Method of acute bacterial enteric infections express-diagnostics

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.

3 dwg, 1 ex

 

This invention relates to the field of medical diagnostics, namely to identify bacterial infections

Acute intestinal infections (AII) are widely distributed among all age groups, while occupy a significant place in the structure of infant mortality. OKA is a group of different diseases (dysentery, salmonellosis, typhoid fever, E. coli infection, and others). In the United States annually 6.5 million cases of foodborne infections and over 9000 deaths. Among bacteriologically decrypted cases in the first place was salmonellosis, then shigellosis (Higashiyama, 2002, Kilgore P.E., 1995). These diseases of bacterial etiology share similar pathogenesis (bacteremia/antigenemia, exposure to toxins, metabolic disorders) and similar clinical manifestations gastrointestinal syndrome, exsicosis, toxemia (Taiwanese, Lavrenova, 1989), which complicates their clinical differentiation. To confirm the diagnosis using bacteriological methods: cultures of faeces, blood, vomit, and serological methods for detection in the dynamics of the level of specific antibodies to the bacteria (V.V. Ivanov, 2002). Due to the huge epidemiological significance of intestinal infections are of special importance accelerated diagnostic methods for the appointment of adequate causal and the pathogen is practical therapy.

A known way of detecting bacterial infections is the test indirect haemagglutination, rnga.

1. In 10 wells of a number of contributing 0.5 ml of 9%solution of sodium chloride.

2. In the 1st hole of a number of contributing 0.5 ml of test serum diluted 1:25, and make a series of twofold dilutions from 1:50 to 1:6400 by moving from hole to hole 0.5 ml and mixing them at least 3-4 times.

3. To each well add 0.2 ml of 1%of the working suspension of diagnosticum. At the same time put the controls.

4. Plate slightly shaken and incubated at 37 ° °1.5-2 hours

5. Incubation at room temperature for 14-18 hours

6. Evaluation of the results

This method allows to obtain good results. However, there are circumstances that reduce its effectiveness: it takes a long time (24 hours), time-consuming and to obtain the results required additional equipment (microplates and spectrophotometer), drugs cannot long keep and take pictures. In some cases the definition of serological markers ineffective: in newborns due to immature immune system, depression of the immune system and in the early stages of the disease, because antibodies are formed otsrochennoe.

The closest to our proposed method is "a Method for the diagnosis of Haemophilus influenzae in histolo the practical preparations" patent for invention №21932004 from 20.11.02, Rahasyam, Ascutney, Mavlanov.

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

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

3. Treated with 0.1% solution of trypsin in 0.1% CaCl2within 20 minutes.

4. Treated with 0.03% hydrogen peroxide for 20 minutes.

5. Washed in two portions of phosphate-saline buffer (pH 7.2)for 5 minutes each.

6. Incubated in 0.3% Tritonx-100 at 37 ° °C for 15 minutes.

7. Put polyclonal rabbit antibodies to ibaselabelprovider. Incubated with polyclonal antibodies at a temperature of 37°C for 45 minutes.

8. Washed in water for 5 minutes.

9. Washed in two portions of phosphate-saline buffer (pH 7.2)for 5 minutes each.

10. Put individuai conjugate DAKO En Vision+System, HRP and incubated for 45 min in a dark chamber at room temperature.

11. Washed in two portions of phosphate-saline buffer (pH 7.2)for 5 minutes each.

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

13. Poorly Domracheva with hematoxylin.

14. Microscopy.

This method allowed the authors to obtain good results. However, a disadvantage of the top the method is that he traumatic as material for research are the biopsies. The conduct of all stages of the way quite a long time. Taking biopsy, fixation, fill in paraffin, preparation of histological sections take at least 6-7 hours and more than 5 hours spent on conducting immunoperoxidase reaction. Due to high background staining when working with paraffin sections decreases the accuracy of the result.

To eliminate these drawbacks can help authors suggest a method for the diagnosis of DCI, based on the detection of antigens of pathogens.

The technical result of the invention consists in reducing time and improving the accuracy of diagnosis. This result is achieved by the fact that the antigen of the pathogen OKA determine indirect immunoperoxidase method according to the invention blood cells (lymphocytic suspended in a volume of 0.05 ml), obtained by centrifugation, put on a glass slide (smear), dried at room temperature, fixed in pure acetone - 3-5 min, then treated with 3% H2About220 minutes, incubated with normal blocking serum (10 minutes), incubated with poly - or monoclonal antibodies to target antigens at a temperature of 37°C for 45 minutes, incubated with polymer detection system (Super Enhacer for 20 min in the dark camerapro room temperature, incubated with SS Label for 30 min in a dark chamber at room temperature), then treated with 3,3-diaminobenzidine the tetrachloride (DAB) and the presence of brown granules in lymphocytes and monocytes indicates the detection of the desired antigen of the pathogen (diagnosis) infection for the timely appointment of etiotropic and pathogenetic therapy. Way well-reproduced, indicating its reliability.

Standard indirect immunoperoxidase method described Golak, Swan-Norden. Introduction to immunocytokine. M: Mecicine, 1986

1 - rinse the slices in a buffer;

2 - handling 0.03% solution of hydrogen peroxide;

3 - rinse buffer;

4 is applied to the first slice specific antibodies;

5 - rinse buffer;

6 - application of a second antibody coupled to horseradish peroxidase;

7 - rinse buffer;

8 - handling 0.03% solution of diaminobenzidine (DUB);

9 - pokraska with hematoxylin;

10 - conclusion in balm. Microscopy.

But evidence from the literature and our own experience, the scheme only in General reflects the progress of the production methods. Depending on the size, structure and nature of detected antigen required detailed design all stages, starting with commit and before the application of diaminobenzidine.

Method proposed by the authors differs in that the detection is possible antigens of pathogens OKA as the object of research was first used lymphocytic suspension.

Empirically it was found that it is necessary to 0.05 ml of the lymphocyte suspension as a minimum for the preparation of smears, holding immunocytochemically painting and adequate assessment of the result.

In the framework of the proposed method first proposed processing smears more concentrated (3%) hydrogen peroxide solution, contributing to more and better blocking of endogenous peroxidase, which is especially important when using lymphocytic mist.

One of the difficult problems is the possible presence of serum non-specific (background) antibodies. To eliminate nonspecific reactions proposed treatment normal (blocking) serum before applying the first antibody.

The use of a detection system of the polymer complex Super Sensitiv Polimer-HRP Detection System production BioGenex, as more sensitive, allowed the use of specific antibodies in low concentrations and to minimize nonspecific background staining that could not be achieved by conventional detection systems. In addition to the above, the detection system does not contain avidin and Biotin, therefore, non-specific staining in the interaction avidin with endogenous Biotin is excluded. Blood is a complex and sensitive subject when conducting immuno is istoritcheskih research. In this regard, the proposed new method for the diagnosis of OKA, based on the detection of antigens of pathogens in blood cells requires a complex methodological techniques. The failure of the latter does not give objective results or distorts the outcome of the studies does not allow for appropriate interpretation of the obtained data.

The proposed method is as follows.

1. Lymphocytes and monocytes, obtained by centrifugation of peripheral blood, put on a glass slide (smear).

2. Dried at room temperature.

3. Fixation in pure acetone 3-5 minutes

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

5. Treatment in 3% H2About220 minutes.

6. Rinse in two portions of phosphate-saline buffer (pH 7.4)for 5 minutes each.

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

8. Drawing on strokes poly - or monoclonal antibodies to target antigens. Incubation with these antibodies at a temperature of 37°C for 45 minutes.

9. Rinsing in water for 5 minutes.

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

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

12. Rinse in two portions FOSFA the but-saline buffer (pH 7.4), 5 minutes each.

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

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

15. Weak pokraska with hematoxylin.

16. Microscopy.

Microscopy stained preparations is carried out in a conventional microscope. The reaction product in the study of lymphocytes and monocytes containing the desired antigen, detected as brown granules intracellularly and on the cell membrane. When evaluating the data, microscopic examination it is important to consider the emergence of artificially changes, simulating a positive fashion. Most often it is the cellular artifacts found on the edge of the smear. In this case, it is necessary to evaluate reactions to other sites.

The necessary controls for the antibodies

To obtain reliable results and to exclude possible non-specific reactions is mandatory to conduct inspections on the quality of the reagents and the specificity of the antisera.

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

2) Nespecificnomu serum eliminate through the widest what about the cultivation of highly concentrated serum and shortening the incubation time. The described method is supported by examples of specific performance.

A study was conducted of material 20 patients with acute intestinal infection in the first 3 days from the onset of the disease, 8 of them was revealed the expression of complex antigen Salmonella, 2 Sh.Sonnei, 5 Sh. Flexneri as monoinfection, 2 was determined antigens Sh. Flexneri and Salmonella, 2 Sh. Sonnei and Salmonella and one Sh. Flexneri and Sh. Sonnei. The obtained results are fully consistent with the data serology and clinical manifestations. In all cases, the results of the study on the identification of antigens of pathogens OKA were obtained after 4 hours from the time of receipt

As an example, here is the micrograph of the stroke lymphocytic suspension with clearly defined by the expression of a complex of Salmonella antigen in lymphocytes and monocytes in a patient Zvyagintsev, 4 years, no history of the disease 5650, FGU NEEDY University, St. Petersburg from 12.10.2006, with distinct manifestations of acute intestinal infections (figure 1).

Micrograph smear lymphocytic suspension with clearly defined expression of antigen Sh. Flexneri in lymphocytes and monocytes of a patient Plicas Days, 2 years, no history of the disease 3347, DEEB No. 3, St. Petersburg from 03.11.2006, with the clinic of acute intestinal infections (figure 2).

Micrograph smear lymphocytic mist with a strong expression of antigen Sh. Sonnei in lymphocytes and m is nocito patient Ephraim A., 4 years, no history 3210, DEEB No. 3, St. Petersburg from 27.10.2006, with clinic severe course of acute intestinal infections (figure 3).

The control group was investigated lymphocytic suspended two patients with pertussis and one patient with CNS CNS infection. In all cases, the Salmonella antigens, Sh. Flexneri, Sh. Sonnei were not found.

Thus, the proposed method allows a high degree of specificity and accuracy to detect the expression of antigens of pathogens OKA smears lymphocytic mist. The developed method has high reliability.

The method is simple enough, it may have wide application in medical practice for the rapid diagnosis of the OKA and accordingly the timely appointment of etiotropic and pathogenetic therapy.

The proposed method has economic significance, since it does not require the use of additional laboratory equipment and can be used as a rapid diagnosis, as it reduces the time of receipt of the up to 3-4 hours.

The method of rapid diagnosis of acute bacterial intestinal infections by conducting immunohistochemical staining for detection of the desired antigen under a light microscope, characterized in that on a glass slide with a thin layer applied 0.05 ml lymphocyte the Oh-suspension the patient's blood, 3-7 min, fixed in acetone, for 3-5 min, treated with 3% H2About2within 20 min, washed, treated with blocking serum for 10 min, incubated with poly - or monoclonal antibodies to the desired antigen for 45 min at 37 t°, then treated with reagents polymer system detection: incubate with Super Enhanced™ Reagent for 20 min, washed, incubated with Poly-HRP Reagent for 30 min; washed, treated with 3,3-diaminobenzidine the tetrachloride 2-3 min and the detection of the desired antigen in lymphocytes, monocytes (not less than 5-8 cells in field of view) as brown granules, diagnose the etiology of acute intestinal infections.



 

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