[Development of criteria for assessing the quality of microbiological studies and for evaluating their efficiency in tuberculosis service facilities and the general health network].

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 state-of-the-art of Russia's Laboratory Tuberculosis Diagnosis Service: basic problems and ways of their overcoming].

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).

[The quality of bacteriological detection and determination of drug sensitivity of Mycobacterium tuberculosis by the participants of the External Clinical Laboratory Study Quality Assessment made by the Federal System in 2002-2003].

The paper presents the results of the assessment of the quality of cultural detection and determination of Mycobacterium tuberculosis (MBT) sensitivity at Russia's public health laboratories, which has been made by the Federal System for External Assessment of the Quality of Clinical Laboratory Studies in 2002-2003. The "Bacteriological Detection" Section involved 102 laboratories, of them 57 laboratories also participated in the section "Detection of Drug Resistance". Sixty-three laboratories operated as part of specialized tuberculosis-controlling service facilities and 39 laboratories worked as part of general medical care therapeutic-and-prophylactic institutions. A set of 5 lyophilized samples containing 2.5 x 10(6) M. tuberculosis H37Rv (n = 1); 2.5 x 10(4) M. tuberculosis H37Rv (n = 2); M. fortuitum (n = 1); E. coli in the mixture with autoclaving-killed M. tuberculosis H37Rv (n = 1). 77% of the laboratories could detect in the cultural sample with a high content of M. tuberculosis, by obtaining a correct result; 85% revealed MBT if only in every three MBT-containing samples; 15% of the laboratories could not find MBT in none of the three positive samples. 12% of the laboratories took the growth of microorganisms in the M. fortuitum-containing sample for that of M. tuberculosis. 58% of the laboratories obtained correct results if only in one sample containing M. tuberculosis H37Rv; 42% reported the detection of resistance to one or several antituberculous drugs in the samples containing the drug-sensitive strain H37Rv (a false drug resistance). All the results of determination of drug resistance (DR) in the M. fortium-containing sample were correct. The findings suggest that it is necessary to increase the professional competence of laboratory personnel to the level that ensures the adequate of fulfillment of the standard protocols for detecting MTB and determining DR, which have been introduced by the Ministry of Health of the Russian Federation through its order No. 109 on March 21, 2003.

Typing of Mycobacterium tuberculosis strains resistant to rifampicin and isoniazid by molecular biological methods.

Mutations in the rpoB, katG, inhA, oxyR/ahpC genes in rifampicin- and isoniazid-resistant M. tuberculosis strains isolated from residents of Moscow, Astrakhan', and Moldova Republic were studied by molecular biological methods (heteroduplex analysis, single strand conformational polymorphism, biochips). Twenty-five combinations of mutations were detected. Some differences in the type distribution of detected mutations were found. The use of biochips is the most perspective method for determining the type of mutation.

[Comparison of nitrate reductase and automatic BACTEC MGIT 960 AST techniques for determining the drug sensitivity of mycobacteria tuberculosis]. / Sravnenie nitratreduktaznogo i avtomatizirovannogo BACTEC MGIT 960 AST metodov opredeleniia lekarstvennoi chuvstvitel'nosti mikobakterii tuberkuleza.

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.

[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.

[Molecular genetic methods for the detection of rifampicin-resistant Mycobacterium tuberculosis strains]. / Molekuliarno-geneticheskie metody vyiavleniia rifampitsinrezistentnykh shtammov Mycobacterium tuberculosis.

RCR-heteroduplex (GDA) and chip methods were used to detect rifampricin-resistant (RR) and rifampicin-sensitive (RS) Mycobacterium tuberculosis (MTB) in the samples from patients (sputum) and in the clinical isolates of MTB from these patients (MB/BacT liquid medium and Lowenstein Jensen's (LJ) solid medium. The efficiency of detecting RR and RS of MTB (from the sputum) is 100 and 92.3% in the chip and GDA tests, respectively. Correlations between GDA (sputum) and drug test (LJ) were 91.7%, that of chip (sputum) and drug test LJ, 88.5%, chip (sputum) and chip clinical isolates (LJ), 100%. The efficacy of GDA and chip in the detection of RR of MTB strains is under discussion.

[Automated methods of culture determination of M. tuberculosis in liquid media]. / Avtomatizirovannye metody kul'tural'nogo opredeleniia M. tuberculosis na zhidkikh sredakh,

A hundred and seventy respiratory samples from patients with different forms of tuberculosis were used to test the efficiency of the automatic liquid culture systems BACTEC MGIT 960 and MB/BacT with inoculation into the standard dense media. All these media provided 47 M. tuberculous isolates, of them 41 (87.2%), 38 (80.9%), and 76.6% on the BACTER 960, MB/BacT, and dense media, respectively. The average time of detection of mycobacterial growth by means of automatic systems was much shorter and equal to 10.7 days on the BACTEC 960 and 18.7 days on the MB/BacT versus 33.2 days on the standard dense medium. In terms of their sensitivity and detection rate, the automatic systems were superior to the dense media widely used in laboratory practice.

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.

[Functional characteristics of the antioxidative system in mycobacteria grown on perfluorodecalin-modified media]. / Osobennosti funktsionirovaniia antiokislitel'noi sistemy mikobakterii, vyrashchennykh na sredakh, modifitsirovannykh perftordekalinom.

The growth of mycobacteria on perfluorodecalin-modified media was shown to be accompanied by distinct alterations in the activity of the antioxidant enzyme system in M. bovis BCG and M. lufu. In M. bovis BCG the levels of glutathione transferase and glutathione peroxidase-hydrogen peroxidase activity are decreased by 45.47% and 100.88%, respectively. In M. lufu, on the contrary, the level of superoxide dismutase is increased by 42.23%, with no changes observed in the levels of glutathione transferase and glutathione peroxidases. The data obtained suggest physiological heterogeneity of mycobacteria and, thus, open prospects for the differential approaches to the problem of increasing the efficacy of in vitro cultivation of various mycobacterial species, including M. leprae.