Laboratory diagnosis of helminthiases. Method for isolating a pure culture of Leishmania obtained by scraping pathological tissues

Simple Methods

macroscopic method. When examining feces, one can detect helminths, their heads, segments, fragments of strobili, which stand out on their own or after deworming. This method is especially recommended for the detection of enterobiasis, taeniasis and taeniarhynchosis.

Small portions of feces are mixed with water in a flat bath or in a Petri dish and, looking at good lighting against a dark background, using a magnifying glass if necessary, remove helminths and all suspicious white formations with tweezers or a pipette. The collected material is transferred to another cup with water or onto a glass slide in a drop of diluted glycerin or isotonic sodium chloride solution for further study.

In the settling method, the entire test portion of feces should be mixed with water in a glass cylinder, then carefully drained upper layer water. This is repeated several times. When the liquid becomes transparent, it is drained, and the precipitate is viewed in small portions in a glass bath or Petri dish, as indicated above.

Microscopic methods are the main method of examining feces to detect eggs or helminth larvae. Various research methods are described below. In order to increase the reliability of the examination, the analyzes can be repeated several times daily or with an interval of 1-3 days.

Native smear method. Native smear is the most common and technically available method faecal studies. In a native smear, eggs and larvae of helminths of all kinds can be found. However, with a small number of eggs in the feces, they are not always found. Therefore, the study of feces only with the help of a native smear is not complete and should be supplemented by enrichment methods. The efficiency of native smear examination is markedly improved when viewing four preparations prepared from a fecal sample on two glass slides without cover slips, which allows to examine a total of approximately the same amount of feces as with the Kato method (see below).

A small amount (the size of a match head) of stirred feces is thinly smeared with a wooden stick on the surface of a glass slide in a drop of 50% glycerin solution. Usually two smears are prepared on one glass. The smear is viewed under a low magnification microscope (ob. 8, app. 7). In doubtful cases, it is covered with a cover slip and examined under high magnification (ob. 40).

To prepare a large native smear, 200-300 mg of feces (the size of a large pea) are ground on a 6x9 cm glass in 15-20 drops of a 50% aqueous solution of glycerin. Viewed under a binocular stereoscopic microscope (vol. 4, approx. 12.5 or vol. 2, approx. 17) in transmitted light without cover slips. In doubtful cases, you can translate the lens to a higher magnification. In such smears, colored large helminth eggs are clearly visible, and transparent eggs of the pygmy tapeworm are somewhat worse. This method is not suitable for detecting small eggs. At the same time, a large volume of the studied material and a large field of view with a high depth of field provide a significant efficiency of this modification compared to a conventional native smear.

The thick cellophane smear (Kato method) is more effective than native smear examination, but also needs to be combined with enrichment methods. Eggs of all types of helminths are detected, however, in order to detect eggs of the pygmy tapeworm (transparent eggs) or opisthorchis (small eggs), the laboratory assistant must be especially careful not to miss them (Fig. 21).

The method is based on the detection of helminth eggs in a thick smear of feces, clarified with glycerin and tinted with malachite green. The hydrophilic cellophane is preliminarily cut into 20 x 40 mm plates and immersed in a Kato mixture (6 ml of a 3% aqueous solution of malachite green, 500 ml of glycerol, 500 ml of a 6% phenol solution). 3-5 ml of the mixture is enough for 100 plates, which are ready for use in a day and can be stored in the same mixture in a well-closed container at room temperature for 6 months. In the absence of malachite green (recommended to reduce eye fatigue of the laboratory assistant) and phenol (disinfectant), only a 50% aqueous solution of glycerin can be used, the effectiveness of the study is not reduced.

Rice. 20. Method for preparing a thick smear of feces with cellophane according to Kato

100 mg of excrement is applied to a glass slide, covered with a cellophane plate treated as above, and pressed down with a rubber stopper so that the excrement does not spread from under the cellophane. Microscopy at low or high magnification of the microscope is carried out no later than 1 hour (in hot weather - 30-40 minutes) after the preparation of the smear. The reason for the opacity of the preparation may be a thick layer of feces, poor processing of the plate in the Kato mixture, insufficient exposure time of the preparation under cellophane. Prolonged clearing with glycerin and excessive drying of the preparation also make it difficult to detect eggs.

Twisting method according to S.S. Shulman. The method was proposed for the detection of helminth larvae in the feces, primarily Strongyloid. Only freshly excreted feces are examined, 2-3 g of which are transferred to a glass jar, stirred with a glass rod in a circular motion with 3-5 times the amount of saline, without touching the walls of the vessel. Eggs and larvae of helminths accumulate in the center. After mixing, the drop at the end of the stick is quickly transferred to a glass slide, covered with a coverslip, and examined under a microscope.

enrichment methods. The enrichment methods are based on the difference in the specific gravity of the eggs and the salt solution used, which makes it possible to detect a small amount of them. If the specific gravity of the eggs is greater than the specific gravity of the liquid, then the eggs are concentrated in the sediment, which is examined under a microscope. This sedimentation method is used for trematode eggs. With a higher specific gravity of the solution, the eggs float to the surface of the liquid, and then the film is examined. These are flotation (floating) methods, they are most effective for detecting hookworm, whipworm and pygmy tapeworm eggs.

flotation methods. The Fulleborn method is based on the floating of helminth eggs in a saturated solution of sodium chloride, which has a high relative density (1.2), which makes it possible to detect eggs with a small number of them. The method is more effective than studying a native smear, although it is more difficult. The advantages of the method are cheapness and availability. It is recommended to combine the study of the native smear and the Fülleborn method.

A saturated solution is prepared by dissolving 400 g of sodium chloride in 1 liter of water while boiling. The relative density of the solution is 1.18-1.22. The solution is stored in a closed bottle. For analysis, 2-3 g of feces are placed in a jar with a volume of 30-50 ml and, while stirring with a stick, a saturated solution of sodium chloride is added almost to the top. A strip of paper quickly removes floating large particles. After 45-60 min. settling with a wire loop, remove the surface film and transfer it to a glass slide in a drop of 50% aqueous glycerol solution. Instead of removing the film with a loop, you can add the solution in the jar to the top, cover with a glass slide, to the surface of which the floating eggs stick. Several preparations are being prepared. Additionally, 2-4 preparations are viewed from the sediment, picking it up with an eye pipette on 2 glass slides. In addition to the surface film, it is also necessary to examine the sediment, since the eggs of trematodes, taeniids, unfertilized eggs of ascaris do not float in this solution. Eggs of a number of helminths do not immediately float in saline solution. So, if the maximum number of eggs of the pygmy tapeworm floats up after 15-20 minutes, then ascaris - after 1.5-2 hours, whipworm - after 2-3 hours.

Thus, the advantages of this method include its cheapness and availability, the disadvantages are the need to view preparations on the surface film and sediment, as well as the duration of settling.

The method of E. V. Kalantaryan is also an enrichment method, but it is more efficient and simpler than the Fülleborn method. A saturated solution of sodium nitrate with a relative density of 1.38 is used. Therefore, the eggs of most helminths float up and are found in the surface film; sediment examination is not required.

To prepare a saturated solution of sodium nitrate, 1 kg of sodium nitrate salt (sodium nitrate) is dissolved in 1 liter of water and boiled until completely dissolved and a film forms on the surface. Without filtration, pour into a dry bottle. In the absence of sodium nitrate, it can be replaced with ammonium nitrate (ammonium nitrate), dissolving 1.7 kg per 1 liter of water. The relative density of the resulting solution is 1.3, which somewhat reduces the efficiency compared to the sodium nitrate solution.

Advantages of the method: the eggs of most helminths quickly emerge and are found in the surface film, which eliminates the need to study the sediment. The disadvantages of the method are the deficiency of sodium nitrate, as well as the fact that trematode eggs, taeniid oncospheres do not float and remain in the sediment. It should be borne in mind that with prolonged (more than 1-2 hours) exposure of feces in a solution, the eggs of some helminths begin to swell and settle, disappearing from the surface film.

sedimentation methods

P. P. Goryachev’s method is based on the principle of egg settling. The smear in this case is light, without coarse impurities, which facilitates the detection of small eggs of trematodes (opisthorchia, etc.). The specific gravity of opisthorch eggs is high, so they do not float in saline solutions.

70-100 ml of a saturated sodium chloride solution is poured into a cylinder with a diameter of 2-3 cm. Separately, thoroughly mix 0.5 g of feces in 20-25 ml of water and carefully filter through a funnel with two layers of gauze into a cylinder on saline, avoiding mixing (so that two clearly demarcated layers are formed). After 2-3 hours, the upper layer with feces is sucked off with a pipette, and the remaining saline solution is left to stand for 12-20 hours or centrifuged. The precipitate is pipetted onto a glass slide, covered with a coverslip and microscoped.

Goryachev's method was proposed for the detection of opistorch eggs and proved to be more effective than native smear examination and the Fülleborn method. Currently, for the diagnosis of opisthorchiasis (clonorchiasis), the methods of Kato and Kalantaryan are recommended as quite effective and technically simpler.

Krasilnikov method. Under the action of surfactants that are part of detergents (detergents), helminth eggs are released from feces and concentrated in the sediment.

A 1% solution of Lotus washing powder is preliminarily prepared. To do this, 10 g of the powder is dissolved in 1 liter of tap water. In the absence of "Lotus" you can use other washing powders, but each of them must be taken as much as it will dissolve without the formation of a precipitate in 1 liter of tap water. 20-30 ml of detergent solution is poured into a glass vessel with a capacity of 30-50 ml, a small portion of excrement is placed there and mixed well. The ratio of feces and solution should be approximately 1:20. The feces must be in the solution for at least a day. During this time, a sediment of 2-3 layers is formed at the bottom. The bottom layer consists of coarse heavy particles, helminth eggs collect in the middle layer, the top layer is whitish-gray flakes. Then, with a pipette, 2-3 drops of liquid are collected from the middle layer and transferred to a glass slide. 2 preparations are prepared on one glass, covered with a coverslip and microscoped.

The Krasilnikov method allows you to detect eggs of all types of helminths excreted with feces.

Ether-formalin sedimentation method and chemical-sedimentation method for high efficiency are very time-consuming, especially during mass examinations, in this regard, it is more expedient to use the ether-acetic method. It allows, after additional processing of the sediment with chemical reagents, practically only helminth eggs to be obtained in it, which facilitates the identification of small trematode eggs. This method turned out to be universal, revealing the eggs of all intestinal helminths, cysts of intestinal protozoa, it can also be used to quantify the intensity of invasion.

Pour 7 ml of a 10% solution into centrifuge graduated tubes. acetic acid and add 1 g of faeces to the 8 ml mark. The faeces are thoroughly mixed with a stick until a homogeneous mixture is formed, and then filtered through two layers of gauze into another centrifuge tube (so that the new tube of the filtered solution again has 8 ml, if less, then you can additionally rinse the funnel with a bandage with a 10% solution of acetic acid, through who filtered the fecal solution). To this tube add 2 ml of ether (up to the 10 ml mark), stopper and shake vigorously for 30 sec. The mixture is centrifuged at 3000 rpm for 1 minute (or 2 minutes at 1500 rpm). The coagulant layer (in the form of a cork in the upper part of the tube) is separated from the walls of the tube with a stick and carefully drained together with the supernatant. The precipitate (usually small, colorless) is applied to glass slides with a pipette, covered with a coverslip and microscoped.

The simplest are divided into 4 classes:

When encysted, the microorganism acquires a rounded shape and becomes covered with a protective shell. In the form of a cyst, protozoa become less susceptible to adverse environmental factors.

Research may include:

Note:There are a lot of varieties of diagnostics, we will consider those types that are most common in clinical laboratory practice.

Private types of diagnostics

In each specific case, the laboratory assistant is tasked with finding a specific pathogen, sometimes others are found along with the main one.

There are 6 species of this microorganism capable of living in the human intestine. Only dysentery amoeba, which occurs in a vegetative form and in the form of cysts, is of clinical importance.

Additionally, immunological methods are used:

• indirect immunofluorescence;
• indirect agglutination (PHA);
• radial immunodiffusion.

Note: serological methods are uninformative and are used only as an addition to the main ones in doubtful cases.

Diagnosis of ciliary (ciliates)

The pathogenic form of microorganisms of this genus is balantidia. This is a microbe that causes balantidiasis - a disease accompanied by an ulcerative process of the large intestine. The causative agent is found in a native smear in the form of a vegetative form and a cyst. The material for the smear (feces and mucus) is taken during a sigmoidoscopy examination and sown on special media.

Diagnostics of flagellates (leishmania, giardia, trypanosomes, trichomonads)

Leishmania, trypanosoma, giardia, trichomonads are dangerous for humans.

Leishmania- microbes causing leishmaniasis, are examined in blood smears, materials bone marrow, scrapings from skin infiltrates. In some cases, in the diagnosis of Leishmania, sowing on nutrient media is used.

Trypanosomes- causative agents of sleeping sickness (American / African trypanosomiasis, or Chagas disease).

The African variant is determined in the initial period in the study of peripheral blood. Pathological microbes during the progression of the disease are found in the material of punctures of the lymph nodes, in advanced stages - in the cerebrospinal fluid.

To diagnose trypanosomes in case of suspected Chagas disease, the test material is examined under a microscope at low magnification. In this case, smears and a thick drop are pre-stained.

Trichomonas(intestinal, oral,) are detected by microscopy of materials taken from the affected mucous membranes.

Identification of sporozoans (malarial plasmodium, causative agent of coccidosis, etc.)

The most common and dangerous species for humans is malarial plasmodium, which has 4 main varieties of the pathogen: the causative agent of three-day malaria, four-day malaria, tropical malaria and oval malaria.

Sexual development of Plasmodium (sporogony) takes place in Anopheles mosquitoes. Asexual (tissue and erythrocyte schizogony) - in the liver tissue and human erythrocytes. These features life cycle must be taken into account in the diagnosis of malarial plasmodium.

So, in the blood of a newly ill patient, germ cells of the sporogony cycle can be found. But at the height of malarial attacks, schizonts appear in large numbers in the blood.

Moreover, in different phases of malarial fever, various forms of plasmodium appear:

• during the period of chill, the blood is filled with merozoites, a kind of schizont;
• at the height of temperature, ring-shaped trophozoites accumulate in erythrocytes;
• the decrease in temperature is characterized by the predominance of amoeboid trophozoites;
• during periods of normal condition, the blood contains adult forms of schizonts.

The study of the causative agent of malaria (malarial plasmodium) is carried out in a smear and in a thick drop.

Note:the diagnosis of malaria in the study of smears and thick blood drops is sometimes erroneous. Blood platelets in some cases may be erroneously classified as a malarial pathogen. Also, fragments of leukocytes and other cells sometimes simulate plasmodium.

Basic research methods for protozoa

Let's take a brief look at the most common research methods for the presence of protozoa.

Diagnosis of protozoa using a native smear and a smear stained with Lugol's solution (in feces)

The drug is prepared from an emulsion of feces in an isotonic solution. Two drops of sodium chloride and Lugol's solution are applied to a glass slide. The test material is added to both compositions with a wooden stick and, after being covered with glass, is viewed at different resolutions of the microscope.

According to certain signs, the found protozoa are registered. For accuracy, 2-3 preparations are prepared from one material. In doubtful cases, the analysis is repeated several times over 2-3 weeks.

The method can detect vegetative and cystic forms:

• lamblia;
• balantidia;
• dysentery amoeba.

Together with pathogenic forms, non-pathogenic protozoa are also determined. Healthy carriers also have luminal and cystic forms.

Important:research in order to avoid inaccuracies and errors should be carried out repeatedly.

The result of the diagnosis of protozoa by the method of a native and stained smear should contain a description of the form of the pathogen (translucent, cyst, tissue).

Research requirements:

• the material taken for analysis (liquid feces) is examined no later than 30 minutes after defecation;
• formed feces must be diagnosed within 2 hours after defecation;
• the material should not contain impurities (disinfectants, water, urine);
• only wooden sticks are used to work with the material, glass ones are not suitable due to slipping of the mucus;
• Sticks must be burned immediately after use.

Conservation method (examination of feces) in the diagnosis of protozoa

The study is carried out by fixing the protozoa with a preservative. The difference between this method and the previous one is that preservatives allow you to save the drug for a long period.

Used preservatives:

• Barrow. Contains preservative ingredients: 0.7 ml sodium chloride, 5 ml formalin, 12.5 ml 96% alcohol, 2 g phenol and 100 ml distilled water. Coloring composition: 0.01% solution of thionine (azure).
• Safarliev's solution. Composition: 1.65 g zinc sulfate, 10 ml formalin, 2.5 g crystalline phenol, 5 ml acetic acid, 0.2 g methylene blue, 100 ml water. This preservative is used in cases where the material must be stored for more than a month.

Empty bottles are filled with a preservative, the material is transferred into them, in proportions of 3: 1, then, if necessary, a dye is added. Evaluation of the results is carried out in the study of 2-3 drugs.

Formalin-ether enrichment method (analysis for the presence of protozoa in feces)

This diagnostic method allows you to separate and concentrate protozoan cysts. The following ingredients are needed for analysis: formalin (10 ml), 0.85 g of isotonic solution, distilled water, sulfuric ether, Lugol solution.

A mixture of biomaterial with the listed liquids is mixed and centrifuged. The precipitate obtained at the bottom of the tube is stained with Lugol's solution and examined for the presence of cysts and vegetative forms.

Leishmania detection method (bone marrow smear)

For the diagnosis of leishmaniasis, reagents are used: a mixture of Nikiforov (sulfuric ether and ethanol), phosphate buffer, Azur-eosin according to Romanovsky.

The bone marrow substance is very carefully placed on a glass slide after special preparation. A microscope with an immersion system is used.

In the acute period of the disease, a large number of Leishmania are found in the punctate.

Note:Sometimes blood cells may resemble processed leishmania, so it is very important for the laboratory technician to be attentive and have sufficient experience for self-examination.

Method for detecting leishmania in a smear from a skin infiltrate

The required reagents are similar to the previous assay.

The test material is obtained from the existing tubercle or ulcerative contents. Scraping with suspicion of leishmaniasis is done very carefully with a scalpel, without blood. Then the preparation is prepared on glass. For the accuracy of the results obtained, several preparations are simultaneously examined.

In the presence of a disease, among the macrophages, fibroblasts, and lymphoid cells present in the test material, Leishmania is also determined.

Method for isolating a pure culture of Leishmania obtained by scraping pathological tissues

With this method of diagnosing the simplest tissue scrapings are placed in a special nutrient medium, in which active reproduction of Leishmania occurs.

Before taking a scraping, the skin is carefully treated with alcohol, then an incision is made in the tubercle, from the bottom of which the contents are removed and placed in a test tube with the medium. The material is taken several times, after which it is placed in different test tubes. Then, in a thermostat at a temperature of 22-24 degrees, cultivation takes place. The results are evaluated under a microscope. This method is used when other, cheaper and faster methods of diagnosing protozoa are ineffective.

You can see how tests for the presence of protozoa are deciphered in practice by a drop of blood by watching a video review:

Lotin Alexander, medical columnist

Methods helminthological research divided into direct and indirect. Direct methods: detection of the helminths themselves, their fragments, eggs, larvae in feces, urine, duodenal secretion, sputum, nasal and vaginal mucus, the contents of the subungual spaces, biopsied tissue pieces. Indirect methods: detection secondary changes arising in the human body as a result of the vital activity of the parasite, serological reactions, general research blood, urine. The most common methods for examining feces are helminthoovoscopic and protozooscopic. When diagnosing, it is impossible to identify by any one method the eggs or larvae of all types of helminths living in digestive system person. Thus, when using the flotation method, trematode eggs and, in some cases, unfertilized roundworm eggs do not float into the surface film (due to the high specific gravity). In feces, it is very rare to find pinworm eggs, teniid oncospheres, which are detected by special research methods: scraping from perianal folds for pinworms and teniids, sedimentation methods for trematodes (opistorch eggs, etc.). Therefore, for a targeted examination of the patient for helminthiases, the doctor in the referral should indicate which helminths should be given the main attention (diagnosis), which will allow the laboratory assistant to choose the appropriate technique for identifying this type of helminth. Feces taken from different places feces in an amount of at least 50 grams (teaspoon) in a clean glass dish should be sent to the laboratory no later than one day after defecation and examined on the day of admission. If necessary, save feces until next day it is placed in a cold place (0-4°C) or filled with one of the preservatives. Before the study, the feces are mixed with a stick so that the helminth eggs are evenly distributed in total mass. If any helminth eggs are found in the preparation, the viewing is not stopped, because. may be double or triple invasion. Monitoring the effectiveness of the treatment of helminthiasis is carried out by examining feces for helminth eggs in 2-3 weeks or 2-3 months after treatment, depending on the detected helminth. Macroscopic methods are used to detect whole sexually mature helminths or their fragments in the feces with the naked eye or with a hand-held magnifying glass. Often on the surface of the feces after defecation, you can see actively crawling pinworms; excreted with feces roundworm; sometimes people themselves notice the discharge of helminths. In patients with diphyllobothriasis, fragments of the strobilus of the tapeworm (in the form of "noodles") can stand out, and in those infected with teniids (pork or bovine tapeworm), segments of helminths (in the form of "white cuts") often leave the feces (in the form of "white cuts") or they actively crawl out of the anus. The macroscopic method is the main one for the differential diagnosis of teniadosis and teniarhynchosis (in combination with a survey). Of the special macroscopic methods, the method of sequential washing of feces is used. Feces are mixed in water to obtain a uniform suspension, after which, under good lighting, they are carefully examined in separate small portions in black photographic cuvettes or on a dark background in Petri dishes. With tweezers or a dissecting needle, all suspicious white particles, large formations suspicious of fragments of helminths are removed and examined under a magnifying glass between two glass slides. Small helminths or heads of cestodes are examined under a magnifying glass in a drop of glycerin or under a microscope. When using this method for the diagnosis of segments of the pork, bovine tapeworm, wide tapeworm, the washed segments are placed between two glasses and, looking at the light under a magnifying glass or a low magnification microscope, determine the species by the structure of the uterus (in a mature segment pork tapeworm 8-12 lateral branches depart from the central trunk, and in the bovine tapeworm 18-32, more often 28-32, in a wide tapeworm, the segments are wider and the uterus is in the center in the form of a "rosette"). If the uterus is poorly visible, then it can first be held for some time in a 50% glycerin solution, after which even the deserted uterine trunks are clearly visible. When determining these cestodes by the structure of the detached heads, they are carefully placed with a neck in a drop of glycerol between glass slides (or covered with a coverslip) and, without squeezing, are examined under a microscope at low magnification.

Microscopic methods are divided into simple, complex and special.

Simple methods include native smear, native smear with Lugol's solution, thick smear under cellophane according to Kato, twisting (according to Shulman) and perianal scraping.

Complex methods are more efficient and are based on the concentration of eggs in preparations. They include pre-treatment of faeces with liquid reagents, as a result of which helminth eggs either precipitate or float to the surface of the liquid.

TO complex methods enrichment methods include:

a) flotation (when the specific gravity of the eggs is less than the specific gravity of the saline solution and the eggs float into the surface film);

b) sedimentation (when the specific gravity of the eggs is greater than the specific gravity of salt solutions and the eggs settle into sediment).

Special methods for detecting eggs and larvae of helminths, cysts and vegetative forms of protozoa are the methods of scraping, flotation, sedimentation, larvoscopy, protozooscopy, the study of bile and the methods of staining smears of feces, sputum, etc.

Sampling and conservation

For research, feces are taken from different places in portions of 50 g and sent to the laboratory in a clean glass or plastic container with a tight lid. Fresh feces are examined (not more than a day old), and in some cases (in the study for strongyloidiasis) immediately after defecation. A number of preservatives for feces containing helminth eggs have been proposed: 4-10% formalin solution, which must be heated to t° 50-60° to prevent the development of hookworm eggs; a mixture of 0.2% aqueous solution of sodium nitrate (1900 ml), Lugol's solution (5 g of iodine, 10 g of potassium iodide, 250 ml of water), formalin (300 ml) and glycerin (25 ml), in which helminth eggs are stored 6-8 months; a mixture of glycerin (5 ml), formalin (5 ml) and water (100 ml); solutions of 1-1.5% detergents "Lotus", "Extra", "Barf", "Tide", etc. (in a weight ratio of feces and detergent solution 1: 5); a mixture of merthiolate 1: 1000 (200 ml), formalin (25 ml), glycerin (5 ml), distilled water (250 ml) with the addition of 0.6 ml of Lugol's solution (based on 1 g of feces per 10 ml of the mixture).

For the diagnosis of helminthiases, macro- and microhelminthoscopic methods for examining feces are used.

Macrohelminthoscopy studies

Settling method

The daily portion of faeces is thoroughly mixed with 5-10 times the amount of water, poured into high glass cylinders (jars, buckets) and left until the suspended particles are completely settled. The upper cloudy layer is carefully drained and clean water is added to the top (repeat several times until the water above the sediment becomes transparent). Having drained the top layer, transfer the precipitate to a cuvette or Petri dish and view it (on a dark background) under a magnifying glass or with the naked eye.

Screening method

Feces mixed with water are placed on the upper sieve of the device, consisting of a system of sieves with holes of decreasing diameter, the device is connected to the water supply network and, by opening the water tap, is washed, and the flowing liquid is discharged into the sewer. Large helminths remain on the upper sieve, while smaller ones linger on the lower ones. The sieves are turned over and, having washed the contents into dark cuvettes, they are viewed with the naked eye or under a magnifying glass.

Microhelminthoscopy studies

It is carried out in order to detect eggs (helmintho-ovoscopic methods - color. Fig. 1) or helminth larvae (helmintho-larvoscopic methods).

Helmintoovoscopic methods

Qualitative methods without enrichment

native smear. A small amount of feces is triturated on a glass slide in a drop of 50% glycerol solution or boiled water. Large particles are carefully removed, the mixture is covered with a coverslip and examined under a microscope (two smears are viewed). It is more convenient and easier to prepare long smears between two glass slides. The native smear is used only in addition to enrichment methods, since it is not effective enough, especially with weak invasions.

Thick smear with cellophane according to Kato(K. Kato, 1954) is very effective method research. Pieces of hydrophilic cellophane (4x2 cm in size) are soaked for 24 hours in a mixture of glycerol (50 ml), 6% phenol solution (500 ml) and 3% aqueous solution (6 ml) of green malachite (the latter is optional). OK. 100 mg of feces are smeared on a glass slide and, covered with a piece of wet cellophane, pressed down with a rubber stopper No. 5. The drug is examined after 30-60 minutes, when it dries slightly and clears up, as a result of which helminth eggs are easy to detect under a low magnification microscope.

Qualitative methods with enrichment (floating and settling methods)

The first are based on the use of saturated solutions of various chemicals. substances in which eggs float due to the difference in specific gravity.

Kofoid-Barber method(Ch.A. Kofoid, M. A. Barber) modified by Fulleborn (F. Fulleborn, 1920). OK. 5 g of feces in a tall and narrow jar (volume 100 ml) are stirred with a wooden stick in 100 ml of a saturated solution table salt, ud. weight to-rogo 1,18 (400 g of salt are dissolved at boiling in 1 l of water). Large particles that have floated to the surface are quickly removed with a stick or piece of paper. After settling the mixture for 45-90 min. a wire loop (0.8-1 cm in diameter) removes the entire surface film, transfers it to a glass slide. When testing for hookworm eggs, the mixture is left to stand for 10-15 minutes. You can remove the film directly with a glass slide, which cover the jar so that it comes into contact with the liquid (a saturated salt solution is added to the edges of the jar). After settling, the slide is removed, quickly turned over and examined under a microscope (without a cover slip) a film with helminth eggs adhering to it. This method well reveals eggs of all nematodes and pygmy tapeworm. Heavy trematode eggs; most cestodes and unfertilized roundworms float poorly, so the sediment is also examined. To do this, after removing the film, the liquid from the jar is quickly drained and a few drops are taken from the sediment with a loop or pipette, transferred to a glass slide, a drop of glycerin is added for clarification and examined under a microscope.

Method E. V. Kalantaryan(1938) Is a more efficient modification of the Fülleborn method. In it, the sodium chloride solution is replaced by a saturated solution of sodium nitrate, sp. weight to-rogo 1.39 (one volume of sodium nitrate is dissolved in an equal volume of water during boiling), the film is removed after 20-30 minutes.

Faust's methods are also applied (E. S. Faust, 1939), Brudastov et al. (1970) and others.

For the deposition of helminth eggs, a chemical is used. substances that dissolve fats and proteins in feces.

Telemann's method (W. Telemann, 1908) as modified by Miyagawa (Y. Miyagawa, 1913). OK. 5 g of feces are ground in a mortar or jar, adding 5 ml of ethyl ether and 50% hydrochloric acid solution; the mixture is filtered through a wire or hair sieve into a test tube and centrifuged. Three layers are formed in the test tube: at the top - ether with dissolved fat, below - hydrochloric acid with dissolved protein substances, in the sediment - insoluble parts of feces and helminth eggs. The upper layers are drained, and water is added to the sediment and centrifuged. A few drops of the precipitate are transferred to a glass slide and examined under a microscope. This method can detect eggs of all types of helminths, but they are sometimes deformed.

Ritchie method (L. S. Kitchie, 1948). To dissolve fats and proteins, formalin and ether are used.

For the deposition of helminth eggs, various detergents can be used. Abroad, the filtration method in special Bell devices has become widespread, which is used to study not only feces, but also urine, blood, etc.

There are a number of methods that combine the principles of flotation and egg settling. Among them are the methods of Lane (C. A. Lane), Rivas (D. Rivas), Gorkina, Darling (S. T. Darling). One of these flotation-sedimentation methods, as well as the method of successive discharges, was proposed by N.V. Demidov (1963, 1965) for research on fascioliasis and dicroceliosis.

Quantitative Methods

Stoll method(N. R. Stoll, 1926). In a graduated wide test tube or flask (with two marks: 56 and 60 ml), decinormal sodium hydroxide solution is poured up to the first mark, feces are added until the liquid level reaches the second mark, thoroughly mixed with a glass rod and, placing 10 glass beads or small pebbles, stopper and shake for 1 min. Quickly, so that the suspension does not settle, 0.075 ml of the mixture (0.005 g of feces) is collected with a graduated pipette, transferred to a glass slide with a grid applied on it, covered with a cover slip and the helminth eggs in the preparation are counted under a microscope; multiplying the resulting number by 200, get the number of eggs in 1 g of feces. For a more accurate result, eggs are counted in two or more preparations and an average is taken. The Stoll method is insensitive for weak invasions. Therefore, it is recommended to count the number of eggs in preparations prepared various methods flotation or sedimentation, subject to the constant use of an equal sample of feces and dishes of the same volume. A thick Kato smear is also used.

Beaver Method(R. C. Beaver, 1950). A standard smear is prepared, the density of which is determined using an electrophotometer, and then all helminth eggs are counted in it.

Helmintolarvoscopic methods

Bermann method(G. Baermann, 1917). 5-10 g of freshly excreted feces are placed on a metal mesh in a glass funnel, at the narrow end of which a rubber tube with a clamp is put on. To avoid contamination of the sediment with fecal particles, it is recommended to place (on the mesh) paper or add animal charcoal or cornmeal to the feces. The funnel is filled with warm water (t ° 45-50 °) until it comes into contact with feces. Due to thermotropy, the larvae actively move to warm water and gradually accumulate in the lower part of the funnel, above the clamp. After 3-4 hours, the clamp is opened, the liquid is lowered into 1-2 test tubes, centrifuged for 2-3 minutes, the top layer is drained, and the precipitate is examined on a glass slide under a microscope.

Method for the detection of miracidia schistosomes. The faeces are washed in the dark at t° 8-10°, the sediment is kept for 45 minutes. in bright light at t ° 28 °, then pour into a dark flask with a tube on the side. The miracidia are concentrated in a transparent side tube from where they can be picked out.

The methods of Fülleborn and others are also used to detect hookworm larvae.

Methods for studying other secretions, as well as tissues and organs

OK. 100 ml of the test urine stand for 30 minutes. in a cylinder and, having removed the upper layer, pour 10-15 ml of sediment into a test tube, centrifuge at 1500 rpm for 1-2 minutes; sediment is examined. It is advisable to collect the entire daily portion of urine.

Urogenital schistosomiasis is diagnosed by identifying miracidia. A portion of fresh urine is centrifuged for 5 minutes, the sediment is poured into a black-colored flask, a transparent glass tube is soldered to the upper part of the cut. Water is added in a ratio of 1:5, 1:10 and placed for 2 hours in a thermostat at t ° 25-30 °. The miracidia emerging from the eggs are visible through a transparent tube with the naked eye in the form of rapidly moving dots. At hron, the form of a schistosomiasis at patients by the end of an urination blood is allocated, but eggs in urine find seldom therefore it is recommended to resort to a biopsy of a bladder.

Sputum examination. The sputum may contain eggs of paragonimus, schistosomes, tominxes, larvae of migrating nematodes, fragments of echinococcal bladder. The entire delivered portion of sputum is carefully examined with the naked eye or under a magnifying glass, all visible scraps of tissue, rust-colored clusters, etc. are selected and examined; then view the entire portion with strokes. Purulent sputum pour an equal amount of 0.5% caustic alkali solution, centrifuge and examine the sediment.

With some helminthiases in sputum, there are characteristic changes. With paragonimiasis, for example, in sputum, clusters of eggs in the form of yellowish lumps can be found, as well as a large amount of mucus, leukocytes, erythrocytes, alveolar cells, Kurschmann's spirals, elastic fibers, Charcot-Leiden crystals. Characteristic rhomboid crystals with pointed ends are also revealed. Availability a large number eosinophils allows to differentiate paragonimiasis from tuberculosis.

Examination of the contents of abscesses and punctates. IN purulent secretions abscesses, as well as in tumors and cysts removed during surgery, you can find helminths, their fragments, larvae and eggs (echinococcus, alveococcus, sparganum, cysticercus, dirofilaria, roundworms, toxocara, paragonimus, etc.). The research technique is common; in some cases, histological sections are prepared from tumor tissues. Purulent contents can be processed by the Telemann method; the clear liquid is examined in the same way as the duodenal contents by adding sulfuric ether. Echinococcal fluid is centrifuged and the sediment is examined for the presence of scolexes and hooks; preparations are stained with carbolfuchsin according to Ziehl-Nelsen.

Blood study. Microfilariae and larvae of migratory nematodes are found in the blood. A drop of blood is taken from a finger or from an earlobe and examined fresh or preparations are made from it.

A drop of blood is placed on a glass slide with a square of vaseline applied and lightly pressed with a cover slip. Under a microscope, microfilariae are seen moving between blood cells. Blood can be placed between two layers of adhesive cellulose tape; microfilariae remain mobile for 6 hours; in a completely dried preparation, they can be distinguished under a microscope within 30 days. Microfilaria species can only be identified in stained smears or thick drops. The prepared preparations are dried, hemolyzed and stained according to Romanovsky - Giemsa, Wright, Ziehl-Nelsen, Leishman, Papanicolaou (see Wright staining method, Romanovsky-Giemsa method, Ziehl-Nelsen method). Having found microfilariae in a drop of blood stained according to Romanovsky-Giemsa, the preparation is additionally stained with Hansen's hematoxylin; after 15-60 min. washed 2 min. in running water. The repainted preparation is differentiated in 0.2% hydrochloric acid solution; the cap of microfilariae turns pale purple, and the nuclear substance of the body turns dark purple.

For weak invasions, it is necessary to examine 2-10 blood ices with an anti-coagulant (for example, with a 5% solution sodium citrate) using one of the following methods.

Knott's method (J. Knott, 1939), modified by Markell and Foge (E. K. Markell, M. Voge, 1965). 2 ml of blood is mixed in a centrifuge tube with 10 cases of 1% acetic acid, centrifuged for 2 minutes. at 1500 rpm; surface layer drained, the precipitate is distributed on several glass slides and examined under a microscope. Preparations can be stained according to Romanovsky-Giemsa or other methods.

Bell Filtration Method (D. R. Bell, 1967). In an apparatus consisting of a stainless steel funnel with a rectangular hole and membrane filters of the same shape, 19 x 42 mm in size, with a pore size of 0.8 to 5 μm, blood is filtered into test tubes, hemolyzed in a mixture of 1 ml of Tipol detergent and 9 ml of fiziol, solution (to speed up the filtration, the apparatus is connected to a vacuum pump). The filter is fixed in boiling distilled water and stained according to Romanovsky-Giemsa or hot Ehrlich hematoxylin. The colored filter is dried in a desiccator or in isopropyl alcohol (successively in 3 cups), clarified on glass with immersion oil and examined under a coverslip. Painted preparations are stored for several weeks. The Bell method is the most effective for quantitative counting of microfilariae in the blood.

The method with Goldsmid's polyvidone is also used.

Skin research. In the skin, microfilariae of the oncho-church and helminth larvae of animals can be found, causing skin form larva migrans(cm.). Skin sections or material obtained by scarification are taken in the thigh, calf, buttocks or at the level of the deltoid muscle. A cone-shaped piece of the epidermis is lifted with an entomological pin and, cut off with a razor, examined on glass in a drop of fiziol solution. At negative result a fresh preparation is re-examined after 10 minutes; larvae are usually localized along the edges of the preparation. The received material can be kept in 2 ml fiziol, solution within 1-2 hours. and examine the sediment. It is recommended to take 5 skin sections from the patient.

It is possible to prepare preparations from blood and tissue fluid released after a strong compression of the sections. Drops are stained according to Mayer, Romanovsky-Giemsa or Delafield's hematoxylin.

Standard Method for Quantifying Invasion. Biopsied area of ​​skin dia. 3-5 mm and weighing at least 1-4 mg weigh, cut into small pieces and count the larvae under a microscope in fiziol. solution on a glass slide; 1-4 larvae in the field of view are marked with a + sign, 5-9 larvae - with a ++ sign, 10-19 - with a +++ sign, 20 or more - with a + + + + sign. Quantitative accounting is more convenient to carry out on stained preparations.

Test for trichinosis, cysticercosis and schistosomiasis

Identification of Trichinella larvae

compression method. A piece of double-headed or calf muscle(near the tendon), taken surgically with asepsis, is split with dissecting needles into separate thin fibers, squeezed between two glass slides in a drop of glycerin so that the preparation is thin and transparent. Trichinella larvae are clearly visible under a microscope with a darkened field of view. Research of several pieces of muscles in the compressoriums used in veterinary-san is effective. practice, especially in special trichinelloscopes.

The Bechman Digestion Method(G. W. Bachman, 1928). 1 and crushed muscles are poured with 60 ml of artificial gastric juice (0.5 g of pepsin; 0.7 ml of concentrated hydrochloric acid; 100 ml of water) and incubated for 18 hours. in a thermostat at t° 37°; the upper layer of the liquid is drained, warm water (t° 37-45°) is added to the precipitate and poured into the Bermann apparatus. An hour later, the liquid is lowered into a test tube, centrifuged and the sediment is examined. If the larvae are enclosed in calcified capsules, they are preliminarily decalcified in a solution of hydrochloric, nitrogen, or sulfuric acid.

bioassay. Pieces of the studied muscles are fed to white mice or rats. After 2-3 days, sexually mature Trichinella can be found in the duodenal contents, and after 2-3 weeks, larvae in the muscles of the diaphragm and tongue.

Histological sections. Pieces of muscles are fixed in Bouin's, Zenker's or other fluids; in the usual way sections are prepared on a microtome and stained with Delafield's hematoxylin.

Identification of cysticerci

A piece of extirpated muscle, connective tissue, etc., is examined with the naked eye, a cysticercus is carefully isolated - a whitish translucent vesicle 1-2 cm in size, crushed between two glass slides in a drop of glycerin and examined under a microscope. To determine the viability of the cysticerci isolated from the tissues, they are kept in a 50% solution of bile for fiziol. solution in a thermostat at t° 37°; after 10-60 min. the head of a viable cysticercus turns outwards. The calcified cysticerci are previously decalcified with a 4% solution of nitric acid for an hour.

Detection of schistosome eggs

At hron, forms of schistosomatosis, when the formation of granulomas prevents the release of eggs from the tissues into the intestinal lumen or into the urinary tract, a biopsy of the rectal mucosa is used, which is performed with a special spoon using a rectoscope, choosing a site with a visible lesion. The biopsy piece is crushed between two glass slides and examined under a microscope. If the result is negative, the pieces are clarified in a 4% solution of caustic alkali and gistol is prepared from them. slices. A biopsy of the mucosa of the jejunum is performed orally and the resulting material is examined by the compression method or gistol, sections are prepared from it. In some cases of urinary genitourinary schistosomiasis, the diagnosis can only be made on the basis of an endovesical biopsy. With hepatolienal form of schistosomiasis, a puncture biopsy of the liver is performed; gistol, sections are prepared from the obtained material and examined under a fluorescent microscope. A fluorescent microscope with a dark field of use and for examining liver tissue for schistosome eggs. When schistosomes affect the female genital tract, the discharge is collected using a vaginal mirror or pieces of the mucous membrane of the cervix are taken with a sharp spoon; the received material is examined under a microscope on glass in a drop fiziol, solution.

Research on enterobiasis and teniidosis

Perianal scraping (recommended to take in the evening, 1 - 1.5 hours after the patient went to bed, or in the morning before toileting). With a match cut obliquely and moistened in a drop of liquid (fiziol, solution, boiled water or 2% solution of bicarbonate soda), applied to a glass slide, do I carefully do it? scraping from the mucous membrane of the anus and the folds around it (from the center outwards). The mucus collected at the end of the match is scraped off with the edge of the cover slip into a drop of liquid on the slide and, covered with the same cover slip, is examined. During mass examinations, when scrapings are taken in children's or other institutions, the used match is placed in a drop of liquid on a slide, after drying, the drops are covered with another slide and delivered to the laboratory, wrapped in paper and secured with an elastic band. For the study of rectal mucus, a special device is used - a Shakhmatov or Ziemann tube, with which mucus is taken from the rectum and smears are examined under a microscope.

Cotton swab method. Patients are given a test tube with a cotton swab on a glass or wooden stick at home; in the morning the patient wipes the perianal folds with a swab moistened boiled water and put it in a test tube with a small amount of water. In the laboratory, swabs are rinsed in the same tube with a new portion of water, and the sediment is examined after centrifugation.

Hall's cellophane method (M. C. Hall, 1937). A square piece of cellophane is reinforced with a rubber band on a glass rod. Scraping is done with dry cellophane and placed in a test tube. In the laboratory, cellophane is slightly shifted from the stick and, cutting off its tip with scissors, straighten it on a glass slide; moistened with a decinormal sodium hydroxide solution, covered with a coverslip and examined under a microscope.

Graham's cellulose tape method (C. F. Graham, 1941). A strip of cellulose tape is pressed with its adhesive side to the perianal folds of the subject, then with the same side to the slide and examined without a cover slip; you can add a drop of toluene. VV Kaledin (1972) proposed to use for these purposes celluloid discs cut from washed x-ray film and moistened with glycerin; disks are examined on a glass slide in a drop of silicate glue. The cellulose tape method can also be used to examine children's underwear.

Subungual scraping. The edges of the nail, the nail bed, the subungual spaces are moistened with a 0.5-1% solution of caustic alkali and wiped with wet cotton swabs. The swabs are placed in centrifuge tubes with the same solution, centrifuged and the sediment is examined.

Object research methods environment on eggs and larvae of helminths (sanitary and helminthological studies)

Research is carried out in order to determine the degree of contamination of objects with eggs and larvae of helminths external environment. The received data use for an assessment a dignity. the state of institutions and enterprises and the effectiveness of ongoing measures to combat helminthiasis.

When studying the degree of contamination of various environmental objects with helminth eggs and larvae and assessing their role in the epidemiology of helminthiasis, it is important to establish not only the number of eggs found, but also their viability and invasiveness, which are determined: a) by appearance, under a microscope; b) staining with various dyes, including luminescent ones; as a rule, live eggs and larvae do not stain; c) cultivation under optimal conditions to the invasive stage; d) infection of laboratory animals (bioassay).

Studies of water and sewage. For one study, 10-25 liters of water (rivers, seas, ponds, swimming pools, water pipes) and 1-2-5 liters of sewage are used.

Method 3. G. Vasilkova (1941). Water or sewage is filtered through membrane ultrafilters in a Goldman apparatus, which consists of a glass or metal funnel with filters connected by a nozzle ring to a Bunsen flask. To speed up the filtration process, a vacuum pump is connected to the apparatus. After filtration, membrane filters are examined under a microscope, having been cleared with a 50% glycerol solution; the accumulated sediment is cleaned off and viewed separately in the form of smears. Filtration devices and other designs are used.

The method of G. Sh. Gudzhabidze and G. A. Yudin (1963) for the study of sewage fluid. 1 liter of liquid is settled for 2 hours in a Lisenko cylinder; the resulting sediment (5-9 ml) is treated as in the study of the soil (see below).

Method N. A. Romanenko (1967). To 1 liter of waste liquid placed in a glass cylinder with a capacity of 1200-1500 ml, add 0.4-0.6 g of aluminum sulfate or ferric chloride (for the purpose of coagulation, which accelerates the process of sedimentation of suspended particles) and after 40 minutes. the mixture is centrifuged for 3 minutes. at 1000 rpm; the top layer is drained, and to dissolve the flakes, add 1-2 ml of a 3% solution of hydrochloric acid, then 150 ml of a saturated solution of sodium nitrate. The sediment is examined like soil.

Soil research

Soil contamination with helminth eggs is determined in order to identify foci of helminthiases and assess the effectiveness of their rehabilitation.

Method 3. G. Vasilkova and V. A. Gefter (1948). 12.5 g of soil are mixed in test tubes (100 ml) made of stainless steel or brass with 20 ml of 5% caustic alkali solution, adding 10 glass beads or small pebbles. The tubes are closed with rubber stoppers and shaken for 20 minutes. in a shaker or by hand. After removing the plugs, the test tubes are centrifuged for 3-5 minutes, the surface liquid is drained, 60-80 ml of a saturated solution of sodium nitrate is added to the sediment and, having thoroughly mixed, centrifuged again for 3-5 minutes. In this surface film with floating eggs is removed by touching the surface of the mixture with a complex loop (5-6 loops connected on a common rod), and transferred to a glass of water; mixed with the same solution, centrifuged; the whole procedure is repeated at least 3 times. The contents of the glass, where the film is transferred, is diluted with water and filtered through membrane filters, which are examined under a microscope in a drop of glycerin.

Method 3. G. Vasilkova and V. A. Gefter modified by A. A. Namitokov (1961) differs from the main method in that instead of studying film preparations, half the contents of the tube are used (each time adding a new portion of a saturated solution of sodium nitrate), filtered and examine filters.

N. A. Romanenko (1968) recommends examining soil samples and sewage sludge for helminth eggs using the apparatus proposed by G. Sh. Gudzhabidze. 50 g of soil are thoroughly mixed for 1 min. in 150 ml of water in centrifuge tubes (capacity 250 ml) with special blades, which are driven by an electric motor. The mixture is centrifuged for 3 minutes. at 1000 rpm, the water is drained off and, adding 150 ml of a saturated solution of sodium nitrate, mix and centrifuge again for 3 minutes. Test tubes with samples are placed in a stand, sodium nitrate solution is added until a convex meniscus is formed, covered with glass slides (10 x 6 cm) and set aside for 10-15 minutes, then the glasses are removed and examined; the procedure is repeated at least 4 times.

Research on larvae of helminths according to the method of Bermann (1917). 200-400 g of soil is placed in a piece of gauze on a metal mesh (with a hole diameter of 1-2 mm) put on the wide part of a glass funnel fixed in a tripod. A rubber tube with a clamp is stretched over the narrow end of the funnel. The funnel is filled with warm (t ° 50 °) water so that the lower part of the mesh with soil comes into contact with water. Due to thermotropy, the larvae actively crawl out into warm water and, settling, accumulate in the lower part of the rubber tube above the clamp. After 3-4 hours, 50 ml of the contents are released from the funnel into a test tube, centrifuged and the sediment is examined.

Research of vegetables, fruits and berries

Investigate mainly vegetables, berries and fruits, eaten without heat treatment. Method 3. G. Vasilkova (1948). 5-10 pieces of vegetables or fruits (approx. 0.5 kg) or 100-200 g of greens (lettuce, green onions) are poured for several hours with water in wide-mouthed glass jars with ground stoppers and shaken for 10-20 minutes. in a shaker or by hand. The water is drained, the test objects are rinsed clean water and all wash water is filtered in the Goldman apparatus; filters are examined by clearing with glycerin. You can tint the filters with 25% Lugol's solution in glycerin; while helminth eggs are stained in Brown color and are easily recognizable among starch grains dyed in Blue colour. Large sediment is treated like soil.

Study of swabs from household items and hands. A glue brush (or a cotton swab wrapped in a piece of nylon fabric) dipped in a 2% solution of bicarbonate of soda is repeatedly carried out over the object or hands being examined, after which it is rinsed in the same solution poured into a test tube. In the laboratory, brushes and swabs are rinsed with clean water; all wash water is centrifuged and the sediment is examined.

Method V. A. Gefter (1960). It is more efficient to collect dust from soft equipment with a vacuum cleaner connected to a funnel, which consists of 2 detachable parts: a membrane filter is placed on the surface of the lower part covered with a metal or plastic mesh and strengthened by putting on upper part funnels. The assembled funnel is attached to the vacuum cleaner hose with a rubber tube. Dust is collected from the object for 3 minutes, after which the filter is removed and replaced with a new one. In the laboratory, the filters are viewed under a microscope, having been cleared with glycerin. If the layer of dust on the filter is large, it is scraped off and viewed as smears or treated like soil.

Immunological diagnostic methods

Possibility of use immunol. methods for diagnosing helminthiases is due to the ability of helminths to produce active antigens that affect the host's immunocompetent cells and stimulate the production of antibodies. The most effective immunodiagnostic methods for intestinal helminthiases, when the secrets and excretions of helminths with antigenic activity, enter directly into the host's blood. Immunol, reactions are used to diagnose ascariasis, trichinosis, filariasis, schistosomatosis, echinococcosis and alveococcosis, cysticercosis, paragonimiasis, the symptom complex larva migrans- (toxocariasis, angiostrongylosis), etc. Apply various serol, tests (precipitation reactions, agglutination, complement fixation, fluorescent antibodies ) and intradermal allergy tests. Antigens are prepared from larvae and mature helminths using saline extracts of homogenates of freshly frozen or lyophilized tissues, as well as various biologically active fluids (fluid from echinococcal blisters, ascaris cavitary fluid, etc.). Due to the fact that helminths have a very complex antigenic structure and in their antigenic mosaic there are components and individual determinants that cross-react with other types of helminths, bacteria, and host antigens, methods are being developed to purify them from nonspecific components. Fractionation is carried out different methods: gel filtration in columns with Sephadex, ion-exchange chromatography on DEAE-Sephadex, treatment with acids, etc. Fractional antigens usually have a higher specificity than whole extracts, while their activity is approximately the same. Immunodiagnostic methods find everything greater application. Reactions are used not only for the most complete and early detection of patients, but also for the study of foci and the study of the epidemiology of helminthiases.

Serological reactions. The reaction of microprecipitation on live larvae is used to diagnose nematodes - the preimaginal phase of ascariasis, ankylostomidosis, trichinosis. The reaction becomes positive 5-10 days after infection (ascariasis, ankylostomidosis) and persists for 90-100 days. The antigen is live nematode larvae isolated from the tissues of experimentally infected laboratory animals. The selected larvae are thoroughly washed from the host proteins with sterile fiziol, solution and distilled water and 10-15 copies each. placed with a Pasteur pipette on a sterile glass slide with a well. Apply 2-3 drops of the test serum, cover with a sterile cover slip, place in a humid chamber (Petri dish lined with moistened filter paper) and incubate for 24-48 hours. in a thermostat at t° 37°. With a positive reaction under a low magnification of the microscope, they are visible around the mouth and anal holes larvae greyish-white, slightly opalescent masses of precipitates of spherical or zigzag shape. The reaction efficiency reaches 85-95%.

The ring precipitation reaction was developed by V. P. Pashuk (1957) for the diagnosis of trichinosis. The reaction becomes positive at the 2-3rd week of illness. Its efficiency reaches 80-90%. The antigen is a powder extract from larvae dried at t° 35° isolated from the muscles of infected animals. In test tubes diam. 0.5 cm pour 0.1 ml of each dilution of the antigen and carefully layer on it (or lower it to the bottom) an equal amount of the test serum so that the liquids do not mix. The tubes are held for 30 minutes. in a thermostat at t ° 37 °, and then 30-60 min. at room temperature. With a positive reaction, a whitish delicate ring appears at the border of contact between the antigen and serum, which easily disintegrates when shaken.

The precipitation reaction in the gel according to Ouchterlony (O. Ouchterlony, 1949) in the micromodification of A. I. Gusev and V. S. Tsvetkov was proposed by I. A. Ginovker, E. A. Zabozlaeva, A. V. Doronin (1970) for the diagnosis of early phases of opisthorchiasis. According to preliminary data, its efficiency is 87%. The antigen is an extract of a homogenate of freshly frozen sexually mature opisthorchis isolated from the liver of cats.

The reaction of indirect hemagglutination (see) with a diagnosticum - a suspension of formalized and tanized ram erythrocytes sensitized by echinococcal fluid - developed

LN Stepankovskaya (1972) for the diagnosis of echinococcosis and alveococcosis.

RNHA with a diagnosticum of formalinized sheep erythrocytes sensitized with an extract of a homogenate of fresh-frozen cysticerci of porcine tapeworm was proposed by L. M. Konovalova (1973) for the diagnosis of cysticercosis of the brain; it is effective in 85% of cases.

RNHA with ascaris diagnosticum is proposed for the diagnosis of the preimaginal phase of ascariasis.

The complement fixation reaction (see) is put according to the usual method and is used to diagnose trichinosis and cysticercosis.

Method of luminescent antibodies (indirect method). This method with defatted dry homogenate of porcine tapeworm cysticerci as an antigen, fixed on a glass slide, was developed by JI. M. Konovalova (1973) for the diagnosis of human cysticercosis. The same reaction with gistol, sections of Trichinella larvae as an antigen was developed for the diagnosis of trichinosis.

E. S. Leykina, V. A. Gefter.

When helminth eggs are found on various environmental objects (soil, water, vegetables, etc.), it is always necessary to determine their viability by appearance, staining with vital dyes, cultivating under optimal conditions and setting up a biological sample, i.e.

Feeding laboratory animals.

Determination of the viability of eggs or larvae of helminths in appearance. Helminth eggs are microscoped first at low magnification, then at high magnification. In deformed and dead eggs of helminths, the shell is torn or bent inward, the plasma is cloudy, loosened. In segmented eggs, cleavage balls (blastomeres) are unequal in size, irregular in shape, and often shifted to one pole. Sometimes there are abnormal eggs, which, having external deformities, develop normally. In living larvae of ascarids, fine granularity is present only in the middle part of the body, as they die, the granularity spreads throughout the body, large shiny hyaline vacuoles appear - the so-called strings of pearls.

To determine the viability of mature eggs of ascarids, whipworms, pinworms, active movements of the larvae should be caused by slightly heating the preparation (to a temperature not exceeding 37 ° C). It is more convenient to observe the viability of ascaris and whipworm larvae after they are isolated from the egg shell by pressing on the cover glass of the preparation with a dissecting needle or tweezers.

In invasive larvae of ascarids, a cap is often seen that has exfoliated at the head end, and in larvae of whipworms that have completed development in the egg, a stylet is found in this place at high magnification. In the dead larvae of helminths, regardless of their location (in the egg or outside it), body decay is noticed. Wherein internal structure the larva becomes lumpy or granular, and the body is cloudy and opaque. Vacuoles are found in the body, and breaks are found on the cuticle.

The viability of teniid oncospheres (bovine, porcine tapeworm, etc.) is determined by the movement of embryos when exposed to digestive enzymes. The eggs are placed on a watch glass with dog gastric juice or artificial duodenal juice. The composition of the latter: pancreatin 0.5 g, sodium bicarbonate 0.09 g, distilled water 5 ml. Watch glasses with eggs are placed in a thermostat at 36-38 ° C for 4 hours. In this case, the living embryos are released from the shells. The shells of living oncospheres also dissolve in acidified pepsin and in alkaline solution trypsin after 6-8 hours in a thermostat at 38 °C.

If teniid eggs are placed in a 1% solution of sodium sulfide, or a 20% solution of sodium hypochloride, or in a 1% solution of chlorine water at 36-38 ° C, mature and live embryos are released from the membranes and do not change for 1 day. Immature and dead oncospheres shrink or swell and increase sharply, and then "dissolve" within 10 minutes - 2 hours. Live embryos of teniids also actively move in a mixture of 1% sodium chloride solution, 0.5% sodium bicarbonate solution and bile at 36- 38 °C.

The viability of scolex echinococci is determined with low heating. To do this, scolexes or brood capsules washed in water are placed in a drop of water on a glass slide with a hole, covered with a coverslip and examined under a microscope with a heating stage at a temperature of 38-39 ° C. If there is no heating table, the preparation is heated using any heat source. At the same time, viable scolexes actively move, reducing or relaxing the suckers, lengthening and shortening the proboscis. If you place scolex in 0.5-1% water solution filicilene at room temperature, then all viable scolexes will quickly turn out and die. Non-viable scolexes do not turn out.

The viability of fasciolia adolescariae collected from plants and other objects of water bodies is checked by examining them on a glass slide in saline under a microscope with a heating stage. When heated, the trematode larvae in the cyst begin to move.

The viability of the eggs of the dwarf tapeworm is determined by the location of the hooks on the embryo.

In living eggs of the pygmy tapeworm, sluggish pendulum-like movements of the protoplasm and almost imperceptible extensions and shifts of the pointed ends of the lateral pair of hooks take place away from the middle pair.

The contractions of the protoplasm of the embryo and the germinal hooks help the embryo to free itself first from the shells of the oncosphere, and then from the outer shell of the egg.

In a live egg, the median pair and lateral pairs of hooks are arranged in parallel; in some eggs, the blades of the lateral pairs are brought together and located at an angle of less than 45° relative to the middle pair of hooks.

The dying embryo convulsively contracts and sluggishly pushes the hooks apart. In the dead embryo, the movement of the hooks stops, and they are arranged in a disorder, sometimes the hooks lateral to the neighboring pair are moved apart at a right, obtuse or acute angle.

Sometimes wrinkling of the embryo, the formation of granularity are observed. A more accurate method is based on the appearance of movements of the oncosphere during a sharp change in temperature: from 5-10 to 38-40 °C.

Determining the viability of immature nematodes should be studied in a humid chamber (Petri dishes), placing ascaris eggs in a 3% formalin solution prepared in an isotonic sodium chloride solution at a temperature of 24-30 ° C, whipworm eggs in a 3% solution of hydrochloric acid at a temperature of 30-35 ° C, pinworm eggs in an isotonic solution of sodium chloride at a temperature of 37 "C. Petri dishes should be opened 1-3 times a week for better aeration and moisten the filter paper again with clean water.

Observations of the development of helminth eggs are carried out at least 2 times a week. The absence of signs of development within 2-3 months indicates their non-viability. Signs of the development of helminth eggs are first the stages of crushing, the division of the contents of the egg into separate blastomeres. During the first day, up to 16 blastomeres develop, which pass into the second stage - morula, etc.

Hookworm eggs are cultured in a glass cylinder (50 cm high and 7 cm in diameter) closed with a stopper. A mixture of equal volumes of sterile sand, charcoal and feces with eggs of hookworm, diluted with water to a semi-liquid consistency, carefully poured into the bottom of the cylinder using a glass tube. During 1-2 days of settling in the dark at a temperature of 25-30 ° C, rhabditoid larvae hatch from the eggs, and after 5-7 days they become already filariform: the larvae crawl up the walls of the cylinder, where they are visible even to the naked eye. Trematode eggs naturally developing in water, such as opisthorchis, diphyllobotriid, fasciol, etc., are placed on a watch glass, a Petri dish or in another vessel, a small layer of ordinary water is poured. When cultivating fasciola eggs, it should be taken into account that they develop faster in the dark, while miracidium is formed in live eggs at a temperature of 22-24 ° C in 9-12 days. When microscopy of developing trematode eggs, miracidium movements are clearly visible. Fasciola miracidium emerges from the egg shells only in the light. When cultivating, the water is changed after 2-3 days.

Hookworm larvae and strongyloid are cultivated on agar in a Petri dish with animal charcoal. After being in a thermostat at a temperature of 26-30 ° C for 5-6 days, the larvae spread over the agar, leaving behind a path of bacteria (Fülleborn method).

Method of Harada and Mori (1955). 7 ml of distilled water are added to test tubes placed in a rack. Take 0.5 g of feces with a wooden stick and make a smear on filter paper (15X150 mm) 5 cm from the left edge (this operation is carried out on a sheet of paper to protect the surface of the laboratory table). Then the strip with the smear is inserted into the tube so that the left end free from the smear reaches the bottom of the tube. The upper end is covered with a piece of cellophane and tightly wrapped with an elastic band. On the test tube write the number and surname of the subject. In this state, the tubes are stored for 8-10 days at a temperature of 28 °C. To study the culture, remove and remove the cellophane cover and remove a strip of filter paper with tweezers. Care should be taken in this case, as a small number of infective larvae can move to the upper end of the filter paper or to the wall of the test tube and penetrate under the surface of the cellophane.

The tubes are placed in a hot water bath at 50°C for 15 minutes, after which the contents are shaken and quickly poured into a 15-mL tube to precipitate the larvae. After centrifugation, the supernatant is removed, and the precipitate is transferred to a glass slide, covered with a coverslip and microscoped under low magnification.

For the differential diagnosis of filariform larvae, it is necessary to use the data presented in Table. 13.

Methods for staining eggs and larvae of helminths. Dead tissues in most cases perceive colors faster than living ones. These features are used in helminthology to determine the viability of eggs and larvae of helminths. However, in some cases, some paints are better perceived by living tissues than by dead ones.

Table 13 Differential Diagnosis filamentous larvae of A.duodenale, N.americanus, Strongyloides stercoralis, Trichostrongylus spp.

For differential recognition of live and dead eggs and larvae, the following paints and methods are used.

Methylene blue leucobase is used to stain living and dead tissues. A living cell or tissue reduces methylene blue to a colorless leucobase; dead tissue does not have this ability, and therefore acquires a color.

For coloring Ascaris eggs, you can use methylene blue in a solution of lactic acid with caustic alkali (methylene blue 0.05 g, caustic soda 0.5 g, lactic acid 15 ml). Live eggs do not perceive color, the embryos of dead eggs turn blue.

The staining method is not applicable to immature roundworm and whipworm eggs; the pigmented shell stains, and therefore it is not visible whether the germ cell inside the egg has stained.

Ascaris larvae are stained with a basic solution of brilliant-cresyl blue paint at a concentration of 1:10 LLC. in the following way: a drop of liquid with ascaris eggs and a drop of the main paint solution are applied to a glass slide. The preparation is covered with a coverslip, which is tightly pressed to the object with light tapping with a dissecting needle. Under a microscope, the number of hatched larvae and the degree of their staining are observed, after which the same preparation is reviewed again after 2-3 hours. Only undeformed larvae that have not stained for 2 hours are considered alive. Dead larvae stain when the shell breaks (partially or completely).

The possibility of staining preparations with iodine solution in determining the viability of eggs of ascaridia birds is indicated. In this case, a 5% alcohol solution of iodine is used as a dye. When it is applied to the drug, the embryos of dead ascarid eggs turn orange within 1-3 seconds. Dead eggs of opisthorchis and oncospheres of bovine tapeworm are stained with a solution of toluidine blue (1:1000), and dead oncospheres of bovine tapeworm are stained with a solution of brilliant-cresyl blue (1:10,000). At the same time, the embryos and shells of both dead and live eggs acquire color. Therefore, after staining, eggs and oncospheres are washed in clean water and additionally stained with safranin (at a dilution of 1:10,000 with a 10% alcohol solution). The alcohol removes the dye from the shells, and the safranin gives them a red color. As a result, living eggs are stained red, the embryo of dead eggs is blue, and the shell remains red. Dead embryos of oncospheres of bovine tapeworm quickly, within a few minutes, are stained bright red or pink with safranin or blue with diamond-cresyl blue at a dilution of 1:4000 or with indigo carmine at a dilution of 1:1000-1:2000.

Living embryos do not change under the influence of these colors even after 2-7 hours.

To determine the viability of pygmy tapeworm eggs, it is recommended to use the following paints: 1) brilliant cresyl blue (1: 8000) - after 1 hour, the oncosphere of dead eggs is especially brightly stained, which stands out sharply against a pale or colorless background of the rest of the egg; 2) safranin: at a dilution of 1:8000 when exposed for 2 hours and 1:5000 for 3-5 hours; 3) 50% solution of pyrogallic acid at a dilution of 1:2 - when exposed for 1 hour at a temperature of 29-30 ° C (the lower the temperature, the longer the staining process).

Live plerocercoids of the broad tapeworm are very well stained with an aqueous solution (1:1000) of neutral mouth for 5-20 minutes. To obtain a persistent pink color that does not disappear within 5 days and does not affect the mobility of plerocercoids, 10 minutes is usually sufficient. The degree of color is controlled by viewing the larvae in pure isotonic sodium chloride solution, for which the plerocercoids are periodically removed from the paint. It is advisable to use methylene blue to stain dead plerocercoids.

R.E. Chobanov et al. (1986) proposed a method for determining the viability of eggs and larvae of helminths using the rubrin pigment obtained by cultivating the fungus Peniciliium rubrum as a dye. To do this, use a 3% aqueous dye solution.

The process of coloring eggs and larvae is completed after 1.5 hours. Non-viable eggs of pinworms, bovine and dwarf tapeworms, ankylostomides, trichostrongylids acquire an intense pink color, larvae of ankylostomids and trichostrongylids become red. A less bright color is observed in the eggs of ascaris and whipworms, since, standing out from the intestines, they already have a dark brown color: Viable eggs and larvae do not stain.

Physico-chemical methods for stimulating the release of miracdia from trematode eggs. The methods were developed by S.M. German and S.A. Beer (1984) to determine the viability of opisthorchia and dicrocelium eggs by exposing the eggs to the reaction medium. If they are alive, miracidium comes out. The methods are based on physicochemical activation of the miracidium hatching gland and stimulation motor activity larvae. Stimulation is achieved by exposing trematode eggs to a special reaction medium in combination with successive methods - creating a temperature difference, drying a suspension of eggs, exposure to a weak current of liquid in the test drop, which contribute to the mass release of miracidia from eggs.

Determination of the viability of opistorch eggs by the method of Herman, Beer. A suspension of eggs in water (tap, settled) is pre-cooled to 10-12 ° C. All subsequent operations are carried out at room temperature (18-22 °C). One drop (about 0.05 ml) of a suspension containing 100-400 eggs is added to a centrifuge tube. The test tubes are placed in a rack for 5-10 minutes to precipitate the eggs. Then narrow stripe filter paper carefully suck off excess water until it is completely removed. 2 drops of the medium are added to the test tube, shaken, the contents are transferred with a pipette onto a glass slide and left for 5-10 minutes, slightly shaking (or placed under a hair dryer) to create weak liquid currents in the suspension drop under study. This operation, which imitates the intestinal peristalsis of a mollusk, allows you to activate the release of miracidia. After that, 2 more drops of the medium are added to the suspension, and then the preparation is microscopically examined using a conventional light microscope (X200). During this time, eggs with viable miracidia should open the lid, while the larva actively emerges into the environment. Due to the presence of ethanol in it, miracidium is immobilized in 3-5 minutes, and then stained with a dye in the medium. As a result, miracidia are easily detected and counted.

Preparation of the reaction medium. The medium is prepared in 0.05 M Tris-HCi buffer under optimum pH conditions of 8.0-9.5. Ethanol is added to the buffer up to 10-13% and dye (safranin, methylene blue and others working within the pH range) up to weak staining liquid (for example, for safranin, its final concentration will be 1:50,000). You can use another buffer that works in the alkaline pH range, such as 0.05 M phosphate (pH 8.5). Therefore, the medium contains 96% ethanol - 12 parts; dye (mother solution) - 1-10 parts; 0.05 M tris-HC! buffer (pH 8.5-9.5) - up to 100 parts. Medium example: 12 parts of 96% ethanol, 1 part of saturated safranin solution, the rest up to 100 parts - 0.05 M Tris-HCl buffer, pH 9.5.

Determination of the viability of dicrocelium eggs by the method of Herman, Beer, Stratan. A drop of suspension containing 100-150 trematode eggs is placed in a centrifuge tube for 1-2 minutes to settle the eggs. The liquid is then carefully dried with a strip of filter paper. Add 1-2 drops of the reaction medium with a Pasteur pipette and incubate in a water bath at 28-30 °C for 2-3 minutes. Composition of the medium: 6 parts of butanol, 94 parts of 0.4% sodium chloride solution or 0.3% potassium chloride solution in distilled water. The eggs in the medium are transferred with a pipette onto a glass slide and left for 1.5-2 hours at room temperature (18-22 ° C), while every 25-30 minutes (as it dries) add 1-2 drops (0.05 ml) solution of butanol in distilled water. After that, the preparation is microscoped at 100-200-fold magnification. Viability is determined by the number of opened eggs with released miracidia. Butanol penetrates through the pores of the egg shell, reaches the miracidia and activates them. Incubation at a marked temperature enhances this process. Butanol at a concentration of 3-7% is detrimental to the miracidium that has emerged from the egg. The transfer of a suspension of eggs from a test tube to a glass slide allows, by the time miracidium exits (after 30-40 minutes), to reduce the concentration of butanol due to volatilization to a safe level (1.5-0.5%). The presence of sodium chloride in the medium at a concentration of 0.1-0.5% (or potassium chloride at a concentration of 0.05-0.4%) determines the activity of the released miracidium. In contrast to the small transparent eggs of opisthorch, the eggs of dicrocelium have a dark-colored shell; they have a clearly visible lid, which is open after the release of the miracidium. Therefore, the viability of dicrocelium eggs is more conveniently assessed by counting eggs that have opened, rather than by staining and counting miracidia.

Luminescent method for the study of eggs and larvae of helminths.

For the first time in helminthological practice, luminescence microscopy methods were applied in 1955. It was reported that luminescence microscopy makes it possible to differentiate living and dead objects without damaging the egg. For fluorescence, not UV rays were used, but the blue-violet part of visible light, with a conventional microscope and glass slides; to the illuminator "OI-18" was applied special set color filters.

It was found that live and dead eggs of roundworms, pinworms, pygmy tapeworms, bovine tapeworms, broad tapeworms and other helminths luminesce differently. This phenomenon is observed both during primary luminescence without the use of dyes, and when stained with fluorochromes (acridine orange, corifosphine, primulin, auroline, berlerin sulfate, tripaflavin, rivanol, quinacrine, etc.).

Unpainted live non-segmented roundworm eggs glow bright green with a yellowish tint; in dead eggs, the shell radiates green light much brighter than dark green germinal; in roundworm eggs with a larva, only the shell appears, while in the dead, both the shell and the larva are bright yellow.

Unpigmented and unsegmented live eggs of pinworms and pygmy tapeworms emit a greenish-yellow light; in dead eggs, the shell intensely luminesces against the background of a dark green embryonic mass. With secondary luminescence (when stained with acridine orange at a dilution of 1:10 OOO and 1:50 OOO from 30 minutes to 2 hours), the shell of living and dead nematodes, trematodes and cestodes luminesces differently.

The shell of living and dead Ascaris lumbricoides, Toxocara leonina, Enterobius vermicularis, Hymenolepis nana, H.fraterna, H. diminuta, T.saginatus, D.latum turns orange-red. Embryos of living Asc. lumbricoides, T.leonina, H.diminuta, D.latum and oncospheres of the bovine tapeworm luminesce in a dull dark green or gray-green color. The dead embryos of these helminth eggs emit a "burning" orange-red light. Live pinworm larvae and toxocars (egg shells) emit a dull gray-green light, and when they die, the color changes from the head end to a “burning” light green, then yellow, orange, and finally bright orange.

When stained with fluorochromes - coryphosphyllum, primulin - in dead eggs of ascarids and whipworms, a glow from lilac-yellow to copper-red is observed. Viable eggs do not luminesce, but turn dark green. Live eggs of the trematodes Paragonimus westermani and Clonorchis sinensis do not luminesce after staining with acridine orange, while dead eggs emit a yellowish green light.

The luminescence method can also be used to determine the viability of helminth larvae. So, fluorochromized with a solution of acridine orange (1:2000) larvae of strongylate, rhabdita glow: live - green (with a tint), dead - bright orange light. Live larvae of Trichinella do not glow or give a weak glow when treated for 10 minutes with solutions of fluorescein isothiocyanate, auramine, etc. Fluorochromized dead larvae (at a concentration of 1:5000) give a bright glow.

Living miracidia that have emerged from the shell emit a dim bluish light with a barely noticeable light yellow corolla of cilia, but 10-15 minutes after death they appear as a bright “burning” light green, and then orange-red light.