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By taking into account the present requirements for the equipping of the laboratories, the authors have drawn up the minimum standard list of equipment, as well as a list of additional equipment for the specialized bacteriological laboratory of a tuberculosis-controlling institution, which performs microbiological studies for the diagnosis and control of chemotherapy for tuberculosis. The specifications and characteristics of the baric types of equipment used to fit out the laboratories under the present conditions are described. Equipping the laboratories in accordance with the draw-up lists is shown to ensure a qualitative, effective, and safe work. Recommendations on how to supply the laboratories with equipment, to make the optimal choice, and to use consumables for tests are given.

The major available indicators of the efficiency of laboratory service activities in diagnosing tuberculosis, including those widely used in international practice and Russian traditional tuberculosis statistical data, are analyzed. The optimal criteria for assessing the quality and efficiency of work of the clinical diagnostic laboratories of the general health network and the bacteriological laboratories of a tuberculosis service in the detection, diagnosis, and chemotherapy monitoring of tuberculosis have been developed. The recommended levels of the indicators, which are to be achieved by well working laboratories, if a well-implemented tuberculosis-controlling program is available, are defined.

The authors have revealed the acutest problems of laboratory tuberculosis diagnosis service in the Russian Federation. They have made their conclusion on this issue and developed the following recommendations: to continue the reorganization of Russia's laboratory tuberculosis diagnosis service; to reconstruct and repair laboratories; to provide them with up-to-date equipment; to make a full-scale training of laboratory workers; to standardize the procedures of microbiological tests for tuberculosis; to organize bacteriological tuberculosis service monitoring in Russia, including monitoring of tuberculosis-controlling facilities; to develop and officially approve a number of normative documents (current standards for the time spent on working operations and criteria for estimating the load on the personnel performing the procedures of a study, new staff lists for laboratories, the level and structure of salaries in the laboratory's personnel).

M. avium and three M. tuberculosis strains were used as an example to study the bacteriostatic activity of the liposomal form of isoniazid in the liquid nutrient medium by serial dilutions. Unlike isoniazid in solution, its liposomal form was found to suppress the multi plication of all study mycobacterial samples and diminishes their viability, but at different concentrations. The liposomes that did not contain the drug exerted an inhibitory effect only on the M. tuberculosis strains H37Rv and Erdman. It is suggested that the change in isoniazid sensitivity results from the effect of liposomal phospholipids on some mycobacterial enzymes. Electron microscopy indicated that the liposomal form of isoniazid is able to penetrate into the alveolar macrophageal cytoplasm of guinea pigs just 30 min after incubation.

Current molecular and epidemiological studies could reveal individual families of Mycobacterium tuberculosis strains. The purpose of the present study was to reveal the spread of Mycobacterium tuberculosis strains of the family Beijing in the Samara Region and to define risk factors for their transmission. This was a cross-sectional populational molecular epidemiological study that showed that the Beijing genotype prevailed among the obtained isolates (66.6%; 586/880) and it was encountered among convicts and young persons (RR 1.3; 95% CI 1.2-1.5 and RR 1.2; 95% CI 1.0-1.3, respectively), which is indicative of active and recent transmission. Multifactorial analysis indicated that male sex (OR 1.5; 95% CI 1.1-1.9), younger age (OR 1.3; 95% CI 1.1-1.7), homelessness (OR 5.6; 95% CI 1.1-6.3), and prior or current confinement (OR 2.0; 95 CI 1.5-2.7) were substantially associated with the risk of contamination with the strain of the Beijung family. Drug resistance, including multidrug resistance, was twice higher among the strains of this family.

The true prevalence rates of multidrug-resistant tuberculosis (MDRT) are unknown for most regions of Russia. This study was conducted in the Samara Region that differs from other regions in the rapid spread of HIV infection. The purpose of this study was to determine the primary and acquired resistance of Mycobacterium tuberculosis (MBT) to first-line antituberculous drugs in patients from civil and penitentiary sectors and to reveal risk factors of drug resistance of MBT. Six hundred patients (309 civilians and 291 prisoners who had been bacteriologically diagnosed as having tuberculosis. The authors have established the following:--in new cases, primary drug resistance is as follows: to isoniazid [38.9% (95% CI, 31.3-36.9%)], to rifampicin [25.9% (95% CI, 19.4-33.4%)] and to MDRT [23.0% (95% CI, 16.7-30.3%)];--in prisoners, the primary resistance of MBT was statistically more significant than in civilians;--male sex, in adequate prior or current treatment for tuberculosis for more than 4 weeks, the presence of fibrocavernous tuberculosis and previous prison stay are essential risk factors of the development of resistance of MBT to both any first-line drug and MDRT;--HIV infection is unassociated with resistance.

The experience of researchers of the Central Research Institute of Tuberculosis, Russian Academy of Medical Sciences, in the pilot areas of Russia has made it possible to perfect approaches to improving a bacteriological diagnosis of tuberculosis in general health care facilities. A decisive role in enhancing the effectiveness of detection of patients with tuberculosis by the Ziel-Nielsen test is played by the following factors: training of laboratory personnel; provision of laboratories with high-grade equipment and reagents; introduction of the methods and techniques unified and approved by the Ministry of Health of the Russian Federation; regular supervision of jurisdictional laboratories; outside control of the quality of studies. Proper organization of examination of patients, including an effectual selection of patients to be examined, the observance of the established frequency of their examination, and provision of the required quality of a taken sample greatly affect the efficiency of a microscopic diagnosis of bacillary patients. In addition, the centralization of performed microbiological tests for tuberculosis in the most trained, certified, and licensed large laboratories exercising a centralized control of the quality of microbiological studies is optimal.

The purpose of the study was to define a role of molecular genetic techniques in the evaluation of the efficacy of short-term chemotherapy in patients with pulmonary tuberculosis. A total of 203 patients with first detected tuberculosis patients who isolated Mycobacterium tuberculosis (MBT) were examined. Microbiological methods (bacterioscopy and seeding) and polymerase chain reaction (PCR) were used to detect MBT in new cases. During chemotherapy, there was a reduction in the number of positive results of both bacterioscopy and PCR, which serves as a sign of abacillation. Following 2 months of chemotherapy, 50% of the patients with negative bacterioscopy and seeding were found to have MBT DNA. In the patients with negative bacterioscopy and seeding, the results of PCR were also negative by month 6. In a group of patients without bacterial isolation, but with positive sputum tests for MBT DNA, the efficiency of chemotherapy was confirmed by the negative results of PCR. The latter is shown to enhance the sensitivity of bacteriological tests for MBT and to reduce the time of obtaining their results.

The accelerated nitrate reductase method (NRM) developed at the Central Research Institute of Tuberculosis versus the automatic assay of drug sensitivity by means of a BACTEC 960 bacteriological analyzer was assessed. NRM was carried out, by using the Lówenstein-Jensen medium for 10 days. It is based on the detection of alive Mycobacteria tuberculosis, by recording their enzymatic activity. The study showed a good agreement of the results obtained by NRM with those obtained on a BACTEC 960 analyzer. Agreements were found for 52 isolates in 47 (90.4%) cases, the results disagreed in the testing of 5 (9.6%) cultures. The results of NRM were identical to those for 21 of the 22 cultures sensitive on a BACTEC 960 device; the coincidence was 95.5%. The sensitivity of NRM ranged from 88.2% (for rifampicin) to 96.3% (for isoniazid) and the specificity did from 96% (for isoniazid) to 100% (for streptomycin, rifampicin, and ethambutol). The positive prognostic value of NRM was 100% (for streptomycin, rifampicin, and ethambutol) and 96.3% (for isoniazid). The negative prognostic value of NRM ranged from 94.6 to 96.8% for individual drugs. The efficiency of NRM (a ratio of the number of correct results to the total number of results) was greater than 0.96, which suggests that this method and the BACTEC MGIT 960 AST technique may be regarded as rather comparable. The testing of NRM versus the automatic BACTEC MGIT 960 AST technique has indicated that the former may be successfully used to determine the sensitivity of Mycobacteria to the critical concentrations of first-line antituberculous agents.

Microbiological and histological studies made in patients with active tuberculosis revealed that 65.5% of cases had revertants from ultraminor forms (UMF) of Mycobacteria tuberculosis (MBT), which are typically bacterial in shape and referred to as representatives of the genus Mycobacterium tuberculosis. Cultivation of the diagnostic material containing MBT UMF the enriched liquid nutrient medium could detect rod-shaped bacterial forms of MBT in 18% of the patients with tuberculosis. Inoculation of the MBT UMF-containing material isolated from patients with tuberculosis to guinea-pigs revealed productive sarcoid-like changes in the organs of the experimental animals in 30.6% of cases. A postmortem study of the organs of experimental animals inoculated the material containing "visible, but not growing" mycobacteria detected paraspecific tissue reactions and infiltration of lung tissue by multiple cells of the macrophageal series.

The practice of the researchers of the Central Research Institute of Tuberculosis in Russia's experimental areas yielded approaches to improving the microbiological diagnosis of tuberculosis in both specialized and general health care facilities. A decisive role in enhancing the efficiency of microbiological identification of patients with tuberculosis is played by the following factors: laboratory staff training, provision of laboratories with qualitative equipment and reagents, introduction of unified methods and those approved by the Ministry of Health of the Russian Federation, regular supervision of jurisdictional laboratories, and outside test quality control. The efficiency of microbiological diagnosis of bacillary patients primarily depends on the expediency of formation of groups of patients under study, the observance of the frequency of examinations, and the assurance of the quality of collected materials. With this, the centralization of microbiological tests for tuberculosis at the most trained, certified, and licensed large laboratories that exercise a centralized control over the quality of microbiological tests.

The authentic data on the drug resistance of Mycobacterium tuberculosis can be obtained only provided that standard laboratory procedures and the unified standardized method for determining the resistance are used along with a compulsory outside quality control over the performance of this test. The supervisory activities of the researchers of the Central Research Institute of Tuberculosis (CRIT), Russian Academy of Medical Sciences, made in some regions of Russia included standardization of methods and their implementation quality control allowed them to reveal and to correct main errors in drug resistance testing. In this connection, this made it to record the true level of M. tuberculosis resistance to essential antituberculous agents in the areas investigated by the researchers of CRIT. Analyzing the data on the drug resistance of M. tuberculosis in the above areas leads to the conclusion that 2000 was marked by rather high rates of primary drug resistance; nearly a fourth of all new and prior cases of pulmonary tuberculosis studied for its drug resistance were carriers of a M. tuberculosis resistant to at least one antituberculous agent.

Luminescence bacterioscopy of capillary blood has been found to be an additional rapid laboratory method that verifies a clinical diagnosis in cases of negative sputum bacterioscopic tests in 48.8% of the adolescent patients. The Boy luminescence microscopy shows that in adolescent tuberculosis patients, Mycobacteria are significantly more frequently present in the capillary blood (72.4%) than in the sputum (30.9%) and bronchial washes (34.8%). The detection of Mycobacteria in the capillary blood has been evidenced by cultural tests of some venous blood samples in the BACTEC 460 TB system.

The studies have showed the frequency and spectrum of drug resistance, determined by the methods of proportions and absolute concentrations, to be virtually equal. This suggests that both methods are of equal diagnostic value. The method of absolute concentrations is rather accurate and similar to that of proportions in sensitivity, by ensuring comprehensive determination of the spectrum of drug resistance. Furthermore, the advantages of this method are its simplicity, accessibility, and cost-efficiency.

The efficiency of tuberculosis control programs is largely determined by methods for rapid diagnosis of the agent. In comparison with the traditional methods, new molecular technologies for characterization of mycobacteria appear to be more promising, because the result can be obtained in almost no time. Sixty-five strains of M. tuberculosis isolated in various regions of Russia were investigated. Drug resistance and strain appurtenance of this sample were determined by classical (absolute concentrations method, IS6110-RFLP) and modern molecular genetic methods (detection of mutations in rpo B gene, DRE-PCR). The spectrum of mutations of the rpoB gene associated with rifampicin resistance was evaluated by direct sequencing. Mutations involving codons 531 (62.7%), 526 (18.6%), and 516 (10.2%) of rpoB gene predominated in the studied sample. The studied strains were discriminated into 52 individual strains by IS6110-RFLP and DRE-PCR typing. Analysis of the resultant genetic variants showed the predominance of M. tuberculosis family W. The efficiency of combined approach to screening for M. tuberculosis is discussed.

The results of determination of drug medium depend on both individual cultural properties of the strains tested and the type of the medium used. The composition of a medium is of significance for some strains: the latter grow in any medium and are characterized by the same spectrum of resistance. However, most drug-resistant strains have higher feeding demands which are not fully met on the lyophilized Löwenstein-Jensen medium. The strains in the lyophilized medium show an incomplete spectrum of resistance and less grow than do those in freshly prepared Löwenstein-Yersen medium.

Combined microbiological investigation of diagnostic material containing granular mycobacteria has revealed cultures-revertants both in tuberculous and sarcoidosis patients. Identification of the isolated cultures provided the conclusion on identity of granular mycobacterial structures discovered both in tuberculous and sarcoidosis patients.

Microbial spectrum in patients with disorders of the upper respiratory tract consisted mainly of streptococci, staphylococci and pneumococci in 1991-1993, 1994 and later, respectively. Most specific for detection of nontuberculous flora was brushing with protecting agar plugs in combination with transport medium and anaerobic culturing. Direct immunofluorescence allows detection of antigens M. chlamydia in 29.4%, M. pneumoniae in 18.5% of cases. In diagnosis of tuberculosis the following new technologies were introduced: bacterioscopy with previous culturing of the material on liquid culture media followed by biological test and polymerase-chain reaction; determination of drug resistance based on nitrate reductase activity of M. tuberculosis indicating resistance of M. tuberculosis on liquid media in 4-7 days, dense egg media in 8-12 days, usage of Popesku medium enables correction of tuberculosis chemotherapy one month after the test initiation; M. tuberculosis identification using Western blot with monoclonal antibodies in some cases for 7 days.

The paper outlines difficulties in tuberculosis diagnosis and effectiveness of various methods of mycobacteria identification. The latter along with direct and indirect determination of mycobacterial drug resistance require perfection. There is a need in investigation of diagnostic samples from tuberculous and respiratory sarcoidosis patients for mycobacterial granular forms. A multimodality diagnostic approach to examination of clinical material from tuberculous patients is advocated.

Mycobacterial variability was studied in active tuberculosis patients from radioactive contamination area subject to close radiation control. For such areas it was noted that the patients discharge altered coccoid mycobacteria stained with routine luminescent dyes. Biological investigations of freshly isolated strains revealed potentiation of M. tuberculosis virulence in patients with respiratory tuberculosis. A large number of patients from the contaminated areas against control demonstrated primary resistance to rifampicin and isoniazid.

The article deals with the practical use of monoclonal antibodies to M. tuberculosis. As shown in this work, the use of monoclonal antibodies permits the determination of antibodies to M. tuberculosis and mycobacterial antigens in the spinal fluid of patients with tuberculous meningitis.

Study of the protein spectra of the mycobacteria of a different taxonomic belonging and isoniazid, streptomycin- and rifampicin-resistant strains has shown typical differences of the protein complexes of the studied strains.

Influence of non-clostridial anaerobic infection on the course of chronic pulmonary tuberculosis caused by intrapleural administration of BCG used in a vaseline-lanolin mixture was studied experimentally on guinea pigs. Administration of Bacteroides fragilis in the presence of a mild tuberculous inflammation aggravates the course of a specific process promoting pleural empyema development, increase in the size of pulmonary granuloma, extension of pathologic changes into the parenchymatous organs and aggravation of a nonspecific inflammatory reaction. Animals with mixed infection exhibited a longer duration and higher level of M. tuberculosis and B. fragilis excretion than those having a mono-infection. The findings suggest M. tuberculosis and B. fragilis synergism.

Soviet chlorhexedin bigluconicum (CHBG) was used for sputum treatment. 129 sputum specimens were investigated. Among them 45 specimens were bacterioscopically negative. The rest contained low, moderate and high numbers of tubercle bacilli. The sputum was incubated on the Löwenstein-Jensen and Finn-II media. Comparison of two treatment methods (with Na3PO4 and CHBG) showed that CHBG had a more sparing effect on tubercle bacilli. The most marked effect was observed with incubation of oligobacillar materials and materials containing low numbers of tubercle bacilli. The highest differences were revealed when the materials were incubated on the Löwenstein-Jensen medium. It was shown that isolation of the cultures from oligobacillar materials increased by 4-14.3 per cent. The growth intensity and rate were higher. The majority of the cultures grew in 3-4 weeks whereas with using Na3PO4 part of the cultures grew during the period between the 6th and the 8th weeks. When the number of tubercle bacilli was high irrespective of the treatment procedure the number of the grown cultures was the same. The germination level was low and amounted to 3.9 per cent after treatment with Na3PO4 and to 2.3 per cent after treatment with CHBG. The method using CHBG is simple, economic and valuable in laboratory practice.

The efficacy of a chemotherapeutic effect of flurenizid, a new antituberculous drug, was experimentally demonstrated. Flurenizid is an aromatic heterocyclic derivative. As estimated on the models of hematogenic disseminated and destructive pulmonary tuberculosis, a chemotherapeutic dose of the drug was 20 and 50 mg per kg body weight of an animal. Phagocytotic stimulation and fibrillogenetic intensification represent one of the mechanisms of its action. The most pronounced efficacy of flurenizid was noted when it was combined with isoniazid.

The activity of aspartate and alanine aminotransferases in mycobacteria of different specific affinity and the degree of resistance to antituberculous drugs were studied. Differences in alanine and aspartic acid transamination processes taking place in mycobacteria with different biological properties were demonstrated.

The results of the present investigation indicate that antituberculosis therapy for a period of 6 months leads to qualitative changes in M. tuberculosis population. This is manifested by the appearance of the filterable forms of M. tuberculosis in pathological material. At the same time these forms retain the initial pathogenicity of M. tuberculosis and induce not only tuberculous, but also nonspecific inflammation. Among the population of these filterable forms organisms carrying the genetic information of the species and capable of replication processes have been detected.

Two series of experiments were made to develop experimental cavernous tuberculosis of the lungs in guinea-pigs. For preliminary sensitization, the Freund adjuvant was used in the first series of experiments, whereas the BCG vaccine was employed in the second series. The cavity was formed after the culture of H37Rv mycobacteria was inoculated into the lungs of the sensitized animals. The animals vaccinated with BCG developed cavernous tuberculosis with a three-layer cavity wall and with the multiplication of the bacterial population. The animals sensitized with the Freund adjuvant after the mycobacterial culture was inoculated into the lungs demonstrated changes of the caseous pneumonia type to form lung tissue destruction. Attempts to develop the three-layer wall with the latter method ended in failure. The application of BCG vaccine enabled the optimal model of cavernous tuberculosis to be obtained.

The specific features of bacterial excretion by patients with pulmonary tuberculosis in the process of chemotherapy, depending on the duration of treatment, have been studied, and the time-course of the excretion of ultramicro forms of mycobacteria by patients with and without caverns in the lungs in the process of chemotherapy has been followed. The results of the detection of M. tuberculosis ultramicro forms with the use of the biological and bacteriological methods indicate that both these methods are highly effective and informative. The method of the direct reversion of ultramicro forms into coccoid ones in Shkol'nikova's culture medium with 10% of plasma added has proved to be the simplest. The injection of sputum filtrates containing filter-passing (ultramicro) forms of mycobacteria into experimental animals induced the development of specific minor tuberculous inflammation of a productive character without the caseation of granulomas or progressing; such inflammation coursed as a latent lympho-hematogenous process.

Two series of experiments were carried out using 66 guinea pigs infected with tuberculosis. In the first experiment generalized tuberculosis was stimulated by means of subcutaneous administration of Mycobacterium tuberculosis H37RV strain at a dose of 0.0001 mg. In the second experiment destructive form of tuberculosis was developed after intrapulmonary administration of the bacteria (1 mg) followed by VCG vaccination. In both experiments the animals were divided into groups with spontaneous form of the infection and the group of animals treated with rifamycin and isoniazid. Total protein, elastolytic activity, cathepsins B and D, free antitryptic activity were estimated in lung homogenates. Generalized and destructive forms of tuberculosis were shown to induce an increase in content of soluble protein as well as in cathepsin D, elastolytic and antitryptic activities in tissues. Destructive form of tuberculosis was characterized by more than 4-fold increase in elastolytic activity and by a distinct activation of cathepsin B. The antibacterial drugs used affected only slightly the biochemical patterns studied in lung tissue.

The experimental study made on 185 guinea pigs revealed that during the chemotherapy of destructive tuberculosis the typical bacterial forms of M. tuberculosis rapidly disappeared, but the cavities of destruction were retained and the presence of the filter-passing forms of M. tuberculosis, along with antigenemia and specific cell-mediated and humoral immunity could be detected. The use of repeated biological subculturing resulted in the reversion of the filter-passing forms of M. tuberculosis to their typical bacterial forms; simultaneously the increase of antigenemia (from subculture to subculture) and the enhancement of immune responsiveness occurred.

A comparative study on the therapeutic value of the II line drugs (ethoxide, prothionamide, cycloserin) and bacteriostatics (ethambutol anf rifampicin), with their action upon experimental tuberculosis produced by streptomycin- or isoniazid-resistant mycobacteria of the bovine type, was carried out. This showed rifampicin and ethambutol to be mose effective. Ethoxide and prothionamide proved more efficient when acting upon isoniazid-resistant strains. Cycloserin turned out to be virtually inactive with regard to both streptomycin- and isoniazid-resistant mycobacteria.

Pathological material, i. e. bronchoalveolar lavage fluid (BALF) and blood plasma from patients with various pathological processes in the lungs was studied. Eighty seven patients with sarcoidosis of the respiratory organs were subjected to complex microbiological investigation and ultrasmall forms of tubercle bacilli were detected in BALF of 67 (77 per cent) of them. Histological examination revealed alveolitis and granulomas of lymphoid-macrophagal nature in the lungs of the experimental animals exposed to the material from the sarcoidosis patients.