[The detection of medication sensitivity of M.tuberculosis isolated from patients with tuberculosis with multiple medication resistance in testsystem "Sensititre MycoTB".]

The test-system "Sensititre MycoTB" was applied to analyze 159 cultures of M.tuberculosis isolated from patients with tuberculosis. According data of investigation in "Bactec 960" they are identified as cultures with multiple medicinal sensitivity. The detailed analysis of results of application of mentioned test-system (mycoTB) demonstrated that significant part of cultures with multiple medicinal sensitivity is sensitive to anti-tuberculosis pharmaceuticals. The significant part of cultures is characterized by "intermediate" sensitivity/resistance than can be established by mentioned test-system. This data permits enlarging possibilities of chemotherapy of tuberculosis.

[DETERMINATION OF CRITICAL CONCENTRATION OF CHEMOTHERAPY DRUGS FOR MYCOBACTERIUM TUBERCULOSIS SENSITIVITY EVALUATION USING Sensititre MyCoTB TEST SYSTEM].

AIM: Determination of critical concentration for chemotherapy drugs, widely used for tuberculosis treatment, for use in Mycobacterium tuberculosis drug sensitivity results evaluation by Sensititre MycoTB test-system. MATERIALS AND METHODS: Minimal inhibiting concentration (MIC) of isoniazid, rifampicin, streptomycin, ethambutol, amikacin, kanamycin, ofloxacin and moxifloxacin against conditionally sensitive and conditionally resistant strains of tuberculosis mycobacteria (TBM), isolated from various diagnostic material, obtained from patients with various forms of lung tuberculosis being treated in MCSPCTC hospital and dispensaries for tuberculosis control of Moscow, were studied in Sensititre MycoTB test system. RESULTS: Critical concentration of chemotherapy drugs for MycoTB test system was determined as a result of the obtained MIC values analysis as the minimal concentration that suppressed growth of 95% of sensitive strains and does not obstruct growth of 95% of resistant. The following MIC values were established: streptomycin--1.0, isoniazid--0.25, rifampicin--1.0, ethambutol--4.0, ofloxacin--2.0, moxifloxacin--0.25, kanamycin--2.5 and amikacin- 1.0 µg/ml. CONCLUSION: The developed critical concentration of the mentioned preparations is currently used for evaluation of sensitivity/ resistance of TBM clinical isolates from MCSPCTC.

[Comparing performance of "TB-BIOCHIP", "Xpert MTB/RIF" and "genotype MTBDRplus" assays for fast identification of mutations in the Mycobacterium tuberculosis complex in sputum from TB patients].

The frequency of mutations causing drug resistance in MTB isolates were studied in the respiratory material obtained from TB-patients in the Moscow Region. In izoniazid-resistant isolates, the most prevalent mutation was found to be the Ser315Thr substitution in the katG gene (15.8%) whereas the most frequent mutations in multidrug-resistant isolates were Ser531Leu and Ser315Thr in the rpoB and katG genes (26.3%), or a combination of these two substitutions with a T15 mutation in the inhA gene (5.3%). We compared performance of three molecular assays--"TB-BIOCHIP" ("BIOCHIP-IMB", Ltd, Russia), Xpert MTB/RIF ("Cepheid", USA) and GenoType MTBDRplus ("Hain Life-science", Germany), with the efficiency of luminescent microscopy, and phenotypic drug-suscepibility testing in an automated system BACTEC MGIT 960 (Becton, Disckinson and Company, USA). Xpert MTB/RIF, TB-BIOCHIP and GenoType MTBDRplus detected MTB in sputum in 92, 78 and 49% of all culture-positive cases, respectively. The agreement between standard cultural data and molecular DST results for Xpert MTB/RIF (resistance towards rifampicin), for TB-BIOCHIP and GenoType MTBDRplus (resistance towards rifampicin and izoniazid) amounted to 100, 97 and 100% respectively. Summing up, Xpert MTB/RIF was concluded to be the most efficient assay for primary detection of MTB, whereas the TB-BIOCHIP was shown to be the only molecular assay sensitive enough for simultaneous detection of MTB DNA and for revealing multidrug resistance in sputum (i.e. resistance to both first-line anti-TB drugs, rifampicin and izoniazid).

[Isolation and identification of non-tuberculosis mycobacteria].

Seeding of 117,579 samples of clinical materials at the Central Microbiological Laboratory, Moscow Research and Practical Centre of Tuberculosis, resulted in isolation of 15,786 (13,4%) mycobacterial cultures, with 15,333 (97.1%) ones being identified as M. tuberculosis complex and 490 (3.1%) as non-tuberculosis mycobacteria (NTM). Samples collected in the Moscow region were dominated by MAC, M. kansasii, M. xenopi, and M. fotuitum. Cultivation on solid and liquid media in an automated regime was shown to be optimal for isolation of NTM. Comparison of mycobacteria identification by microbiological methods and by high performance liquid chromatography revealed excellent agreement between the results (96.1%), with the latter technique taking less time to obtain them. Agreement between identification of acid-resistant mycobacterial cultures using the MAIS-dif test system and microbiological methods was estimated at 93.5%. IMS biochips may also be used to detect species composition of myobacteria because only one case of disagreement with the results of microbiological methods was documented in the study of 108 cultures by the two techniques.

[Analysis of genetic predisposition to pulmonary tuberculosis in native Russians].

Tuberculosis (TB) is one of the most important concerns of public health. There is evidence suggesting that genetic status is responsible for predisposition to infectious diseases including TB. To determine genetic risk factors of TB development, the frequencies of polymorphisms of genes CYP1A1, CYP2D6, CYP2C9, CYP2C 19, GSTT1, GSTM1, NAT2, MDR1, and NRAMP1 in 73 TB patients and 352 healthy individuals were determined by allele-specific hybridization using microarray technology. The TB patients have shown a significant increase in the frequency of the null GSTT1 genotype (OR = 3.26, 95% CI = 1.91 - 5.55, p = = 0.000028) as well as the double null GSTT1/GSTM1 genotype (OR = 4.05, 95% CI = 2.14 -7.65, p = = 0.000034) compared to the group of healthy donors. It was shown that the NAT2*5/*5 genotype in combination with the "null" GSTT1 and the double "null" GSTT1/GSTM1 genotypes was observed significantly more often in the TB patients than in the control sample. Thus the examined GSTT1, GSTM1 and NAT2 gene polymorphisms may potentially alter the risk of TB development in ethnic Russians and are of interest for further research using larger cohorts of patients.

[The detection rate of different species of non-tuberculous mycobacteria in Moscow].

The centralized microbiological laboratory of the Moscow Scientific-and-Practical Center for Tuberculosis Control (MSPCTC) conducted studies to isolate and identify mycobacterial cultures from clinical materials (sputum, bronchial and bronchoalveolar lavages) that had come from Moscow tuberculosis facilities in August 2005 to November 2008. The isolated mycobacterial cultures were identified by microbiological studies, high-performance liquid chromatography, and restriction fragment length polymorphism analysis of the hsp65 gene by means of the MAIS Dif test system developed at the MSPCTC. During the investigation, 117579 inoculations were made; 15786 (13.4%) of mycobacterial cultures were isolated, which included 15333 (97.1%) M. tuberculosis complex (MAC) cultures and 490 non-tuberculous mycobacteria (NTM) ones that amounted to 3.1% of the total number of the mycobacterial cultures isolated in this period. Two thirds of mycobacteria were referred to as slowly growing NTM (according to the Runyon classification; among them there were predominant MAC (32.40%), M. kansasii (14.7%), and M. xenopi (13.9%). Among the rapidly growing mycobacteria, M. fortuitum (21%) and M. chelonae/abscessus complex (6.5%) were isolated. MAC was frequently detected in all the examined administrative districts of the city However, among the slowly growing NTMs, M. kansasii was identified in the South and West districts even at a greater rate; M. xenopi ranked first in the West district. Among the rapidly growing NTM, M. fortuitum was most common in all districts. M. chelonae/abscessus complex was next in the East district (these NTMs were undetectable in other districts).

[Effectiveness of new diagnostic drug Diaskintest in children for tuberculosis diagnostic].

DST was ascertained to have a high sensitivity in virtually all patients with tuberculosis and a positive reaction was first noted in the infected. With stabilization and regression, the response to DST was much less pronounced than that in clinical and primary infection (that to the Mantoux test being more evident). DST showed its use as a marker of active tuberculosis not only in its local forms, but also in latent tuberculous infection. This makes it possible to apply DST when preventive treatment is performed. The agent may be used to monitor the progress of treatment. DST has a high specificity--healthy individuals had a negative response to DST while the Mantoux test was positive in many cases. The high specificity of DST was suggested by the fact that the persons vaccinated with (this caused BCG ostitis) had a negative reaction to DST while the Mantoux test was positive in all cases BCG-vaccinated BCG. The findings warrant the use of DST for the differential diagnosis of tuberculosis and BCG-associated complications and the possibility of differentiating postvaccinal and infection allergy in children and adolescents.

[Clinical trials of the new skin test Diaskintest for the diagnosis of tuberculosis].

A new reagent for a skin test given the name Diaskintest has been designed for the screening diagnosis of tuberculosis and preclinical and clinical trials conducted. Preclinical trials were carried out on 315 laboratory animals (guinea-pigs, albino mice). The reagent Diaskintest was ascertained to be nontoxic, to have no sensitizing properties, to be safe and specific, and to induce no positive reactions in BCG-vaccinated animals and healthy guinea-pigs. Its specific activity was comparable with that of the national reference--purified tuberculin PPD-L-2. With progression of tuberculous lesions, the guinea-pigs showed higher responses to Diaskintest dilution and the BCG-vaccinated animals lacked responses to Diaskintest with increased delayed type hypersensitivity. The clinical trial was permitted by the Federal Service for Surveillance in Health Care and Social Development of the Russian Federation. Clinical trials were conducted in 150 persons. The safety, specificity, sensitivity of Diaskintest were first examined in the clinical studies and its action was compared with the results of tuberculin skin test (Mantoux test) with 2 TE of PPD L-2. Diaskintest was ascertained to be highly sensitive when given in a dose of 0.2 microg in 0.1 ml. In patients with active tuberculosis and new cases of Mycobacterium tuberculosis infection, the agent induced a positive skin reaction (a papule of more than 10 mm) in 98-100% of cases (p < 0.05). The agent caused no reaction associated with BCG vaccination. The specificity of the test was 93-100% with 95% significance. The rate of overexuberant reactions (vesicular necrotic changes, lymphangitis, and lymphadenitis) was 4-14% with 95% significance. Tuberculosis patients with significant immunopathological disorders might have no skin sensitivity to Diaskintest, as to PPD L-2 (a negative test). The findings substantiate the use of Diaskintest for mass epidemiological surveys for the differential diagnosis of tuberculosis and BCG vaccination-associated complications. The agent may be also used to evaluate the activity of the process in patients with tuberculosis and the efficiency of treatment in combination with other methods and to make a differential diagnosis of tuberculosis.

[Tuberculosis and HIV infection: detection, follow-up, treatment: Moscow data].

The paper describes the follow-up and treatment of patients with tuberculosis concurrent with HIV infection in Moscow in 2004-2005. Major epidemiological parameters, such as morbidity, mortality, and prevalence of this comorbidity, are given. Analysis of these indices suggests that the epidemic situation associated with tuberculosis concurrent with HIV infection became worse in the past 2 years. As compared with 2004, in 2005 the number of such patients increased from 294 to 445, including that of first detected patients rose from 123 to 174. In this group of patients, there was a preponderance of young males aged 29 to 39 years. Most patients with this pathology suffered from drug addiction and alcoholism and other concomitant diseases. The bulk of them were unemployed and disabled. In the HIV-infected, the clinical forms of tuberculosis were severe with a predominance of acute and disseminated processes; the rate of drug resistance, including multidrug resistance, was high, which made treatment difficult and resulted in high mortality.

[Criteria for clinical recovery and the evaluation of the efficiency of treatment in patients with respiratory tuberculosis in Moscow].

Criteria for clinical cure have been analyzed and the efficiency of treatment evaluated in patients with respiratory tuberculosis in Moscow. If today's official criteria for the efficiency of treatment have been ascertained to preserve in treating the first detected patients, a phthisiological service will completely lose an objective control of this section of work in several years. The only criterion that gives some idea of the results of treatment in first detected patients is currently to transfer them to dispensary register group III. However, when the results of interim treatment stages are unknown, conditions are created for utter arbitrariness in the time of the transfer (this happens at present). It is necessary to hold today's existing major criteria (cessation of bacterial isolation and closure of decay cavities) for evaluating the efficiency of treatment in first detected patients (who are largely inpatients) and to add one more criterion (disappearance of Xray signs of active tuberculosis manifestations); moreover, the magnitude of major or minor residual tuberculous changes should be, of course, taken into account after a basic course of chemotherapy.

[Potentialities of laboratory diagnosis in tuberculous meningitis].

The results of examination of 84 patients with tuberculosis of the central nervous system were used to make comparative clinical and laboratory studies. They revealed that lymphocytosis detected in the cerebrospinal fluid (CF), particularly the prevalence of lymphocytes (more than 50%), and decreased levels of chlorides in the cytogram were of value in the comprehensive diagnosis of tuberculous meningitis. The detection of CF Mycobacterium tuberculosis (MBT) (applying the whole currently available set of methods) is an absolute criterion for the diagnosis of tuberculous meningitis (however, with, unfortunately, few number positive results). The detection of mycobacterial DNA, antigens, and tuberculosis antibodies is an impotent component of a diagnostic complex for tuberculous meningitis. The determination of cytosis, protein, glucose, chlorides, lymphocytic subpopulations, soluble gamma-interferon mediators, mycobacteria, DNA, MBT antigens, and tuberculosis antibodies in SF is essential in treating tuberculous meningitis.

[New technologies in the determination of drug susceptibility in Mycobacterium tuberculosis]. / Novye tekhnologii opredeleniia lekarstvennoi chuvstvitel'nosti Mycobacterium tuberculosis.

A variety of mutations in the genes rpoB, katG, inhA, ahpC, kasA was studied by using different molecular biological methods (conformational polymorphism of single-chain fragments, heteroduplex analysis, biochips) in rifampicin- and isoniazid-resistant Mycobacterium tuberculosis (MBT) strains isolated from patients with pulmonary tuberculosis. Twenty-nine mutation combinations were identified in the MBT strains. The use of biochips is the most promising method for identifying the type of mutations responsible for the simultaneous resistance to rifampicin and isoniazid. Detection of several MBT strains in one patient requires the use a combination of molecular biological and microbiological studies.

[Influence of social factors on tuberculosis mortality, effectiveness of sociomedical preventive measures in Moscow in the 20th century]. / Vliianie sotsial'nykh faktorov na smertnost' ot tuberkuleza, éffektivnost' mer mediko-sotsial'noi zashchity v Moskve v 20m stoletii.

The correlations between the time series of weather values of sociodemographic, socioeconomic, and epidemiological parameters of tuberculosis (death rates) in the 20th century were studied. Analysis of population indices confirmed the association of migratory processes and the intensity of the tuberculosis epidemic situation, as well as the significance of these processes for Moscow, the necessity of their notification for assessing the sociodemographic situation to plan tuberculosis-controlling measures. The study has indicated that there is a relationship of the salary/wage that determines the income of the bulk of the population to tuberculosis mortality rates and that regulation of a migratory process, population density, and improvement of living conditions are of considerable importance in the prevention of this disease. Comparative analysis of the data in the periods when the whole contemporary rather effective complex of tuberculosis-controlling measures (1953-2000) and when the above complex were only formed (1910-1953) has demonstrated the economic efficiency of the above measures. It is stated that the system of sociomedical protective measures is a real factor that may rather effectively withstand the poor social conditions promoting the spread of tuberculosis.

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.