[Development and use of a domestic test system for diagnosis of tuberculous infection on the basis of quantitative analysis of interferon-gamma induction in whole blood samples in vitro, by using specific recombinant antigens].

A test system was developed to detect tuberculous infection by qualitative analysis of interferon-gamma (IFN-gamma) in the plasma samples after 20-24-hour incubation of whole blood samples in the presence of Mycobacterium tuberculosis (MBT) antigens: tuberculin PPD and a mixture of the MBT-specific recombinant antigens ESAT-6 and CFP-10. The analysis used 3 test tubes each containing 1 ml of heparinized venous blood, one of which served as a control; the other two test tubes were employed to measure antigen-induced IFN-gamma production. Whether this test system might be used to determine primary tuberculous infection was studied in 277 children and adolescents. The threshold diagnostic IFN-gamma induction level determined in the test tube containing a mixture of the antigens ESAT-6 and CFP-10 was ascertained. Postvaccine allergy was detectable if there was IFN-gamma induction in the test tube containing tuberculin and if there was no diagnostic IFN-gamma level in that containing the antigens ESAT-6 and CFP-10. The diagnostic sensitivity of detection of primary tuberculous infection was 97.6% with 94.4% specificity, which enabled this condition to be differentiated from postvaccine allergy. The level of antigen-induced IFN-gamma may be lower in relatively disseminated forms of pulmonary tuberculosis.

[In vitro interferon-gamma induction in whole blood samples is a test for tuberculous infection in children and adolescents].

By applying the interferon-gamma (IFN-gamma) induction technique in the whole blood samples exposed to short-term (22-24-hour) incubation in the presence of Mycobacterium tuberculosis antigens--PPD tuberculin and specific recombinant ESAT-6 lacking in the cells of vaccine BCG and other non-tuberculous mycobacteria, the authors studied the groups of children and adolescents with a negative Mantoux test (n = 31), with postvaccine BCG allergy (n = 40), as well as patients with primary tuberculous infection (n = 84) and those with pulmonary tuberculosis (n = 44). Patients with primary tuberculous infection and a high sensitivity (94%) and a high specificity (97%) may be differentiated from children and adolescents with postvaccinal allergy when the recombinant ESAT-6 antigen and the critical IFN-gamma level (greater than 70 pg/ml) detectable in the plasma samples after incubation with the antigen. It has been also shown that in adolescents with local forms of pulmonary tuberculosis specific IFN-gamma induction may be suppressed in number of cases, which is ascribed to decreased specific immunity.

[Significance of determination of gamma-interferon in the diagnosis of tuberculous exudative pleurisy].

The pleural fluid concentration of gamma-interferon (gamma-IFN) was studied in 44 patients with exudative pleurisy. The tuberculous nature of exudative pleurisy was established in 25 patients on the basis of X-ray study, a follow-up, microbiological study of pleural exudate specimens, and morphological studies of biopsy specimens obtained at video-assisted thoracoscopy or surgery. The the pleural exudate concentrations of gamma-IFN were over 300 pg/ml (mean 1019 +/- 161 pg/ml) in 19 out of 21 patients with exudative pleuritis in a phase of active inflammation. The patients with the fibrous outcome of pleurisy of tuberculous etiology and those with exudative pleurisy of non-tuberculous etiology, including that in malignancies and papapneumonic pleurisy, were observed to have lower concentrations of gamma-IFN (mean 118 +/- 16 pg/ml). With the discriminating level of above 300 pg/ml, the sensitivity and specificity of the test were 90.5 and 100%, respectively.

[Polymerase chain reaction to determine and control drug-resistant Mycobacterium tuberculosis strains].

Real-time polymerase chain reaction was used to develop a one-stage procedure for molecular genetic analysis of Mycobacterium tuberculosis (MBT) DNA in order to determine mutations associated with drug resistance to the antituberculous agents: isoniazid and rifampicin. To analyze the spread of drug-resistance of the causative agent of tuberculosis in Russia, two thousand MBT strains were studied in 24 regions of all the federal districts. Testing 1406 MBT strains isolated by first detected and untreated patients revealed multidrug resistance (MDR) in 21.9% of cases. MRD was detected in 58.5% of the previously treated patients with MDR. The agreement of molecular genetic analysis of drug resistance with the results of cultural tests of 1096 strains was 94%.

[Use of polymerase chain reaction for the diagnosis and evaluation of chemotherapy for pulmonary tuberculosis].

Polymerase chain reaction (PCR) using a new procedure for preparing sputum samples for DNA isolation on the basis of immunomagnetic separation of mycobacteria was employed to examine sputum samples from 141 patients with first diagnosed pulmonary tuberculosis and 510 diagnostic materials from patients with nonspecific lung disease. In 47 patients with pulmonary tuberculosis, sputum bacterial isolation was followed up, by using PCR and conventional microbiological studies during regular, every 6-7-week, examination. The sensitivity of PCR employing the above procedure for preparing the samples averaged 66% versus 48% when a cultural study was applied. Its specificity was 99.4%. Bacterial isolation cessation with the use of PCR well correlated with the clinical and X-ray trends in the disappearance of the signs of a tuberculous process.

[Multiplication of Mycobacterium tuberculosis in the organs of mice with a single exposure to cyclophosphamide]. / Razmnozhenie mikobakterii tuberkuleza v organakh myshei pri odnokratnom vozdeistvii tsiklofosfamidom.

The injection of cyclophosphamide, used as an immunomodulating agent in a dose of 100 mg/kg, into mice infected with M. tuberculosis induced an increase (a virulent culture) or a decrease (a culture with low virulence) in the multiplication of mycobacteria in the spleen. In mice infected with a virulent culture and protected from infection with streptomycin for 1 week cyclophosphamide induced a considerable decrease in the number of viable mycobacteria in the lungs by days 18-20 after infection.

[Determination of antibodies to mycobacterial antigens in tuberculosis by erythrocyte immunoadsorption with a rosette marker]. / Opredelenie antitel k antigenam mikobakterii pri tuberkuleze metodom éritroimmunoadsorbtsii s rozetochnym markerom.

A solid-phase enzyme immunoassay system for the determination of antibodies to mycobacterial antigens, based on the method of erythrocyte immunoadsorption in microchambers for immunological reactions, has been developed. To detect antibodies specifically bound with the solid-phase antigen, the affinity rosettes of Staphylococcus aureus strain Cowan I, carrying protein A, with erythrocytes conjugated with human gamma globulin have been used. The significant correlation of the titers of 34 sera, determined by means of erythrocyte immunoadsorption, with extinction values obtained in the solid-phase enzyme immunoassay of antibodies to Mycobacterium tuberculosis has been established. The coincidence of the results in 92% of cases has been noted.

[Obtaining hybridomas producing monoclonal antibodies with different specificities against Mycobacterium tuberculosis of the human and bovine types]. / Poluchenie gibridom, produtsiruiushchikh monoklonal'nye antitela s razlichnoi spetsifichnost'iu k mikobakteriiam tuberkuleza chelovecheskogo i bych'ego vidov.

A procedure for isolation of hybridomes producing monoclonal antibodies (McAB) to tubercle bacilli is described. Specificity of the McABs was studied with the solid phase radioimmune and immunoenzyme tests. Supernatant of tubercle bacilli destroyed with ultrasound was used as antigens. The McABs did not practically react with antigens of the tubercle bacilli atypical forms. Five ascitic monoclonal hybridomes were isolated. Four of them produced antibodies with selective specificity to antigens of bovine tubercle bacilli (M. bovis-8 and BCG) and one produced antibodies to antigens of human tubercle bacilli (H37Rv).

[Generalized tuberculosis after kidney transplantation]. / Generalizovannyi tuberkulez posle transplantatsii pochki.

Tuberculosis is one of severe infectious complications in patients on hemodialysis and after kidney transplantation. Incidence of disseminated and generalized forms is high, whereas clinical symptoms are weak and nonspecific. An aggressive generalized form of tuberculosis was observed in a kidney transplant recipient. M. tuberculosis, the antigen and DNA were registered only a few days before death. Disseminated foci in the lungs were seen on CT image only in the agonal period in spite of multiple x-ray investigations. Thus, our experience and experience of other investigators evidence that if recipients of renal transplant have fever of unknown genesis and do not respond to standard antibiotic therapy, tuberculosis should be suspected and a course of specific antituberculosis therapy should be started as early as possible.

[The effectiveness of Mycobacteria tuberculosis isolation by polymerase chain reaction: results of a randomized trial]. / Effektivnost' obnaruzheniia mikobakterii tuberkuleza legkikh metodom polimeraznoi tsepnoi reaktsii (rezul'taty randomizirovannogo issledovaniia).

The results of testing sputum for Mycobacterium tuberculosis by different methods: polymerase chain reaction (PCR), luminescence microscopy, and culture tests were compared. Clinical sputum samples were studied in 62 patients with pulmonary tuberculosis and 72 patients with non-specific diseases of the lung. The specificity of PCR was 98.7%. The sensitivity of PCR was 59.6%; that of cultural tests was 43.8%.

[Direct genetic analysis of the rifampicin resistance of M. tuberculosis isolates in sputum samples]. / Priamoi geneticheskoi analiz rezistentnosti k rifampitsinu izoliatov M. tuberculosis v obraztsakh mokroty.

The paper shows a rapid method for diagnosing the resistance of Mycobacterium tuberculosis to rifampicin in the testing of clinical sputum samples. The sputum samples from 12 patients ineffectively treated for pulmonary tuberculosis were treated by the immunomagnetic mycobacterial separation technique; polymerase chain reaction was used to perform the amplification and direct sequencing of the gene fragment rho poB by identifying the mutations responsible for mycobacterial rifampicin resistance. Other equal parts of the same sputum samples were cultured on liquid medium for 5 days and subsequently examined in the same manner and also cultured on the Löwenstein-Jensen solid medium, followed by the determination of rifampicin sensitivity by the routine procedure. Routine examination revealed 7 cases of rifampicin resistance. Short-term (5-day) cultivation of sputum samples, followed by a molecular genetic study, also established rifampicin resistance in all the 7 cases of the 12 tested samples.

[An immunologic method for identification of M. tuberculosis and M. bovis (BCG) based on the use of monoclonal antibodies]. / Immunologicheskii metod identifikatsii M. tuberculosis i M. bovis (BTsZH) na osnove primeneniia monoklonal'nykh antitel.

Monoclonal antibodies interacting with specific selectivity with human tubercle bacilli (monoclonal antibodies of the cell producing strain 62D) and bovine tubercle bacilli (BCG) (monoclonal antibodies of the cell producing strain 60D) were produced with hybridoma technology. A rapid, productive and safe procedure for identification of M. tuberculosis and M. bovis (BCG) was developed on the basis of solid phase enzyme immunoassay. The immunological procedure was compared with the biochemical (niacin) method for identification of mycobacteria. The immunological procedure was shown to be reliable and promising for practical use.

[Effectiveness of liposome-incorporated streptomycin in experimental tuberculosis in mice]. / Effektivnost' streptomitsina, vkliuchennogo v liposomy, pri éksperimental'nom tuberkuleze u myshei.

The chemotherapeutic efficacy of streptomycin incorporated into liposomes was studied on mice with experimental tuberculosis. Streptomycin incorporated into liposomes was prepared with the detergent method using purified egg lecithin, sodium cholate and streptomycin sulfate. The level of streptomycin incorporation into liposomes was 250-399 micrograms of streptomycin base per 1 mg lecithin. The size of the liposome was determined by electron microscopy. It was 40-80 nm. BALB/c and (CBA X C57B1(6)) F1 mice infected with M. tuberculosis H37Rv (0.1 mg of half-moist mass per mouse, intravenously) were used in the experiments. It was shown that the liposome-incorporated streptomycin injected intravenously on the 3rd or the 4th day (20 or 25 mg/kg) or 3 times on the 4th, 7th and 10th or the 12th days (50 or 67.5 mg/kg a day) after the infection inhibited the growth of M. tuberculosis in the spleen tissue, while analogous doses of streptomycin solution had no such an effect. With respect to the lung tissue, the antibacterial effect of the liposome-incorporated streptomycin was not potentiated. When the liposome-incorporated streptomycin was administered 3 times, the life-span of the animals was increased and the loss of the body weight was retarded as compared to the results of an analogous treatment course with the use of streptomycin solution. The acute toxicity of intravenous streptomycin incorporated into liposomes was lower as compared to that of streptomycin solution.

[Pharmacokinetics of dihydrostreptomycin, after intravenous administration as a liposomal preparation, in the blood serum and tissues of intact mice and those with generalized tuberculosis]. / Farmakokinetika digidrostreptomitsina posle vnutrivennogo vvedeniia liposomal'nogo preparata v syvorotke krovi i tkaniakh intaktnykh i porazhennykh generalizovannym tuberkulezom myshei.

Distribution of 3H-dihydrostreptomycin ( DHS ) in the blood serum and organs of intact mice and mice with generalized tuberculosis was studied. The antibiotic was administered in the form of a liposomal preparation and solution. It was shown that the distribution pattern of DHS administered in the form of a liposomal preparation corresponded to that of liposomes, which were the drug carriers. The serum levels of DHS injected intravenously in the form of a liposomal drug in a dose of 1 mg per mouse in the intact animals were 19 and 17 times higher 1 and 3 hours after injection, respectively as compared to those provided by the solution. In animals with generalized tuberculosis, the respective figures were 5 and 1.9 times. The levels of DHS in the liver provided by the liposomes were 4-6 times higher at all the time intervals in both the groups of animals. The antibiotic levels in the spleen of intact and infected animals were 2.5-3 and 8-12 times higher, respectively. In the lungs of intact animals, the antibiotic levels were 2 times higher 1 hour after administration. In infected animals, the levels of the antibiotic in the lung tissue were 3 times higher in 1 and 3 hours. The DHS levels in the kidneys of infected and intact animals were similar after drug administration in the form of a solution and liposomal preparation. The extremely high difference in the DHS levels in the spleen tissues of the mice with generalized tuberculosis after drug administration in the form of a solution and liposomal preparation may be of a significant importance for sanation of the organ lymphoid system affected with tuberculosis.

[Immunoenzyme analysis for the identification of Mycobacterium bovis by using monoclonal antibodies]. / Immunofermentnyi analiz dlia identifikatsii mikobakterii tuberkuleza bych'ego tipa s pomoshch'iu monoklonal'nykh antitel.

Three monoclonal antibodies (MAb) were obtained after fusion of mouse BALB/c splenocytes immunised with gamma-irradiated field strains M. bovis and cells of mouse myeloma. Mab specificity was determined at enzyme immunoassay of the bacteria. The antibodies were able to identify various epitopes of the protein (molecular mass under 31 kD). One of the antibodies obtained served the basis for the test-system intended for rapid identification of M. bovis. The system requires 0.5-1 mg of the bacterial mass.

[Value of molecular biological methods in the diagnosis of urogenital tuberculosis]. / Znachenie molekuliarno-biologicheskikh metodov v diagnostike mochepolovogo tuberkuleza.

Enzyme immunoassay (EIA) was used to study the diagnostic value of determination of serum Mycobacterium tuberculosis (MBT) antibodies in 41 patients with active urinary tract tuberculosis, 14 with inactive tuberculosis and 140 with nontuberculous diseases of the urinary system. The sensitivity of EIA was 73% with 88.6% sensitivity. Polymerase chain reaction revealed the causative agent of tuberculosis in the urine samples of 16 out of 17 patients with active urinary tract tuberculosis, negative tests were observed in 4 cured patients and 39 patients with nontuberculous diseases of the urinary tract (100% specificity).

[Production of liposomal preparations of streptomycin and dihydrostreptomycin]. / Poluchenie liposomal'nykh preparatov streptomitsina i digidrostreptomitsina.

The effect of the conditions of the formation of liposomal preparations of streptomycin and dihydrostreptomycin on incorporation of the antibiotics into the liposomes was studied. The liposomes were obtained with the detergent (cholate) method modified by the authors. The modification implies preliminary application of a 20 per cent antibiotic buffer solution on columns for gel filtration of a mixed mycellar antibiotic solution (20 per cent). The volume ratio of the preliminary applied buffer solution and the mixed mycellar solution is higher than 4:1. The new procedure is simple, readily reproduced, providing formation of the liposomal preparations characterized by high levels of the antibiotic incorporation (about 300 micrograms per 1 mg of lecithin). The preparations are stable with regard to the antibiotic release. The liposomes do not aggregate on storage for a long period (more than 6 months). The liposomal preparations thus formed may be useful as intravenous dosage forms of the antibiotics.

[Determination of antituberculous antibodies in the cerebrospinal fluid by an immunoenzyme assay method in patients with tuberculous meningitis]. / Opredelenie v spinnomozgovoi zhidkosti protivotuberkuleznykh antitel metodom immunofermentnogo analiza u bol'nykh tuberkuleznym meningitom.

Possibilities of using enzyme immunoassay to identify specific antibodies to M. tuberculosis in the cerebrospinal fluid of 44 patients with tuberculous meningitis and 81 cases with other diseases of the central nervous system were analysed. The assay sensitivity and specificity in an active period of tuberculous meningitis made up 100 and 97.5%, respectively. The findings permit one to recommend the enzyme immunoassay for an early diagnosis of tuberculous meningitis.

[The use of highly dispersed iron particles for increasing the sensitivity of the luminescent microscopic method of determining Mycobacterium tuberculosis]. / Primenenie vysokodispersnykh ferrochastits dlia povysheniia chuvstvitel'nosti liuminestsentno-mikroskopicheskogo metoda opredeleniia mikobakterii tuberkuleza.

A highly dispersed ferromagnetic powder obtained by a plasmochemical method (particle size was 100-500 A) was treated by means of an ultrasonic disperser: suspension was added to the trisodium phosphate homogenized and neutralized sputum (0.3 mg of the initial powder per 1 ml of sputum) of patients with various forms of pulmonary tuberculosis. The sputum was then incubated at slight stirring for 40 min and centrifuged; the precipitate was used to prepare smears which were stained with auramine; mycobacteria were detected by luminescence microscopy. The ferromagnetic suspension was found to increase luminescence microscopy sensitivity to 85.4%. The efficacy of the method was 30.8% more than that in the cultivation of infectious material in solid nutrient media.

Detection of rifampicin-resistant Mycobacterium tuberculosis strains by hybridization and polymerase chain reaction on a specialized TB-microchip.

Two alternative methods for identification of rifampicin-resistant strains of Mycobacterium tuberculosis on biological microchips are developed. The methods are based on detection of point mutations and other rearrangements in the rpoB gene region determining rifampicin resistance. Hybridization on TB-microchip detects 30 mutant variants of DNA in rifampicin-resistant strains (about 95% of all resistant forms). Allele-specific microchip PCR shortens the duration of analysis to 1.5 h. These methods can be used in clinical diagnostic laboratories for evaluating drug resistance/sensitivity of tuberculosis agent and for monitoring of the efficiency of antibiotic therapy.