RU2362995C1 - Way of blood protozoan diseases diagnostics - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine area, in particular to laboratory diagnostics. For realisation of a way of diagnostics of blood protozoan disease a blood sampling is performed, the blood is centrifugated, applied in the form of a drop on 2 separate glasses having 3.3 mcl of blood plasma. Thus on the first glass 1.7 mcl of the erythrocytes taken from the top fraction of blood, and on the second - 1.7 mcl of the erythrocytes taken from the bottom fraction of blood are added to the plasma. The smears are form by means of glass, fixed, painted using a stain, washed from excess of a stain using the distilled water, dried and investigated by means of optical microscopy with an immersion. At revealing endoglobular includings of cherry-lilac colour in the erythrocytes, the amazed segmented neutrophils and monocytes, the blood protozoan infection is diagnosed.

EFFECT: rising of accuracy of blood protozoan diseases diagnostics and reduction of time of the analysis.

4 cl, 2 ex

Description

The invention relates to the field of humanitarian and veterinary medicine and is intended for laboratory diagnosis of blood parasitic infections in humans, agricultural and small pets.

For the diagnosis of blood parasitic infections (hemosporidioses) in humanitarian and veterinary medicine, the cytological method (Romanowsky-Giemsa or Wright-Giemsa smears staining), the polymerase chain reaction (PCR) method, a biological test on laboratory animals, such as golden hamsters, and serological diagnostic methods are used : complement binding reaction (CSC or CFT-complement fixation test), indirect immunofluorescence reaction (IFAT-indirect fluorescent antibody test), competitive inhibition - enzyme-binding immunosorbent method (CI-ELISA - competitive-i nhibition enzyme-linked immunosorbemt assay) [1-5].

The disadvantage of the above serological diagnostic methods for the clinical manifestation of the disease is the limited time periods for the formulation of reactions, the complexity and high cost, as well as the lack of full reproducibility of the results by several methods. Many laboratories lack equipment and reagents, as well as qualified personnel for the formulation of sophisticated modern techniques, such as PCR analysis (DNA polymerase chain reaction method). Biological test on laboratory animals is long in time - 6 weeks; CSC - in fresh cases (in the early stages), the disease is negative, active 2-3 weeks after the disease, after which the titer drops after 3 months. no longer has practical significance [5]; IFAT - gives positive results 7-10 days after the onset of the disease, CI-ELISA - designed only for cattle. Cytological methods for staining native blood according to Romanowsky-Giemsa or Wright-Giemsa are uninformative with low parasitemia.

The closest to the claimed method, the prototype is a method for the diagnosis of blood parasitic infections [6], which includes the following successive stages.

5 ml of venous blood are sampled, followed by centrifugation at 1000-3000 rpm for 10-15 minutes and 6-10 μl of blood plasma and 3-5 μl of concentrated bottom sediment are taken into a separate tube and thoroughly mixed. From the obtained suspension, 2-3 smears are prepared (5 μl per glass) with a special polished glass, the width of which is twice as narrow as a slide, and dried in air until 2-3 minutes dry. The smears are fixed in methanol for 10 minutes and dried in air for 5 minutes, stained with a 20% solution of a standard dye azure-eosin pH 5.0 for 15-30 minutes, washed under running tap water and washed from excess dye of methylene azure in a bath with distilled water in replaceable water for 15-20 minutes, after which it is dried in air for 15-20 minutes. Microscopy of smears is carried out in transmitted light with immersion. The search for red blood cells affected by blood parasitic infections is carried out from the beginning of the smear - the site of the largest accumulation of red blood cells with parasites and further along the upper and lower edges of the smear. If endoglobular inclusions of cherry-lilac color are detected in orange red blood cells, a blood-parasitic disease is diagnosed.

The time required to detect blood parasitic infections with a blood smear microscopy is 3 to 10 minutes. The total time spent on diagnosis is from 75 to 115 minutes.

The method is simple and does not require expensive equipment and reagents, but it has several disadvantages. It is not always possible to take 5 ml of blood, especially in sick individuals and small animals. The use of plasma in the amount of 6-10 μl and 3-5 μl of the concentrated bottom of the sediment with the preparation of 2-3 smears of 5 μl per glass significantly increases the cost of laboratory equipment (tubes) and the time of preparation and study of the smears themselves. In addition, red blood cells are not taken into account in the initial phase of infection with babesia or affected by small parasite species (about 0.1-0.5 μm), which are lighter and have a higher membrane charge, therefore they do not settle, but are in the upper blood fraction. The use of unfiltered concentrated dye solution can lead to the precipitation of large particles of paint on the smear, which greatly complicates the procedure for subsequent examination of the blood smear. Drying the brushstrokes in air and at high humidity takes extra time, so drying the brushstrokes is easier and faster with a hairdryer with a stream of cold air.

An object of the invention is to increase the accuracy of diagnosis and reduce the time for analysis.

The technical task is achieved by the proposed method, which consists in the following.

Take a sample of 0.5-1.5 ml of blood in a syringe or tube with an anticoagulant. The test blood is centrifuged for 5-10 min at 3500-5000 rpm, 3.3 μl of blood plasma is applied to two glass slides in the form of a drop. On one of them, 1.7 μl of erythrocyte mass taken from the boundary layer with plasma (upper blood fraction) is added, and on the other - 1.7 μl of erythrocyte mass taken from the lower blood fraction. Drops on the glass are thoroughly mixed, which allows you to maintain the viscosity of native blood (2 to 1 ratio) and significantly increase the number of red blood cells affected by babesia in smears. Get 2 smears (5 μl per glass). Brushstrokes are made with special polished glass, the width of which is two times narrower than a glass slide, and dried in air. Then the smears are fixed in methanol and dried in a stream of cold air using a hairdryer. Next, the smears are stained with a 20% solution of a standard dye azure-eosin (Romanovsky-Giemsa) pH 5.0, and the concentrated dye is filtered before use through a paper filter or filter paper to remove undissolved paint particles from it. The smears are washed with distilled water so that salts from tap water do not settle on the smear, and washed from the excess dye of methylene azure in a bath with distilled water in replaceable waters. Then the smears are dried in a stream of cold air using a hairdryer. Microscopy of smears is carried out in transmitted light with immersion. The search for red blood cells affected by blood parasitic infections is carried out from the beginning of the smear - the site of the largest accumulation of red blood cells with parasites, and then along the upper and lower edges of the smear. Under microscopy, erythrocytes have an orange color, and endoglobular inclusions have a cherry-lilac color, in individual trophozoites a contoured nucleus is released. If endoglobular inclusions of cherry-lilac color are detected in yellow red blood cells, a blood-parasitic infection is diagnosed.

The time required to detect blood parasitic infections by microscopy of a blood smear is from 3 to 5 minutes. The total time for diagnosis of the proposed method is from 55 to 68 minutes.

The defining differences of the proposed method from the prototype are:

- the test blood is pre-centrifuged at 3500-5000 rpm, which allows you to halve the time for this stage and quickly sediment most of the red blood cells with babesias, which occupy most of the cell volume, to the bottom of the tube in 5-10 minutes;

- after centrifugation, 3.3 μl of blood plasma is applied in droplet form on two slides, 1.7 μl of erythrocyte mass from the upper blood fraction is added to one of them, and 1.7 μl of erythrocyte mass from the lower blood fraction is added to the other and mixed thoroughly, which allows preserving viscosity of native blood (ratio 2 to 1) and significantly increase the number of red blood cells affected by babesia in smears.

Before use, the concentrated dye is, in particular, filtered through a paper filter or filter paper to remove undissolved ink particles from it.

The dye from the smears is washed off mainly with distilled water, which avoids the loss of insoluble salts of iron, calcium and magnesium on the smear when using tap water.

The principle of diagnostics is based on the fact that red blood cells affected by intraerythrocytic parasites (babesia, malarial plasmodium, anaplasmas and other hemosporidioses) have a sedimentation rate greater due to a decrease in the charge of the erythrocyte membrane [7] than in normal unaffected red blood cells. Conglomerates (adhering erythrocytes) are formed in the blood due to the release of cytoadhesive ligands by hemosporidia onto the erythrocyte membrane [8]; therefore, they can be easily precipitated by centrifugation. Red blood cells with early stages of hemosporidia and ring-shaped forms of parasites rise up to the layer of white blood cells bordering the plasma. A smear prepared from the upper fraction of red blood cells significantly increases the accuracy of direct determination of the parasite in the blood. If parasitemia in native blood is low (1 affected red blood cell per 20-100 μl or more), then a study of 5 μl of native blood can produce a false negative result. Moreover, the affected blood due to the presence of cytoadhesive ligands by the number of parasites has a heterogeneous structure: one drop of blood from the collected blood volume may not contain hemosporidia, and in another drop of blood the number of infected red blood cells will be high. Blood sampling is carried out in a minimum representative volume in the amount of 0.5-1.5 ml in a syringe or test tube with any anticoagulant (heparin, Trilon B, sodium citrate), which allows using the proposed method to view a blood volume of 5 μl, which corresponds to 100-300 in reality drops of blood.

The inventive method allows you to quickly and accurately determine the presence of parasites in the blood.

The inventive method diagnosed blood parasitic infections in dogs (n = 180) and cats (n = 48). Diagnosis was made after treatment in city veterinary hospitals and clinics, where a blood test showed a negative result for babesiosis, however, the pet owners again applied for a diagnosis of chronic dermatitis, local and total alopecia, animal fatigue, anorexia, functional cardiovascular failure, inflammatory conditions on the auricle, middle and inner ear, hematomas of the auricle, with progressive cachexia, lymphadenitis, lymphoma and, allergic reactions, bronchitis and pneumonia, lesions of the central and peripheral nervous system, arthritis, glomerulo- or pyelonephritis, anemia, enteritis and intestinal bleeding, immunodeficiency states, yellowness and anemicity of the skin, conjunctivitis and kerato inflammatory diseases, etiology. Examination of smears from native blood under a microscope showed the presence of rare very small inclusions inside red blood cells, as well as V-shaped and dot structures typical of Bartonella, on membranes and inside red blood cells from 0 to 1 in the field of view. When processing blood according to the proposed method, erythrocytes with large trophozoites inside and clear contour nuclei in them were detected in the smear, which indicated the presence of babesias in the blood in a state of active reproduction. In the red blood cells from the upper fraction, small babesias from 0.5 to 1-3 μm in size were present. In some animals, several types of babesia could be distinguished simultaneously morphologically. The number of bartonella on red blood cells increased to 8-10 and more than 100 in individual fields of view.

The inventive method diagnosed blood parasitic infections in 750 people with various chronic diseases of unknown etiology and with a number of symptoms identical to those observed in animals. 525 people in smears from native blood, affected by babesia of red blood cells, had from 1 for the entire smear to 5 in separate fields of vision. Bartonella was found in an amount of 1 to 3 in a smear. When processing blood by the claimed method, the number of detected babesias in the smear increased to 25-75 in the field of view. In 225 people in 2 smears from native blood, not a single red blood cell affected by babesia was found. After preparation of smears from the lower fraction of centrifuged blood, it was possible to identify from 1 to 9 affected red blood cells in 2-4 visual fields (latent, weak parasitemia) and from 1 to 7 affected red blood cells in the upper fraction). The number of detected bartonellas on red blood cells and inside increased to 1-4 and more than 100 in separate fields of vision. After centrifugation, some smears revealed anaplasmas in red blood cells, rickettsia in segmented neutrophils, monocytes and lymphocytes.

The method is illustrated by the following examples of specific performance.

Example 1

A dog of the breed German Shepherd named Uldari - a female at the age of 7 years. A blood test from a vein by a standard method gave a negative result.

A study of venous blood of the claimed method. For this purpose, 0.5 ml of blood was drawn into a syringe with heparin, the test blood was centrifuged for 5 min at 5000 rpm, and 3.3 μl of blood plasma was applied to two glass slides in the form of a drop. On one of them, 1.7 μl of erythrocyte mass taken from the upper blood fraction was added, and on the other - 1.7 μl of erythrocyte mass taken from the lower blood fraction. The drops on the glass were thoroughly mixed, formed two smears, fixed them in methanol (10 min) and dried them in a stream of cold air using a hairdryer. Then the smears were stained with a 20% solution of the standard dye azure-eosin pH 5.0 for 15 minutes. The concentrated dye was filtered through a filter paper before use. The smears were washed with distilled water, washed from excess dye of methylene azure in a bath with distilled water in replaceable water for 15 minutes and dried. Microscopy of smears was performed in transmitted light with immersion. The search for red blood cells affected by blood parasitic infections was carried out from the beginning of the smear - the site of the largest accumulation of red blood cells with parasites and further along the upper and lower edges of the smear. Under microscopy, erythrocytes had an orange color, and endoglobular inclusions had a cherry-lilac color, a contoured core was isolated in individual trophozoites. A study of venous blood of the claimed method allowed us to identify from 10 to 30 red blood cells affected by babesia and from 1 to 3 bartonella in separate fields of view in the upper and lower fractions of the centrifuge.

Example 2

Patient A., 43 years old, a donor with experience of 5 years. When examining a blood smear prepared according to a standard technique, blood parasites (hemosporidia) were not detected.

A study of venous blood of the claimed method. For this, 1.5 ml of blood was drawn into a test tube with Trilon B, the test blood was centrifuged for 10 min at 3500 rpm, and 3.3 μl of blood plasma was applied to two glass slides in the form of a drop. On one of them, 1.7 μl of erythrocyte mass taken from the upper blood fraction was added, and on the other - 1.7 μl of erythrocyte mass taken from the lower blood fraction. The drops on the glass were thoroughly mixed, formed two smears, fixed them in methanol (10 min) and dried them in a stream of cold air using a hairdryer. Then the smears were stained with a 20% solution of the standard dye azure-eosin pH 5.0 for 20 minutes. The concentrated dye was filtered through filter paper before use. The smears were washed with distilled water, washed from the excess dye of methylene azure in a bath with distilled water in replaceable water for 20 minutes and dried. Microscopy of smears was performed in transmitted light with immersion. The search for red blood cells affected by blood parasitic infections was carried out from the beginning of the smear - the site of the largest accumulation of red blood cells with parasites and further along the upper and lower edges of the smear. Under microscopy, erythrocytes had an orange color, and endoglobular inclusions had a cherry-lilac color, a contoured core was isolated in individual trophozoites. The venous blood test by the claimed method allowed us to identify from 15 to 32 red blood cells affected by babesia in the field of view of the lower fraction of centrifuged red blood cells and from 11 to 23 affected red blood cells in the upper fraction. Rickettsia have been identified in segmented neutrophils and monocytes.

Using this method will significantly reduce the time spent on a high-quality diagnosis of blood parasitic infections, allow specialists in humanitarian and veterinary medicine to efficiently, timely and quickly identify patients with acute and chronic course of a blood parasitic disease, donors with latent carriage of infection or asymptomatic course of the disease, conduct preliminary monitoring of donated blood before blood transfusion.

The method is technically simple, effective and available in any laboratory for the qualitative diagnosis of blood parasitic infections.

Information sources

1. Krause P.J. et al. Comparison of PCR with Blood Smear and Inoculation of Small Animals for Diagnosis of Babesia microti Parasitemia. - J. Clinical Microbiology. - 1996. - P.2791-2794.

2. Avarzed A. et al. Monoclonal Antibody against Babesia equi: Characterization and Potential Application of Antigen for Serodiagnosis. - J. Clinical Microbiology. - 1998 .-- P.1835-1839.

3. Boustani M.R., Gelfand J.A. Babesiosis. - Clinical Infectious Diseases. - 1996. - P.611-614.

4. Lodes M.J., Houghton R.L., Bruinsma E.S. et al. Serological expression cloning of novel immunoreactive antigens of Babesia microti. - Infection of immunity. - 2000. - V.68. - N.5 - P.2783-2790.

5. Azhinov S.A. Dynamics of CSC in hemosporidiosis of horses // Tr. Rostovsk reg. N.-i. vet. experience. Art. - 1955. - issue 11. - S.197-200.

6. RF patent No. 2218565. “Method for the diagnosis of blood-parasitic diseases”, cl. G01N 33/48, publ. 12/10/2003.

7. Kudryavtsev A.A., Kudryavtseva L.A. Clinical hematology of animals. - M .: Kolos, - 1974. - 399 p.

8. Allred D.R. Antigenic variation in babesiosis: is there more than one ′ why ′ // Microb. Infect. - 2001. - Vol.3 - P.481-491.

Claims (4)

1. A method for the diagnosis of blood parasitic infections, including blood sampling, preliminary centrifugation, applying the bottom of the red blood cells in the form of a drop on a glass slide, smear formation using glass, the width of which is 2 times narrower than the glass slide, fixing the smear on the glass, air drying , staining the smear with dye, washing the smear from excess dye, drying the smear after staining in air and subsequent blood testing using optical microscopy with immersion, characterized in that the blood is preliminarily they are centrifuged completely at 3500-5000 rpm, applied to 2 separate glasses of 3.3 μl of blood plasma, while 1.7 μl of red blood cells taken from the upper blood fraction are added to the first glass to the plasma, and 1, 7 μl of red blood cells taken from the lower blood fraction, and when detecting endoglobular inclusions of cherry-lilac color in red blood cells, affected segmented neutrophils and monocytes, they diagnose a blood-parasitic infection. 2. The method according to claim 1, characterized in that the concentrated dye solution is filtered through a paper filter or filter paper before use. 3. The method according to claim 1, characterized in that the smears are dried in a stream of cold air using a hairdryer. 4. The method according to claim 1, characterized in that the dye from the smears is washed off with distilled water.