Diagnosis of protozoal infections
Jun 16, 2018
Not all protozoa( Protozoa) that parasitize a person are pathogens.To detect the type of pathogen and confirm the clinical diagnosis, one can not do without methods of laboratory diagnosis.The simplest, settled in the human body, can be in different stages of its development.Contents: Classes of parasites and biomaterials under study Specific diagnostics
Parasite classes and biomaterials
The simplest are divided into 4 classes:
- Rhizopoda - represented in humans by amoeba;
- flagellata( flagellata) - the main pathological representative - leishmania;
- ciliary( ciliata) infusoria - the most common causative agent of the disease - balantidium;
- sporozoa( sporozoa) - a dangerous parasite of this group - malarial plasmodium.
The microorganism acquires a rounded shape when encysted and is covered with a protective coating.In the form of cysts, protozoa become less susceptible to adverse environmental factors.
Localization of pathogenic microorganisms can be in different organs and tissue structures.Therefore, the choice of material for research depends on where the parasite can be.
The following may be exposed to the study:
- bone marrow;
- the contents of abscesses( in the liver, lungs, brain);
- contents of cavities( abdominal, pleural, pericardial);
- mucus and exudates of the intestines, fistulas.
Note: variety of diagnostics is very much, we will consider those species that are most common in clinical laboratory practice.
Particular types of diagnostics
In each specific case, the lab technician is given the task of finding a particular pathogen, sometimes others are found along with the main one.
Diagnosis of Kornozzheki( Amoeba)
There are 6 species of this microorganism capable of dwelling in the human intestine.Clinical significance is only dysentery amoeba, occurring in a vegetative form and in the form of cysts.
The main way to detect a parasite is to study it in a native( unprocessed) biological preparation.
In addition, immunological methods are used:
- indirect immunofluorescence;
- indirect agglutination( PHA);
- radial immunodiffusion.
Note: serological methods are of little informative and are used only as a supplement to the main ones in doubtful cases.
Diagnosis of ciliary( infusoria)
The pathogenic form of microorganisms of this genus - balantidium.It is a microbe that causes balantidiasis, a disease accompanied by a ulcerative process of the large intestine.The causative agent is found in the native smear in the form of vegetative form and cyst.Material for the smear( feces and mucus) is taken with a sigmoidoscopy study and is sown to special media.
Diagnosis of flagellates( leishmania, lamblia, trypanosomes, trichomonads)
For a person are the danger of leishmania, trypanosomes, lamblia, trichomonads.
Leishmania - microbes that cause leishmaniasis, are examined in blood smears, bone marrow materials, scrapings from skin infiltrates.In some cases, in the diagnosis of leishmania, sowing is applied to nutrient media.
Tripanosomes are causative agents of sleeping sickness( American / African trypanosomiasis, or Chagas disease).
The African variant is determined in the initial period in the study of peripheral blood.Pathological microbes in the progression of the disease are found in the material of puncture of the lymph nodes, in advanced stages - in the cerebrospinal fluid.
The causative agent of American trypanosomiasis in the acute phase of the disease can be found in the flagellate form.Subsequently, the paratyzes are found in muscle cells, myocardium, in the neurons of the central nervous system.
For the diagnosis of trypanosomes with suspected Chagas disease, the test material is examined under a microscope at low magnification.In this case, strokes and a thick drop are pre-stained.
Giardia, the leading parasitic lifestyle in the duodenum is found in bile or in analysis of feces( cysts).
Trichomonas( intestinal, oral, vaginal) are detected by microscopy of materials taken from the affected mucous membranes.
Detection of sporophores( malarial plasmodium, causative agent of coccidosis, etc.)
Sporuncles are parasitic forms that inhabit the cells, tissues and cavities of the body .
The most common and dangerous for human species is the malarial plasmodium, which has 4 main types of pathogens: the causative agent of three-day malaria, four-day malaria, tropical malaria and malaria oval.
The sexual development of plasmodium( sporogony) takes place in mosquitoes Anopheles.Bessless( tissue and erythrocytic schizogonia) - in the liver tissue and human erythrocytes.These features of the life cycle are necessarily taken into account in the diagnosis of malarial plasmodium.
For example, a newly ill patient in the blood can detect the sex cells of the sporogony cycle.But at the height of malarial seizures in the blood in a large number of schizonts appear.
Moreover, different forms of plasmodium manifest themselves in different phases of the malarial fever:
- during the chill period the blood is filled with merozoites, a variety of schizonts;
- at the height of temperature in erythrocytes accumulate ring-shaped trophozoites;
- decrease in temperature is characterized by the predominance of amoeboid trophozoites;
- during periods of normal blood condition contains adult forms of schizonts.
Investigation of the pathogen of malaria( malarial plasmodium) is carried out in a smear and in a thick drop.
Morphological forms of parasites for clear differentiation are analyzed by specific staining.This variant of "visualization" makes it possible to disassemble individual morphological features of one or another type of microorganism.
Note: malaria diagnosis when examining smears and thick drops of blood is sometimes mistaken.The blood platelets in some cases can be mistakenly attributed to the malarial pathogen.Also sometimes plasmodial fragments of leukocytes and other cells simulate.
Basic methods for investigating the simplest
Briefly introduce the most common methods of research for the presence of protozoa.
Diagnosis of the simplest method of the native smear and smear stained with Lugol's solution( in the stool)
The preparation is prepared from an emulsion of stool in isotonic solution.On the slide are applied two drops of sodium chlorine and a number of Lugol's solution.In both compositions, the test material is added with a wooden rod and, after covering with glass, is scanned at different resolutions of the microscope.
By certain characteristics, the detected protozoa are recorded.For accuracy 2-3 preparations from one material are prepared.In doubtful cases, the analysis is repeated several times during 2-3 weeks.
The method can detect vegetative and cystic forms:
- dysentery amoeba.
Together with pathogenic forms, non-pathogenic protozoa are also identified.Also, healthy carriers have luminal and cystic forms.
Important: studies should be carried out repeatedly to avoid inaccuracies and errors.
The result of diagnosis of the simplest method of native and colored smear should contain a description of the form of the pathogen( luminal, cyst, tissue).
Requirements for research:
- The material collected for analysis( liquid feces) is examined no later than 30 minutes after defecation;
- the formulated feces should be diagnosed within 2 hours after defecation;
- in the material should not be impurities( disinfectants, water, urine);
- for working with material use only wooden sticks, glass are not suitable due to slipping slime;
- chopsticks after use immediately it is necessary to incinerate.
Conservation method( faecal examination) for the diagnosis of protozoa
The study is carried out by fixing protozoa with a preservative.The difference between this method and the previous one is that the preservatives keep the drug for a long period.
- Barrow.Contains preserving ingredients: 0.7 ml of sodium chloride, 5 ml of formalin, 12.5 ml of 96% alcohol, 2 g of phenol and 100 ml of distilled water.Coloring composition: 0.01% solution of thionine( azura).
- Safarliev's solution.Composition: 1.65 g of zinc sulfate, 10 ml of formalin, 2.5 g of crystalline phenol, 5 ml of acetic acid, 0.2 g of methylene blue, 100 ml of water.This preservative is used in cases where the material should be stored for more than a month.
Preservative fill empty vials, they carry the material in 3: 1 proportions, then add the dye if necessary.Evaluation of the results is performed with 2-3 drugs.
Method of formalin-ether enrichment( analysis for the presence of protozoa in the feces)
This diagnostic method allows you to separate and concentrate the protozoan cysts.For the analysis, the following ingredients are needed: formalin( 10 ml), 0.85 g isotonic solution, distilled water, sulfur ester, Lugol solution.
A mixture of biomaterial with the listed liquids is mixed and centrifuged.The precipitate obtained at the bottom of the tube is stained with Lugol's solution and examined for cysts and vegetative forms.
Method for finding leishmania( bone marrow smear)
For the diagnosis of leishmaniasis, reagents are used: a mixture of Nikiforov( sulfuric ether and ethyl alcohol), phosphate buffer, Azur-eosin by Romanovsky.
The bone marrow substance is very carefully placed on a slide after special preparation.A microscope with an immersion system is used.
In the acute period of the disease, a large number of leishmanias are found in the punctate.
Note: sometimes blood cells may resemble the processed Leishmania, so it is very important for the lab technician to be attentive and have sufficient experience for self-study.
Detection method for leishmania in a smear from a skin infiltrate
The required reagents are similar to the previous analysis.
The test material is obtained from an existing tubercle or ulcerative contents.Soskob with suspicion of leishmaniasis is done very accurately with a scalpel, without blood.Then the preparation is prepared on the glass.For the accuracy of the results obtained, several drugs are simultaneously examined.
In the presence of disease among the macrophages, fibroblasts, and lymphoid cells present in the material under study, leishmanias are also determined.
Method for isolating a pure culture of leishmania obtained by scraping pathological tissues
With this method of diagnosis, the simplest scraping of tissues is placed in a special nutrient medium in which active reproduction of leishmanias takes place.
Before removing the scrap, the skin is carefully treated with alcohol, then the incision of the tubercle is made, 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, in a thermostat at a temperature of 22-24 degrees, cultivation takes place.The results are evaluated under a microscope.This method is used when other, cheaper and faster ways to diagnose protozoa are ineffective.
You can see how in practice the tests for the simplest blood drop are deciphered, you can look at the video review:
Lotin Alexander, medical reviewer