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The systems approach to anti-tuberculosis medicine Prof. Vadim M. Govorun

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.

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The systems approach to anti-tuberculosis medicine Prof. Vadim M. Govorun

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  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 ? Research Power Genomics Transcriptomics Proteomics Bacterial proteogenomic profiling as a modern tool of the medical microbiology Practical Resources Microbiology Practical Genomics Practical Proteomics NEW information about object

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

  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 (2002-2006 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 PracticalGenomics Practical Proteomics Microbiology • Phenotype Assay: • morphology • growth features • drug susceptibility • Genomic Assay: • identification • genotyping • drug resistance determination • Proteomic Assay: • protein profiles • identification • typing Cultural methods PCR, sequencing, minisequencing, MALDI-TOF MS MALDI-TOF MS

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

  11. Transfer of technologies Research Institute for Physical–Chemical Medicine Scientific potential, newtechnologies • Fine-tuningof 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 sensitivitymonitoring program : year 2004 Murmansk St. Petersburg Arkhangelsk Tver Moscow Tula Ryazan Yekaterinburg N. Novgorod Khabarovsk Samara Stavropol Irkutsk

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

  14. Establishment oftuberculosis monitoring system Decree of Russian Federation Governmentfrom 10th ofMay 2007 № 280 about Federal Target program«Prevention and control ofsocially significantdiseases (years 2007-2011)» 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 % New problem: emergency of the ExtensivelyDrug-ResistantTuberculosis (XDR-TB) strains Deaths from TB (inc HIVinfected) ~ 3 million peoples per year

  16. Tuberculosis in Russia • TB morbidity 83 per 100000 population • Infant (before 14th yr) morbidity 16 per 100000 population • New ss+ TB cases 32 per 100000 population • Deaths from TB 22 per 100000 population • Prevalence MDR in new cases 20 - 33% • (45% in some regions !!!) New problem: emergency of the ExtensivelyDrug-ResistantTuberculosis (XDR-TB) strains Prevalence XDR among MDR in Russia ~14%

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

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

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

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

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

  23. Distribution amino acid substitutions in RRDR of rpoB gene among M.tubercolosis strains in different Russian regions Distribution amino acid and nucleotide substitutions in KatG and fabG promoter 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. Genotyping of M. tuberculosis MDR/XDR THE MAIN GOAL: TO PREVENT SECONDARY RESISTANT CASES OF TB EPIDEMIOLOGICAL MONITORING BASED ON MODERN MOLECULAR GENETIC METHODS

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

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

  29. Genotyping of M. tuberculosisSpoligotypingVNTR/MIRU typing • 18th European Congress of Clin. Microb. Infect. Dis. -2008. • 39th Union World Conference on Lung Health. -2008.

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

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

  32. Goal 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.

  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 Med. statistic Patient Ecology Topography 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 ofclinical strains of M. TUBERCULOSIS

  38. 14% from MDR

  39. Genomic projectstrains under investigation AI and KY genotypes are endemic for Russian Federation Among KY-strains MDR and XDR are prevalent

  40. Genomic project - methodology • 454 Life Sciencestechnology • ABI PRISM 3700 Genetic Analyzers

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

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

  43. Grants and funding Research works were partially supported by Ministry of Public Health of Russian Federation Research works were partially supported by Bruker Daltonics,Germany (Development Contract No. BDALIPCM 270505).

  44. Thank you for attention, Any questions? 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, 119992, Moscow, Russia. govorun@hotmail.ru tel. (495) 246-77-21

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