Tuberculin Diagnosis

Tuberculin Diagnostics - a set of diagnostic tests to determine the specific sensitization of the body to mycobacteria tuberculosis using tuberculin-autoclaved culture filtrate mycobacterium tuberculosis. Tuberculin is classified as incomplete antigens - haptens, which are not capable of causing disease or developing immunity to it, but cause a specific response related to delayed-type allergy. At the same time, tuberculin has a high specificity, even in very large dilutions. The emergence of a specific reaction to tuberculin is possible only if the organism is pre-sensitized with mycobacteria as a result of spontaneous infection or BCG vaccination.

By its chemical composition, tuberculin is a complex drug containing Tuberculoproteins, polysaccharides, lipids, nucleic acids, stabilizers and antiseptics. The biological activity of tuberculin, provided by tuberculoprotein, is measured in tuberculin units (TE) and is standardized against the national standard. The national standard, in turn, should be compared with the international standard. In international practice, PPD-S (tuberculin Zeybert or standard-tuberculin) is used.

At present, the following forms of PPD-L (domestic purified tuberculin Linnikova) are produced in the country:

• allergen tubercle purified liquid in standard dilution (purified tuberculin in standard dilution) is ready-to-use tuberculin, used for mass and individual tuberculin diagnostics;
• allergen tubercle cleared dry for cutaneous, subcutaneous and intradermal application (dry purified tuberculin) - a powdered preparation (dissolving in the applied solvent), used for individual tuberculin diagnostics and for tuberculin therapy only in anti-tuberculosis facilities.

The purpose of the Mantoux test

If the human body is previously sensitized to mycobacteria tuberculosis (spontaneous infection or as a result of BCG vaccination), then in response to the introduction of tuberculin, a response-specific reaction arises that is based on the mechanism of HRT. The reaction begins to develop 6-8 hours after the introduction of tuberculin in the form of various manifestations of an inflammatory infiltrate, the cellular basis of which is formed by lymphocytes, monocytes, macrophages, epithelioid and giant cells. The triggering mechanism of HRT is the interaction of the antigen (tuberculin) with the receptors on the surface of the effector lymphocytes, resulting in the release of mediators of cellular immunity that involve macrophages in the process of antigen destruction. Some cells die, secreting proteolytic enzymes, which have a damaging effect on the tissue. Other cells accumulate around lesions. The time of development and the morphology of the reactions in any method of application of tuberculin do not differ fundamentally from those in intradermal administration. The peak of the reaction of GZT is 48-72 h, when its non-specific component is minimal, and the specific reaches a maximum.

Tubercular diagnostics is divided into mass and individual.

The purpose of mass tuberculin diagnostics is a population survey on tuberculosis. Tasks of mass tuberculin diagnostics:

• identification of tuberculosis patients of children and adolescents;
• identification of persons. Who are at risk of tuberculosis for follow-up at a phthisiatrician (people newly infected with mycobacteria tuberculosis with a "bend" of tuberculin samples, with an increase in tuberculin samples, with hyperergic tuberculin samples, with tuberculin samples that have been at a moderate and high level for a long time) necessity - for preventive treatment;
• selection of children and adolescents for booster revaccination;
• definition of epidemiological indicators for tuberculosis (population infection, annual risk of infection).

For mass tuberculin diagnostics, only Mantoux with 2 TE is used. Using only purified tuberculin in standard dilution.

In order to select children and adolescents for the revaccination of BCG, the Mantoux test with 2 TE. According to the calendar of preventive vaccinations, are performed in the decreed age groups of 7 years (zero and first grades of secondary school) and at 14 years (eighth and ninth classes). Revaccination is performed uninfected previously, clinically healthy individuals with a negative reaction to the Mantoux test.

Individual tuberculin diagnostics are used for individual examinations. Objectives of individual tuberculin diagnostics:

• differential diagnosis of postvaccinal and infectious allergies (HRT);
• Diagnosis and differential diagnosis of tuberculosis and other diseases;
• the definition of the "threshold" of individual sensitivity to tuberculin;
• determination of tuberculosis activity;
• evaluation of treatment effectiveness.

When carrying out individual tuberculin diagnostics, use various tuberculin tests with dermal, intradermal, subcutaneous injection of tuberculin. For various tuberculin samples, both purified tuberculin in standard dilution (allergen tubercle purified in standard dilution) and dry purified tuberculin (allergen tubercle purified dry) are used. Purified tuberculin in standard dilution can be used in anti-tuberculosis facilities, children's polyclinics, somatic and infectious hospitals. Dry purified tuberculin is allowed for use only in anti-tuberculosis facilities (tuberculosis dispensary, tuberculosis hospital and sanatorium).

Technique of research and evaluation of results

Drugs of tuberculin PPD-L are injected into the human body skin, intradermally and subcutaneously. The route of administration depends on the type of tuberculin test.

Graduated skin test of Grinchar and Karpilovsky

GKP is a cutaneous tuberculin test with 100%, 25%, 5% and 1% solutions of tuberculin. To obtain a 100% solution of tuberculin, 2 ampules of dry purified tuberculin PPD-L in 1 ml of solvent are consistently diluted, subsequent solutions of tuberculin are prepared from the resulting 100% solution. To obtain a 25% solution from an ampoule with a 100% solution with a sterile syringe, 1 ml is collected and poured into a sterile dry vial. Another 3 ml of solvent is added by another sterile syringe, the bottle is shaken thoroughly, 4 ml of a 25% solution of tuberculin is prepared. To get a 5% solution of tuberculin from a vial of a 25% solution with a sterile syringe, 1 ml is taken and transferred to another sterile dry vial, then 4 ml of solvent is added, shaken and 5 ml of a 5% solution of tuberculin, etc.

On a dry skin of the inner surface of the forearm, previously treated with 70% ethanol, sterile pipettes are applied by drop of tuberculin of varying concentrations (100%, 25%, 5%, 1%) so that the tuberculin concentration decreases from the ulnar fold in the distal direction. Below a drop of 1% solution of tuberculin is applied a drop of solvent without tuberculin as a control. For each solution of tuberculin and for monitoring, separate labeled pipettes are used. The skin of the forearm is stretched from below with the left hand, then the ospothrivial pen breaks the integrity of the surface layers of the skin in the form of a scratch of 5 mm length, conducted through each drop in the direction of the longitudinal axis of the arm. Scarification is performed first through a drop of the solvent, then sequentially through 1%, 5%, 25% and 100% solutions of tuberculin by rubbing the tuberculin 2-3 times with the flat side of the feather after each scarification to penetrate the drug into the skin. The forearm is left open for 5 minutes to dry. For each examinee a separate sterile pen is used. On the site of scarification appears a white cushion, indicating sufficient time for absorption of tuberculin. After that, the remains of tuberculin are removed with sterile cotton wool.

Evaluate the HCUC for NA. Shmelev in 48 hours. There are the following reactions to HCV:

• anergic reaction - no response to all solutions of tuberculin;
• nonspecific reaction - slight reddening at the place of application of 100% solution of tuberculin (very rare);
• normal reaction - moderate sensitivity to large concentrations of tuberculin, lack of response to 1% and 5% solutions of tuberculin:
• giperergicheskaya reaction - responses to all concentrations of tuberculin infiltrates increase with increasing concentration of tuberculin, possible vesiculo-necrotic changes, lymphangitis, screenings;
• equalization reaction - approximately the same size of infiltrate for all concentrations of tuberculin, large concentrations of tuberculin do not elicit an adequate response;
• a paradoxical reaction is a smaller intensity of the reaction to higher concentrations of tuberculin, more intense reactions to small concentrations of tuberculin.

Equalizing and paradoxical reactions are also called inadequate reactions to HCV. Sometimes inadequate reactions to HCV are attributed to hyperergic reactions.

GKP has a differential diagnostic value when clarifying the nature of tuberculin allergy. Postvaccinal HRT is characterized by normal adequate responses, whereas in the case of IA, the response to HCV may be hyperergic, equalizing, or paradoxical. In the early period of primary infection ("turn"), taking place with functional changes, paradoxical, egalitarian reactions are observed.

At practically healthy children, favorably transferred a primary tubercular infection. GKP is also normal.

The SCP has a great importance for the differential diagnosis of tuberculosis and other diseases, for determining the activity of the tuberculosis process. In patients with active tuberculosis, hyperergic, equalizing and paradoxical reactions are more common. Heavy course of tuberculosis can be accompanied by energetic reactions.

The decrease in susceptibility to tuberculin according to the HCF (the transition from hyperergic reactions to normal, from inadequate to adequate, from energetic to positive normal) in tuberculosis patients against antibacterial treatment testifies to the normalization of the organism's reactivity and the effectiveness of therapy.

Intradermal test with various dilutions of tuberculin

The initial solution of tuberculin is prepared by mixing an ampoule of dry purified tuberculin PPD-L (50 thousand TE) with a solvent ampoule, the main dilution of tuberculin is 50 thousand TE in 1 ml. The drug should be dissolved for 1 minute, until a clear and colorless solution. The first dilution of tuberculin is prepared by adding 4 ml of solvent to the ampoule with the main dilution (1000 TE are obtained in 0.1 ml of the solution). The second dilution of tuberculin is prepared by adding to the 1 ml of the 1st dilution 9 ml of the solvent (100 TE are obtained in 0.1 ml solution). All subsequent dilutions of tuberculin (before the 8th) are prepared in a similar way. Thus, dilutions of tuberculin correspond to the following doses of tuberculin in 0.1 ml of solution: 1 st dilution - 1000 TE, 2 - 100 TE, 3 - 10 TE, 4 - 1 TE. 5 th - 0,1 TE, 6 th - 0,01 TE. 7th - 0.001 TE. 8th - 0.0001 TE.

The Mantoux test with different dilutions of tuberculin is carried out in the same way. As a production with 2 TE. For each dilution using a separate syringe and needle. One test is performed on a single forearm with two dilutions of tuberculin at a distance of 6-7 cm from each other. At the same time, you can place the third test with another dilution of tuberculin on the other forearm. Evaluate the sample after 72 h:

• negative reaction - absence of papula and hyperemia, presence of only a knock-off reaction (0-1 mm);
• doubtful reaction - a papule less than 5 mm or hyperemia of any size;
• positive reaction - papule 5 mm or more.

Titration (determination of the threshold of sensitivity to tuberculin) is completed when a positive reaction to the smallest dilution of tuberculin is achieved. Positive reactions to high dilutions of tuberculin with doses of 0.1 TB. 0.01 TE, etc. Indicate a high degree of sensitization of the body and usually accompany active tuberculosis. Negative reaction to 100 TE in the overwhelming majority of patients with a probability of 97-98% allows to reject the diagnosis of tuberculosis or to exclude the infectious nature of allergy.

The overwhelming majority of patients and infected persons with the presentation of cutaneous and intradermal tuberculin samples reveal only a local reaction to tuberculin. In isolated cases, common reactions are noted for the Mantoux test with 2 TE. Such patients are subject to a thorough clinical and radiological examination. Even more rarely observed focal reactions.

Subcutaneous tuberculin test of Koch

The subcutaneous tuberculin test of Koch is a subcutaneous injection of tuberculin.

In children's practice, Koch's test is usually started with 20 TE. For this, 1 ml of purified tuberculin in standard dilution or 0.2 ml of 3rd dilution of dry purified tuberculin is injected subcutaneously without taking into account a preliminary study of the threshold for sensitivity to tuberculin.

A number of authors first dose of 20 TE for Koch test is recommended for the normal character of Mantoux sample with 2 TE and negative or weakly positive reaction for 100% solution of tuberculin in HCV. With a negative reaction to the Koch test with 20 TE, the dose is increased to 50 TE. And then to 100 TE. In children with hyperergic reactions to the Mantoux test with 2 TE, the Koch test begins with the introduction of 10 TE.

In response to Koch's test, local, general and focal reactions develop.

• Local reaction occurs at the site of injection of tuberculin. The reaction is regarded as positive with a size of infiltrate of 15-20 mm. Without a common and focal reaction, it is of little informative.
• Focal reaction - changes after the introduction of tuberculin in the focus of tuberculosis lesion. Along with clinical and roentgenological signs, it is advisable to study sputum, bronchial flushing waters before and after the introduction of tuberculin. Positive focal reaction (the increase in clinical symptoms, the increase in perifocal inflammation during X-ray examination, the appearance of bacterial excretion) is important both for the differential diagnosis of tuberculosis with other diseases, and for determining the activity of the tuberculosis process.
• The general reaction is manifested in the deterioration of the body as a whole (body temperature, cellular and biochemical blood composition).
  • The temperature response is considered positive if there is an increase in body temperature by 0.5 ° C compared to the maximum before subcutaneous administration of tuberculin (it is advisable to conduct the thermometry 3 hours 6 times a day for 7 days - 2 days before the sample and 5 days against the background of the sample ). In the vast majority of patients, an increase in body temperature is observed on the 2nd day, although a later increase on the 4th-5th day is possible.
  • After 30 minutes or 1 hour after subcutaneous administration of tuberculin, the absolute number of eosinophils decreased (FA Mikhailov's test). After 24-48 h, ESR increases by 5 mm / h, the number of stab neutrophils by 6% or more, the lymphocyte count decreases by 10% and platelets by 20% or more (Bobrov's test).
  • After 24-48 h after subcutaneous administration of tuberculin, the albumin-globulin coefficient decreases due to a decrease in albumin content and an increase in α ₁, α ₂ and γ globulins (Rabukhin-Ioffe protein-tuberculin test). This test is considered positive when the indicators change not less than 10% of the initial level.

Alternative methods

In addition to tuberculins used in vivo, in vitro preparations have been developed for which tuberculins or various antigens of mycobacteria are used.

For the detection of antibodies to mycobacteria of tuberculosis, a diagnosticum is issued erythrocyte tubercular antigenic dry - rams' erythrocytes, sensitized with a phosphatide antigen. Diagnostics is designed to conduct indirect hemagglutination (RNGA) reaction in order to detect specific antibodies to the mycobacterium antigens of tuberculosis. This immunological test is used to determine the activity of the tuberculosis process and control of treatment. To determine antibodies to mycobacteria tuberculosis in the blood serum of patients, an immune-enzyme test system is also designed - a set of ingredients for carrying out ELISA. Used for laboratory confirmation of the diagnosis of tuberculosis of various localization, evaluation of the effectiveness of treatment, and the decision to assign specific immunocorrection. The sensitivity of ELISA for tuberculosis is low, it is 50-70%, the specificity is less than 90%, which limits its use and does not allow the use of a test system for screening tuberculosis infection.

PCR-test systems are used to detect mycobacteria.

[1], [2], [3], [4], [5], [6], [7],

Contraindications to Mantoux test

Contraindications to Mantoux test with 2 TE:

• skin diseases, acute and chronic infectious and somatic diseases (including epilepsy) during the period of exacerbation;
• allergic conditions, rheumatism in acute and subacute phases, bronchial asthma, idiosyncrasy with severe cutaneous manifestations during exacerbation;
• quarantine for childhood infections in children's groups;
• interval less than 1 month after other preventive vaccinations (DTP vaccination against measles, etc.).

In these cases, the Mantoux test is carried out 1 month after the disappearance of clinical symptoms or immediately after quarantining.

There are no absolute contraindications to conducting dermal and intradermal tests with tuberculin. It is not recommended their setting during the period of exacerbation of chronic allergic diseases, with exfoliative dermatitis, pustular skin diseases, during acute respiratory infections.

Subcutaneous administration of tuberculin is undesirable in patients with active rheumatic process, especially with heart damage, with exacerbation of chronic diseases of the digestive system.

[8], [9], [10], [11], [12]

Factors affecting the result of the Mantoux test

The intensity of the tuberculin reaction depends on many factors. In children, the sensitivity to tuberculin is higher than that of adults. In severe forms of tuberculosis ( meningitis, miliary tuberculosis, caseous pneumonia ) often indicate a low sensitivity to tuberculin due to a pronounced inhibition of the reactivity of the body. Some forms of tuberculosis ( tuberculosis of eyes, skin), on the contrary, are often accompanied by high sensitivity to tuberculin.

The intensity of the reaction to 2 TE depends on the frequency and multiplicity of revaccinations against tuberculosis. Each subsequent revaccination leads to an increase in sensitivity to tuberculin. In turn, a decrease in the frequency of revaccinations of BCG leads to a decrease in the number of positive results for the Mantoux test in 2 times, hyperergic - in 7 times. Thus, revocation of revaccinations helps to reveal the true level of infection of children and adolescents with mycobacteria of tuberculosis, which, in turn, allows for full coverage of BCG with revaccination of adolescents at the required time.

The dependence of the intensity of the Mantoux reaction on the magnitude of the postvaccinal sign of BCG was revealed. The more postvaccinal scar, the higher the sensitivity to tuberculin.

In helminthic invasions, hyperthyroidism, acute respiratory diseases, viral hepatitis, chronic foci of infection, sensitivity to tuberculin is increased. In addition, up to 6 years, IA (HRT) is more pronounced in older children.

Increased susceptibility to tuberculin is observed when Mantoux test is administered within 1 day to 10 months after vaccinations against childhood infections (DTP, DTP-M, ADS-M, measles, parotoxin vaccine). Previously, negative reactions become questionable and positive, and after 1-2 years they again become negative. Therefore, the tuberculosis diagnosis is planned either before the prophylactic vaccination against childhood infections, or not earlier than 1 month after vaccination.

Less pronounced reactions to tuberculin are recorded in the summer. The intensity of tuberculin reactions decreases with feverish conditions, oncological diseases, viral childhood infections, during menstruation, in the treatment of glucocorticoid hormones, antihistamines.

Evaluation of tuberculin test results may be difficult in areas with a significant spread of weak sensitivity to tuberculin caused by atypical mycobacteria. Differences in the antigenic structure of various types of mycobacteria cause a different degree of severity of skin reactions when using different antigens. When carrying out a differentiated test with different types of tuberculin, the most pronounced reaction is caused by tuberculin, prepared from the type of mycobacteria that the organism is infected with. Such drugs are usually called sensitins.

Negative reaction to tuberculin is called tuberculin anergy. Possible primary energy - the lack of response to tuberculin in uninfected individuals, and secondary energy, developing in infected individuals. Secondary energy, in turn, can be positive (as a variant of biological cure for tuberculosis infection or a state of immunoanergy, observed, for example, in the case of "latent microbism") and negative (in severe forms of tuberculosis). Secondary energy also occurs in lymphogranulomatosis, sarcoidosis, many acute infectious diseases (measles, rubella, mononucleosis, whooping cough, scarlet fever, typhoid, etc.), with avitaminosis, cachexia, neoplasms.

Children and adolescents with hyperergic sensitivity to tuberculin following the results of mass tuberculin diagnostics are the group of the most endangered tuberculosis and require the most thorough examination from a phthisiatrician. The presence of hyperergic sensitivity to tuberculin is most often associated with the development of local forms of tuberculosis. With tuberculin hypertension, the risk of tuberculosis is 8-10 times higher than with normal reactions. Particular attention should be paid to children infected with mycobacteria of tuberculosis, in the presence of hyperergic reactions and contact with tuberculosis patients.

In each individual case, it is necessary to study all the factors that affect the sensitivity to tuberculin, which is of great importance for diagnosing, choosing the right medical tactics, the method of patient management and its treatment.

[13], [14], [15], [16], [17], [18]