[The development of laboratory diagnostic of leprosy using polymerase chain reaction].

The purpose of study is to develop techniques of laboratory diagnostic of leprosy on the basis of polymerase chain reaction. The techniques were worked through such as extraction of DNA from various clinical material (bioptates and scarifcate of skin, scrapes from mucous surface of nose and trophic ulcers, blood serum). The systems of oligonucleotides were constructed to 16S pRNA and probe for identifcation of Mycobacterium leprae in real-time format for amplifer DT-96 ("The DNA-Technology", Russia). The optimal regimen of amplifcation was worked out. To control polymerase chain reaction processing a system of internal control was introduced. The evaluation of specifcity of test-system was implemented using 17 strains of various types of mycobacteria and clinical samples from 32 patients with leprosy and 15 healthy individuals being in family contact with patients with leprosy. The 100% sensitivity was established concerning detection of Mycobacterium leprae in bioptates of skin using polymerase chain reaction technique as compared with standard bacterioscopic technique (88.9%). In all other clinical samples, a higher sensitivity of developed test-system on the basis of polymerase chain reaction was detected too. The proposed test-system has higher sensitivity and specifcity that permits to resolve an issue of fast identifcation of Mycobacterium leprae. It can be applied in epidemiological studies at investigation of prevalence of agent of disease.

DMSO-Free Cryopreservation of Human Umbilical Cord Tissue.

Human umbilical cord represents a source of multipotent stromal cells of a supreme therapeutic potential. The cells can be isolated from either fresh or cryopreserved umbilical cord tissues. DMSO is a cryoprotectant most commonly used for preservation of umbilical cord tissues; however, cyto- and genotoxicity of this compound is evident and well documented. In the present study we performed successful cryopreservation of the umbilical cord tissue using other cryoprotectants: propylene glycol, ethylene glycol, and glycerol. Of these, 1.5 M ethylene glycol and 20% glycerol turned out to be the best in terms of the preservation of living cells within the frozen tissue, early onset of migration of these cells out of the thawed explants, and overall efficacy of multipotent stromal cell isolation. Cryobanking of tissues can improve availability of multiple cell products for medical purposes and promote the development of personalized medicine.

Association of polymorphisms of HLA-DRB1 and TNF-308 G/A with radiographic joint damage in patients with early rheumatoid arthritis with high inflammatory activity, treated according to the principle of "Treat to target" (REMARKA study).

AIM: To clarify the association between HLA-DRB1 and TNFα (-308G>A) genes polymorphism and joint destruction/further progression during 12 months of the follow-up period (FUP) in patients with early (<6 months), active, predominantly antibodies to cyclic citrullinated peptide (ACCP) and rheumatoid factor (RF)-positive rheumatoid arthritis (RA) treated according to "Treat to target" strategy. MATERIALS AND METHODS: The study included 85 patients with early RA and duration of symptoms <6 months. All patients were initially assigned to subcutaneous methotrexate (MTX) with rapid dose escalation to 20-25 mg/week. Combination MTX + biological therapy, mainly adalimumab, was used when MTX was ineffective. Joint destruction was assessed by Sharp-Van der Heijde modification scoring method at baseline and after 12 months FUP. Real time polymerase chain reaction (PCR-RT) was used for TNFα gene polymorphism (-308G>A) genotyping. Low resolution PCR-RT with subsequent sequence-based typing of *04 were performed to study HLA-DRB1 gene polymorphism. The HLA-DRB1*01, *04:01, *04:04, *04:05, *04:08, *10 alleles were categorized as SE+ (Shared Epitope) alleles. RESULTS: As for TNFα gene polymorphism, it was demonstrated that the number of narrowings and total Sharp score values were almost twice as high at baseline in GG genotype carriers as compared to GA genotype carriers (р<0,005, and р<0,004 respectively). Similar association was found after 12mo FUP. The progression of joint destruction, assessed as the change (∆) in the number of erosions, joint space narrowings and the total score, was statistically significantly associated with HLA-DRB1*(SE) genotypes: the carriers of SE (SE+/SE+) double-dose had more advanced progression as compared to (SE+/SE-)/(SE-/SE-) carriers (р<0,028, р<0,019, р<0,035 respectively). CONCLUSION: Our data suggest that HLA-DRB1 (SE+) gene and TNFα (-308G>A) polymorphisms are associated with the progression of radiographic joint destruction in early, active RA patients managed according to "Treat to target" stratagy.

[Conditionally pathogenic microorganisms in patients with bisphosphonate jaw osteonecrosis].

The objective of the study was to define treatment strategy in cases of facial bones bisphosphonate induced osteonecrosis based on the study of the role of conditionally pathogenic oral microorganisms. Three typical clinical cases of bisphosphonate osteonecrosis of the facial bones were analyzed and 15 conditionally pathogenic oral microorganisms were identified in these patients using real-time PCR in saliva, wound and bone samples. A comparative analysis was carried out with purulent-inflammatory diseases of maxillofacial area. The study results proved an important role of conditionally pathogenic microorganisms of the oral cavity in the development of osteonecrosis of the facial bones. Wide range of bacterial species was identified in osteonecrosis of the facial bones patients. While bone tissue is most exposed to microbial communities, surgical treatment results in effective rehabilitation for a long period.

[ASSOCIATION OF POLYMORPHISM GENES OF SURFACTANT PROTEINS IN PATIENTS WITH INFLUENZA].

AIM: Evaluate role of gene polymorphisms of surfactant proteins in susceptibility and severity of influenza infection course in representatives of Moscow population. MATERIALS AND METHODS; 320 influenza patients, infected with various influenza virus strains, and 115 healthy individuals (control group),, were included into the study. Human DNA samples genotyping for determination of SFTPA2 gene rs1965708 and rs1059046, SFTPB gene rs1130866 polymorphisms was carried out using a modified method of "adjacent samples". RESULTS: Most of the individuals of the control group and influenza patients are carries of alleles and genotypes rs1965708 and rs1059046 of SFTPA2 gene, rs1130866 of SFTPB gene, that have, based on scientific literature data, shown association with severe course of influenza A(H1N1) pdm09 and other inflammatory diseases of the respiratory tract. Generally, significant differences in frequency of occurrence of unfavorable genotypes CC rs1965708, AA rs1059046 of SFTPA2 gene and CC rs1130866 of SFTPB gene in influenza patients in comparison with individuals of the control group were not detected, that gives evidence on a high (from 19 to 51%) prevalence of these genotypes in the studied population. Allele C and genotype CC rs1965708 of SFTPA2 gene, allele A and genotype AA rs1059046 of SFTPA2 gene, allele C and genotype CC rs1130866 of SFTPB gene did not shown an association with severe course of A(H1N1) pdm09 influenza. The following pathology registered in most (88%) of the patients with severe course of influenza A (H1N1)pdm09: diseases of cardiovascilar (44%), endocrine (36%) and respiratory (12%) systems. CONCLUSION: Because in most of the deceased patients due to severe course of A (H1N1)pdm09 influenza, diseases of cardiovascular, respiratory and endocrine system were detected, and an association of unfavorable disease outcome with the studied genetic markers was not detected, dominating risk factor of development of severe course and lethal outcome for A(H1N1)pdm09 influenza in the studied cohort was comorbidity.

[Efficacy of anti-neuraminidase drugs application during and after an influenza pandemic].

The emergent 2009 A(H1N1) pandemic brought into acute focus the problem of choosing the most effective anti-influenza drugs for successive influenza infection spreading control. Oseltamivir and zanamivir, influenza virus neuraminidase inhibitors (NAIs), were recommended by the WHO experts for the treatment and prevention of influenza, including that caused by pandemic strains. A major concern regarding the use of specific antiviral compounds is the emergence of the drug-resistant strains. Oseltamivir carboxylate and zanamivir IC50 values were equal to 0.3-5.2 microM for the most of A(H1N1)pdm09 pandemic strains and 1.6-8.6 microM for the strains of influenza B virus in cell-based ELISA assay (2009-2010 season). All the studied strains of influenza A(H1N1 ) pdm09 (151) and B (22) viruses were sensitive to NAIs (2009-2011 seasons). For the first time in Russia oseltamivir-resistant A(H1N1) pdm09 influenza virus was isolated from the patient on the 5th day of a treatment course of this drug.

[The specific features of the cocirculation of influenza viruses in the 2010-2011 postpandemic period according to the results of activities of the D. I. Ivanovsky Research Institute of Virology, Ministry of Health and Social Development of Russia].

The paper gives the results of monitoring the circulation of influenza viruses in the 2010-2011 season, that covers the second year of circulation of pandemic A(H1N1)v virus strains, and their interaction with seasonal A (H3N2) and B strains. Unlike the previous season, the beginning of an increase in morbidity was recorded in January 2011; its peak in the most of contiguous areas was noted at 5-7 weeks of 2011, with its further decline to threshold levels at week 11 of 2011. Preschool and school children were most involved in the epidemic process. Three influenza virus strains (A(H1N1)v, A(H3N2), and B) were found to circulate. Differences were found in the level of participation of the isolated strains in individual areas of the Russian Federation. Detailed typing of the isolated strains determined the compliance of the vast majority of them with vaccine viruses. The pandemic influenza A(H1N1)v virus strains retained their susceptibility to oseltamivir and were resistant to rimantadine. The participation of non-influenza acute respiratory viral infection pathogens was estimated as follows: 11.9% for parainfluenza viruses, 5.9% for adenoviruses, and 3.5% for PC viruses, and 0.7% for pneumonia Mycoplasma, which was comparable with the previous epidemic seasons.

[Immunogenetics of type 1 diabetes mellitus--from fundamental ideas to medical practice].

The review of studies of Russian researchers on theoretical and practical aspects of genetic predisposition to type 1 diabetes associated with immunity: HLA and not HLA genes. Most important for practical public health outcomes are evidence that HLA-genetic predisposition to type 1 diabetes is associated with the DRB1-genotype, consisting entirely of variants DRB1-genes associated with the development of T1D. It was also established that CTLA4 gene has an independent predictive value for T1D.

[The development and testing of a molecular biological test system for DNA detection of anthrax pathogen by real-time polymerase chain reaction assay].

The paper presents the results of the development and testing of a molecular biological test system for DNA detection of anthrax pathogen (Bacillus anthracis) by real-time polymerase chain reaction assay. The test system has shown high sensitivity, specificity, and reproducibility of results of analysis, as exemplified by aqueous suspensions of daily agar cultures of Bacillus anthracis strains, related and heterologous species of microorganisms, and clinical materials of experimental animals. There is evidence for the persistence of the basic characteristics of the test system when stored at 22 +/- 2 degrees C for 12 months.

[Molecular genetic studies of the susceptibility of epidemic influenza A(H1N1) virus strains isolated in the 2006-2009 seasons in Russia to oseltamivir (Tamiflu)].

Oseltamivir (Tamiflu) is recommended by WHO experts as a drug to treat and prevent of influenza and to create stocks if its new pandemic variant occurs. The susceptibility of influenza viruses to oseltamivir was studied by polymerase chain reaction-based techniques detecting specific mutations in the neuraminidase gene. The increase in the number of oseltamivir-resistant influenza viruses, isolated from the Russian Federation, with type 1 neuraminidase H274Y mutation from 49% (2007-20008) to 92% (2008-2009) did not depend on the frequency of oseltamivir use. Full correlation of the results obtained by various techniques allows them to be used to monitor the susceptibility of influenza viruses to oseltamivir.

[Sensitivity of the epidemic and pandemic influenza virus strains to zanamivir (Relenze) in in vitro experiments].

The paper presents the results of the first Russian experience in evaluating the sensitivity of the epidemic and pandemic influenza virus strains, circulating in the period 2009-2010, to the anti-neuraminidase drug zanamivir. A complex of studies, including enzyme immunoassay, fluorometric assay and partial sequence of the neuraminidases (NA1 and NA2) from influenza A virus strain, was applied. The findings Indicate that all the test strains, including those resistant to oseltamivir, were susceptible to zanamivir. The latter is recommended by the WHO for the prevention and treatment of influenza in pregnant women.

[Accuracy of a real-time polymerase-chain-reaction assay for a quantitative estimation of genetically modified sources in food products].

The accuracy of a real-time polymerase-chain-reaction assay for genetically modified sources in food products was determined using two official test systems (kits) of primers and samples. These kits were recommended by the Federal Center of State Sanitary and Epidemiological Surveillance (Russian Ministry of Health) and the European Commission. We used the following three models of thermocyclers: iCycler iQ (BioRad, United States), Rotor-Gene 3000 (Corbett Research, Australia), and DT-322 (DNA-Technology, Russia). Studies of samples that contained 1% genetically modified sources showed that the error of a quantitative assay for genetically modified sources in food products corresponds to 20-30% and does not depend on the kit type and the thermocycler model used.