RU2315315C2 - Method for early diagnostics of tuberculosis infection in children - Google Patents

Abstract

FIELD: medicine, phthisiotherapy.

SUBSTANCE: for earlier diagnostics of tuberculosis infection in children it is necessary to detect the level of lymphocytic proliferation in whole blood microcultures according to blast-transformation reaction (BTR) with allergen tuberculin (PPD-L) in 6 dilutions, evaluate the index of stimulation of specific cell response according to detecting stimulation index. At values of stimulation index being below 3.2 one should diagnose the absence of infecting with tuberculosis mycobacteria. At stimulation index being above 3.2 one should diagnose the infecting with tuberculosis mycobacteria, and at this value being above 12.1 - active tuberculosis process should be stated upon. The innovation enables to increase the quality and the accuracy of earlier diagnostics.

EFFECT: higher accuracy and efficiency of diagnostics.

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Description

The invention relates to medicine, mainly to phthisiopediatrics, can be used to diagnose primary infection, latent activity of tuberculosis in children and assess the effectiveness of specific therapy.

Known immunological method for determining anti-tuberculosis antibodies - enzyme-linked immunosorbent assay (ELISA). In the diagnosis of tuberculosis, serological methods, including ELISA, did not live up to all expectations, since it was not possible to detect specific M. tuberculosis antigens, for which most patients develop the corresponding antibodies. Existing enzyme immunoassay systems have a specificity of 90-94% (6-10% false-positive results in healthy individuals or patients with non-tuberculosis diseases), while sensitivity (the number of positive results in patients with active tuberculosis) is on average about 70%. Therefore, talking about the independent significance of this method for the differential diagnosis of respiratory tuberculosis is not yet possible (Diagnosis and chemotherapy of respiratory tuberculosis. A manual for doctors. - M .: Medicine and life, 2003. - 48 p.), As well as for differential diagnosis of Mycobacterium tuberculosis infection and paraallergy, ELISA sensitivity in infected individuals is 23.2% (Nekrasov S.V. Diagnostic and prognostic significance of determining anti-tuberculosis antibodies during infection and disease of pulmonary tuberculosis in children and adults: Abstract of thesis .... Candidate of Medical Sciences. - M., 1999. - 23 p.).

The reaction of blast transformation of lymphocytes (RBTL) with tuberculin (PPD-L) is known to assess the state of specific cellular immunity, used in the clinic (especially in combination with a number of other immunological tests) for the differential diagnosis of tuberculosis, assessing the activity of its course and detecting minimal activity of tuberculous lesions . The mechanism of this reaction is the transformation of lymphocytes into blast cells under the action of a specific mitogen - tuberculin. The reaction consists of 3 stages (Abramova Z.P., Dubrovskaya N.A., Eremeev V.V., Averbakh M.M. Comparative determination of the proliferative activity of lymphocytes in the macro- and microvariant of the blast transformation reaction in patients with tuberculosis. Actual laboratory issues diagnosis of tuberculosis in the experiment and the clinic. - M., 1989. - S.114-116):

1. the allocation of lymphocytes (or leukocyte mass);

2. cultivation of lymphocytes (leukocytes) with PPD-L;

3. evaluation of the results.

The disadvantages of the method: a low percentage of detecting an increased level of blast formation (positive results) can be explained by the loss of lymphocytes at the stage of their isolation (at fecol-verographin). For the optimal course of RBTL, the presence of macrophages is necessary, therefore, the use of highly purified lymphocytes is not recommended (Lebedev K.A., Ponyakina I.D. Immunogram in clinical practice. - M .: Nauka, 1990. - 224 p.).

The objective of the invention is to improve the quality and reduce the time of early diagnosis of tuberculosis infection in children.

The problem is solved in that in a method for early diagnosis of tuberculosis infection in children, including assessing the state of specific cellular immunity using the reaction of blast transformation of lymphocytes with tuberculin, characterized in that RBTL is carried out using microcultures of whole blood with a tuberculosis allergen (PPD-L), use 6 dilutions and in parallel with phytohemagglutinin (PHA), assess the level of specific cellular response by determining the stimulation index and with indicators of the stimulation index enee 3.2 diagnose the absence of infection with Mycobacterium tuberculosis, with stimulation index of more than 3.2 diagnose infection with Mycobacterium tuberculosis, and the exponent a 12.1 - active tubercular process.

The developed reaction based on RBTL (modified RBTL with PPD-L) allowed to increase the information content of this immunological test. During the test, the loss of T-lymphocytes and other blood cells participating in the reaction (macrophages) is excluded, several doses of antigen (6 dilutions) are used, in parallel with tuberculin, the reaction is carried out with a non-specific mitogen - phytohemagglutinin (as an indicator of secondary immunodeficiency). The possibility of using a small volume of blood (200 μl). Evaluation of the method is carried out according to the degree of activity of inclusion of ³ H-thymidine (using an automatic scintillation counter for this).

RBTL is performed using microcultures of whole blood with PHA and in parallel with PPD-L without isolation of lymphocytes. Heparinized blood (25 IU of heparin per 1 ml of blood) was cultured in round-bottom immunological plates, in complete growth medium (ORS) (medium 199 containing 10% fetal calf serum, L-glutamine (2 mM), HEPES (10 mM), gentamicin sulfate 50 μg / ml) - control blood samples. Experimental blood samples were cultured in ORS containing PHA (0.06 ml) for 120 hours and in ORS containing PPD-tuberculin (0.06 ml) in 6 dilutions (50; 25; 12.5; 6.25; 3.1; 1.55 μg / ml), also for 120 hours. 18 hours before the end of cultivation, 1 μCi ³ H-thymidine is introduced into the cell cultures (control and experimental). At the end of the cultivation, blood cells are collected by a semi-automatic cell collector on glass fiber filters and dried. The count of radioactivity in imp./min is carried out in scintillation fluid using a "Betta-2" camera. Assessment of the level of specific cellular response is carried out by determining the stimulation index (IS) according to the formula:

It was found that the result of a lymphocyte stimulation index of up to 3.2 is negative, more than 3.2 is positive.

In the studies, the specificity and sensitivity of mRBTL with PPD-L was determined. High specificity was established - 97.9% and method sensitivity - 97.8%. The share of true results of MRBTL with PPD-L was 97.8 ± 1.1%.

We studied the informative value of the proliferation rate of lymphocytes during stimulation with tuberculin (mRBTL with PPD-L) in 279 children. Among them, in 117 (41.9%) children 2-17 years of age, respiratory tuberculosis was confirmed radiologically, including in the infiltration phase in 72 (61.5%), in the decay phase in 6 (5.1%), phase resorption and compaction in 11 (9.4%), calcification in 16 (13.7%) and extrapulmonary localization. Infection was diagnosed in 107 (38.4%) children of preschool and primary school age, with an unspecified infected 24 (8.6%). Healthy, not responding to tuberculin or with questionable results of the Mantoux test with 2 TE PPD-L - 31 (11.1%) children of preschool age, of which 9 children were examined in dynamics after 6-12 months. In total, 293 analyzes were performed as part of the study.

The results are shown in table 1, as can be seen from the table, the level of proliferative response of lymphocytes to tuberculin in children uninfected, infected (including during the "turn") and patients with tuberculosis was significantly different (p = 0,000).

When examining children with tuberculosis in the infiltration phase, before treatment, proliferation of lymphocytes for tuberculin in all (100%) was expressed, while the results of the phases of the tuberculosis process before treatment did not differ significantly (p = 0.302).

Table 1. Observation groups n The result of MRTL with PPD-L, stimulation index M ± m Confidence interval Not infected 29th 2.1 ± 0.2 * 1,1-3-1 Patients with tuberculosis in the infiltration phase before treatment 25 16.6 ± 2.0 * 6.7-26.5 Patients with tuberculosis, regardless of the phase of the process before treatment 42 14.6 ± 1.4 * 5.3-23.9 Mycobacterium tuberculosis infected (tuberculin-positive) 107 7.4 ± 0.4 * 3,5-11,3 The period of early tuberculosis infection ("turn") 71 7.7 ± 0.5 * 3.3-12.1 Mycobacterium tuberculosis infected for over a year 36 6.7 ± 0.4 4.1-9.3 * p = 0,000

Analyzing the dynamics of proliferation of lymphocytes to tuberculin during treatment, it was found that during the first 2 months there was no significant decrease in average values (p> 0.05), during the third month of treatment a decrease of 43% from the initial value was noted, but not significantly ( p> 0.05). A statistically significant difference (p = 0.005) with the initial level was observed only in patients who received anti-TB therapy (2-3 drugs) for 6 months or more.

When examining 24 children from the diagnostic “0” group, 14 children had a stimulation index lower than 3.1, which corresponded to the level of uninfected people. Repeated studies were conducted for 6 children (control after a year), while mildly proliferated tuberculin remained in all examined patients. In 11 children with a low level of stimulation index, a decrease in sensitivity to tuberculin (dubious test results) in the Mantoux reaction with 2 TE PPD-L after preparation for the test was also noted; in 3 children, a positive result was preserved.

Thus, the positive results of the Mantoux test with 2 TE PPD-L with mild proliferation of tuberculin were regarded by us as a manifestation of paraallergy and accounted for 12 ± 2.7%.

We found a high level of correlation between the Mantoux test with 2 TE PPD-L and mRBTL with PPD-L (r = 0.71 according to Spearman and r = 0.91 is the sign correlation coefficient).

The obtained results of studies of MRBTL with PPD-L suggest that children with a positive tuberculin breakdown, whose stimulation index is not more than 3.1, should be classified as uninfected, and a positive Mantoux reaction with 2 TE PPD-L should be regarded as para-allergy (with 3 years). Children with a positive Mantoux test with 2 TE PPD-L and with a stimulation index of more than 3.2 are considered infected with Mycobacterium tuberculosis. In children with a positive Mantoux test with 2 TE PPD-L and with a stimulation index of more than 12.1, evaluate the results as an active tuberculosis process despite the absence of radiological data.

The following extracts from case histories can serve as examples of informativeness in determining the level of specific cellular response in primary tuberculosis infection in children.

Example 1. Dmitry K., 2 years 5 months, inpatient card No. 17/03, was admitted to the hospital on 01.28.03 for treatment. Clinical diagnosis: Tuberculosis of the intrathoracic lymph nodes of the bronchopulmonary group in the infiltration phase, Office - (epidemiological No. 18604).

Since December 2002, contact has been established with his mother. An examination in January 2003 revealed a hyperergic reaction to tuberculin (Mantoux test with 2 TE PPD-L) - an infiltrate of 15 mm with necrosis, which coincided with the first positive result. There were no complaints upon receipt, but there was a manifestation of intoxication in the form of pale skin, a deficiency of body weight, and palpation revealed peripheral lymph nodes enlarged to a second size in 5 groups. For other organs and systems without features. Upon admission, the level of specific cellular response in mRTBL with PPD-L was determined; the stimulation index was 17.4.

An X-ray examination revealed pathological changes characteristic of tuberculosis only 10 months after the start of treatment.

The treatment was carried out with phtivazide and pyrazinamide. Monitoring the results of the Mantoux test with 2 TE PPD-L was carried out after 5 months - 12 mm papule. In dynamics, after 6 months of treatment, the stimulation index was 5.5.

Thus, the activity of primary tuberculosis infection and the effectiveness of chemotherapy were confirmed using MRTL with PPD-L.

Example 2. Adena S., 10 l. 11 months, inpatient card No. 141/03, was admitted to the hospital on 10.11.2003 with the aim of eliminating tuberculosis of the urinary system.

From birth, was observed by a nephrologist about a congenital anomaly of MVS. From the age of 6, she was observed by a TB specialist regarding a “turn” of tuberculin samples; preventive chemotherapy was not carried out. Contact has not been established. BCG was vaccinated at birth, 5 mm scar. Mantoux test with 2 TE PPD-L in September 2003 - 11 mm infiltrate.

Upon admission to the hospital, no manifestations of tuberculous intoxication were observed. Examination of organs and systems revealed no pathological changes. A Koch test was performed with 50 TE PPD-L, the result is negative.

The level of specific cellular response was determined in MRBTL with PPD-L, the stimulation index was 4.8, which corresponds to the level of infection with tuberculosis mycobacteria.

Thus, based on the data obtained, tuberculosis was excluded. The child was discharged from the hospital on 05.12.2003 with a clinical diagnosis: Congenital malformation of MVS. Chronic pyelonephritis, recurrent course. Neurogenic bladder dysfunction.

Example 3. Olga G., 14 years old, hospital patient card No. 77/03, was admitted to the hospital on 06/18/2003 to clarify the diagnosis and treatment. Clinical diagnosis: Tuberculosis of the urinary system: parenchymal tuberculosis of the kidneys, bilateral, MBT ±?

Since 2001, he has been observed by a TB specialist due to an increase in tuberculin samples; once during urine culture, mycobacterium tuberculosis was detected, and family contact was established. BCG vaccination at birth and revaccination at 7 years of age, one 3 mm scar. Mantoux test with 2 TE PPD-L in May 2003 - 15 mm infiltrate.

Upon admission to the hospital, no manifestations of tuberculous intoxication were observed. Examination of organs and systems revealed no pathological changes. A Koch test was performed with 50 TE PPD-L, the result is dubious. An X-ray examination of the chest organs revealed no pathology. On excretory urograms, pathological changes in the kidneys and urinary tract were not detected.

The level of specific cellular response in mRTTL with PPD-L was determined, the stimulation index was 22.9, which confirmed the specificity of the infection and its activity.

The treatment was carried out with isoniazid, rifampicin, streptomycin. Positive clinical dynamics were noted.

Example 4. Vera M., 6 l. 8 months, an individual baby card f. No. 026 / y, entered the sanatorium garden on 03.10.2002 for sanatorium treatment. Clinical diagnosis: Tubinfection? Lymphadenopathy of the intrathoracic lymph nodes of unknown etiology. Dysmetabolic nephropathy.

Since 2001, she has had intermittent contact with an uncle who has pulmonary tuberculosis. Vaccination at birth, 6 mm scar. For 3 years, tuberculin diagnosis was not carried out. Mantoux test with 2 TE PPD-L in August 2001 - an infiltrate of 8 mm. Registered with a TB specialist since July 2002 in the “0” group.

Upon admission to the sanatorium garden complaints of frequent colds, violation of the stool (tendency to diarrhea). On examination, the skin was clean, of a normal color, the tongue was covered with a whitish coating, there were carious teeth, hypertrophy of the tonsils up to size II. On palpation, multiple peripheral lymph nodes increased to size II-III were determined in 3 groups (antero-cervical, posterior-cervical, axillary). Lymph nodes were densely elastic consistency, not soldered to the surrounding tissue, painless. During auscultation, systolic murmur was heard at Botkin's point. For other organs and systems without features. Mantoux test with 2 TE PPD-L - infiltrate 9 mm. The level of specific cellular response was determined in MRBTL with PPD-L; the stimulation index was 2.6, which indicated the absence of tuberculosis infection. During an X-ray examination of the chest organs in a direct projection, the roots were enriched, the structure of the right root was fuzzy, and no pathological changes were revealed in the lateral projection.

Within 3 months, preparation for the Mantoux test was carried out, control in November 2002 - 3 mm infiltrate (the result during the second examination in April 2003 was the same). When x-ray control in November 2002, pathology was not detected. In a control immunological study, after four months, unexpressed proliferation of tuberculin remained (stimulation index 2.0).

Thus, the absence of infection was confirmed, and the changes that took place were regarded as a manifestation of a nonspecific infection.

In our opinion, given the high specificity and sensitivity of the method, the use of mRBTL with PPD-L in clinical practice in combination with other methods will improve the quality of diagnosis of specific inflammation, evaluate the effectiveness of specific treatment, reduce the time of diagnosis of infectious allergies, therefore, conduct timely prevention of local forms of tuberculosis in children.

Claims (1)

A method for early diagnosis of tuberculosis infection in children, including assessing the state of specific cellular immunity using the reaction of blast transformation of lymphocytes (RBTL) with tuberculin (PPD-L), characterized in that RBTL is carried out using microcultures of whole blood with a tuberculosis allergen (PPD-L) in 6 dilutions, the level of specific cellular response is assessed by determining the stimulation index, and if the stimulation index is less than 3.2, the absence of mycobacterium tuberculosis infection is diagnosed, etc. and a stimulation index of more than 3.2 diagnoses infection with Mycobacterium tuberculosis, and with an indicator of more than 12.1 - active tuberculosis process.