The patient’s sputum is a mixture of the secretion of the glands of the mucous membrane of the trachea and bronchi, serous effusion from pathologically altered vessels, sometimes particles of caseous masses, necrotic granulation and lung tissue, as well as salt impurities. Sputum is secreted in many diseases of the lungs and bronchi.
In tuberculosis institutions, patients collect sputum in individual containers, a quarter of the container is filled with a solution of chloramine, where it is partially disinfected. The containers are graduated, and this makes it possible to determine the daily amount of sputum.
- Bacterioscopic method
- Flotation method
- Fluorescent microscopy
- Phase contrast microscopy
- Investigation of flushing from the bronchi
- Examination of urine, feces, cerebrospinal and pleural fluid for MBT
- Biological method
- Accelerated methods for the search for mycobacteria
- Molecular genetic methods
Macroscopically, in patients with pulmonary tuberculosis, sputum has a mucous or mucopurulent character, odorless, homogeneous, does not form layers. The amount of sputum may vary. At the onset of pulmonary tuberculosis, sputum is not excreted, then it can be excreted in the morning by separate spitting, later its amount can reach 100-200 ml.
After macroscopic examination, bacterioscopic, bacteriological and biological examination of sputum is carried out.
Bacterioscopic method
The bacterioscopic method covers direct bacterioscopy of smears from pathological material stained according to Ziehl-Neelsen, bacterioscopy by flotation, luminescent microscopy, phase contrast microscopy.
Bacterioscopic examination of sputum begins with the preparation of a smear. To do this, sputum is mixed with a solution of sodium hydroxide and centrifuged to obtain a sediment. The precipitate is transferred to a glass slide with glass rods. The second glass slide is evenly placed: covered with a cover glass. The drug is examined under a microscope. In it you can see leukocytes, elastic fibers, and with special staining – atypical cells and various bacteria.
Mycobacteria stain positive for Gram. To detect MBT, a sputum smear is prepared, dried, fixed over the flame of an alcohol lamp, and then stained according to Ziehl-Neelsen. Coloring is done first with a carbolic solution of fuchsin. Mycobacterium tuberculosis does not perceive color well, therefore, a solution of fuchsin is applied to the preparation in large quantities, heated over the flame of an alcohol lamp until steam appears. Then the paint is drained, the preparation is washed in water and discolored in a 5% sulfuric acid solution or in a mixture of ethyl alcohol and a hydrochloric acid solution. At the same time, all bacteria and morphological elements of sputum, except for Mycobacterium tuberculosis, are colorless. The smear is washed with running water, and then methylene blue is applied for 1-2 minutes. After that, the paint solution is drained, the smear is washed and dried. The preparation is studied through an immersion microscope system, for which a drop of immersion oil is applied to the preparation in order to create the same environment between the objective lens and the preparation. Mycobacterium tuberculosis under the microscope can be seen stained red.
The Ziehl-Nielsen staining method makes it possible to identify Mycobacterium tuberculosis in cases where 1 cm3 of sputum contains about 5,000-10,000 MBT, provided that 300 fields of view are viewed. With a small amount of MBT in the sputum, the bacterioscopic method is ineffective.
Sputum examination should be carried out for three consecutive days, and if the result is negative, the flotation method should be used.
Flotation method
It is based on the fact that when shaking two liquids with different specific gravity, it is easier for the liquid to float to the surface simultaneously with Mycobacterium tuberculosis in suspension. The essence of the technique is that an aqueous suspension is prepared with hydrocarbons (xylene, benzene) and MBT. A creamy foam with MBT will flow to the surface of the water, which is sucked off with a pipette and applied to a glass slide. The foam layer on the glass slide is dried and a new foam layer is applied from the flask. So foam is layered 5-6 times, after which the smear is fixed and stained according to Ziehl-Neelsen.
Fluorescent microscopy
Fluorescent microscopy is an effective method for detecting Mycobacterium tuberculosis. The essence of the method lies in the ability of Mycobacterium tuberculosis, stained with special dyes (auramine, rhodamine), to glow when irradiated with ultraviolet rays. The advantage of the luminescent microscopy method is the possibility of conducting research at lower magnifications of the microscope, which provides viewing in a short time of a larger number of fields of view than with conventional microscopy. The possibility of detecting mycobacterium tuberculosis increases by 10-15% compared to conventional bacterioscopy and by 8% compared to the flotation method.
Use a fluorescent microscope at low magnification. On a dark background, golden yellow Mycobacterium tuberculosis is visible.
Phase contrast microscopy
This is the only microscopic research method that allows you to observe mycobacteria and their biologically modified forms in a living state. To conduct such a study, a special phase-contrast device is used.
Cytological examination of sputum in patients with pulmonary tuberculosis reveals a small number of neutrophils in the stage of significant degeneration against the background of caseous detritus, accumulation of mononuclear cells, Pirogov-Langhans giant cells, and sometimes eosinophils.
In cases where there is no sputum or it is excreted in a very small amount, the patient is prescribed an expectorant mixture or irritating inhalations. In addition, in cases where there is no sputum, bronchial lavage is examined.
Investigation of flushing from the bronchi
It is performed by a doctor. To do this, the patient on an empty stomach is lubricated with the root of the tongue, the back wall of the pharynx and the epiglottis with a 1% solution of dicaine. Then, with a laryngeal syringe, 5-10 ml of isotonic sodium chloride solution is poured into the trachea. The patient immediately develops a strong cough and an isotonic solution of sodium chloride is introduced and coughed up on a Petri dish. Also, bronchial lavage (BAS – bronchoalveolar lavage) is obtained using BAL – broncho-alveolar lavage, which is performed during bronchoscopy. BAZ is examined by the flotation method or by the seeding method. The study of flushing from the bronchi allows additional detection of MBT in 5-10% of cases.
Examination of urine, feces, cerebrospinal and pleural fluid for MBT
In clinical practice, MBT often has to be manifested in urine, feces, cerebrospinal and pleural fluid. A urine test for MBT is carried out in cases where, during the study of the sediment, at least 15 leukocytes are found in each field of view.
To detect MBT, urine is repeatedly centrifuged, each time layering new portions from the urine sediment onto a glass slide. The smear is stained according to the Diehl-Neelsen method. The detection of Mycobacterium tuberculosis in the urine indicates the presence of kidney tuberculosis.
To study urine according to the flotation method, let it stand, then 20-30 ml of sediment is processed, as usual, without heating in a water bath. The absence of MBT in purulent urine and this does not exclude the presence of kidney tuberculosis. In such cases, it is necessary either to inoculate it on nutrient media, or to apply the biological method of research.
A study of feces for MBT should be carried out in cases of suspected intestinal tuberculosis. At the same time, it must be remembered that in patients who secrete MBT and partially swallow sputum, MBT can be detected by examining feces and unaffected intestines, since Mycobacterium tuberculosis is resistant and does not always die under the influence of digestive juices. In the feces of patients with tuberculosis, the intestines often contain mucus, blood, and impurities of pus.
The detection of MBT in the cerebrospinal fluid is often of decisive importance in the diagnosis of tuberculous meningitis. However, the bacterioscopic method can detect MBT only in 10-20% of patients with tuberculous meningitis. The study is carried out according to the following methodology. A test tube with cerebrospinal fluid is placed for 8-10 hours in a cool place. If a delicate fibrin film is formed in it (it consists of cells and MBT), it is applied to a glass slide and a preparation is prepared according to the Ziehl-Neelsen method. In cases where the film is not formed, the drug is prepared from the sediment after centrifugation.
To detect MBT in pleural exudate, punctures and discharge from fistulas, a preparation is prepared in the same way as in the study of sputum.
Bacteriological examination consists of inoculation of the material on nutrient media and differentiation of the MBT culture from acid-resistant saprophytes. This method can detect Mycobacterium tuberculosis, when 1 ml of the material contains 20-100 microbial cells. Test tubes with the medium are placed in a thermostat at a temperature of 37 ° C. The first colonies of mycobacteria may appear on the 18-30th day, and sometimes after 2-3 months. Bacteriological examination is carried out simultaneously with bacterioscopic three times.
The inoculation of the test material is carried out on special media after pre-treatment. The most commonly used media are: Levenshtein-Jensen, Finn-2, Middlebrook, Ogawa, etc., which contain substances that inhibit the growth of foreign microorganisms. Preliminary processing of pathological material consists in the destruction of the accompanying bacterial flora. Sowing material from the bronchi, as well as sowing urine, is done from a centrifugate or sediment. At the same time, urine, if it is not contaminated, can not be processed.
Pretreatment of biological material
Several pretreatment methods exist to prepare for culture of sputum, pleural exudate, and cerebrospinal fluid.
The Mazur method consists in processing the pathological material with 3-4 ml of a 2% sulfuric acid solution in a sterile test tube. The tube is shaken for 3 minutes, after which the material is applied to the egg media with a sterile platinum loop.
Petrov’s method: sputum is treated with 4% sodium hydroxide solution for 15 minutes and then centrifuged.
Biological method
This is the inoculation of pathological material into animals. Guinea pigs or white mice are injected with the test material at a dose of 2-3 ml into the inguinal region or into the abdominal cavity. To do this, it is first treated with a 3-8% solution of hydrochloric acid, washed well (otherwise tissue necrosis may occur at the injection site, even the death of the animal from acid exposure is possible).
Uncontaminated urine and CSF can be inoculated without pretreatment. In the presence of virulent MBT strains in the pathological material, the animal becomes ill with tuberculosis and dies 1-2 months after infection. If the MBT masses have weakened virulence, the animal dies later or may not die at all. In such cases, the animal is slaughtered, and the diagnosis is established after an autopsy, macro- and microscopic detection of tuberculous tubercles. The diagnosis can be made on the basis of intravital examination of an enlarged regional lymph node or infiltrate that has arisen at the site of inoculation of pathological material.
In recent years, for the differentiation of mycobacteria, the gas-liquid chromatography method has been used, based on the high ability of various compounds (amino acids, nucleic acids, fats, carbohydrates, etc.). This does not exclude the stage of cultivation of the pathogen on nutrient media, which can be attributed to the disadvantages of the method.
Accelerated methods for the search for mycobacteria
Accelerated methods for detecting mycobacteria include detecting MBT using an indicator tube BBL MOIT. The growth of Mycobacterium tuberculosis occurs within 4-10 days. BBL MOIT tubes contain broth and a fluorescent compound that reacts to oxygen dissolved in the broth. The initial concentration of oxygen does not give a bright glow. When oxygen is absorbed by the MBT culture, bright fluorescence is observed at the bottom of the tube. This result is considered positive. If there is no fluorescence or it is insignificant, the result is negative. The readings of the test tubes are taken into account daily, starting from the second day of incubation.
Recently, ELISA and molecular genetic methods have been used to diagnose tuberculosis.
Molecular genetic methods
Methods for identifying MBT using monoclonal antibodies obtained by hybridoma technology have appeared. Now there are more than 100 monoclonal antibodies to the epitopes of the main MBT antigens. The use of these antibodies in enzyme immunoassay makes it possible to very quickly differentiate the type of pathogen, but so far the method is used in scientific development to identify the causative agent of tuberculosis, which is most common.
Among the molecular genetic methods for diagnosing tuberculosis, the method of DNA probes and polymerase chain reaction (PCR) is most often used. These methods are based on the principle of complementarity of nucleotide bases in the construction of a double-stranded DNA molecule. During DNA probing, if the sample contains a specific region of mycobacteria DNA under study, a hybrid (double-stranded fragment) of the DNA of the test and the DNA probe is formed.
The basis of the PCR principle is the multiple increase in a DNA fragment. Amplification (multiple doubling of the bacterial gene) requires the presence of two primers (seeds) – a small single-stranded DNA fragment that connects to the complementary region of one of the template DNA chains. Subsequently, this chain is completed by DNA polymerase. An increase in the amount (by 106 times) of the amplified fragment turns out to be electrophoresis in agar gel. Accounting for the results of reactions is carried out under the plates of the transilluminator in ultraviolet light,
DNA probing allows for the determination of mycobacteria in the diagnostic material within 2-4 days, and PCR – for 4-6 hours with the highest sensitivity of the methods – 10-100-1000 cells in the test sample.