RU2267786C2 - METHOD FOR in vitro EVALUATING GERMINAL ECHINOCOCCUS ELEMENTS VIABILITY - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves evaluating germinal elements ability for building micro acephalocystes. When testing, other harmful factors influence upon germinal Echinococcus elements is restricted to minimum. Reversible transformability in protoscolices arisen after treatment with formalin at 32-38°C during 12-48 h. A situation approximating clinical cases is modeled when protoscoleces treated with scolexocidal agents fall into favorable conditions.

EFFECT: simplified low cost method.

1 dwg

Description

The invention relates to medicine, namely to surgery of echinococcosis. In order to prevent relapse of echinococcal disease during surgical intervention by neutralizing the germinal elements of echinococcus in the fibrous cavity and on the surface of the patient’s tissues, a number of chemical agents and physical factors are used. As such agents, 10% iodoformoglycerol emulsion, 2-5% tincture of iodine, 3% boric acid solution, furatsillin solution 1: 5000, rivanol 1: 1000, 1-15% aqueous and glycerin formalin solutions, 1% dioxidine, various antibiotics, 3-30% sodium chloride solutions, 80-100% glycerin, hot saline, betadine, etc. [1, 2, 3]. Most of the above preparations were not widely used due to high toxicity to host tissues or weak neutralizing activity.

The use of certain physical factors (laser, X-ray irradiation, ultraviolet radiation, UHF, plasma flow, local hyperthermia, cryotherapy, low frequency ultrasound) did not give encouraging clinical results, although experimental data showed their high efficiency [2].

Currently, the search for effective methods of disinfecting the walls of the fibrous capsule of hydatid echinococcus, thoracic and abdominal cavities, especially with the breakthrough of echinococcal cysts that do not have a damaging effect on body tissues, especially the lung and peritoneal cover, continues to be relevant.

The key point of such studies is the objectivity of the criteria for assessing the viability of the germinal elements of the parasite, the effect of which is aimed at neutralizing them. These are: the motor activity of protoscolexes at a temperature of 37-40 ° C, the amount of calcareous bodies and the morphological structure of the parenchyma, the crown of the hooks and cuticular membrane [3]. In addition, the ability of protoscolexes to stain in the native preparation and cause the formation of echinococcal cysts in laboratory animals when staging a biological sample is evaluated.

The disadvantages of the above criteria include the difficulty of registering emerging changes in the native drug (with the exception of the cessation of movement), as well as the need for constant and prolonged monitoring of the same field of view. At the same time, the native preparation has an additional damaging effect of temperature fluctuations and drying. In addition, the above criteria are only indirect signs by which it is necessary to judge the inability of the processed material to cause the development of the disease when it enters the patient’s tissue. A biological sample is required [3], for which laboratory animals highly sensitive to the disease and long-term (several months) follow-up are required.

Closest to our invention is the method proposed by F.P. Kovalenko et al. (Kovalenko F.P., Rasulov S.M., Hakberdiev P.S. et al. Method for the determination of viable echinococcus protoscolexes in an in vitro sample. - Copyright certificate No. 1635961. Bull. No. 11 of 1991). This method is taken as a prototype.

The prototype method is as follows.

On a glass slide, two drops of a suspension of the studied protoscolexes (after exposure to the test agent) in physiological saline are placed. The first drop is control, the second is experimental. Using filter paper, saline is removed from the second drop and one drop of glycerol is applied to the protoscolexes. Both drops are covered with a coverslip and immediately subjected to microscopy at a small magnification of the microscope. In living protoscolexes under the influence of glycerol, body contracture is observed. At the same time, protoscolexes are reduced in size 1.5-2 times compared with the control, lose their rounded shape, acquire irregular shapes, the parenchyma of the body of protoscolexes is enlightened, the calcareous bodies become invisible. In appearance, protoscolexes resemble crystals. These changes occur within 1-2 seconds and are observed within 30-40 minutes after the addition of glycerol. In the dead protoscolexes with the addition of glycerol, these changes are not observed when compared with the control.

The disadvantages of the prototype should include the following points.

1. The criterion used - the appearance of contracture in living protoscolexes and the absence of such in the dead - are only an indirect sign by which it is necessary to judge the effectiveness of the antiparasitic effect and the inability of the treated germ elements to cause the disease, i.e. form cysts.

2. Registration of reduction and immobilization are possible only for protoscolexes, in which in the initial state the movements are noticeable and active. In most intact protoscolexes, movements in the echinococcal cyst are hardly noticeable, and in brood capsules they are generally difficult to detect. After exposure to agents with hypertonic properties (3-30% NaCl, alcohol, 80-100% glycerol, etc.), protoscolexes are already in a state of contracture, which limits the use of the method.

3. After exposure to a scolexocidal agent in a clinical situation, protoscolexes are placed on favorable conditions for their development. In this case, the reversibility of changes in protoscolexes is not excluded, which is not taken into account in the prototype. On the contrary, protoscolexes are additionally exposed to adverse factors, which may entail a false positive assessment of the effectiveness of the effect.

The aim of the invention is the creation of an objective and close to a biological test method for testing methods for the neutralization of germinal elements of echinococcus, used to prevent relapse of the disease.

SUMMARY OF THE INVENTION

Observing sterility, a suspension from a sediment from the freshly taken contents of a hydatid cyst, once washed in physiological saline, is placed in a test tube. Microscopy of the native preparation from the sediment is performed. Suitable material for the experiment is material in which mature protoscolexes and brood capsules are present.

0.2-0.5 ml of suspension of the germinal elements are placed in test tubes with the tested drugs, including physiological saline (for control) or they are affected by the tested physical factors for the required amount of time. After the necessary exposure, the germinal elements are washed with physiological saline and the vials with them are placed in a thermostat at a temperature of 32-38 ° C for 12-48 hours. All procedures are carried out in compliance with sterility.

At the end of the incubation period, the presence and quantity of microacephalocysts in each vial is assessed. In the control bottle, for the most part of protoscolexes, microacephalocysts are formed from the stem (see drawing: native preparation, magnification 8 × 8; pos. 1 - protoscolex, pos. 2 - microacephalocyst).

The effect on the germinal elements of echinococcus is considered effective if microacephalocysts have not formed.

An example of a specific use of the proposed method

The method proposed as an invention is used to study the antiparasitic activity of 3% formalin upon exposure for 2 minutes.

Observing sterility, a suspension was placed in a test tube from a precipitate of freshly extracted contents of the patient’s hydatide cyst, which was washed once in physiological saline. At microscopy, the sediment contains mature protoscolexes and brood capsules.

0.5 ml of suspension of germ elements was placed in a test tube with 3% formalin and 0.9% NaCl solution (control). After the necessary exposure, the germinal elements were washed 4 times with warm saline and the vials with them were placed in a thermostat at a temperature of 32-38 ° С. All procedures were carried out in compliance with sterility.

After 12-48 hours of incubation, microscopy of the contents of the sediment from the vials evaluated the presence and amount of microacephalocysts in each vial. In the control bottle, for the most part of protoscolexes, microacephalocysts up to 1/3-1 / 2 of the protoscolex volume were formed from the peduncle. In a bottle with germinal elements treated with 3% formalin, microacephalocysts were not detected.

Signs that differ from the prototype

In contrast to the known methods for studying the effectiveness of the damaging effects of various methods on the germinal elements of echinococcus carried out in vitro, including in the prototype, the proposed method assesses the preservation of the ability of protoscolexes to form microacephalocysts (the initial stage of an echinococcal cyst) after exposure to test scolixocidal agents, thereby the sample is close to a biological sample.

The beneficial effect of the invention

The key point of research on the search for optimal scoliotic agents is the objectivity of the criteria for assessing the viability of the germinal elements of the parasite, the effect of which is aimed at neutralizing them. To date, the known criteria are the motor activity of protoscolexes at a temperature of 37-40 ° C, the amount of calcareous bodies and the morphological structure of the parenchyma, the crown of the hooks and cuticular membrane [1]. In addition, the ability of protoscolexes to stain in the native preparation and cause the formation of echinococcal cysts in laboratory animals when staging a biological sample is evaluated. With the exception of the last criterion (biological test), all other criteria are indirect signs of the inability of the treated germ elements to cause the disease (echinococcosis), because microorganisms may be characterized by the properties of the regeneration of an organism from a separate part of them or several cells.

Evaluation of the ability to form microacephalocysts from germ elements, taken into account by the proposed method, is a more objective criterion that is closer to a biological sample, compared with other criteria evaluated in vitro.

In addition, when testing, the effect of other damaging moments on the test germinal elements of echinococcus, which occurs with other methods (staining, temperature fluctuations, etc.), is minimized. The method takes into account the possibility of reversibility of changes in protoscolexes that occur after exposure to agents. Moreover, a situation close to clinical is simulated, in which protoscolexes treated with scolexocidal agents fall into favorable conditions.

The method is simple to implement, does not require additional equipment and high material costs.

Information taken into account

RU 1635961, 03.23.1991. - prototype.

1. Biryukov Yu.V. et al. Treatment of the cavity of cysts with hydroactive echinococcosis. Surgery, 1984, No. 5, pp. 27-29.

2. Gilevich M.Yu. The choice of method for treating the cavity of the fibrous capsule with echinococcectomy. Surgery, 1984, No. 4, pp. 74-76.

3. Kovalenko F.P. et al. The effect of low-frequency ultrasound on the viability of the germinal elements of the larvocystic chamber and multichamber echinococci in an in vitro experiment. Honey. parasitol., 1986, No. 6, p.31-36.

Claims (1)

A method for assessing the preservation of viability of echinococcus germ elements after exposure to a physical or chemical factor in vitro and microscopy, characterized in that the echinococcus germ elements, treated with a chemical or physical factor, are incubated at a temperature of 32-38 ° C for 12-48 hours, after which assess the viability by the presence and number of developed microacephalocysts.