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The systems approach to anti-tuberculosis medicine Prof. Vadim M. Govorun Research Institute for Physical–Chemical Medicine of Ministry of Public Health.

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Presentation on theme: "The systems approach to anti-tuberculosis medicine Prof. Vadim M. Govorun Research Institute for Physical–Chemical Medicine of Ministry of Public Health."— Presentation transcript:

1 The systems approach to anti-tuberculosis medicine Prof. Vadim M. Govorun Research Institute for Physical–Chemical Medicine of Ministry of Public Health of Russian Federation

2 XXI century - high throughput technologies for investigation of clinically relevant microorganisms are developed - genomic - proteomic - transcriptomic - metabolomic Proteins RNA DNA

3 What can we do for practice? What are the current main questions for infection medicine?

4 Clinically relevant microorganisms investigation by molecular methods Microbial species identification Molecular studies of drug-resistance – PCR – sequencing – SNP scanning Molecular epidemiology monitoring – Serotyping (proteotyping) – genotyping – VNTR analysis

5 morphology growth features drug susceptibility identification genotyping drug resistance determination protein profiles identification typing Microbiology Practical Genomics Practical Proteomics Transcriptomics Research Power Genomics Proteomics NEW i nformation about object Practical Resources ? Bacterial proteogenomic profiling as a modern tool of the medical microbiology

6 Molecular typing Genetic markers of drug resistance detection Species identification Bacterial strains differentiation DNA sequencing DNA chip technique Data accumulation and analysis Novel mechanisms of drug resistance discovery Proteomic research MALDI mass spectrometry

7 How it started? Establishment of gonococcus monitoring system Decree of Russian Federation Government from 13th of November 2001 N 790 about Federal Target Program «Prevention and control of social-related diseases ( years)» Subprogram «About measures of prevention of further spreading of sexually transmitted diseases»

8 Project main idea Complex use of bacteriological, serological methods and post-genomic technologies Development and introduction of high-throughput measuring-computational platform for sexual transmitted disease monitoring and conduction of effective measures aimed to reduce amount of infected and ill individuals

9 Practical resources Practical Genomics Microbiology Practical Proteomics Phenotype Assay: morphology growth features drug susceptibility Cultural methods Genomic Assay: identification genotyping drug resistance determination PCR, sequencing, minisequencing, MALDI-TOF MS Proteomic Assay: protein profiles identification typing MALDI-TOF MS

10 Development and introduction of leading-edge technologies of diagnostics and prediction + Reflex IV (Bruker Daltonics) MALDI-TOF MS DNA array PCR

11 Transfer of technologies Research Institute for Physical–Chemical Medicine Scientific potential, new technologies Fine-tuning of technology Introduction to practice Establishment and control of clinicodiagnostic laboratories Personnel training Regional laboratories (practical use)

12 Regions of the Russian Federation – participants of N.gonorrhoeae sensitivity monitoring program : year 2004 Khabarovsk Moscow Tver Murmansk Arkhangelsk Yekaterinburg Irkutsk Stavropol N. Novgorod TulaRyazan Samara St. Petersburg

13 Regions of the Russian Federation – participants of N.gonorrhoeae antibiotic-resistance monitoring program: year 2005 Moscow N. Novgorod Stavropol Murmansk Samara Yekaterinburg St. Petersburg Arkhangelsk Irkutsk Kazan Kirov Pskov Cheboksary Ryazan Astrakhan Vladivostok Omsk Saratov Khabarovsk Novosibirsk Kaluga Syktyvkar Chelyabinsk Izhevsk Krasnoyarsk Krasnodar Penza Chita Kostroma Orenburg Rostov-on-Don Kaliningrad Ufa Tumen Perm Voronezh

14 Establishment of tuberculosis monitoring system Decree of Russian Federation Government from 10 th of May 2007 № 280 about Federal Target program «Prevention and control of socially significant diseases (years )» Subprogram «Tuberculosis»

15 Tuberculosis in the world  People infected  2 billion  New TB cases 8.8 million  New ss+ TB cases 3.9 million  Change incidence rate 1% per year  Prevalence HIV in new adult cases 12 %  Prevalence MDR in new cases 3.2 % Deaths from TB (inc HIV infected) ~ 3 million peoples per year New problem: emergency of the Extensively Drug-Resistant Tuberculosis (XDR-TB) strains

16 Tuberculosis in Russia  TB morbidity  83 per population  Infant (before 14 th yr) morbidity  16 per population  New ss+ TB cases  32 per population  Deaths from TB  22 per population  Prevalence MDR in new cases % (45% in some regions !!!) New problem: emergency of the Extensively Drug-Resistant Tuberculosis (XDR-TB) strains Prevalence XDR among MDR in Russia ~14%

17 TB problems for Russian Federation Threat of XDR-strains appearance and spreading Enormous level of MDR spreading High morbidity level

18 Integration scheme Research Institutes Research Institute for Physical-Chemical Medicine Ministry of Public Health of Russian Federation The Institute of Cytology and Genetics Novosibirsk Tuberculosis Research Institute Ural Research Institute for Phthisiopulmonology, Ekaterinburg Central Tuberculosis Research Institute, Moscow Development the monitoring and control systems of the TB (including drug-resistance) spreading Technology development and technology transfer Data flow Information supporting and data analysis (math modeling, statistic, data bases)

19 Molecular epidemiology of TB Molecular studies of drug-resistance – sequencing – InnoLipa – Biochip – MALDI-ToF mass-spectrometry based technology Genotyping of M. tuberculosis – IS 6110 typing – Spoligotyping – VNTR/MIRU typing

20 Mass-spectrometry based minisequencing methodMass-spectrometry based direct protein profiling Systems for detect genetic markers of drug resistance Monitoring of M. tuberculosis resistance spread Systems for fast detect Mycobacterium, using unique protein profiles Inter- and intra species differentiation for epidemiology, evolution studies, studies of microbial populations MALDI ToF MS based platform for practice Research Institute for Physical-Chemical Medicine

21 Method: primer extension reaction followed by MALDI ToF MS 1.PCR 2.Primer extension reaction 3.MALDI-ToF MS measuring TETRAD DNA ENGINE (MJ Research, Inc.) Judgment about resistance Genotyping Reflex IV (Bruker Daltonics) Microflex (Bruker Daltonics) Biological samples DNA extraction Purification Dephosphorylation Analysis of mass spectra. Conclusion about the presence of known nucleotide mutations

22 Central region (n=383) Ural region (n=310) West Siberian region (n=283) Three regions from which 976 M. tuberculosis strains were collected. Tuberculosis clinical isolates genetic markers of drug resistance monitoring (2006 – 2008) DNA of M. tuberculosis strains were collected from: Central Region of Russia (383), Ural Region (310), West Siberian Region (283). Reflex IV (Bruker Daltonics) microflex (Bruker Daltonics) Total – 976 strains were studied

23 Distribution amino acid and nucleotide substitutions in KatG and fabG promoter among M.tubercolosis strains in different Russian regions Distribution amino acid substitutions in RRDR of rpoB gene among M.tubercolosis strains in different Russian regions

24 Molecular studies of drug-resistance MALDI-ToF mass-spectrometry based technology

25 Molecular studies of drug-resistance MALDI-ToF mass-spectrometry based technology

26 MDR/XDR THE MAIN GOAL: TO PREVENT SECONDARY RESISTANT CASES OF TB EPIDEMIOLOGICAL MONITORING BASED ON MODERN MOLECULAR GENETIC METHODS Genotyping of M. tuberculosis

27 Molecular genetic typing method based on 24 Variable Number of Tandem Repeats (VNTR) loci. High throughput system Size fragment analysis by ABI prism™ 3100 Epidemiological typing by VNTR and spoligotyping Research Institute for Physical-Chemical Medicine

28 24 MIRU (micobacterial interspersed repetitive units) were selected for VNTR analysis Central Region 100 M. tuberculosis strains collected from Central Region of Russia. VNTR/MIRU-typing HGDI=0.97

29 Genotyping of M. tuberculosis Spoligotyping VNTR/MIRU typing 18th European Congress of Clin. Microb. Infect. Dis th Union World Conference on Lung Health

30 GIS technologies Application of geographical informational systems in health service Development of the software complex for computer modeling and designing in the area of the post-genome systems biology The Institute of Cytology and Genetics

31 Universal High-throughput technological platforms for typing and monitoring Research Institute for Physical-Chemical Medicine The Institute of Cytology and Genetics National microbiological and molecular genetic monitoring and control systems of the TB spread. (Geographic Informational Systems – GIS) Novosibirsk Tuberculosis Research Institute Ural Research Institute for Phthisiopulmonology, Ekaterinburg Central Tuberculosis Research Institute, Moscow New technologies adoption

32 Management of collection, analysis, and visualization of data in epidemiology. Prognosis of distribution of epidemics. GIS enables - visualization of spatial data; - storage of information in the database; - complex analysis of heterogeneous data. GIS provides an instrument for extracting reference information and for drawing up of accounts in accordance with the needs of decision making. Goal

33 Tasks: To reveal geographic distribution of disease penetration To analyze of spatial and temporal trends, causative agents of diseases To find the gaps in immunization To compose databases with simple for the user data access and management To model and forecast epidemics To plan interventions To monitor results of intervention To plan resources and supplies of medicines To visualize information by using maps via the Internet

34 Database “Epidemiology”: accumulation, storage, and analysis of information Pathogen Patient Ecology Topography Statistic Med. statistic Operative data

35 Results of modeling Modeling of gonorrhea distribution (unfavorable scenario)

36 Изменение числа заболеваний (в процентах) Scabies in children (temporal dynamics)

37 Genomic Project –resequencing of clinical strains of M. TUBERCULOSIS

38 14% from MDR

39 Genomic project strains under investigation R-№ RFLP genotype Phenotype RIFINHEMBSTRPzETHAMICAPOFL R-894AI SSSSSSSSSSusceptible R-849KY RRRRRRRRRXDR R-898KY SRRRSRRSSPolyresistent R-975KY RRRRRSRRSMDR AI and KY genotypes are endemic for Russian Federation Among KY-strains MDR and XDR are prevalent

40 Genomic project - methodology 454 Life Sciences technology ABI PRISM 3700 Genetic Analyzers

41 Perspectives Global system for molecular and epidemiological monitoring of infection diseases. 1.N. gonorrhoeae (complite) 2.M. tuberculosis (finished) 3.S. aureus (MRSA) (in progress) 4. Hospital-acquired infection (in progress)

42 Collaboration Central Tuberculosis Research Institute, Russian Academy of Medical Sciences, Moscow, Russia Aleksey V. Kuz’min, Sofia N. Andreevskaya, Elena E. Larionova, Tat’yana G. Smirnova, Larisa N. Chernousova Ural Research Institute for Phthisiopulmonology, Ekaterinburg, Russia Eugeny Yu. Kamaev, Sergey N. Skorniakov. Novosibirsk Tuberculosis Research Institute of Ministry of Public Health of Russian Federation, Novosibirsk, Russia Vladimir N. Kinsht, Andrey G. Cherednichenko. The Institute of cytology and genetics, RAS

43 Research works were partially supported by Bruker Daltonics, Germany (Development Contract No. BDALIPCM ). Grants and funding Research works were partially supported by Ministry of Public Health of Russian Federation

44 For further information please contact: Prof. Vadim M. Govorun Research Institute for Physical–Chemical Medicine of Ministry of Public Health of Russian Federation Malaya Pirogovskaya St, 1a, , Moscow, Russia. tel. (495) Thank you for attention, Any questions?


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