[The structure of pathogenic germline variants in colorectal cancer in Moscow patients]. / Struktura patogennykh germinal'nykh variantov pri kolorektal'nom rake v vyborke patsientov Moskvy.

OBJECTIVE: Describe the structure of pathogenic germline variants and clinical and anatomical features in colorectal cancer patients in Moscow. MATERIAL AND METHODS: The whole genome sequencing results of patients with suspected hereditary cancer syndrome were evaluated. All identified genetic variants were validated using Sanger sequencing. RESULTS: The study included 238 patients with colorectal cancer, 41/238 (17.2%) patients have pathogenic germline variants associated with hereditary cancer syndromes or increased cancer risk. Lynch syndrome accounts for 8% of all colorectal cancer cases (19/238), and familial adenomatous polyposis - 1.7% (4/238). 5 new genetic variants were described for the first time in a Russian colorectal cancer patients: MLH1 c.1921dup (p.Leu641fs), APC c.2929C>T (p.Gln977Ter), PMS2 c.327del (p.Ala110LeufsTer2), MSH2 c.1857dup (p. Val620CysfsTer24), ATM c.895G>T (p.Glu299Ter). In 197 of 238 patients, no significant variants were identified or variants with an uncertain clinical underlying cause were identified. CONCLUSION: According to the results of the study, an earlier manifestation of a malignant neoplasm and a more frequent occurrence of high-grade carcinomas in the presence of pathogenic germline mutations were noted compared to the group of patients without clinically significant varianrs, while in the group with identified mutations, the frequency of regional and distant metastasis was not increased.

[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).

[Criteria for determining the adaptation of children to studying loads].

Three hundred and two Krasnoyarsk gymnasium students were examined to define criteria for determining the adaptation to high schooling loads. The values of the emotional status and adaptive processes were studied in children with varying resistance and varying mastering capacities; the impact of cardiovascular parameters on their adaptive process was defined The criteria for estimating the adaptation to high schooling loads were the presence or absence of acute chills and neurotic reactions, cardiovascular performance, and psychoemotional status.

[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.

[Comparative data on the use of high performance liquid chromatography to identify mycobacteria isolated on liquid and solid media].

Two hundred and forty-two mycobacterial cultures isolated from clinical materials on the Lwenstein-Jensen solid medium and 208 cultures in the automatic Bactec MGIT 960 system (on the modified Middlebrook 7H9 liquid medium) were examined. Of them, there was M. tuberculosis (85 and 82, respectively), M. kansasii (26 and 24), MAC (46 and 38), M. xenopi (24 and 20), M. fortuitum (26 and 22), and M. chelonae/abscessus complex (20 and 18). Identification of mycobacterial cultures isolated on the solid medium by microbiological assay and high performance liquid chromatography (HPLC) showed the agreement of results of the cultures isolated on the liquid medium in 95.5 and 97.2%, respectively; that by these techniques revealed rapidly growing nontuberculosis mycobacteria in 95.8 and 95.2% of cases, respectively; slowing growing mycobacteria in 91.7 and 97.8%, and M. tuberculosis in 96.5 and 97.6%. Mycobacterial isolation on the Middlebrook 7H9 liquid medium in the automatic Bactec MGIT-960 system takes a shorter time than that on the solid (Lwenstein-Jensen) medium. The microbiological identification of mycobacteria lasts as long as 3 weeks while the use of HPLC reduces its time to 24 hours. The efficiency of HPCL does not depend on whether mycobacterial cultures are isolated on the solid or liquid media.

[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).

[Study of the sensitivity of non-tuberculous mycobacteria isolated on the solid and liquid media to antituberculous agents].

The centralized bacteriological laboratory of the Moscow Scientific-and-Practical Center for Tuberculosis Control Department of Public Health of the city of Moscow, examined the susceptibility of non-tuberculous mycobacteria (NTM) isolated and studied on the solid (Levenstein-Jensen) and liquid (Middlebrook 7H9 in the BACTEC-960 automatic system) media. A total of 159 cultures obtained from the solid medium and 132 from the liquid one were studied; of them there were 4 and 5 M. chelonae cultures, respectively, M. flavescense (n=6 and 4), M. fortuitum (n=34 and 28), MAC (n=52 and 44), M. xenopi (17 and 14), M. kansasii (n=41 and 35). Among the slowly-growing NTM, MAC was found to be resistant to streptomycin, isoniazid, rifampicin, and ethambutol in most cases. M. kansasii was sensitive to all basic antituberculous agents (ATAs) in most cases. M. xenopi was resistant to rifampicin in two thirds and to streptomycin, isoniazid, and ethambutol in a half of cases. Among the rapidly growing NTM, M. fortuitum was resistant to the study basic ATAs in most cases. No significant differences were found in the susceptibility/resistance of NTM to the basic ATAs depending on whether they had been isolated and their susceptibility evaluated on the solid and liquid media. Moreover, the duration of the entire study using the liquid medium was 2-3 times shorter.

[Study of the drug-sensitivity of nontuberculous mycobacteria].

The central bacteriological laboratory of the Moscow Research-and-Practical Center for Tuberculosis Control studied the sensitivity of nontuberculous mycobacteria (NTM) to a number of antituberculous drugs (streptomycin, isoniazid, rifampicin, ethambutol, kanamycin, ethionamide, cycloserine, ofloxacin) by the absolute concentration method (after isolating the cultures on both solid and liquid nutrient media). A total of 160 cultures, including M. chelonae (n = 4), M. flavescense (n = 6), M. fortuitum (n = 34), M. avium complex (MAC) (n = 52), M. xenopi (n = 18), M. kansasii (n = 41), M. marinum (n = 2), M. malmoense (n = 1), M. simiae (n = 1), and M. gastri (n = 1), were explored. MAC was found to be resistant to streptomycin, isoniazid, rifampicin, ethambutol, and ethionamide in most cases, to ofloxacin in a nearly half of cases, to canamycin and cycloserine in a third of cases. In most cases, M. kansasii was sensitive not only to first-, but also second-line chemical drugs. M. xenopi was resistant to rifampicin in two thirds of cases and to streptomycin, isoniazid, ethambutol, and ethionamide in a half of cases. This species of NTM was more sensitive to kanamycin, cycloserine, and oflaxacin. In the majority of cases, M. fortuitum was resistant to streptomycin, isoniazid, rifampicin, ethambutol, ethionamide, kanamycin, and cycloserine and sensitive to ofloxacin.

[Isolation of mycobacteria on different growth media and their identification].

AIM: To compare results of isolation of mycobacteria on different growth media. MATERIALS AND METHODS: From August 2005 to September 2008 Central Bacteriological Laboratory of MSPCTC performed 111,029 inoculations of clinical samples, isolated 14,513 (13.5%) cultures of mycobacteria, of which 14,095 (97.1%) belonged to M. tuberculosis complex and 418 (2.9%)--to non-tuberculous mycobacteria (NTM). RESULTS: Two thirds of isolated NTM belonged to slowly growing NTM (Ranyon classification), of which bacteria from M. avium complex as well as M. kansasii and M. xenopi predominated. M. fortuitum was the most frequently isolated between rapidly growing NTM. CONCLUSION: For isolation and identification of NTM the optimal was inoculation on at least 2 media: solid agar (Middlebrook 7H11) or egg medium and liquid medium (Middlebrook 7H9, in automated system BACTEC MGIT 960), each of which has its own advantages.

[Identification of mycobacteria by high-performance liquid chromatography].

AIM: To compare results of mycobacteria identification by bacteriologic methods as well as by high-performance liquid chromatography (HPLC). MATERIALS AND METHODS: Two hundred and eighty strains of mycobacteria isolated from respiratory specimens and identified by bacteriologic methods and HPLC were studied. RESULTS: It was established that results of HLPC use were highly correlated with results of microbiologic methods of mycobacteria identification: for identification of M. tuberculosis complex the correlation was 97.0%, for nontuberculous (NTM) slowly growing mycobacteria--95.3%, for quickly growing NTM--96.2% (overall--96.1%). Results of identification of mycobacteria by HPLC were ready in significantly shorter time-frame (during 24 hours). CONCLUSION: HLPC method could be recommended for identification of mycobacteria in bacteriologic reference laboratories.

[Rapid identification of mycobacteria using laser fluorescence].

AIM: To develop rapid method of identification of mycobacteria based on laser fluorescence. MATERIALS AND METHODS: Characteristics of laser-induced fluorescence of 19 bacteria species, including 17 species of mycobacteria, were studied. Identification of microorganisms was performed using measurement of spectral-fluorescent characteristics. RESULTS: Library of spectral-fluorescent characteristics of mycobacteria in different concentrations ratios and associations was created, which formed the basis of database for identification of mycobacteria by laser-fluorescent method. Principles of diagnostic algorithm of indication and differentiation of mycobacteria using this method were developed. Effect of myramistin for increasing the intensity of mycobacteria fluorescence, account of the diffracting characteristics of medium for adjustment of spectral characteristics of mycobacteria and processing of data by factor analysis are needed. Efficacy of the method was 80 - 90%. CONCLUSION: Principles of rapid identification of mycobacteria and their associations developed on the basis of laser-fluorescent method are experimentally founded and tested on unknown cultures of mycobacteria and objectively prove the possibility to apply this method for express identification of mycobacteria belonging to M. tuberculosis complex as well as non-tuberculous mycobacteria.

SrTiO(3):Cr nanocrystalline powders: size effects and optical properties.

The crystal structure, optical absorption, and photoluminescence of chromium impurity centers were studied in nanocrystalline SrTiO(3):Cr (0.1 mol%) powders with average particle size within the range 13-100 nm prepared by the Pechini-type polymeric sol-gel method. Only the presence of a cubic perovskite phase of O(h)(1) symmetry was proved for the powders at room temperature, by means of x-ray diffraction. The lattice constant a = 3.910 Å, larger than that of bulk SrTiO(3) crystals (a = 3.905 Å), was found for nanoparticles with the size about 20 nm. The optical absorption edge and the zero-phonon R-line ([Formula: see text]) of luminescence of the octahedral Cr(3+) centers shifted to higher energies with decreasing nanoparticle size. These size effects were regarded as intrinsic to SrTiO(3). An unusual and large temperature shift of the R-line position very similar to the 'dielectric related' one of the bulk crystals was observed for all powders, evidencing their quantum paraelectric behavior. However, the powders with the average particle size about 13 and 20 nm did not reveal completely reproducible behavior of the R-line position at low temperatures. This instability was considered a possible manifestation of a low-temperature phase transition in small enough SrTiO(3) nanoparticles.

[Use of molecular genetic studies to determine the species of the mycobacteria mais and M. tuberculosis complex].

The genus Mycobacterium currently comprises more than 90 species of Mycobacterium, of which a third is able to induce human diseases. With a rise in the incidence of diseases induced by non-tuberculosis mycobacteria, tuberculosis caused by M. bovis that is characterized by a severe cause and a high frequency of poor outcomes cannot be remembered. The species of mycobacteria should be identified to establish a diagnosis and to prescribe adequate chemotherapy. For this purpose, cultural, biochemical, chromatographic, and molecular genetic studies are conducted. The present study using the hsp65 gene restriction fragment length polymorphism test on museum mycobacterial strains and strains isolated from the diagnostic material of patients with suspected tuberculosis by means of Hind61 restrictase has provided a clear differentiation of the restriction profiles of MAIS complex mycobacteria and some other species of non-tuberculosis mycobacteria. To determine the species of representatives of M. tuberculosis complex (M. tuberculosis, M. bovis, BCG M. bovis), the authors have successfully used the test system "TUB-dif" developed at the Institute of Molecular Genetics, Russian Academy of Sciences, by applying the chain polymerase reaction of the senX3-regX3 region.

Identification of mycobacteria of the MAIS Complex and M. tuberculosis by restriction fragment length polymorphism analysis of hsp65 gene.

Restriction fragment length polymorphism analysis of hsp65 gene was performed on museum strains of mycobacteria using Hin6I restrictase. Study of restriction profiles allowed us to distinguish mycobacterial species of the MAIS complex and several strains of nontuberculous mycobacteria.

[Specific antisera to mycobacteria]. / Spetsificheskie antisyvorotki k mikobakteriiam.

Antisera to several Mycobacterium species (M. bovis, M. avium, M. kansasii and M. smegmatis), capable of reacting with homologous antigen diluted not below 1:16000 in the ELISA test, have been obtained. These antisera have been found to cross-react with other Mycobacterium species. Specific antisera to M. bovis, M. avium, M. kansasii and M. smegmatis have been obtained by the multiple exhaustion method on Mycobacterium antigens giving the maximum cross reaction and used for the isolation of antibodies to these Mycobacterium species. In ELISA exhausted antisera produce reactions in the dilution 1:500. Antisera (antibodies) to M. bovis react with the determinant of the antigen with a molecular weight of 25-26 kD, antisera (antibodies) to M. kansasii react with the determinant of the antigen with a molecular weight of 72 kD, those to M. smegmatis react with the determinant of the antigen with a molecular weight of 38 kD and those to M. avium, with the determinant of the antigen with a molecular weight of 16-18 kD.