Tuberculin diagnostics

Tuberculin diagnostics is a set of diagnostic tests for determining specific sensitization of the body to Mycobacterium tuberculosis using tuberculin - an autoclaved filtrate of Mycobacterium tuberculosis cultures. Tuberculin is classified as an incomplete antigen - hapten, which is not capable of causing disease or the development of immunity to it, but causes a specific response related to delayed-type allergy. At the same time, tuberculin has high specificity, acting even in very large dilutions. The occurrence of a specific reaction to tuberculin is possible only under the condition of preliminary sensitization of the body to Mycobacterium as a result of spontaneous infection or BCG vaccination.

In its chemical composition, tuberculin is a complex preparation containing tuberculoproteins, polysaccharides, lipids, nucleic acids, stabilizers and antiseptics. The biological activity of tuberculin, provided by the tuberculoprotein, is measured in tuberculin units (TU) and standardized relative to the national standard. The national standard, in turn, must be compared with the international standard. In international practice, PPD-S (tuberculin Seibert or standard tuberculin) is used.

Currently, the following forms of PPD-L (Linnikova's domestic purified tuberculin) are produced in the country:

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

The purpose of the Mantoux test

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

Tuberculin diagnostics are divided into mass and individual.

The purpose of mass tuberculin diagnostics is to screen the population for tuberculosis. The tasks of mass tuberculin diagnostics:

• identification of children and adolescents with tuberculosis;
• identification of individuals at risk of developing tuberculosis for subsequent observation by a phthisiatrician (individuals infected with tuberculosis mycobacteria for the first time with a "turn" in tuberculin tests, with an increase in tuberculin tests, with hyperergic tuberculin tests, with tuberculin tests that have been at a moderate and high level for a long time), if necessary - for preventive treatment;
• selection of children and adolescents for BCG revaccination;
• determination of epidemiological indicators for tuberculosis (infection rate of the population, annual risk of infection).

For mass tuberculin diagnostics, only the Mantoux test with 2 TE is used, using only purified tuberculin in a standard dilution.

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

Individual tuberculin diagnostics are used to conduct individual examinations. The goals of individual tuberculin diagnostics are:

• differential diagnosis of post-vaccination and infectious allergy (PVA);
• diagnostics and differential diagnostics of tuberculosis and other diseases;
• determination of the “threshold” of individual sensitivity to tuberculin;
• determination of the activity of the tuberculosis process;
• evaluation of treatment effectiveness.

When conducting individual tuberculin diagnostics, various tuberculin tests are used with cutaneous, intradermal, subcutaneous administration of tuberculin. For various tuberculin tests, both purified tuberculin in standard dilution (purified tuberculosis allergen in standard dilution) and dry purified tuberculin (purified dry tuberculosis allergen) are used. Purified tuberculin in standard dilution can be used in anti-tuberculosis institutions, children's clinics, somatic and infectious disease hospitals. Dry purified tuberculin is approved for use only in anti-tuberculosis institutions (anti-tuberculosis dispensary, tuberculosis hospital and sanatorium).

Research technique and evaluation of results

Tuberculin PPD-L preparations are administered to the human body cutaneously, intradermally and subcutaneously. The route of administration depends on the type of tuberculin test.

Graduated skin test of Grinchar and Karpilovsky

The GKP is a skin tuberculin test with 100%, 25%, 5% and 1% tuberculin solutions. To obtain a 100% tuberculin solution, 2 ampoules of dry purified tuberculin PPD-L are successively diluted in 1 ml of solvent, and subsequent tuberculin solutions are prepared from the resulting 100% solution. To obtain a 25% solution, 1 ml is drawn from an ampoule with a 100% solution using a sterile syringe and poured into a sterile dry vial. 3 ml of solvent is added using another sterile syringe, the vial is thoroughly shaken, and 4 ml of a 25% tuberculin solution is obtained. To obtain a 5% tuberculin solution, 1 ml is drawn from a vial with a 25% solution using a sterile syringe and transferred to another sterile dry vial, then 4 ml of solvent is added, shaken and 5 ml of a 5% tuberculin solution is obtained, etc.

On dry skin of the inner surface of the forearm, pre-treated with 70% ethyl alcohol solution, drop by drop of tuberculin of different concentrations (100%, 25%, 5%, 1%) are applied with sterile pipettes so that the concentration of tuberculin decreases from the elbow crease in the distal direction. Below the drop with 1% tuberculin solution, a drop of solvent without tuberculin is applied as a control. Separate marked pipettes are used for each tuberculin solution and for the control. The skin of the forearm is stretched from below with the left hand, then the integrity of the superficial layers of the skin is violated with a smallpox pen in the form of a scratch 5 mm long, drawn through each drop in the direction of the longitudinal axis of the arm. Scarification is performed first through a drop of solvent, then successively through 1%, 5%, 25% and 100% tuberculin solutions, rubbing the tuberculin 2-3 times with the flat side of the pen after each scarification to allow the preparation to penetrate the skin. The forearm is left open for 5 minutes to dry. A separate sterile pen is used for each subject. A white ridge appears at the scarification site, indicating that there is sufficient time for the tuberculin to be absorbed. After this, the remaining tuberculin is removed with sterile cotton wool.

The GCP is assessed according to N.A. Shmelev after 48 hours. The following reactions to GCP are distinguished:

• anergic reaction - lack of response to all tuberculin solutions;
• non-specific reaction - slight redness at the site of application of 100% tuberculin solution (extremely rare);
• normergic reaction - moderate sensitivity to high concentrations of tuberculin, no reaction to 1% and 5% tuberculin solutions:
• hyperergic reaction - responses to all concentrations of tuberculin, the size of infiltrates increases as the concentration of tuberculin increases, vesicular-necrotic changes, lymphangitis, and screenings are possible;
• equalizing reaction - approximately the same size of infiltrate for all concentrations of tuberculin, high concentrations of tuberculin do not cause an adequate response;
• paradoxical reaction - less intense reaction to high concentrations of tuberculin, more intense reactions to low concentrations of tuberculin.

Equalizing and paradoxical reactions are also called inadequate reactions to GKP. Sometimes inadequate reactions to GKP are referred to as hyperergic reactions.

GKP has differential diagnostic value in determining the nature of tuberculin allergy. Post-vaccination GRT is characterized by normergic adequate reactions, whereas in IA the reaction to GKP may be hyperergic, equalizing or paradoxical. In the early period of primary infection ("turn"), occurring with functional changes, paradoxical, equalizing reactions are observed.

In practically healthy children who have successfully survived the primary tuberculosis infection, the GKP can also be normergic.

The GKP is of 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. Severe tuberculosis may be accompanied by energetic reactions.

A decrease in sensitivity to tuberculin according to the GKP data (transition from hyperergic reactions to normergic, from inadequate to adequate, from energetic to positive normergic) in patients with tuberculosis against the background of antibacterial treatment indicates the normalization of the body's reactivity and the effectiveness of therapy.

Intradermal test with different dilutions of tuberculin

The initial tuberculin solution is prepared by mixing an ampoule of dry purified tuberculin PPD-L (50 thousand TU) with an ampoule of solvent; the basic dilution of tuberculin is obtained - 50 thousand TU in 1 ml. The preparation should be dissolved for 1 min, until the solution is clear and colorless. The first dilution of tuberculin is prepared by adding 4 ml of solvent to the ampoule with the basic dilution (1000 TU in 0.1 ml of solution is obtained). The second dilution of tuberculin is prepared by adding 9 ml of solvent to 1 ml of the 1st dilution (100 TU in 0.1 ml of solution is obtained). All subsequent dilutions of tuberculin (up to the 8th) are prepared in a similar way. Thus, the dilutions of tuberculin correspond to the following doses of tuberculin in 0.1 ml of solution: 1st dilution - 1000 TE, 2nd - 100 TE, 3rd - 10 TE, 4th - 1 TE. 5th - 0.1 TE, 6th - 0.01 TE. 7th - 0.001 TE. 8th - 0.0001 TE.

The Mantoux test with different dilutions of tuberculin is performed in the same way as the test with 2 TE. For each dilution, use a separate syringe and needle. On one forearm, a test is performed with two dilutions of tuberculin at a distance of 6-7 cm from each other. At the same time, a third test with another dilution of tuberculin can be performed on the other forearm. The test is evaluated after 72 hours:

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

Titration (determination of the sensitivity threshold to tuberculin) is completed upon achieving a positive reaction to the smallest dilution of tuberculin. 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. A negative reaction to 100 TE in the vast majority of patients with a probability of 97-98% allows us to reject the diagnosis of tuberculosis or exclude the infectious nature of the allergy.

In the vast majority of patients and infected individuals, only a local reaction to tuberculin is detected when performing skin and intradermal tuberculin tests. In isolated cases, general reactions are noted to the Mantoux test with 2 TE. Such patients are subject to a thorough clinical and radiological examination. Focal reactions are observed even more rarely.

Koch's subcutaneous tuberculin test

Koch's subcutaneous tuberculin test is a subcutaneous injection of tuberculin.

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

A number of authors recommend the first dose of 20 TE for the Koch test in the case of a normergic Mantoux test with 2 TE and a negative or weakly positive reaction to 100% tuberculin solution in the GKP. In the case of 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.

• A local reaction occurs at the site of tuberculin injection. The reaction is considered positive when the infiltrate size is 15-20 mm. Without a general and focal reaction, it is of little information.
• Focal reaction - changes after the introduction of tuberculin in the focus of tuberculosis lesion. Along with clinical and radiological signs, it is advisable to examine sputum, bronchial washings before and after the introduction of tuberculin. A positive focal reaction (increase in clinical symptoms, increased perifocal inflammation in radiological examination, the appearance of bacterial excretion) is important both in the differential diagnosis of tuberculosis with other diseases and in determining the activity of the tuberculosis process.
• The general reaction is manifested in a deterioration in the condition of the body as a whole (body temperature, cellular and biochemical composition of the blood).
  • The temperature reaction is considered positive if there is an increase in body temperature by 0.5 °C compared to the maximum before the subcutaneous administration of tuberculin (it is advisable to conduct thermometry every 3 hours 6 times a day for 7 days - 2 days before the test and 5 days after the test). 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.
  • Thirty minutes or one hour after subcutaneous administration of tuberculin, a decrease in the absolute number of eosinophils is noted (F.A. Mikhailov test). After 24-48 hours, ESR increases by 5 mm/h, the number of band neutrophils by 6% or more, the content of lymphocytes decreases by 10% and platelets by 20% or more (Bobrov test).
  • After 24-48 hours after subcutaneous administration of tuberculin, the albumin-globulin coefficient decreases due to a decrease in the 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 by at least 10% from the initial level.

Alternative methods

In addition to tuberculins used in vivo, preparations have been created for use in vitro, for the production of which tuberculins or various mycobacterial antigens are used.

To detect antibodies to Mycobacterium tuberculosis, a diagnosticum, erythrocyte tuberculosis antigen dry, is produced - sheep erythrocytes sensitized with a phosphatide antigen. The diagnosticum is intended for conducting an indirect hemagglutination reaction (IHA) in order to detect specific antibodies to antigens of Mycobacterium tuberculosis. This immunological test is used to determine the activity of the tuberculosis process and monitor treatment. An enzyme immunoassay test system is also intended to determine antibodies to Mycobacterium tuberculosis in the blood serum of patients - a set of ingredients for conducting ELISA. used for laboratory confirmation of the diagnosis of tuberculosis of various localizations, assessing the effectiveness of treatment, and deciding on the appointment of 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 the test system for screening tuberculosis infection.

PCR test systems are used to detect mycobacteria.

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Contraindications to the Mantoux test

Contraindications to the Mantoux test with 2 TE:

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

In these cases, the Mantoux test is performed 1 month after the disappearance of clinical symptoms or immediately after the quarantine is lifted.

There are no absolute contraindications to skin and intradermal tuberculin tests. It is not recommended to perform them during periods of exacerbation of chronic allergic diseases, exfoliative dermatitis, pustular skin diseases, or during acute respiratory infections.

Subcutaneous administration of tuberculin is undesirable in patients with an active rheumatic process, especially with heart disease, and during exacerbation of chronic diseases of the digestive organs.

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Factors Affecting the Mantoux Test Result

The intensity of the tuberculin reaction depends on many factors. Children have a higher sensitivity to tuberculin than adults. In severe forms of tuberculosis ( meningitis, miliary tuberculosis, caseous pneumonia ), low sensitivity to tuberculin is often noted due to severe suppression of the body's reactivity. Some forms of tuberculosis ( eye tuberculosis, skin tuberculosis), 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 tuberculosis revaccinations. Each subsequent revaccination entails an increase in sensitivity to tuberculin. In turn, a decrease in the frequency of BCG revaccinations leads to a decrease in the number of positive results for the Mantoux test by 2 times, hyperergic - by 7 times. Thus, the cancellation of revaccinations helps to identify the true level of infection of children and adolescents with tuberculosis mycobacteria, which, in turn, allows for full coverage of adolescents with BCG revaccination within the required time frame.

The dependence of the Mantoux reaction intensity on the size of the post-vaccination BCG mark was revealed. The larger the post-vaccination 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 (GTH) is more intensely expressed in older children.

Increased sensitivity to tuberculin is observed when the Mantoux test is administered within 1 day to 10 months after vaccinations against childhood infections (DPT, DPT-M, ADS-M, measles, mumps vaccines). Previously negative reactions become questionable and positive, and after 1-2 years they become negative again. Therefore, tuberculin diagnostics are planned either before preventive vaccinations against childhood infections, or not earlier than 1 month after vaccinations.

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

Evaluation of tuberculin test results may be difficult in areas where low sensitivity to tuberculin caused by atypical mycobacteria is widespread. Differences in the antigen structure of different types of mycobacteria cause different degrees of skin reactions when using different antigens. When conducting a differential test with different types of tuberculin, the most pronounced reactions are caused by tuberculin prepared from the type of mycobacteria with which the body is infected. Such preparations are usually called sensitins.

A negative reaction to tuberculin is called tuberculin anergy. Primary anergy is possible - no reaction to tuberculin in uninfected individuals, and secondary anergy, developing in infected individuals. Secondary anergy, 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 anergy also occurs in lymphogranulomatosis, sarcoidosis, many acute infectious diseases (measles, rubella, mononucleosis, whooping cough, scarlet fever, typhoid, etc.), in vitamin deficiencies, cachexia, neoplasms.

Children and adolescents with hyperergic sensitivity to tuberculin based on the results of mass tuberculin diagnostics are the group most at risk of tuberculosis and require the most thorough examination by a phthisiatrician. The presence of hyperergic sensitivity to tuberculin is most often associated with the development of local forms of tuberculosis. With tuberculin hyperergy, the risk of tuberculosis is 8-10 times higher than with normergic reactions. Particular attention should be paid to children infected with Mycobacterium tuberculosis, with hyperergic reactions and contact with patients with tuberculosis.

In each individual case, it is necessary to study all factors influencing sensitivity to tuberculin, which is of great importance for making a diagnosis, choosing the correct medical tactics, method of patient management and treatment.

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