a distance of 15 mm from one another. 2 μl of the standard - purified secretory IgA with a protein concentration of 2 mg / ml, in dilutions of 1: 1, 1: 2, 1: 4, 1: 8 and the studied secrets are introduced into the wells with a microsyringe. The plates are incubated in a humid chamber for 24 hours at room temperature + 20 ° C. At the end of the incubation, the plates are washed for 2 days with physiological saline and stained with amide black. The diameter of the precipitation rings is measured with an accuracy of 0.1 mm with a compass gauge, a vernier scale or a special ruler manufactured by Beringwerke. Using a graph paper, a calibration curve is built, plotting on the ordinate axis the squares of the radius of the precipitation rings in mm obtained with different dilutions of the standard, and on the abscissa axis the concentrations of the standard - secretory IgA in mg / ml corresponding to these rings. To correctly interpret the results, each secret is examined in parallel with serum against serum IgA, using the same standard. When comparing the results obtained with these two antisera, the total IgA level in secret (the value determined with the serum against serum IgA) is judged, the level of secretory IgA (the value determined with the antiserum against secretory IgA) and the level of serum IgA (the difference between indicated values).

When the sIgA content in saliva is lower than in healthy children - 0.4 + 0.05 - a severe course of infectious diseases of the oropharynx is predicted.

Example 1. Patient K., 8 years old, was admitted to the children's infectious diseases department with a preliminary diagnosis: "Oropharyngeal diphtheria." Upon receipt in the saliva, 0.44 mg / ml sIgA was determined, which is higher than in healthy children. Final diagnosis: "Localized oropharyngeal diphtheria, mild."

Example 2. Patient A., 10 years old, was admitted to the children's infectious diseases department with a preliminary diagnosis: "Diphtheria of the oropharynx, toxic form." In saliva, the sIgA level was 0.15 mg / ml ”, which is twice lower than in healthy children. The final diagnosis: "Diphtheria of the oropharynx, toxic 1 degree, severe. Complication: myocarditis, NK 1 tbsp.".

Example 3. Patient Yu., 8 years old, was admitted to the children's infectious diseases department with a preliminary diagnosis: "Contact with a patient with diphtheria." In saliva, the sIgA level was 0.53 mg / ml, which is much higher than in healthy children. The final diagnosis: "Bacterial carriage of toxigenic diphtheria bacillus", which indicates a favorable course of infection.

Example 4. Patient D., 7 years old, was admitted to the children's infectious ward with a preliminary diagnosis: "Lacunar angina." In saliva, the sIgA level was 0.27 mg / ml, which is lower than in healthy children. The final diagnosis: "Lacunar angina of streptococcal etiology, severe form."

Example 5. Patient S., 5 years old, was admitted to the children's infectious diseases department with a preliminary diagnosis: "SARS, lacunar tonsillitis, lymphadenitis." In saliva, the sIgA level was 0.23 mg / ml, which is significantly lower than in healthy children. Final diagnosis: Epstein-Barr infectious mononucleosis etiology, severe. "

The proposed method was tested in 6 children’s infectious diseases department of the city hospital No. 1 of Rostov-on-Don for 133 patients with various forms of oropharyngeal diphtheria and 10 bacterial carriers. The results are presented in tables 1 and 2:

As can be seen from these tables, the carriers showed the highest sIgA in saliva, exceeding that in healthy children. With localized forms of oropharyngeal diphtheria, which are mild forms of infection, the content of sIgA in saliva is the same as in healthy children (there is no significant difference). In severe subtoxic, toxic forms, sIgA content is significantly lower than in healthy children. Significant differences are noted in the younger age group (3-7 years) (p <0.01). With the combined form of diphtheria of the oropharynx and larynx, the content of sIgA in saliva is sharply reduced, especially in children 7-14 years old, compared with normal values (p <0.01). Thus, the more severe the infection, the lower the sIgA in saliva.

The advantages of the proposed method are that the research material is saliva, in the simplicity of its collection. The method eliminates the long preparatory stage associated with a live culture of microorganisms, does not require a special regime for research, strict rules for septic and antiseptic. A quantitative method that is accurate.

Claims (1)

A method for predicting the severity of the course of infectious diseases of the oropharynx in children, characterized in that the amount of secretory immunoglobulin A in saliva is determined by the method of radial immunodiffusion in the gel and a severe course of the disease is predicted at values below 0.4 ± 0.05 mg / ml.