Helminthological methods. Microscopic examination of stool

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, protozoa become less susceptible to unfavorable factors environment.

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. Clinical significance Only the dysenteric amoeba, which occurs in vegetative form and in the form of cysts, has.

Additionally, immunological methods are used:

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

Note: serological methods are not very informative 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 that cause leishmaniasis are examined in blood smears and 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. Pathological microbes as the disease progresses are found in the material of lymph node punctures, 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 malarial fever manifests itself various shapes plasmodium:

• 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 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.

IN acute period diseases are detected in the punctate a large number of leishmania.

Note:Sometimes the blood cells may resemble processed Leishmania, so it is very important for the laboratory technician to be careful and have sufficient experience to examine them independently.

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.

If there is a disease among the macrophages, fibroblasts present in the test material, lymphoid cells Leishmania is also detected.

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 Leishmania actively multiplies.

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 quick ways Diagnosis of protozoa is 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

Stools are examined using two methods:

1. Macroscopic – detect helminths, their heads, segments, strobila fragments. Small portions of feces are mixed with water in a flat bath or Petri dish and viewed in good light on dark background, using a magnifying glass if necessary. All suspicious formations are transferred with tweezers to another cup with water or onto a glass slide in a drop of diluted glycerol.

With the method defending The portion of feces being tested is mixed with water in a glass cylinder, and after settling, the top layer of water is drained. This is repeated several times. When the liquid becomes transparent, it is drained and the sediment is examined in a Petri dish.

2. Microscopic – to detect eggs and larvae of helminths. There are many research methods.

1). Native smear – the most common and technically accessible research method. Eggs and larvae of all helminths can be detected. However, with a small number of eggs it is not always possible to find them. Therefore, the enrichment method is used.

1). Fülleborg method – this is an enrichment method based on the floating of helminthiasis eggs in a saturated NaCl solution (1.2 – density; 400 g of NaCl per 1 liter of water; 40% NaCl solution). The method is more effective than the native smear. 2-5 g of feces are placed in glass jars and filled with NaCl solution, stirred and after 45 minutes, remove the resulting film with a metal loop, place a drop of glycerin on a glass slide. Examine under a microscope. The disadvantage of the method is the slow emergence of eggs of various helminths, dwarf tapeworm - after 15-20 minutes, roundworm - 1.5 hours, whipworm - 2-3 hours.

2) Kalantaryan method – also an enrichment method, but a saturated solution of NaNO 3 (1.38 density) is used. Most eggs float; sediment testing is not required. The disadvantage is that keeping eggs in solution for a long time leads to the fact that some eggs begin to swell and settle to the bottom, disappearing from the surface film.

3. Goryachev method – based on the principle of egg deposition, detection of small trematode eggs. A saturated NaCl solution is used as a solution and 3-4 ml of feces solution is carefully layered on top. After 15-20 hours, trematode eggs settle to the bottom. The liquid is drained and the sediment is placed on a glass slide and under a microscope.

4. Shulman twisting method – to detect helminth larvae in feces. Only freshly excreted feces are examined. 2-3 g are placed in a glass jar and a 5-fold amount of water is added, quickly stirred with a stick without touching the walls of the jar - 20-30 minutes, then the stick is quickly removed, and a drop of liquid at the end is transferred to a glass slide and microscoped.

5. Berman method – is based on the ability of helminth larvae to migrate towards heat, and serves to identify them in feces.

6. Harada and Mori method (method of rearing larvae) and is recommended for testing for hookworm infections. The method is based on the fact that in heat and on damp filtered paper, hookworm eggs develop into filariform larvae, which can be easily detected. 15 g of feces are applied to the middle of a strip of filtered paper; the paper with feces is placed in a jar so that the lower end is immersed in water and the upper end is secured with a stopper. The jar is kept in a thermostat at 28 0 C for 5-6 days. Filariform larvae develop during this time and descend into the water. The liquid is examined under a magnifying glass. If it is difficult to detect, the liquid is centrifuged, having first killed the larvae by heating to 60 0. The laboratory assistant must wear gloves.

7. Methods for enterobiasis – identification of pinworm eggs and bovine tapeworm.

a) scraping from the perianal folds – with a cotton swab tightly wound on a wooden stick and moistened with a 50% glycerin solution. In the laboratory, the swab is washed off with 1-2 drops of a 50% aqueous solution of glycerin.

b) sticky mite method (Graham method)

The adhesive tape is applied to the perianal folds, then the adhesive layer is applied to a glass slide and examined under a microscope.

C) scraping using eye rods (Rabinovich method). For perianal scraping, glass eye rods are used, the wide part of which is covered with a special glue, which allows pinworm eggs to be retained.

Examination of blood, bile, sputum and muscles

Blood microscopy reveals filaria larvae.

Examination of sputum - paraganim eggs, roundworm larvae, necator, strongyloid, elements of echinococcal bladder.

Muscle examination - if trichinosis is suspected, the muscles of the patient or corpse are examined, as well as the meat that presumably caused the person to become infected. For the purpose of trichinoscopy, the muscle is cut into small pieces and placed in a compressorium, these are two wide, thick glasses that crush the muscles and Trichinella larvae are found in the form of capsules - a compression method.

Digestion method - the muscles are filled with artificial gastric juice (hydrochloric acid solution and pepsin). The muscles are digested, and the larvae are easily identified. Determination of the intensity of invasion: number of larvae up to 200 per 1 g muscle tissue– moderate intensity of invasion; up to 500 – intense; over 500 – super-intensive invasion.

Serological methods

Chapter III. Diagnosis of helminthiasis and methods of helminthological research

It is necessary to examine for helminthiasis all patients seeking medical help, and especially patients turning to a pediatrician, therapist and neurologist with complaints of side effects. gastrointestinal tract, nervous system and with anemia. If the doctor is not always able to use laboratory research methods, then every medical worker providing care in an outpatient clinic or hospital is required to interview the patient about the isolation of helminths.

If there are clinical indications given in the relevant chapters, the diagnosis should be clarified using laboratory methods for testing for helminthiases.

Due to the predominance of intestinal helminthiases, the greatest practical significance has a stool examination.

Methods for testing stool for helminthiasis

The stool is delivered to the laboratory in a clean glass container (about a quarter cup of stool taken from different places one serving); During a routine examination, it is allowed to deliver stool to the laboratory in matchboxes or splint boxes.

To control deworming, the entire portion of feces collected after administration is delivered (as prescribed by the doctor). anthelmintic and laxative (in large closed glass jars, buckets).

Microscopic examination feces is the main method for diagnosing intestinal helminthiases; it should always be preceded by a general macroscopic examination of feces to detect segments of large cestodes, pinworms, roundworms, etc.

The stool should be fresh or canned (in a 5% formaldehyde solution), since drying dramatically changes the structure of the eggs. In addition, when feces stand, the eggs of some helminths (for example, hookworms) develop rapidly, which makes diagnosis difficult.

According to the instructions of the USSR Ministry of Health, it is necessary to examine stool simultaneously using the Fulleborn method and the native smear.

Native smear

Native smear: a small piece of feces (about the size of a pea), taken with a match, glass or wooden stick from different places in the delivered portion, is thoroughly ground on a glass slide in a drop of 50% glycerol solution or in saline solution or in water. Cover with a coverslip, pressing lightly on the latter (with a dissecting needle). The smear should be thin, transparent and uniform. It is used only as an addition to other methods that provide enrichment of the drug. At least two drugs should be reviewed.

In order to detect helminth larvae (as well as their eggs), a native smear is made as follows (according to Shulman): 2-3 g of feces are thoroughly mixed by “twisting” glass rod into an emulsion with five times the amount of pure water or saline. During stirring, the larvae accumulate near the glass rod, so immediately after the end of stirring, a drop of the emulsion is quickly transferred with a glass rod onto a glass slide, covered with a coverslip and examined. S. D. Lyubchenko (1936) proved that the twisting method is more effective than the smear method, especially with regard to roundworm eggs. Based on the work of S. D. Lyubchenko, we consider it advisable to replace the smear method with the twisting method.

Fulleborn method

Fulleborn method: 5-10 g of feces taken from different places are placed in a jar with a capacity of 50-100 ml and thoroughly rubbed with a glass or wooden stick in a saturated solution table salt(400 g of this salt is dissolved in 1 liter of water, heated to a boil and filtered through a layer of cotton wool or gauze; the solution is used cold: specific gravity 1.2). The solution is added gradually until a uniform suspension is obtained, and the total amount of solution added should be approximately 20 times more quantity feces. To mix feces, Fulleborn recommended using tea glasses, but it is more convenient to prepare a suspension in ointment jars with a capacity of 50-100 ml, using two jars for each analysis (or in cups with a capacity of 100 ml).

Immediately after preparing the suspension, large particles that have floated to the surface (plant formations, undigested food debris, etc.) are removed from the surface with a spatula, a metal scoop or a piece of clean paper, after which the mixture is left to stand for 1-1.5 hours. After this time, the entire film is removed from the surface of the mixture by touching a wire or platinum loop (flat) with a diameter of no more than 1 cm, bent at a right angle; The film is shaken onto a glass slide and covered with a coverslip. Place 3-4 drops under each cover slip (18x18 mm). In total, at least 4 preparations should be prepared (one cover glass for each preparation). The loop is heated over a fire and washed with water after each analysis.

Using the Fulleborn method, eggs of all nematodes (with the exception of unfertilized roundworm eggs) and dwarf tapeworm eggs are quickly and easily detected.

The Berman method is used to examine feces for helminth larvae (for strongyloidiasis). This method is as follows: 5 g of feces on a metal mesh (a milk strainer is convenient for this purpose) is placed on a glass funnel attached to a tripod. A rubber tube with a clamp is placed on the lower end of the funnel.

The mesh with feces is lifted and water heated to approximately 50° is poured into the funnel so that the lower part of the mesh with feces is immersed in water. The larvae actively move into the water and accumulate in the lower part of the rubber tube. After 2-4 hours, the clamp is opened and the liquid is drained into one or two centrifuge tubes.

After centrifugation for 1-2 minutes top part the liquid is quickly drained, and the sediment is applied in drops onto glass slides and examined under cover slips or distributed in a thin layer onto 2-3 large glasses and then examined without cover slips.

The Berman method is also used to test soil for the presence of hookworm larvae.

Stoll method

The Stoll method is used to determine the intensity of invasion. A decinormal solution of caustic soda is poured into a special glass flask to the 56 cm 3 mark, and then feces are added until the liquid level reaches 60 cm 3, i.e. 4 cm 3. After shaking with glass beads, 0.075 ml of the mixture is taken for examination and examined under one or two ordinary coverslips. The resulting amount is multiplied by 200 to obtain the number of eggs contained in 1 cm 3 of feces.

Study of duodenal contents

Duodenal juice and bladder bile obtained in the usual way using probing (and gallbladder bile and after a reflex from the gallbladder), thoroughly mix with an equal volume of ethyl ether; the mixture is centrifuged, after which the sediment is examined under a microscope. In addition to sediment, flakes floating in the liquid, which may contain helminth eggs, must be examined microscopically. When testing gastric juice and vomit for helminth eggs, you can use the same technique.

Examination of duodenal juice and stomach contents should be carried out if helminthic diseases of the liver, gall bladder (opisthorchiasis, fascioliasis, dicroceliosis) and duodenum (strongyloidiasis) are suspected.

Sputum examination

The sputum is ground on a glass plate, tightly covered with another glass plate and examined with the naked eye against a light and black background, as well as under a magnifying glass in transmitted light. Individual pieces of sputum (“rusty” accumulations, tissue scraps, etc.) are applied in a thin layer to a glass slide, tightly covered with a coverslip and examined under low and high magnification microscope.

a) To diagnose cutaneous cysticercosis, subcutaneous tissue or muscle, an aseptically excised piece of the relevant tissue is examined first with the naked eye. Areas of tissue are moved apart using dissecting needles in order to detect a vesicle visible to the naked eye - a cysticercus (photo A); its length is 6-20 mm, width 5-10 mm. When a vesicle suspicious for cysticercus is detected, it is crushed between two glass slides and examined under a microscope. Cysticercus (Cistycercus cellulosae) is determined by the presence of a scolex with four suckers and a corolla of hooks (photo B).

Photo. A - cysticerci with scolex turned outward; B - Head of a pork tapeworm.

b) To diagnose trichinosis, an aseptically excised piece of muscle (biceps or gastrocnemius) is carefully crushed in a 50% glycerin solution into the finest fibers using dissecting needles. The crushed muscles are compressed between two glass slides and examined under a low-power microscope in a darkened field of view. It is recommended to test muscles for trichinosis no earlier than on the 8th day of the disease. Trichinella larvae are found in the muscles in a coiled position: they are enclosed in lemon-shaped capsules.

Photo. A - Trichinella larvae in muscles; B — Calcified capsules of Trichinella.

X-ray

Most often, fluoroscopy is used to diagnose echinococcosis and, less commonly, cysticercosis. Cysticerci are detected by fluoroscopy only after calcification (in cases of long-term disease). Behind last years Fluoroscopy is also used to diagnose ascariasis both in the early larval stage and partly in the intestinal stage.

During the period of migration of roundworm (and hookworm) larvae, unstable, sometimes multiple inflammatory foci are detected in the lungs; at the same time, significant eosinophilia appears in the blood.

Sexually mature roundworms are clearly visible on fluoroscopy of the intestines of affected individuals. This method, despite its complexity and cumbersomeness, should be used as an additional method for diagnosing ascariasis in cases with a negative scatological analysis. According to E. S. Geselevich, of 180 patients with ascariasis identified by fluoroscopy, 54 had no ascaris eggs found in their stool (see).

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 setting up a biological sample, 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. Segmented eggs have crushing balls (blastomeres) of unequal size, irregular shape, often shifted to one pole. Sometimes there are abnormal eggs that, having 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, and pinworms, you should call active movements larvae by lightly heating the preparation (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. In this case, the internal structure of the larva becomes blocky 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 digestive enzymes. The eggs are placed on watch glass With gastric juice dogs 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 outer shell eggs.

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% hydrochloric acid solution 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, is 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.

Test tubes are placed in 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 differential diagnosis 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.

Staining Ascaris larvae with a basic solution of brilliant cresyl blue paint at a concentration of 1:10 000 is carried out as follows: a drop of liquid with Ascaris eggs and a drop of the basic 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, 5% is used as a dye. alcohol solution Yoda. 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, turn bright red or pink color safranin or blue brilliant cresyl blue at a dilution of 1:4000 or 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 of the motor activity of the larva. 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 hair dryer) 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 up to 10-13% and a dye (safranin, methylene blue and others working within the pH range) are added to the buffer until the liquid is slightly colored (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 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; A special set of color filters was used for the OI-18 illuminator.

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 emits a green light much brighter than the dark green embryonic shell; 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 colored dark green color. 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.

Ershova I.B., Osychnyuk L.M., Mochalova A.A., State Institution "Lugansk State Medical University", Department of Pediatrics with Childhood Infections.
The article was published in the journal “Actual Infectology”, No. 2 (3), 2014.
Information resource “Zaslavsky Publishing House” www.mif-ua.com

The article outlines methods for diagnosing helminth infestations: microscopic, linked immunosorbent assay, serological, polymerase chain reaction, bioresonance diagnostics, hemoscanning, as well as instrumental (ultrasound and X-ray examination, computed tomography) and laboratory, of indirect significance ( clinical analysis blood, blood test for liver function tests, stool test for dysbacteriosis). The advantages of the methods, their disadvantages, and the reliability of the data obtained are described. Questionnaires for patients are proposed to identify the risk of helminth infection. The advantages of the methods, their disadvantages, and the reliability of the data obtained are described. Questionnaires for patients are proposed to identify the risk of helminth infection.

Helminths have a negative effect on the human body. They lead to allergization, the development of polyhypovitaminosis, macro- and microelementosis, impaired hematopoiesis and vascular permeability, and hormonal imbalance. Helminth infections contribute to the formation chronic diseases(cholecystitis, cholelithiasis, pancreatitis, colitis, diabetes, bronchial asthma, atopic dermatitis), psycho-emotional disorders ( chronic fatigue, irritability, anxiety, hyperactivity in children), anemia, etc. With prolonged helminth infestation, it can develop secondary immunodeficiency.

The alertness of doctors regarding helminth infections among the population is currently insufficient, and prevention is reduced to the treatment of identified infested patients.

Diagnosis of helminthiases is based on clinical, epidemiological and laboratory data. Signs such as asthenic syndrome, recurrent urticaria, impaired regeneration of the skin and mucous membranes, difficult to treat atopic dermatitis and broncho-obstructive syndrome, polylymphadenopathy and hepatosplenomegaly of unknown origin, adenoid vegetations II–III degrees, “geographical” tongue, reduced or selective appetite, unstable stool, may indicate the presence of helminths.

During a serological study, the presence of antibodies to helminths is determined (reliability is about 60%): if echinococcosis, cysticercosis, trichinosis, toxocariasis is suspected, indirect hemagglutination reactions, latex agglutination, complement fixation, and immunofluorescence are widely used.
Not in all cases, determination methods specific antibodies have sufficient specificity and reliability. The antigenic composition of the helminth depends not only on the species, but also on the stage; going through a complex development cycle from egg to adult, helminths change their antigenic composition. In addition, somatic antibodies are used in immunodiagnostic reactions, and in the host’s body antibodies are produced mainly to the excreta and secretions of the helminth. Nonspecific sensitization of the body, the commonality of some antigens of trematodes, protozoa and humans create a high proportion of false-positive reactions in titers below reliably diagnostic ones.

Method for determining helminths using polymerase chain reaction is highly specific and highly sensitive, but due to its high cost and complexity it cannot be used for screening when, for example, it is necessary to examine a group of children from child care facility.

The immune system does not always react (recognize and destroy) to the presence of helminths in the body. This is because some helminths have a durable and chemically resistant capsule or are coated with a substance that is not recognized by the immune system; localized in tissues that are most protected from inflammatory reactions, for example, in spinal cord; many types of them in digestive tract secrete anti-enzymes, which saves them from death; have longer duration life (for years, and sometimes until the death of the person himself); feed on glycolysis of net carbohydrates; have devices such as suction cups, hooks, etc., which facilitate fixation inside the body; many species have sexual reproduction, in which gene information is exchanged, which leads to an increase in the heterogeneous population and a decrease in vulnerability; have high level fertility.

At ultrasonic , x-ray examination abdominal organs , computed tomography can be identified indirect signs helminthiases: hepatosplenomegaly, unevenness of the liver and spleen parenchyma due to small hyperechoic signals, increased The lymph nodes in the gates of the spleen and the helminths themselves (echinococci, balls of intestinal helminths, etc.).

Hemoscanning - qualitative blood examination using a powerful dark-field microscope, you can see the state of blood cells (shape, size, activity, color, etc.), the presence of nonspecific elements and substances - all this directly or indirectly indicates the presence of helminths in the body. The image is displayed on the monitor screen using the built-in video camera in the microscope. This diagnostic method is highly reliable.

Indirect laboratory signs helminthiasis there may be anemia, basophilia, eosinophilia, increased levels of aspartate aminotransferase. Thus, with toxocariasis, a leukemoid reaction of eosinophils (more than 20%) is detected against the background of a persistent allergic syndrome (atopic dermatitis with severe itching and resistance to traditional therapy, severe bronchial asthma). Inhibition of normal E. coli in a stool test for dysbiosis may also indicate possible helminthiasis.

Taking into account the prevalence of helminthiases, we offer questionnaire to determine the risk of helminth infection.

• Swimming in freshwater bodies of water.
• Do not wash your hands before eating with soap and hot water.
• Drink water from unverified sources.
• You eat homemade lard with streaks of meat.
• Do you eat lightly salted fish?
• You eat medium-cooked meat (with blood).
• Do you eat lightly salted, non-factory prepared caviar?
• Don't wash chicken eggs with soap.
• Do not wash bananas, oranges, tangerines before eating.
• Fertilize your garden with manure.
• Eat vegetables straight from the garden.
• You eat fruits and berries straight from the garden.
• Eat fallen fruits.
• Do not pour boiling water over all your greens to make salads.
• Store carrots in sand taken from the yard.
• Walk barefoot on the grass.
• Family members had helminthic infestations.
• The family has a dog or cat.

For each answer " Yes" - 2 points, " Sometimes" - 1, " No" - 0. With a score of 0–5, the probability of infection is negligible, 6–12 - infection is possible, 13–25 - high probability, more than 25 points - very high. With the last two results, regular examination and, possibly, preventive treatment are necessary.

Because the clinical manifestations helminth infections are not always specific, and in the initial stages - nonspecific, we offer a questionnaire for patients for self-diagnosis helminthiasis.

• There is itching in the anus in the morning.
• Nausea in the morning when brushing teeth.
• Peeling of fingers or toes with peeling of layers of skin.
• Allergic skin rash, itchy skin.
• Peeling and swelling in the eyelid area.
• Increased fatigue, lethargy, drowsiness.
• Increased feeling of hunger.
• Feeling of discomfort in the stomach.
• Loss of body weight.
• The presence of several chronic diseases of the gastrointestinal tract, joints, and bronchopulmonary system.
• Poor health, lack of official diagnosis, long-term ineffective treatment.
• Periodic rise in temperature, accompanied by muscle and joint pain.

Answer " Yes"at least 2-3 questions indicate a high probability of helminth infection.

CONCLUSIONS

1. On modern stage There are no laboratory methods for testing for helminth infections that are 100% reliable.

2. The polymerase test has the greatest reliability in diagnosing helminthiasis chain reaction and bioresonance diagnostics.

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