RU2582946C1 - Method of diagnosing low-invasive trematodoses of hepatobiliary system - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is intended for diagnostics of trematode infection. In laboratory assessment 10 ml of 5 % acetic acid is added to 2 g of faeces. Suspension is centrifuged at 1,500 rpm during 1.5 minutes. Obtained deposit is mechanically halved by pouring off the upper fluffy halve. 5 ml of 1 % acetic acid and 4 ml of diethyl ether are added to the rest of the deposit which is centrifuged again at 1,500 rpm during 2 minutes. Resultant deposit is analysed.

EFFECT: disclosed method is highly effective for primary diagnostics of low-invasive hepatic trematode infection, as well as for parasitologic recovery monitoring.

1 cl, 6 ex

Description

The invention relates to medicine, namely to gastroenterology, and can be used in laboratory diagnosis of helminthiasis of the gastrointestinal tract, in particular hepatic trematodiasis.

Trematodoses of the human hepatobiliary system are a group of natural focal biohelminthoses, anthropozoonoses caused by trematodes, whose obligate parasitic zones in humans are intrahepatic bile ducts, extrahepatic bile ducts, and pancreatic ducts.

From the practice of medicine, the method of acetic ether sedimentation (UES) is known, which is used as a special method for the diagnosis of trematodoses (including opisthorchiasis), including for low-grade invasions (Parasitological methods for the laboratory diagnosis of helminthiases and protozozoses. Methodical instructions. MUK 4.2.735 -99. Ministry of Health of Russia. M., 2003. pp. 13-15).

The essence of the resistivity method is that a mixture of feces and diethyl ether emulsified with a solution of acetic acid in a ratio of 3: 2 is subjected to centrifugation for the purpose of layer-by-layer deposition (sedimentation). The sediment after removal of the supernatant and the “fecal plug” contains eggs and larvae of helminths, cysts and oocysts of protozoa, as well as a small amount of fecal particles with a specific gravity of more than 1, i.e. heavier than water. When using the UES method, it is possible to detect helminth eggs at their low concentration in the biomaterial (1 or more eggs in 1 g of feces). However, helminthiases are widespread, in which the concentration of eggs in feces is lower than the threshold for this method (low-invasive hemodobiliary hemodobiliary thermostoses). In addition, the presence of artifacts makes it difficult to find and visualize small eggs of trematodes. Attempts to increase the volume of the studied feces more standard in the resistivity test (more than 1 g) lead to an increase in the volume of the obtained sediment, an increase in the content of artifacts in the sediment, and the inability to obtain a thin and transparent smear.

As a prototype adopted the well-known chemical sedimentation method (CSM) study of feces, which is the closest to the proposed (Parasitological methods for the laboratory diagnosis of helminthiasis and protozooses. Guidelines. MUK 4.2.735-99. Ministry of Health of Russia. M., 2003. P. 15-16). The chemical sedimentation method has an advantage over the analogue (resistivity) method in that it allows to reduce the number of artifacts in the obtained smears by additional processing of the sediment with chemical reagents. The essence of the CSM lies in the fact that the stage, which is fundamentally similar to the resistivity analysis, is preceded by an additional stage of flotation deposition, which consists in the fact that the fecal suspension emulsified with acetic acid is centrifuged in a mixture with a saturated solution of sodium nitrate with a specific gravity of 1.15. After centrifugation, the supernatant is removed, the resulting precipitate is re-centrifuged in a mixture with a solution of acetic acid and diethyl ether (similar to resistivity). In sensitivity, it does not exceed the resistivity, because the amount of feces tested is 0.5-1 g, but

The aim of the invention is to improve the diagnosis of low invasive trematodoses of the hepatobiliary system.

The goal of the invention is achieved by adding 10 ml of 5% acetic acid to 2 g of feces, the suspension is centrifuged at 1500 rpm for 1.5 minutes, the supernatant is drained together with the upper half of the precipitate, to the remaining half of the precipitate add 5 ml of 1% acetic acid and 4 ml of diethyl ether, re-centrifuged at 1500 rpm for 2 minutes, the resulting precipitate was examined.

The proposed method achieves the following positive effect:

1. The sensitivity of the method increases, because the number of studied feces is 2-4 times more than in the prototype.

2. An additional stage of primary sedimentation allows you to mechanically (by draining) to reduce the sediment volume by half or more, additionally emulsify the suspension of feces and discolor it. The obtained smears are thinner, discolored, transparent, visualization of small (less than 20 microns), with a thin shell, slightly stained eggs is facilitated.

3. Compared with the prototype, cheaper reagents are used (acetic acid instead of nitric acid sodium).

4. The complexity is reduced (there is no stage of preparation of a working solution of nitric acid sodium with a given specific gravity, ie, batchwise dissolution of the reagent in hot water with constant heating on the stove and constant stirring until complete dissolution, as well as the need to regularly monitor the gradually decreasing specific gravity with a hydrometer weight of prepared working solution).

5. Diagnostic time is reduced (1 centrifugation for 1.5 minutes instead of 5 minutes).

Thus, the proposed method is more efficient compared to the prototype.

The proposed method is as follows:

Stage 1: 10 ml of 5% acetic acid is poured into a single-use graduated to 12 ml plastic bottle, 2 g of feces are placed, bringing the level to the mark of 12 ml, stirred with a glass rod. Shake vigorously until a uniform suspension is obtained. Allow to stand for 2 minutes. Filter the suspension through a double layer of gauze into a graduated centrifuge tube.

Stage 2: Centrifuged for 1.5 minutes at 1500 rpm. The supernatant is drained.

Then remove the upper loose drained part of the precipitate by turning the tube so that the precipitate is reduced by half. In the case of a precipitate of uniform density, the upper half of the precipitate is removed with a glass rod. Helminth eggs, cysts and protozoan oocysts, being heavier than water, remain centrifuged at the bottom of the sediment.

Stage 3: To the remaining part of the precipitate (about 1 ml) add 5 ml of 1% acetic acid, stir, add 4 ml of diethyl ether. Close the cork. Shake the tube vigorously, keeping it horizontal for 30 seconds. Centrifuged again for 2 minutes at 1,500 rpm. In this case, 3 layers are formed: the upper one is the “fecal plug”, the middle one is the supernatant with ether, and the lower one is the sediment. Then, the fecal plug is carefully separated from the walls of the tube with a glass rod, then the supernatant is drained along with ether and the fecal plug. A drop of 50% glycerol is added to the remaining precipitate, stirred, evenly distributed on a glass slide. The finished smear is microscopic.

The proposed method is used for the initial diagnosis of helminthiases and protozoa of the gastrointestinal tract in the Labmed clinical laboratory from 2008 to the present and has established itself as a reliable and at the same time quite cheap and not difficult, especially in diagnostically difficult cases: if you suspect low invasive conditions, mixed invasions, incomplete parasitological cure.

The claimed advantages of the proposed method for mass and continuous practical use allowed us to come to the following results:

1. The sensitivity of the proposed method is higher, so the detection of low-invasive trematodoses becomes better. An increase in the detection of trematodoses of the hepatobiliary system in the examined contingent leads to the conclusion about the prevalence of invasions of low and extremely low degree, since one invasion with medium and high degree of invasion accounts for at least 20-30 invasive low degree.

Comparative sensitivity of the proposed and analogue (UES) methods with respect to trematodoses of the hepatobiliary system:

the number of examined simultaneously by both methods 94 the number of detected invasions when using resistivity 38 the number of detected invasions using the proposed method 49

2. The technical feasibility of laboratory diagnosis of invasions of low and extremely low degree changes the idea of the structure of parasitic morbidity. It was found that invasion by opisthorchidiasis in the Republic of Tatarstan is higher than invasion by all other helminthiases, including larval (excluding enterobiosis), taken together.

3. When using an analogue and a prototype, an attempt to increase the volume of the studied feces leads to insufficient thinness and transparency of smears, respectively, visualization and identification of small eggs of opisthorchids is technically difficult. This is excluded when using the proposed method. Detected persistent morphological differences between eggs suggest the prevalence of at least 6-7 species and subspecies of trematodes.

4. The proposed method can significantly increase the frequency of occurrence of small (less than 20 microns), poorly stained eggs with a thin shell, non-visualizable structure of miracidia, poorly distinguishable lid, which is difficult when using the analogue and prototype.

5. The proposed method allows us to detect the relationship between egg morphology and sensitivity to standard deworming methods.

6. The proposed method is gaining value for controlling parasitological cure. So, of the 20 treated, in which the proposed control method gave a positive result (egg detection), analog methods (resistivity tests) gave a positive result in 14 treated, in 6 - a negative result.

The following clinical observations illustrate the value of detecting low and extremely low invasions:

Example 1. Patient S., 10 years old. Referred by an allergist with a diagnosis of recurrent Quincke edema. Recurrent urticaria. Parasitological diagnosis: low invasive trematodosis of the hepatobiliary system (metrorchosis).

Example 2. Patient K., 46 years old. Directed by a gastroenterologist with a diagnosis of hr. hepatitis of unspecified etiology with cholestasis syndrome, cytolytic syndrome, moderate hepatosplenomegaly. Parasitological diagnosis: mixed-invasion of a low degree (opisthorchiasis + metrorchosis). Chr. secondary infection with the Epstein-Barr virus.

Example 3. Patient C, 36 years old. Directed by a dermatologist with a diagnosis of papillomatosis of smooth skin. Parasitological diagnosis: hr. low invasive opisthorchiasis. Acquired Immune System Dysfunction. Chr. papillomavirus infection.

Example 4. Patient K., 5 years old. Referred by an ophthalmologist with a diagnosis of recurrent fibrinous plastic uveitis. Parasitological diagnosis: hr. low invasive opisthorchiasis. Chr. giardiasis. Secondary immunodeficiency state. Chr. herpes virus + cytomegalovirus infection. Chr. chlamydial infection.

Example 5. Patient P, 52 years old. Directed by a gastroenterologist with a diagnosis of hr. cholecystitis, pancreatitis. Chr. enterocolitis of unspecified etiology. Parasitological diagnosis: hr. low invasive opisthorchiasis. Chr. giardiasis. Chr. isosporosis.

Example 6. Patient C., 12 years old. Directed by an allergist with a diagnosis of bronchial asthma, atopic, moderate. Allergic rhinosinusitis year-round. Atopic dermatitis is limited. Parasitological diagnosis: hr. low invasive opisthorchiasis. Chr. giardiasis. Toxocariasis, visceral form.

Application of the proposed method.

The double sedimentation method is intended for the initial diagnosis of low-invasive hepatic trematodoses, as well as for monitoring parasitological cure.

Claims (1)

A method for the diagnosis of low-invasive tremadoses of the hepatobiliary system, including double centrifugation of fecal suspension followed by discharge of the supernatant, characterized in that 10 ml of 5% acetic acid is added to 2 g of feces, the suspension is centrifuged at 1500 rpm for 1.5 min, the supernatant is drained along with the upper half of the precipitate, 5 ml of 1% acetic acid and 4 ml of diethyl ether are added to the remaining half of the precipitate, centrifuged again at 1500 rpm for 2 min, the resulting precipitate was studied blowing.