Method for predicting ophthalmoherpes

FIELD: medicine, ophthalmology.

SUBSTANCE: one should detect the content of tumor necrosis factor alpha in acute period, moreover, the above-mentioned factor should be determined in lacrimal liquid on the 11th - 15th d against the onset of herpetic ophthalmoinspection, at its content ranged 176-250 pcg/ml prediction is considered to be favorable, and from the value of 300 pcg/ml and higher - as unfavorable.

EFFECT: higher accuracy of prediction.

2 ex

 

The invention relates to medicine, in particular to ophthalmology, and can be used in medical practice for predicting the course of ophthalmic herpes.

Methods of prediction of ophthalmic herpes based on the study of immunopathological changes in this disease involving abnormalities in the number and range of subpopulations of lymphocytes and cytokine changes the status of the patient (Webmachine, 1997; A.Biglino, 1996).

There is a method of prognosis of ophthalmic herpes (Webmachine, Tonkonogov, Anabuki. Ophthalmological journal, 1981, No. 3, p.142-143) by defining subpopulations of lymphocytes method rosethorne with sheep erythrocytes. The disadvantages of this method are its complexity, subjectivity in the evaluation of the results and the small number of simultaneously ongoing research.

About the nature of the flow and its forecast judged on functional activity of blood cells, determined by the level of their synthesis of cytokines, one of which is tumor necrosis factor α (TNF-α). This cytokine exerts pyrogenic properties, portrays the response of acute phase of inflammation and affects the induction of autoimmune reactions.

The closest in technical essence is "the Way to prevent postoperative complications of penetrating corneoscleral wounds of the eye" (Ossipova et al. In the party of ophthalmology, 1998, No. 3, pp.28-32), the authors of which is shown pathogenetic role of one of the cytokines TNF-α single determination of its contents in serum and tear fluid of patients with corneoscleral wounds.

The disadvantage of this method is that the authors predicted infectious complications of penetrating corneoscleral wounds eye on the content of tumor necrosis factor α in the serum of patients, which is an invasive method. According to this method in the determination of tumor necrosis factor α in the serum of patients prognosis complications after corneoscleral wounds is carried out at a later date (up to 1.5 years), and the frequency efficiency of the forecast is 25-75% of cases.

The technical result of the invention is increasing the frequency and reducing the time of predicting the course of ophthalmic herpes on the basis of the determination of tumor necrosis factor α in the tear fluid of patients during the acute period (11-15 day of onset) of herpetic keratitis.

The technical result is achieved by determining the content of tumor necrosis factor α in tears in the acute period of the disease, characterized in that the identification of the content of tumor necrosis factor α in the tear fluid at 11-15 days from the beginning of Zabol the cation within 176-250 PCG/ml, is assessed as favourable prognosis of the ophthalmic herpes, and when its concentration from 300 PCG/ml and more unfavorable.

The advantage of the proposed method for the prognosis of herpes of the eye is the definition of the content of TNF-α in tears in the acute period (11-15 days) ophthalmic herpes. Confirmation of the proposed method is coincident indicators concentrations of these cytokines in the tear fluid with the clinical course of ophthalmic herpes on average, more than 92% of cases.

The method is as follows.

Lacrimal fluid is picked up by the introduction of the conjunctival SAC in the amount of 1 ml.

Determination of the level provospalitelna TNF-αin the tear fluid of patients spend enzyme-linked immunosorbent method with the use of horseradish peroxidase as indicator enzyme using test systems ProCon (LLC “Protein component”, St. Petersburg).

In the microplate with immobilized antibodies contribute 100 ál of standards TNF-α and 100 μl samples of tear fluid. The tablet is incubated for 1 hour at +37°With continuous shaking, then washed three times with buffer and twice with distilled water. Conducts a full aspiration of the remaining fluid.

In each of the wells of the microplate make 100 ál conjugate with streptavidin horseradish peroxidase, diluted with buffer. The Board is incubated for 30 min at +20-25° With continuous shaking. Fluid from the cells were removed and washed three times with buffer, then twice with distilled water. The tablet is dried by tapping the surface of the lab table covered with a filter paper.

All wells contribute 100 ál of substrate solution with the dye. The microplate incubated for 10-15 minutes at room temperature protected from the direct rays of the place on the shaker. There is the development of blue color. The reaction is stopped by adding 50 μl of sulfuric acid to each well. The total time of production research 2.5-3 hours.

Analysis is carried out with the use of automatic photometer “Multiscan” at a wavelength of 450 nm, set the zero absorbance in the hole with standard 0. The results are expressed in PCG/ml Normal levels of the studied cytokines in the tear fluid in healthy individuals (control group), as a rule, does not exceed 125 PCG/ml

In 63 patients with ophthalmic herpes was studied content provospalitelna TNF-α in tears in the acute period of the disease. Tear fluid was taken by the introduction of the conjunctival SAC in the amount of 1 ml.

Determining the level of TNF-α in the tear fluid of patients was carried out by enzyme-linked immunosorbent method with the use of horseradish peroxidase as indicating the enzyme.

In the microplate with immobilized antibodies contributed 100 ál of standards TNF-α and 100 μl samples of the lacrimal fluid of patients. Tablet incubated for 1 hour at +37°With continuous shaking, then washed three times with buffer and twice with distilled water. Undergoing a complete aspiration of the remaining fluid.

In each of the wells of the microplate made of 100 ál conjugate with streptavidin horseradish peroxidase, diluted with buffer. Tablet incubated for 30 min at +20-25°With continuous shaking. Fluid from the cells was removed and washed three times with buffer, then twice with distilled water. The tablet was dried by tapping the surface of the lab table covered with a filter paper.

All wells were made in 100 µl of substrate solution with the dye. The microplate was incubated for 10-15 minutes at room temperature protected from the direct rays of the place on the shaker. Watched the development of blue color. The reaction was stopped by adding 50 μl of sulfuric acid to each well.

Analysis was performed using an automatic photometer “Multiscan” at a wavelength of 450 nm, setting the zero absorbance in the hole with standard 0. The results were expressed in PCG/ml

34 (53,9%) of 63 patients the content of TNF-α in the tear fluid at 11-15 days from N. the beginning of the disease was identified within 176-250 PCG/ml However, 31 (91.2 per cent) of them clinically, the disease was progressing favorably, and by the end of the 3rd week of the inflammatory process was docked. 3 (8,8%) patients for ophthalmic herpes was characterized as unfavorable, since within 3 weeks remained inflammatory response with gradual transition into the deeper layers of the cornea. Only towards the end of the 4th week was marked subsidence of the inflammatory process in the eye.

29 (46,1%) of 63 patients with ophthalmic herpes concentration of TNF-α in tears in the same period of the disease was 300 PCG/ml or more. Negative for herpes ophthalmopathy (more than 30 days) was observed in 27 (93.1%) are of them, 2 (6,9%) reported short (up to 14 days) favorable course.

Thus, the proposed method for prediction of ophthalmic herpes was correct in 58 out of 63 patients, which was significantly in 92% of cases.

Example 1. Patient U. the hospital treatment for herpes tree keratitis. Determination of cytokines in the tear fluid were carried out on the above-described method. In the midst of disease for 12 hours, the content of TNF-α in the tear fluid was equal 376,2 PCG/ml on the basis of which predicted an unfavorable course. Indeed, in the development of the disease, in spite of antiviral treatment, it was noted by agenie the deep layers of the cornea with involvement in the inflammatory process of the choroid. Was delivered the final clinical diagnosis: corneal ulcers (herpes), acute iridocyclitis. The patient was hospitalized for 27 days. Thus, the high content of TNF-α in tear fluid (376,2 PCG/ml) in the acute stage indicates an unfavorable prognosis, course of ophthalmic herpes.

Example 2. Patient K. Diagnosis: regional herpetic keratitis. Determination of cytokines in the tear fluid and serum were carried out according to the above-described method. On the 15th day from the onset, the concentration of Pro-inflammatory TNF-α in the lacrimal fluid - 185 PCG/ml On the basis set within the above-specified interval (176-250 PCG/ml) values of TNF-α in the tear fluid of the patient is predicted favorable for ophthalmic herpes. Subsidence of the inflammatory process, resorption of infiltration of the cornea, were noted to 19 days after onset of the disease, deep optical medium eyes were calm, which indicates the confirmation of the previously suggested a favorable prognosis of development of ophthalmic herpes.

Thus, the proposed method for prediction of ophthalmic herpes allows to evaluate the clinical course of this disease by changing the content of TNF-α in the tear fluid of patients. The prognosis of the disease is favorable when the level of TNF-αin the lacrimal fluid is STI within 176-250 PCG/ml and an unfavorable when its concentration from 300 PCG/ml and above.

The method of prediction of ophthalmic herpes by determining the content of tumor necrosis factor α in the acute period of the disease, characterized in that the tumor necrosis factor α determine in tear fluid on the 11-15 day from the beginning of herpetic ophthalmopathy, when its content within 176-250 PCG/ml, the prognosis of the disease is assessed as favourable, and from 300 PCG/ml and more unfavorable.



 

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