RU2329507C1 - Method of acute bacterial enteric infections express-diagnostics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medical diagnostics. For purpose of acute bacterial enteric infections diagnostics, lymphocytic suspension is tested for acute enteric infection (AEI) agent antigen by indirect immunoperoxidase method. Centrifugated blood cells are applied on glass slide (smear), dried up at room temperature, fix in pure acetone, and then processed in 3% H₂O₂ for 20 minutes. Blood cells are incubated in blocking normal serum, incubated with poly- or monoclonal antibodies to required antigenes at t 37°C. Then they are processed with reagents of polymeric detection system: Super Enhacer™ Reagent and further with Poly-HRP Reagent. Then they are processed with 3,3-diaminobenzidine-tetrachloride (DAB). Brown granules in lymphocytes and monocytes indicate required antigen, namely infection diagnostics.

EFFECT: method application provides increased accuracy and reduced time of diagnostics of acute bacterial enteric infections.

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Description

This invention relates to the field of medical diagnosis, namely the detection of bacterial infections

Acute intestinal infections (acute intestinal infections) are widespread among all age groups, while occupying a significant place in the structure of infant mortality. AEI is a group of diseases of various etiologies (dysentery, salmonellosis, typhoid fever, intestinal coli infection, etc.). In the United States, 6.5 million cases of foodborne infections and over 9,000 deaths are reported annually. Among bacteriologically deciphered cases, salmonellosis followed by shigellosis (G.I.Osipova, 2002, Kilgore P.E., 1995) came first. These diseases of bacterial etiology combine a similar pathogenesis (bacteremia / antigemia, exposure to toxins, metabolic disorders) and similar clinical manifestations - intestinal syndrome, exicosis, toxicosis (T.E. Ivanovskaya, L.V. Leonova, 1989), which complicates their clinical differentiation. To confirm the diagnosis, bacteriological research methods are used: stool, blood, vomit cultures, as well as serological methods to detect the dynamics of the level of specific antibodies to bacteria (V.V. Ivanova, 2002). Due to the enormous epidemiological significance of intestinal infections, accelerated diagnostic methods for the appointment of adequate etiotropic and pathogenetic therapy are of particular importance.

A known method for detecting bacterial infections is the reaction of indirect hemagglutination of RNGA.

1. In 10 holes of a row make 0.5 ml of a 9% solution of sodium chloride.

2. 0.5 ml of the test serum diluted 1:25 is added to the 1st well of the series and a series of double dilutions from 1:50 to 1: 6400 is made by transferring 0.5 ml from the well to the well and mixing them for at least 3 -4 times.

3. To each well add 0.2 ml of a 1% working suspension of the diagnosticum. At the same time they put in control.

4. The plates are shaken slightly and incubated at a temperature of 37 ° C for 1.5-2 hours

5. Incubation at room temperature for 14-18 hours

6. Evaluation of the results

This method allows you to get good results. However, there are circumstances that reduce its effectiveness: it takes a lot of time (24 hours), is time-consuming and requires additional equipment (microplates and a spectrophotometer) to obtain results, it is impossible to store and photograph the preparations for a long time. In some cases, the determination of serological markers is ineffective: in newborns due to an unformed immune system, with inhibition of the immune system and in the initial stage of the disease, since antibodies are formed delayed.

Closest to our proposed method is the "Method for the diagnosis of hemophilic infection in histological preparations" patent for invention No. 21932004 from 11/20/02, R. A. Nasyrov, A. S. Kvetnaya, M. V. Mankov.

1. Histological paraffin sections of tissues are dewaxed in three portions of xylene, 15 minutes each, at a temperature of 60 ° C.

2. Wash in phosphate-buffered saline (pH 7.2-7.4) for 3-4 minutes.

3. Treat with a 0.1% solution of trypsin in 0.1% CaCl ₂ for 20 minutes.

4. Process 0.03% hydrogen peroxide for 20 minutes.

5. Wash in two portions of phosphate-buffered saline (pH 7.2), 5 minutes each.

6. Incubate in 0.3% TritonX-100 at a temperature of 37 ° C for 15 minutes.

7. Apply polyclonal rabbit antibodies to ribosylribitolphosphate. Incubate with polyclonal antibodies at 37 ° C for 45 minutes.

8. Washed in water for 5 minutes.

9. Wash in two portions of phosphate-buffered saline (pH 7.2), 5 minutes each.

10. Apply DAKO En Vision + System, HRP anti-species conjugate and incubate for 45 minutes in a dark chamber at room temperature.

11. Wash in two portions of phosphate-buffered saline (pH 7.2), 5 minutes each.

12. The material is treated in an aqueous solution containing 0.05% 3,3-diaminobenzidine tetrachloride (DAB) and 0.025% hydrogen peroxide for 3 minutes.

13. Weakly stain with hematoxylin.

14. Microscopy.

This method allowed the authors to get good results. However, the disadvantage of this method is that it is traumatic, since biopsy samples serve as the material for the study. Carrying out all the steps of the method is quite long in time. Taking the biopsy specimen, fixing it, pouring it into paraffin, preparing histological sections takes at least 6-7 hours and more than 5 hours is spent on the immunoperoxidase reaction. Due to the high background staining when working with paraffin sections, the accuracy of the result is reduced.

The elimination of these disadvantages can be helped by the authors proposed method for the diagnosis of acute intestinal infections, based on the identification of pathogen antigens.

The technical result of the invention is to reduce the time and increase the accuracy of diagnosis. This result is achieved in that the antigen of the OCI pathogen is determined by the indirect immunoperoxidase method and, according to the invention, blood cells (lymphocyte suspension in a volume of 0.05 ml) obtained by centrifugation are applied to a glass slide (smear), dried at room temperature, fixed in pure acetone - 3-5 min, and then treated with 3% H ₂ O ₂ for 20 minutes, incubated with normal blocking serum (10 minutes), incubated with poly- or monoclonal antibodies to desired antigens at 37 ° C for 45 minutes, incubating t with a polymer detection system (Super Enhacer for 20 min in a dark chamber at room temperature, incubated with SS Label for 30 min in a dark chamber at room temperature), then 3,3-diaminobenzidine is treated with tetrachloride (DBA) and the presence of brown granules in lymphocytes and monocytes indicates the identification of the desired pathogen antigen (diagnosis of infection) for the timely appointment of etiotropic and pathogenetic therapy. The method is well reproducible, which indicates its reliability.

The standard scheme of the indirect immunoperoxidase method is described by J. Polak, S. Van-Norden. Introduction to immunocytochemistry. M .: Metsitsina, 1986

1 - washing sections in the buffer;

2 - treatment with a 0.03% hydrogen peroxide solution;

3 - washing in buffer;

4 - application to the slice of the first specific antibodies;

5 - washing in buffer;

6 - application of second antibodies associated with peroxidase;

7 - washing in buffer;

8 - treatment with a 0.03% solution of diaminobenzidine (DAB);

9 - dyeing with hematoxylin;

10 - the conclusion of the balm. Microscopy.

But as the literature data and our own experience show, the above scheme only in general terms reflects the progress of the methodology. Depending on the size, structure and nature of the detected antigen, a detailed development of all stages is necessary, from fixing to applying diaminobenzidine.

The method proposed by the authors is characterized in that lymphocyte suspension was used for the first time to identify antigens of the causative agents of acute intestinal infections as an object of study.

Empirically, it was found that 0.05 ml of lymphocytic suspension is required as the minimum sufficient amount for smear preparation, immunocytochemistry and adequate assessment of the result.

In the framework of the proposed method, the first treatment of smears with a more concentrated (3%) hydrogen peroxide solution is proposed, which contributes to a more complete and high-quality blocking of endogenous peroxidase, which is especially important when using lymphocytic suspension.

One of the difficult problems is the possible presence of non-specific (background) antibodies in serum. To eliminate the non-specific reaction, treatment with normal (blocking) serum is proposed before applying the first antibodies.

The use of the Super Sensitiv Polimer-HRP Detection System polymer complex produced by BioGenex as a more sensitive polymer system made it possible to use specific antibodies in low concentrations and to minimize the background of non-specific staining, which could not be achieved with conventional detection systems. In addition to the above, this detection system does not contain avidin and biotin, therefore, nonspecific staining during the interaction of avidin with endogenous biotin is excluded. Blood is a complex and sensitive object during immunohistocytochemical studies. In this regard, the proposed new method for the diagnosis of acute intestinal infections, based on the identification of pathogen antigens in blood cells, requires a set of developed methodological techniques. Failure to comply with the latter does not give objective results or distorts the final result of research, and does not allow an appropriate interpretation of the data obtained.

The proposed method is as follows.

1. Lymphocytes and monocytes obtained by centrifugation of peripheral blood are applied to a glass slide (smear).

2. Dry at room temperature.

3. Fixation in pure acetone 3-5 minutes.

4. Wash in phosphate-buffered saline (pH 7.4) for 3-4 minutes.

5. Processing in 3% H ₂ O ₂ 20 minutes.

6. Rinsing in two portions of phosphate-buffered saline (pH 7.4), 5 minutes each.

7. Incubation in normal blocking serum (Power Block) 10 minutes.

8. Application of smears of poly - or monoclonal antibodies to the desired antigens. Incubation with these antibodies at 37 ° C for 45 minutes.

9. Rinse in water for 5 minutes.

10. Rinse in two portions of phosphate-buffered saline (pH 7.4), 5 minutes each.

11. Application on smears Super Enhacer. Incubation for 20 min in a dark chamber at room temperature.

12. Washing in two portions of phosphate-buffered saline (pH 7.4), 5 minutes each.

13. Application on smears SS Label. Incubation for 30 min in a dark chamber at room temperature.

14. Processing the material in an aqueous solution containing 0.05% 3,3-diaminobenzidine tetrachloride (DAB) and 0.025% hydrogen peroxide for 3 minutes.

15. Weak coloring with hematoxylin.

16. Microscopy.

Microscopy of stained preparations is carried out in a conventional microscope. The reaction product in the study of lymphocytes and monocytes containing the desired antigen is detected in the form of brown granules intracellularly and on the cell membrane. When evaluating microscopic findings, it is important to consider the occurrence of artifacts that mimic a positive coloration. Most often, these are cellular artifacts that are detected along the edge of the smear. In this case, an assessment of the reaction in neighboring areas is necessary.

Necessary controls for antibodies used

To obtain reliable results and to exclude the possibility of nonspecific reactions, a prerequisite is to carry out controls on the quality of the reagents and the specificity of the antiserum.

1) To control the specificity of the antiserum, a non-immune serum or buffer is applied as the first layer (the results must be negative).

2) Serum non-specificity is eliminated by maximally diluting highly concentrated serum and shortening the incubation time. The described method is supported by examples of specific performance.

A study of the material of 20 patients with acute intestinal infection in the first 3 days from the onset of the disease was carried out, in 8 of them the expression of the Salmonella complex antigen was detected, in 2 Sh. Sonnei, in 5 Sh. Flexneri as a monoinfection; in 2, Sh antigens were determined. Flexneri and Salmonella, in 2 Sh. Sonnei and Salmonella and in one Sh. Flexneri and Sh. Sonnei. The results obtained completely coincided with the data of serological studies and clinical manifestations. In all cases, the results of a study to identify antigens of the causative agents of acute intestinal infections were obtained after 4 hours from the moment of receipt of the material

As an example, we give a micrograph of a smear of lymphocytic suspension with clearly defined expression of the Salmonella complex antigen in lymphocytes and monocytes in a patient Zvyagintsev, 4 years old, case history No. 5650, Federal State Research Institute for Nuclear Research Roszdrav, St. Petersburg dated October 12, 2006, with severe manifestations of acute intestinal infection (figure 1).

Micrographs of a smear of lymphocyte suspension with clearly defined expression of Sh. Flexneri in the lymphocytes and monocytes of a patient D. Plykays, 2 years old, case history No. 3347, DIB No. 3, St. Petersburg dated 03.11.2006, with a clinic of acute intestinal infection (figure 2).

Microphotographs of a smear of lymphocytic suspension with pronounced expression of Sh. Sonnei in the lymphocytes and monocytes of the patient A. Efremov, 4 years old, case history No. 3210, DIB No. 3, St. Petersburg dated 10.27.2006, with a clinical presentation of a severe course of acute intestinal infection (Fig. 3).

As a control group, a lymphocytic suspension of two patients with whooping cough and one patient with herpetic neuroinfection was studied. In all cases, antigens Salmonella, Sh. Flexneri, Sh. Sonnei were not detected.

Thus, the proposed method allows with a high degree of specificity and reliability to detect the expression of antigens of the causative agents of acute intestinal infections in smears of lymphocytic suspension. The developed method has high reliability.

The method is quite simple, it can be widely used in practical health care for rapid diagnosis of acute intestinal infections and, accordingly, the timely appointment of etiotropic and pathogenetic therapy.

The proposed method is of economic importance, because it does not require the use of additional laboratory equipment and can be used as express diagnostics, as it reduces the time to obtain the result to 3-4 hours.

Claims (1)

A method for the rapid diagnosis of acute bacterial intestinal infections by immunohistocytochemical staining to detect the desired antigen under a light microscope, characterized in that 0.05 ml of the patient’s blood lymphocytic suspension is applied on a glass slide with a thin layer, fixed in acetone after 3-7 minutes, during 3-5 minutes, treated with 3% H ₂ O ₂ for 20 minutes, washed, treated with blocking normal serum for 10 minutes, incubated with poly- or monoclonal antibodies to the desired antigen for 45 minutes at t 37 °, then treated with reagents of the polymer detection system: incubated with Super Enhanced ™ Reagent for 20 minutes, washed, incubated with Poly-HRP Reagent for 30 minutes; washed, treated with 3,3-diaminobenzidine tetrachloride for 2-3 minutes and if the desired antigen is detected in lymphocytes, monocytes (in at least 5-8 cells in the field of view) in the form of brown granules, the etiology of acute intestinal infection is diagnosed.

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