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

Simple Methods

macroscopic method. When examining feces, you can find helminths, their heads, segments, and fragments of strobila, released independently or after deworming. This method is especially recommended for identifying enterobiasis, taeniasis and taeniarynchosis.

Small portions of feces are mixed with water in a flat bath or in a Petri dish and, viewing 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 glycerol or isotonic sodium chloride solution for further study.

With the settling method, the entire portion of feces being tested should be mixed with water in a glass cylinder, then carefully drained upper layer water. This is repeated several times. When the liquid becomes clear, it is drained, and the sediment is examined in small portions in a glass bath or Petri dish, as indicated above.

Microscopic methods are the main way to examine feces to detect helminth eggs or larvae. The various research methods are described below. In order to increase the reliability of the examination, tests 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 fecal studies. In a native smear you can detect eggs and larvae of helminths of all types. However, with a small number of eggs in the feces, they are not always found. Therefore, stool examination using only a native smear is not complete and should be supplemented with enrichment methods. The efficiency of examining a native smear is markedly improved by viewing four slides prepared from a stool sample on two slides without cover slips, allowing a total of approximately the same amount of stool to be examined as with the Kato method (see below).

A small amount (the size of a match head) of mixed 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 glass and examined under high magnification (magnification 40).

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

Thick smear with cellophane (Kato method) is more effective than studying a native smear, but also requires combination with enrichment methods. Eggs of all types of helminths are detected, however, in order to detect eggs of dwarf tapeworm (transparent eggs) or opisthorchid (small eggs), the laboratory technician 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, cleared with glycerin and tinted with malachite green. Pre-hydrophilic cellophane is cut into plates measuring 20 x 40 mm and immersed in Kato mixture (6 ml of a 3% aqueous solution of malachite green, 500 ml of glycerin, 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-sealed 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), you can use only a 50% aqueous solution of glycerol, the effectiveness of the study is not reduced.

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

100 mg of feces is applied to a glass slide, covered with a cellophane plate treated as indicated above and pressed down with a rubber stopper so that the feces do 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 preparing the smear. The reason for the opacity of the drug may be a thick layer of feces, poor processing of the plate in the Kato mixture, or insufficient period of exposure of the drug 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 is proposed for the detection of helminth larvae, primarily strongyloides, in feces. 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 a 3-5-fold amount of physiological solution, without touching the walls of the vessel. Eggs and larvae of helminths accumulate in the center. After mixing is completed, 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. Enrichment methods are based on the difference in the specific gravity of eggs and the saline solution used, which allows them to be detected in small quantities. If the specific gravity of the eggs is greater than the specific gravity of the liquid, then the eggs are concentrated in a 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 the eggs of hookworms, whipworms and dwarf tapeworms.

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 in small quantities. The method is more effective than studying a native smear, although it is more complicated. The advantages of the method are low cost and accessibility. It is recommended to combine the study of the native smear and the Fulleborn 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. To carry out the 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 is used to quickly remove large floating particles. After 45-60 minutes. After 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 from the sediment are examined, collecting it with an eye pipette onto 2 glass slides. In addition to the surface film, it is also necessary to examine the sediment, since the eggs of trematodes, taeniids, and unfertilized roundworm eggs do not float in this solution. The eggs of a number of helminths do not immediately float to the surface in a salt solution. So, if the maximum number of dwarf tapeworm eggs emerges after 15-20 minutes, then ascaris - after 1.5-2 hours, whipworm - after 2-3 hours.

Thus, the advantages of this method include its low cost 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 effective and simpler than the Fulleborn method. A saturated solution of sodium nitrate with a relative density of 1.38 is used. Therefore, the eggs of most helminths float and are found in the surface film; examination of the sediment 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 filtering, 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 slightly reduces the efficiency compared to a sodium nitrate solution.

Advantages of the method: eggs of most helminths float quickly and are found in the surface film, which eliminates the need to examine the sediment. The disadvantages of the method are the deficiency of sodium nitrate, as well as the fact that trematode eggs and taeniid oncospheres do not float and remain in the sediment. It must be taken into account that when feces are kept in solution for a long time (more than 1-2 hours), 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. In this case, the smear turns out to be light, without coarse impurities, which makes it easier to detect small eggs of trematodes (opistorchid, 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, carefully stir 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 for a saline solution, avoiding stirring (so that two clearly demarcated layers are formed). After 2-3 hours, the top 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 sediment is pipetted onto a glass slide, covered with a coverslip and examined under a microscope.

Goryachev's method was proposed for detecting opisthorchid eggs and turned out to be more effective than the study of a native smear and the Fulleborn method. Currently, for the diagnosis of opisthorchiasis (clonorchiasis), the methods of Kato and Kalantaryan are recommended as quite effective and technically simpler.

Krasilnikov's method. Under the influence of surfactants included in detergents, helminth eggs are released from feces and concentrated in sediment.

A 1% solution of Lotus washing powder is preliminarily prepared. To do this, 10 g of powder is dissolved in 1 liter of tap water. If Lotus is not available, you can use others washing powders, but you need to take as much of each of them as will dissolve without forming sediment 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 to solution should be approximately 1:20. Feces should be in the solution for at least 24 hours. During this time, a sediment of 2-3 layers forms 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 with high efficiency are very labor-intensive, especially during mass examinations; therefore, it is more advisable 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, detecting eggs of all intestinal helminths, cysts of intestinal protozoa, and can also be used to quantify the intensity of invasion.

7 ml of a 10% solution is poured into centrifuge graduated tubes acetic acid and add 1 g of feces to the 8 ml mark. The feces 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 test tube of the strained solution again contains 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). Add 2 ml of ether to this test tube (up to the 10 ml mark), stopper it and shake vigorously for 30 seconds. The mixture is centrifuged at 3000 rpm for 1 minute (or 2 minutes at 1500 rpm). The coagulant layer (in the form of a plug in the upper part of the test tube) is separated from the walls of the test tube with a stick and carefully drained together with the supernatant liquid. The precipitate (usually small, colorless) is pipetted onto glass slides, covered with a coverslip, and examined microscopically.

Protozoa are divided into 4 classes:

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

The following may be subject to research:

Note:There are many types 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 discovered along with the main one.

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

Additionally, immunological methods are used:

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

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

Diagnostics of ciliated (ciliates)

The pathogenic form of microorganisms of this genus is balantidium. This is a microbe that causes balantidiasis, a disease accompanied by an ulcerative process of the large intestine. The pathogen is detected in the 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.

Diagnosis of flagellates (Leishmania, Giardia, Trypanosomes, Trichomonas)

Leishmania, trypanosomes, lamblia, and trichomonas are dangerous for humans.

Leishmania– microbes, causing leishmaniasis, are examined in blood smears, materials bone marrow, scrapings from skin infiltrates. In some cases, when diagnosing leishmania, culture 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 during the study of peripheral blood. As the disease progresses, pathological microbes are found in the material of lymph node punctures, and in advanced stages - in the cerebrospinal fluid.

To diagnose trypanosomes if Chagas disease is suspected, the material being examined is examined under a microscope at low magnification. In this case, the smears and 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 the malarial plasmodium, which has 4 main types of pathogen: the causative agent of three-day malaria, four-day malaria, tropical malaria and malaria ovale.

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

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

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

• during the chill period, the blood fills with merozoites, a type of schizont;
• at elevated temperatures, ring-shaped trophozoites accumulate in erythrocytes;
• a decrease in temperature is characterized by a predominance of amoeba-like 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:Diagnosis of malaria by examination of smears and thick drops of blood is sometimes erroneous. Blood platelets in some cases may be mistakenly attributed to the malarial pathogen. Also sometimes fragments of leukocytes and other cells simulate plasmodium.

Basic methods for studying protozoa

Let's briefly 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 stool)

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

The protozoa found are recorded based on certain characteristics. For accuracy, prepare 2-3 preparations from the same material. In doubtful cases, the analysis is repeated several times over 2-3 weeks.

The method can detect vegetative and cystic forms:

• lamblia;
• balantidium;
• dysenteric amoeba.

Along with pathogenic forms, non-pathogenic protozoa are also identified. Also in healthy carriers there are luminal and cystic forms.

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

The result of diagnosing protozoa using the native and stained smear method should contain a description of the form of the pathogen (luminal, cyst, tissue).

Research requirements:

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

Preservation method (stool examination) for diagnosing protozoa

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

Preservatives used:

• 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 thionin (azura).
• Safarliev's solution. Ingredients: 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 a 3:1 ratio, then dye is added if necessary. The results are assessed by studying 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 the analysis: formaldehyde (10 ml), 0.85 g of isotonic solution, distilled water, sulfuric ether, Lugol's solution.

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

Method for detecting Leishmania (bone marrow smear)

To diagnose leishmaniasis, the following reagents are used: Nikiforov’s mixture (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.

During 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 careful and have sufficient experience to conduct independent research.

Method for detecting leishmania in a smear from skin infiltrate

The required reagents are similar to the previous analysis.

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

In the presence of the disease, leishmania is also detected among the macrophages, fibroblasts, and lymphoid cells present in the test material.

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

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

Before taking the scraping, the skin is thoroughly treated with alcohol, then an incision is made into the tubercle, from the bottom of which the contents are removed and placed in a test tube with medium. The material is taken several times, after which it is placed in different test tubes. Then cultivation occurs in a thermostat at a temperature of 22-24 degrees. The results are assessed 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 using a drop of blood are deciphered in practice by watching the video review:

Lotin Alexander, medical columnist

Methods helminthological research are divided into direct and indirect. Direct methods: detection of helminths themselves, their fragments, eggs, larvae in feces, urine, duodenal secretions, sputum, nasal and vaginal mucus, contents of subungual spaces, biopsied pieces of tissue. Indirect methods: identification 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 helminth-ovoscopic and protozooscopic. When diagnosing, it is impossible to identify 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 their high specific gravity). It is very rare to find pinworm eggs and taeniid oncospheres in feces, which are detected using special research methods: scraping from the perianal folds for pinworms and taeniids, sedimentation methods for trematodes (opisthorchid eggs, etc.). Therefore, for a targeted examination of a patient for helminth infections, the doctor in the referral must indicate which helminths should be focused on (diagnosis), which will allow the laboratory assistant to choose the appropriate technique for identifying this type of helminth. Feces taken from different places stool in an amount of at least 50 grams (teaspoon) in a clean glass container must be sent to the laboratory no later than 24 hours after defecation and examined on the day of receipt. If it is necessary to preserve stool until next day it is placed in a cold place (0-4°C) or filled with one of the preservatives. Before examination, the stool is mixed with a stick so that the helminth eggs are evenly distributed in total mass. If eggs of any helminth are found in the preparation, viewing is not stopped, because there may be double or triple invasion. Monitoring the effectiveness of treatment of helminth infections is carried out by examining feces for helminth eggs 2-3 weeks or 2-3 months after treatment, depending on the detected helminth. Macroscopic methods are used to detect whole mature helminths or their fragments in feces with the naked eye or using a hand magnifying glass. Often, actively crawling pinworms can be seen on the surface of feces after defecation; roundworms are excreted in feces; Sometimes people themselves notice the passage of helminths. In patients with diphyllobothriasis, fragments of tapeworm strobili may be excreted (in the form of “noodles”), and in those infected with taeniids (pork or bovine tapeworm), segments of helminths often leave with feces (in the form of “white clippings”) or they actively crawl out of the anus. The macroscopic method is the main one for the differential diagnosis of taeniasis and taeniarynchosis (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 against a dark background in Petri dishes. Using tweezers or a dissecting needle, remove all suspicious white particles, large formations suspicious for fragments of helminths, and examine them under a magnifying glass between two slides. Small helminths or cestode heads are examined under a magnifying glass in a drop of glycerin or under a microscope. When using this method for diagnosing the segments of the pork, bovine tapeworm, and wide tapeworm, the washed segments are placed between two glasses and, looking at the light under a magnifying glass or low magnification microscope, the species is determined by the structure of the uterus (in a mature segment pork tapeworm 8-12 lateral branches extend from the central trunk, and in the bovine tapeworm there are 18-32, more often 28-32; in the wide tapeworm, the segments are wider and the uterus in the center is in the form of a “rosette”). If the uterus is hard to see, then it can first be kept for some time in a 50% glycerin solution, after which even the empty trunks of the uterus can be clearly seen. When identifying these cestodes by the structure of the detached heads, they are carefully placed with the neck in a drop of glycerin between slides (or covered with a coverslip) and, without squeezing, examined under a microscope at low magnification.

Microscopic methods are divided into simple, complex and special.

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

Complex methods are more effective and are based on the concentration of eggs in the preparations. They involve pre-treatment of feces 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 eggs is greater than the specific gravity of saline solutions and the eggs settle into sediment).

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

Sampling and preservation

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 (no more than a day old) are examined, and in some cases (when testing for strongyloidiasis) immediately after defecation. A number of fecal preservatives containing helminth eggs have been proposed: 4-10% formalin solution, which must be heated to a temperature of 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 iodine, 10 g potassium iodide, 250 ml water), formalin (300 ml) and glycerin (25 ml), in which helminth eggs are preserved 6-8 months; a mixture of glycerin (5 ml), formalin (5 ml) and water (100 ml); solutions of 1-1.5% detergents “Lotos”, “Extra”, “Barf”, “Tide”, etc. (in the 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 (at the rate of 1 g of feces per 10 ml of mixture).

To diagnose helminthiases, macro- and microhelminthoscopic methods for examining feces are used.

Macrohelminthoscopy studies

Settling method

A daily portion of feces is thoroughly mixed with 5-10 times the amount of water, poured into tall glass cylinders (jars, buckets) and left until suspended particles have 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 clear). After draining the top layer, transfer the sediment to a cuvette or Petri dish and view it (against 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, it is washed, and the flowing liquid is drained into the sewer. Large helminths remain on the upper sieve, while smaller ones are retained on the lower ones. The sieves are turned over and, after washing the contents into dark cuvettes, they are examined with the naked eye or under a magnifying glass.

Microhelminthoscopy studies

Carry out for the purpose of detecting eggs (helminth-ovoscopic methods - color. Fig. 1) or helminth larvae (helminth-ovoscopic methods).

Helminthoovoscopic methods

Qualitative methods without enrichment

Native smear. A small amount of feces is ground on a glass slide in a drop of 50% glycerin solution or boiled water. Large particles are carefully removed, the mixture is covered with a coverslip and examined under a microscope (two smears are examined). 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 for weak infestations.

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 glycerin (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 is smeared on a glass slide and, covered with a piece of damp cellophane, pressed down with a rubber stopper No. 5. The preparation is examined after 30-60 minutes, when it dries slightly and becomes clear, 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 differences 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 (100 ml volume) are stirred with a wooden stick in 100 ml of a saturated solution table salt, beat its weight is 1.18 (400 g of salt is dissolved by boiling in 1 liter of water). Large particles that float to the surface are quickly removed with a stick or piece of paper. After settling the mixture for 45-90 minutes. Use a wire loop (0.8-1 cm in diameter) to remove all the surface film and transfer 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, 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 the film with helminth eggs adhering to it is examined under a microscope (without a cover glass). This method is good at identifying eggs of all nematodes and dwarf tapeworms. Heavy fluke 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 glycerol is added for clarification and examined under a microscope.

Method E. V. Kalantaryan(1938) Is a more effective modification of the Fulleborn method. In it, the solution of table salt is replaced by a saturated solution of sodium nitrate, sp. the weight is 1.39 (one volume of sodium nitrate is dissolved in an equal volume of water when boiled), the film is removed after 20-30 minutes.

The methods of Faust (E. S. Faust, 1939), Brudastova et al. are also used. (1970) and others.

Chemicals are used to deposit helminth eggs. substances that dissolve fats and proteins in feces.

Telemann method (W. Telemann, 1908) 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% saline solution; the mixture is filtered through a wire or hair sieve into a test tube and centrifuged. In the test tube, 3 layers are formed: 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 precipitate and centrifuged. A few drops of sediment 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). Formaldehyde and ether are used to dissolve fats and proteins.

Various detergents can be used to precipitate helminth eggs. The filtration method in special Bell devices has become widespread abroad, which is used to study not only feces, but also urine, blood, etc.

There are a number of methods combining the principles of flotation and egg sedimentation. These include the methods of S. A. Lane, D. Rivas, Gorkina, and S. T. Darling. One of these flotation-sedimentation methods, as well as the method of sequential drains, was proposed by N.V. Demidov (1963, 1965) for research on fascioliasis and dicroceliosis.

Quantitative methods

Stoll method(N. R. Stoll, 1926). A decinormal solution of sodium hydroxide is poured into a graduated wide test tube or flask (with two marks: 56 and 60 ml) to the first mark, add feces until the liquid level reaches the second mark, mix thoroughly with a glass rod and, placing 10 glass beads or small pebbles, stopper and shake for 1 minute. Quickly, so that the suspension does not settle, take 0.075 ml of the mixture (0.005 g of feces) with a graduated pipette, transfer it to a glass slide with a grid applied to it, cover it with a coverslip and count the helminth eggs in the preparation under a microscope; By multiplying the resulting number by 200, the number of eggs in 1 g of feces is obtained. For a more accurate result, count the eggs in two or more preparations and take the average. The Stoll method is insensitive for mild 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 portion of feces and dishes of the same volume. A thick Kato smear is also used.

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

Helmintholarvoscopic methods

Behrmann method(G. Baermann, 1917). 5-10 g of freshly excreted feces are placed on a metal mesh in a glass funnel, with a rubber tube with a clamp on the narrow end. To avoid contamination of the sediment with feces particles, it is recommended to place paper (on the mesh) or add animal charcoal or corn flour to the feces. The funnel is filled with warm water (t° 45-50°) until it comes into contact with feces. Due to thermotropism, the larvae actively move into 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 drained into 1-2 test tubes, centrifuged for 2-3 minutes, the top layer is drained, and the sediment is examined on a glass slide under a microscope.

Method for identifying schistosome miracidia. Feces are washed in the dark at a temperature of 8-10°, the sediment is kept for 45 minutes. in bright light at t° 28°, then pour into a dark flask with a straw on the side. The miracidia are concentrated in a transparent side tube, from where they can be selected.

To identify hookworm larvae, the methods of Fulleborn et al. are also used.

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

OK. 100 ml of test urine is left to stand for 30 minutes. in a cylinder and, having removed the top layer, pour 10-15 ml of sediment into a test tube, centrifuge at 1500 rpm for 1-2 minutes; the 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-painted flask, with a transparent glass tube soldered to the top. Add water in a ratio of 1:5, 1:10 and place in a thermostat at 25-30° for 2 hours. The miracidia emerging from the eggs are visible through a transparent tube to the naked eye in the form of rapidly moving dots. In chronic, a form of schistosomiasis, patients release blood towards the end of urination, but eggs are rarely found in the urine, so it is recommended to resort to a bladder biopsy.

Sputum examination. The sputum may contain eggs of paragonimus, schistosomes, tominxes, larvae of migrating nematodes, and 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 accumulations, etc. are selected and examined; then scan the entire portion with smears. Purulent sputum pour an equal amount of 0.5% caustic alkali solution, centrifuge and examine the precipitate.

In some helminthiases, sputum is observed characteristic changes. With paragonimiasis, for example, in the sputum one can find accumulations of eggs in the form of yellowish lumps, as well as a large amount of mucus, leukocytes, red blood cells, alveolar cells, Kurschmann spirals, elastic fibers, Charcot-Leyden crystals. Characteristic diamond-shaped crystals with pointed ends were also revealed. Availability a large number eosinophils allows one to differentiate paragonimiasis from tuberculosis.

Examination of the contents of abscesses and punctates. IN purulent discharge abscesses, as well as in tumors and cysts removed during surgery, helminths, their fragments, larvae and eggs (echinococcus, alveococcus, sparganum, cysticercus, dirofilaria, roundworm, toxocara, paragonimus, etc.) can be found. The research technique is usual; in some cases, histological sections are prepared from tumor tissue. Purulent contents can be treated using the Telemann method; The clear liquid is examined in the same way as the duodenal contents, adding sulfuric ether. The echinococcal fluid is centrifuged and the sediment is examined for the presence of scolex and hooks; The preparations are stained with carbolfuchsin according to Ziehl-Neelsen.

Blood test. Microfilariae and larvae of migrating nematodes are found in the blood. A drop of blood is taken from a finger or earlobe and examined fresh or preparations are prepared from it.

A drop of blood is placed on a slide with a square of Vaseline applied and lightly pressed with a coverslip. Under a microscope, microfilariae are visible moving between blood cells. The blood can be placed between two layers of adhesive cellulose tape; microfilariae remain motile for 6 hours; in a completely dried preparation they can be distinguished under a microscope within 30 days. Microfilariae 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-Neelsen, Leishman, Papanicolaou (see Wright staining method, Romanovsky-Giemsa method, Ziehl-Neelsen method). Having detected microfilariae in a drop of blood stained according to Romanovsky-Giemsa, the preparation is additionally stained with Hansen’s hematoxylin; in 15-60 minutes. wash for 2 minutes. in running water. The recolored preparation is differentiated in a 0.2% saline solution; The microfilaria sheath is painted pale violet, and the nuclear substance of the body is dark violet.

For mild infestations, it is necessary to examine 2-10 pieces of blood 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 Voge (E.K. Markell, M. Voge, 1965). 2 ml of blood is mixed in a centrifuge tube with 10 parts of 1% acetic acid, centrifuged for 2 minutes. at 1500 rpm; surface layer drained, the sediment is distributed on several glass slides and examined under a microscope. Preparations can be stained using Romanovsky-Giemsa or other methods.

Bell filtering 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, measuring 19 x 42 mm, with a pore size of 0.8 to 5 μm, blood hemolyzed in a mixture of 1 ml of tipol detergent and 9 ml fiziol solution (to speed up filtration, the device is connected to a vacuum pump). The filter is fixed in boiling distilled water and stained with Romanovsky-Giemsa or Ehrlich's hot hematoxylin. The colored filter is dried in a desiccator or in isopropyl alcohol (sequentially in 3 cups), cleared on glass with immersion oil and examined under a coverslip. Colored preparations last for several weeks. The Bell method is most effective for the quantitative recording of microfilariae in the blood.

The Goldsmid polyvidone method is also used.

Skin examination. In the skin you can find microfilariae of onchocerci and larvae of animal helminths that cause cutaneous form larva migrans(cm.). Skin sections or material obtained by scarification are taken from the thigh, calf, buttock or 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 physiol solution. At negative result a fresh preparation is re-examined after 10 minutes; The larvae are usually localized at the edges of the preparation. The resulting material can be kept in 2 ml of fiziol solution for 1-2 hours. and examine the sediment. It is recommended to take 5 skin sections from the patient.

Preparations can also be prepared from blood and tissue fluid released after strong compression of the sections. Drops are stained according to Mayer, Romanovsky-Giemsa or Delafield's hematoxylin.

Standard method for quantifying infestations. Biopsied skin area diam. 3-5 mm and weighing at least 1-4 mg, weigh, cut into small pieces and count the larvae under a microscope in physiol. solution on a glass slide; 1-4 larvae in the field of view are indicated by a + sign, 5-9 larvae by a ++ sign, 10-19 by a +++ sign, 20 or more by a + + + + sign. It is more convenient to carry out quantitative calculations on colored preparations.

Testing for trichinosis, cysticercosis and schistosomiasis

Detection of Trichinella larvae

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

Bechman's 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 left for 18 hours. in a thermostat at t° 37°; the top layer of liquid is drained, warm water (t° 37-45°) is added to the sediment and poured into a Behrmann apparatus. After an hour, the liquid is drained into a test tube, centrifuged and the sediment examined. If the larvae are enclosed in calcified capsules, they are first decalcified in a solution of hydrochloric, nitrogen, or sulfuric acid.

Bioassay. Pieces of the muscles being studied 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 can be found in the muscles of the diaphragm and tongue.

Histological sections. Muscle pieces are fixed in Bouin's, Zenker's or other liquid; in the usual way Sections are prepared on a microtome and stained with Delafield's hematoxylin.

Detection of cysticerci

A piece of extirpated muscle, connective tissue, etc. is examined with the naked eye, the 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 cysticerci isolated from tissues, they are kept in a 50% bile solution for physiol. solution in a thermostat at t° 37°; in 10-60 minutes. the head of the viable cysticercus turns outward. Calcified cysticerci are preliminarily decalcified with a 4% solution of nitric acid for an hour.

Detection of schistosome eggs

In hron, forms of schistosomiasis, 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, selecting an area with a visible lesion. The biopsied 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 histol is prepared from them. slices. A biopsy of the jejunal mucosa is performed orally and the resulting material is examined using the compression method or histol sections are prepared from it. In some cases of genitourinary schistosomiasis, the diagnosis can only be made on the basis of endovesical biopsy. In the hepatolienal form of schistosomiasis, a puncture biopsy of the liver is performed; From the resulting material, histol and sections are prepared and examined under a fluorescent microscope. Fluorescence microscope with a dark field is also used for examining liver tissue for schistosome eggs. If the female genital tract is affected by schistosomes, the discharge is collected using a vaginal speculum or pieces of the mucous membrane of the cervix are taken with a sharp spoon; the resulting material is examined under a microscope on glass in a drop of fiziol solution.

Testing for enterobiasis and taeniasis

Perianal scraping (it is recommended to take it in the evening, 1 - 1.5 hours after the patient goes to bed, or in the morning before toileting). With a match, cut obliquely and moistened in a drop of liquid (physiol, solution, boiled water or 2% solution of bicarbonate of soda), applied to a glass slide, 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 cleaned off with the edge of a cover glass into a drop of liquid on a glass slide and, covered with the same cover glass, 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 glass slide; after the drop has dried, it is covered with another glass slide and delivered to the laboratory, wrapped in paper and secured with an elastic band. To study rectal mucus, they use a special device - a Shakhmatov or Ziemann tube, with which they take mucus from the rectum and examine the smears under a microscope.

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

Hall's cellophane method (M. S. Hall, 1937). A square piece of cellophane is secured with an elastic band on a glass rod. The scraping is made with dry cellophane and placed in a test tube. In the laboratory, the cellophane is slightly moved from the stick and, after cutting off its tip with scissors, it is straightened on a glass slide; moistened with decinormal sodium hydroxide solution, covered with a coverslip and examined under a microscope.

Graham's cellulose tape method (S. F. Graham, 1941). A strip of cellulose tape is pressed with the adhesive side to the perianal folds of the subject, then with the same side to a glass slide and examined without a cover glass; You can add a drop of toluene. V. V. Kaledin (1972) for these purposes proposed using celluloid disks cut from washed X-ray film and moistened with glycerin; the discs 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, nail bed, and subungual spaces are moistened with a 0.5-1% solution of caustic alkali and wiped with damp cotton swabs. The swabs are placed in centrifuge tubes with the same solution, centrifuged and the sediment examined.

Methods for studying objects environment for eggs and larvae of helminths (sanitary and helminthological studies)

Research is carried out to determine the degree of contamination of objects with eggs and larvae of helminths external environment. The obtained data is used to assess dignity. the condition of institutions and enterprises and the effectiveness of measures taken to combat helminthiasis.

When studying the degree of contamination of various environmental objects with eggs and larvae of helminths and assessing their role in the epidemiology of helminth infections, 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 are not painted; c) cultivation under optimal conditions until the invasive stage; d) infection of laboratory animals (bioassay).

Water and sewerage studies. For one study, use 10-25 liters of water (rivers, seas, ponds, swimming pools, water supply) and 1-2-5 liters of sewage.

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

Method of G. Sh. Gudzhabidze and G. A. Yudin (1963) for studying sewer fluid. 1 liter of liquid is left for 2 hours in a Lisenko cylinder; the resulting sediment (5-9 ml) is processed as in soil testing (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, accelerating 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 helminthiasis and assess the effectiveness of work on their improvement.

Method 3. G. Vasilkova and V. A. Gefter (1948). 12.5 g of soil is mixed in test tubes (volume 100 ml) made of stainless steel or brass with 20 ml of a 5% solution of caustic alkali, adding 10 glass beads or small pebbles. The tubes are sealed with rubber stoppers and shaken for 20 minutes. in a shaking apparatus or manually. Having removed the stoppers, the tubes are centrifuged for 3-5 minutes, the surface liquid is drained, 60-80 ml of a saturated sodium nitrate solution is added to the sediment and, after mixing thoroughly, centrifuged again for 3-5 minutes. 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; mix with the same solution, centrifuge; The entire procedure is repeated at least 3 times. The contents of the cup into which the film is transferred are 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, as modified by A. A. Namitokov (1961), differs from the main method in that instead of examining film preparations, half the contents of the test tube are used (each time adding a new portion of a saturated sodium nitrate solution), filtered and examine the 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 is thoroughly mixed for 1 minute. in 150 ml of water in centrifuge tubes (250 ml capacity) with special blades driven by an electric motor. The mixture is centrifuged for 3 minutes. at 1000 rpm, drain the water and add 150 ml of saturated sodium nitrate solution, 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 left for 10-15 minutes, then the glasses are removed and examined; the procedure is repeated at least 4 times.

Research on helminth larvae using the method of Behrmann (1917). 200-400 g of soil are placed in a piece of gauze on a metal mesh (with holes 1-2 mm in diameter) placed on the wide part of a glass funnel mounted on a tripod. A rubber tube with a clamp is stretched onto the narrow end of the funnel. The funnel is filled with warm (t° 50°) water so that the lower part of the mesh with the soil comes into contact with the water. Due to thermotropism, the larvae actively crawl 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 examined.

Research of vegetables, fruits and berries

They study 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 with water for several hours in wide-necked glass jars with ground stoppers and shaken for 10-20 minutes. in a shaking apparatus or manually. The water is drained, the objects under study are rinsed clean water and all flush water is filtered in a Goldmann apparatus; The filters are examined by clearing them with glycerin. You can tint filters with 25% Lugol's solution in glycerin; in this case, helminth eggs are stained Brown color and they are easily recognized among the starch grains, colored in Blue colour. Large sediment is treated like soil.

Study of washouts from household items and hands. A glue brush (or a cotton swab wrapped in a piece of nylon fabric) soaked in a 2% solution of bicarbonate of soda is repeatedly passed over the object or hands being examined, and then 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 effective to collect dust from soft equipment using a vacuum cleaner connected to a funnel; the edges consist 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 it on top part funnels. The assembled funnel is connected 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 examined under a microscope, cleared with glycerin. If the layer of dust on the filter is large, it is scraped off and viewed in the form of smears or treated like soil.

Immunological diagnostic methods

Possibility of using immunol. methods for diagnosing helminthiasis 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 are for intestinal helminthiasis, when the secretions and excreta of helminths, which have antigenic activity, enter directly into the host’s blood. Immunol reactions are used for the diagnosis of ascariasis, trichinosis, filariasis, schistosomiasis, echinococcosis and alveococcosis, cysticercosis, paragonimiasis, larva migrans symptom complex (toxocariasis, angiostrongylosis), etc. Various serol tests are used (precipitation reactions, agglutination reactions, complement fixation, fluores cing antibodies ) and intradermal allergy tests. Antigens are prepared from larvae and mature helminths using salt extracts of homogenates of fresh frozen or lyophilized tissues, as well as various biologically active liquids (liquid from echinococcal blisters, abdominal fluid of roundworms, etc.). Due to the fact that helminths have a very complex antigenic structure and their antigenic mosaic contains 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 higher specificity than whole extracts, but their activity is approximately the same. Immunodiagnostic methods find everything greater application. Reactions are used not only for the most complete and early identification of patients, but also for studying foci and studying the epidemiology of helminthiases.

Serological reactions. The microprecipitation reaction on living larvae is used to diagnose nematodes - the preimaginal phase of ascariasis, hookworm disease, and trichinosis. The reaction becomes positive 5-10 days after infection (ascariasis, hookworm disease) and persists for 90-100 days. The antigen is live nematode larvae isolated from the tissues of experimentally infected laboratory animals. The isolated larvae are thoroughly washed from the host proteins with sterile physiol, solution and distilled water and 10-15 copies each. placed using a Pasteur pipette on a sterile glass slide with a well. Apply 2-3 drops of the test serum, cover with a sterile cover glass, place it in a humid chamber (Petri dish lined with moistened filter paper) and leave for 24-48 hours. in a thermostat at t° 37°. If the reaction is positive, under low magnification microscope, visible around the mouth and anal holes larvae are grayish-white, slightly opalescent masses of spherical or zigzag-shaped precipitates. 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 2-3 weeks of illness. Its effectiveness reaches 80-90%. The antigen is a powder extract from larvae dried at 35°, isolated from the muscles of infected animals. In test tubes dia. 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 test tubes are kept for 30 minutes. in a thermostat at t° 37°, and then 30-60 minutes. at room temperature. With a positive reaction, a whitish delicate ring appears at the interface between the antigen and serum, which easily disintegrates when shaken.

The precipitation reaction in gel according to Ouchterlony (O. Ouchterlony, 1949) in micromodification by 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 effectiveness is 87%. The antigen is an extract of a homogenate of fresh frozen sexually mature opisthorchis isolated from the liver of cats.

The reaction of indirect hemagglutination (see) with diagnosticum - a suspension of formalinized and tanized sheep erythrocytes, sensitized with echinococcal fluid - has been developed

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

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

RNGA with ascaris diagnosticum is proposed for diagnosing the preimaginal phase of ascariasis.

The complement fixation reaction (see) is performed according to the usual method and is used for the diagnosis of trichinosis and cysticercosis.

Luminescent antibody method (indirect method). This method, with defatted dry homogenate of pork 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 histol, sections of Trichinella larvae as an antigen was developed for the diagnosis of trichinosis.

E. S. Leikina, V. A. Gefter.

When helminth eggs are detected on various environmental objects (soil, water, vegetables, etc.), it is always necessary to determine their viability by appearance, staining with vital paints, cultivation in optimal conditions and performing a biological test, i.e.

Feeding to laboratory animals.

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

To determine the viability of mature eggs of roundworms, whipworms, pinworms, active movements of the larvae should be caused by slightly heating the drug (to a temperature not exceeding 37 ° C). It is more convenient to observe the viability of roundworm 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 roundworm larvae, a sheath is often seen peeling off at the head end, and in whipworm larvae that have completed development in the egg, a stylet is found in this place at high magnification. In dead helminth larvae, regardless of their location (in the egg or outside it), body decay is noticed. Wherein internal structure The larva becomes clumpy or granular, and the body becomes cloudy and opaque. Vacuoles are found in the body, and breaks are found on the cuticle.

The viability of taeniid oncospheres (bovine, pork tapeworm, etc.) is determined by the movement of the embryos when exposed to them digestive enzymes. The eggs are placed on a watch glass with the dog's 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. At the same time, living embryos are freed from their 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 you place taeniid eggs 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 living embryos are released from the membranes and do not change within 1 day. Immature and dead oncospheres shrink or swell and increase sharply, and then “dissolve” within 10 minutes - 2 hours. Living taeniid embryos 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 of echinococci is determined with low heating. To do this, scolex 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 drug is heated using any heat source. At the same time, viable scolex actively move, contracting or relaxing the suckers, lengthening and shortening the proboscis. If you place the scolex at 0.5-1% water solution filicilene at room temperature, then all viable scolex will quickly turn out and die. Non-viable scolex are not everted.

The viability of Adolescaria fascioli collected on plants and other objects of water bodies is checked by examining them on a glass slide in physiological solution under a microscope with a heating stage. When the trematode larvae are heated, they begin to move.

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

In living eggs of the dwarf tapeworm, sluggish pendulum-like movements of the protoplasm and almost imperceptible moving apart and moving of the pointed ends of the lateral pair of hooks to the sides from the middle pair occur.

Contractions of the protoplasm of the embryo and embryonic hooks help the embryo to free itself first from the membranes of the oncosphere, and then from the outer shell of the egg.

In a living egg, the median pair and lateral pairs of hooks are located parallel; in some eggs, the blades of the lateral pairs are close together and located at an angle of less than 45° with respect to the middle pair of hooks.

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

Sometimes wrinkling of the embryo and the formation of granulation 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.

Determination of the viability of immature nematodes should be studied in a humid chamber (Petri dishes), placing roundworm eggs in a 3% formaldehyde 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 into an isotonic sodium chloride solution at a temperature of 37 °C. Petri dishes should be opened 1-3 times a week for better aeration and the filter paper should be re-wetted 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, dividing 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 hookworm eggs, diluted with water to a semi-liquid consistency, are carefully poured onto 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, rhabditiform larvae hatch from the eggs, and after 5-7 days they become filariform: the larvae crawl up the walls of the cylinder, where they are visible even to the naked eye. The eggs of trematodes, such as opisthorchiids, diphyllobothriids, fasciolae, etc., that naturally develop in water, are placed on a watch glass, a Petri dish or in another vessel, and 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 living eggs at a temperature of 22-24 °C after 9-12 days. When microscopying developing trematode eggs, the movements of the miracidium are clearly visible. Miracidium fasciola emerges from the egg shell only in the light. During cultivation, the water is changed after 2-3 days.

Hookworm and strongyloid larvae are cultured 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 crawl across the agar, leaving behind a path of bacteria (Fulleborn method).

Method of Harada and Mori (1955). Add 7 ml of distilled water to test tubes placed in a rack. Using a wooden stick, take 0.5 g of feces 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 bench). Then the strip with the smear is inserted into the test tube so that the left end free of the smear reaches the bottom of the test tube. The upper end is covered with a piece of cellophane and tightly wrapped with an elastic band. The number and surname of the person being examined is written on the test tube. In this state, the tubes are stored for 8-10 days at a temperature of 28 °C. To study the culture, remove the cellophane cover and remove a strip of filter paper with tweezers. Care should be taken when doing this as small numbers of infective larvae may move to the top end of the filter paper or to the side of the tube and penetrate under the surface of the cellophane.

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

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

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

Table 13 Differential diagnosis filar-shaped larvae of A.duodenale, N.americanus, Strongyloides stercoralis, Trichostrongylus spp.

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

Leukobase methylene blue is used to stain living and dead tissues. A living cell or tissue reduces methylene blue into a colorless leukobase; dead tissue does not have this ability, so it acquires color.

To color roundworm eggs, you can use methylene blue in a solution of lactic acid with caustic alkali (0.05 g methylene blue, 0.5 g sodium hydroxide, 15 ml lactic acid). Living eggs do not perceive color; the embryos of dead eggs turn blue.

The staining method is not applicable for immature eggs of roundworms and whipworms; the pigmented shell is stained, and therefore it is not visible whether the germ cell inside the egg is colored.

Ascaris larvae are stained with a basic solution of brilliantcresyl blue paint at a concentration of 1:10 000. in the following way: a drop of liquid with roundworm eggs and a drop of the main paint solution are applied to a glass slide. The preparation is covered with a coverslip, which is pressed tightly to the specimen while lightly tapping with a dissecting needle. The number of emerging larvae and the degree of their staining are observed under a microscope, after which the same preparation is examined again after 2-3 hours. Only undeformed larvae that have not stained for 2 hours are considered alive. Dead larvae are stained when the shell breaks (partially or completely).

The possibility of staining preparations with iodine solution is indicated when determining the viability of avian roundworm eggs. In this case, a 5% alcohol solution of iodine is used as a dye. When it is applied to the preparation, the embryos of dead Ascaridia eggs turn orange within 1-3 s. 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 living eggs acquire color. Therefore, after staining, eggs and oncospheres are washed in clean water and additionally stained with safranin (diluted 1:10,000 in a 10% alcohol solution). Alcohol removes the color from the shells, and safranin gives them a red color. As a result, living eggs turn red, the embryo of dead ones turns blue, and the shell remains red. Dead embryos of bovine tapeworm oncospheres quickly, within a few minutes, are stained bright red or pink with safranin or blue with brilliant 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 dyes even after 2-7 hours.

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

Living plerocercoids of the tapeworm are very well stained with an aqueous solution (1:1000) of neutralrot for 5-20 minutes. To obtain a persistent pink color that does not disappear within 5 days and does not affect the motility of plerocercoids, 10 minutes is usually sufficient. The degree of coloring is controlled by viewing the larvae in a pure isotonic sodium chloride solution, for which purpose 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 helminth eggs and larvae using “rubrin” pigment, obtained by cultivating the mold 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, hookworms, trichostrongylids acquire an intense pink color, hookworm and trichostrongylid larvae become red. A less bright color is observed in the eggs of roundworms and whipworms, since, when released from the intestines, they already have a dark brown color: Viable eggs and larvae are not colored.

Physico-chemical methods for stimulating the release of miraculid from trematode eggs. Methods were developed by S.M. German and S.A. Beer (1984) to determine the viability of opisthorchid and dicrocelium eggs by exposing the eggs to a reaction medium. If they are alive, the miracidium is released. The methods are based on the 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 sequential techniques - creating a temperature difference, drying a suspension of eggs, exposure to a weak flow of liquid in the test drop, which contribute to the massive release of miracidia from the eggs.

Determination of the viability of opisthorchid eggs using the method of Herman and 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 (approximately 0.05 ml) of a suspension containing 100-400 eggs is added to a centrifuge tube. The tubes are placed in a rack for 5-10 minutes to sediment the eggs. Then narrow strip Using filter paper, carefully suck off excess water until it is completely removed. Add 2 drops of medium to the test tube, shake, transfer the contents with a pipette onto a glass slide and leave for 5-10 minutes, shaking slightly (or place under a hairdryer) to create weak liquid currents in the test drop of suspension. This operation, which imitates the peristalsis of the mollusk intestine, allows one to activate the release of miracidia. After this, 2 more drops of the medium are added to the suspension and then the preparation is microscoped using a conventional light microscope (X200). During this time, the lid of eggs with a viable miracidium should open, and the larva actively emerges into the environment. Thanks to the presence of ethanol in it, the miracidium is immobilized after 3-5 minutes, and then stained with a dye found 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 at optimal pH conditions of 8.0-9.5. Ethanol is added to the buffer to 10-13% and a dye (safranin, methylene blue and others that work within the pH range) to weak staining liquid (for example, for safranin its final concentration will be 1:50,000). You can use another buffer that works in alkaline pH limits, for example 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-NS! buffer (pH 8.5-9.5) - up to 100 parts. Example of a medium: 12 parts of 96% ethanol, 1 part of a 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 using 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 sediment the eggs. The liquid is then carefully dried using a strip of filter paper. Add 1-2 drops of the reaction medium using a Pasteur pipette and incubate in a water bath at 28-30 °C for 2-3 minutes. Medium composition: 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), with 1-2 drops (0.05) added every 25-30 minutes (as they dry). ml) of butanol solution in distilled water. After this, the preparation is examined under a microscope at 100-200x magnification. Viability is determined by the number of opened eggs with miracidia released. Butanol penetrates through the pores of the egg shell, reaches the miracidia and activates them. Incubation at the noted temperature enhances this process. Butanol in a concentration of 3-7% is detrimental to the miracidium emerging from the egg. Transferring a suspension of eggs from a test tube to a glass slide allows, by the time the miracidium is released (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. Unlike the small transparent eggs of opisthorch, dicrocelium eggs have a dark-colored shell; they have a clearly visible cap, which opens after the miracidium emerges. Therefore, it is more convenient to assess the viability of dicrocelium eggs by counting the opened eggs, rather than by staining and counting miracidia.

Luminescent method for studying helminth eggs and larvae.

For the first time in helminthological practice, fluorescent microscopy methods were used in 1955. It was reported that fluorescent 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; applied to the OI-18 illuminator special set color filters.

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

Uncolored, live, unsegmented roundworm eggs glow bright green with a yellowish tint; in dead eggs the shell radiates green light much brighter than the dark green germinal one; In roundworm eggs with a larva, only the shell appears, and in dead eggs, both the shell and the larva are bright yellow.

Unpigmented and unsegmented live eggs of pinworms and dwarf tapeworms emit a greenish-yellow light; In dead eggs, the shell luminesces intensely against the background of a dark green embryonic mass. With secondary luminescence (when stained with acridine orange at a dilution of 1:10,000 and 1:50,000 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 is colored orange-red. Embryos of living Asc. lumbricoides, T.leonina, H.diminuta, D.latum and bovine tapeworm oncospheres fluoresce in a dull dark green or gray-green color. The dead embryos of these helminth eggs emit a "burning" orange-red light. Living larvae of pinworms and toxocara (egg shells freed) emit a dim gray-green light; 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 - coryphosphilus, primulin - the dead eggs of roundworms and whipworms exhibit a glow from lilac-yellow to copper-red. Viable eggs do not luminesce, but are painted dark green. Live eggs of the trematodes Paragonimus westermani and Clonorchis sinensis do not luminesce when stained with acridine orange, but dead eggs emit a yellowish-green light.

The luminescence method can also be used to determine the viability of helminth larvae. Thus, strongylate and rhabditatus larvae fluorochromed with a solution of acridine orange (1:2000) glow: living ones - green (with a tint), dead ones - with a bright orange light. Living Trichinella larvae do not glow or give a weak glow when treated for 10 minutes with solutions of fluorescein isothiocyanate, auramine, etc. Fluorochromed dead larvae (at a concentration of 1:5000) give a bright glow.

Living miracidia emerging 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 with a bright “burning” light green and then orange-red light.