the systems approach to anti-tuberculosis medicine prof. vadim m. govorun
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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 of Russian Federation. - PowerPoint PPT PresentationTRANSCRIPT
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
XXI century - high throughput technologies for investigation of clinically relevant microorganisms are developed- genomic- proteomic- transcriptomic- metabolomic
Proteins
RNA
DNA
What can we do for practice?
What are the current main questions for infection
medicine?
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
• 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 informationabout object
Practical Resources
?
Bacterial proteogenomic profiling as a modern tool of the medical microbiology
Molecular typingGenetic markers of drug
resistance detection
Species identificationBacterial strains differentiation
DNA sequencingDNA chiptechnique
Data accumulation and analysis
Novel mechanisms of drug resistance discovery
Proteomic research
MALDI mass spectrometry
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»
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
Practical resources
Practical GenomicsMicrobiology Practical Proteomics
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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
Development and introduction of leading-edge technologies of diagnostics and prediction
+
Reflex IV (Bruker Daltonics)
MALDI-TOFMS
DNA array
PCR
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)
Regions of the Russian Federation – participants of N.gonorrhoeae sensitivity monitoring program :
year 2004
Khabarovsk
Moscow
Tver
Murmansk
Arkhangelsk
Yekaterinburg
Irkutsk Stavropol
N. Novgorod
Tula Ryazan
Samara
St. Petersburg
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
Establishment of tuberculosis monitoring system
Decree of Russian Federation Government from 10th of May 2007 № 280 about Federal Target program
«Prevention and control of socially significant diseases (years 2007-2011)»
Subprogram «Tuberculosis»
Tuberculosis in the world People infectedPeople infected 2 billion2 billion New TB cases New TB cases 8.8 million 8.8 million New ss+ TB cases New ss+ TB cases 3.9 million 3.9 million Change incidence rate Change incidence rate 1% per year 1% per year Prevalence HIV in new adult cases 12 %Prevalence HIV in new adult cases 12 % Prevalence MDR in new casesPrevalence MDR in new cases 3.2 %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
Tuberculosis in Russia
TB morbidityTB morbidity 83 per 100000 population83 per 100000 population
Infant (before 14Infant (before 14thth yr) yr) morbiditymorbidity 16 per 100000 population16 per 100000 population
New ss+ TB cases New ss+ TB cases 32 per 100000 population 32 per 100000 population Deaths from TBDeaths from TB 22 per 100000 population22 per 100000 population Prevalence MDR in new casesPrevalence MDR in new cases 20 - 33% 20 - 33%
(45% in some regions !!!)(45% in some regions !!!)
New problem: emergency of the Extensively Drug-Resistant Tuberculosis (XDR-TB) strains
Prevalence XDR among MDR in Russia ~14% Prevalence XDR among MDR in Russia ~14%
TB problems for Russian Federation
•Threat of XDR-strains appearance and spreading
•Enormous level of MDR spreading
•High morbidity level
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
Informati
on supportin
g
and data an
alysis
(math
modeli
ng,
statist
ic,
data base
s)
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
01002003004005006007008009001000
Streptococcus pneumoniae_1765_n23 Streptococcus pneumoniae_965_n3 Streptococcus pneumoniae_1779_n23 Streptococcus pneumoniae_42697P Streptococcus pneumoniae_1538_n23 Streptococcus pneumoniae_340_n7 Streptococcus pneumoniae_613_n3 Streptococcus pneumoniae_1243Zil Streptococcus pneumoniae_64336_2P Streptococcus pneumoniae_135VL Streptococcus pneumoniae_328VL Streptococcus pneumoniae_1688_n3 Streptococcus pneumoniae_1349_n3 Streptococcus pneumoniae_95VL Streptococcus pneumoniae_97VL Streptococcus pneumoniae_1010_n3 Streptococcus pneumoniae_2992_n9 Streptococcus pneumoniae_1373_n3 Streptococcus pneumoniae_531SP Streptococcus pneumoniae_635iar Streptococcus pneumoniae_1874Irk Streptococcus pneumoniae_608P Streptococcus pneumoniae_36P Streptococcus pneumoniae_540_n7 Streptococcus pneumoniae_661Irk Streptococcus pneumoniae_653Irk Streptococcus pneumoniae_414Irk Streptococcus pneumoniae_350T Streptococcus pneumoniae_49619 Streptococcus pneumoniae_4972Irk Streptococcus pneumoniae_64004P Streptococcus viridans_2462SP Streptococcus viridans_2693SP Streptococcus viridans_5045SP Streptococcus viridans_2733SP Streptococcus pneumoniae_265VL Streptococcus pneumoniae_3103Irk Streptococcus pneumoniae_522SP Streptococcus pneumoniae_523SP Streptococcus pneumoniae_63073P
Score Oriented Dendrogram for Streptococcus _db
Distance Level
1010_n3_gki (0.0000)1243Zil_gki (0.0000)
2462SP_gki (0.0000)2693SP_gki (0.0000)2733SP_gki (0.0000)5045SP_gki (0.0000)
350T_gki (0.0124)1765_n3_gki (0.0000)1874Irk_gki (0.0000)2992_n9_gki (0.0000)36P_gki (0.0000)522SP_gki (0.0000)63073P_gki (0.0000)635iar_gki (0.0000)
4972Irk_gki (0.0000)64336_2P_gki (0.0000)
49619_gki (0.0014)523SP_gki (0.0000)531SP_gki (0.0000)95VL_gki_1 (0.0000)3103Irk_gki (0.0000)
340_n7_gki (0.0000)608P_gki (0.0000)64004P_gki (0.0000)653Irk_gki (0.0000)661Irk_gki (0.0000)
540_n7_gki (0.0024)414Irk_gki (0.0017)
1349_n3_gki (0.0000)135VL_gki (0.0000)1373_n3_gki (0.0000)1538_n23_gki (0.0000)1688_n3_gki (0.0000)1779_n23_gki (0.0000)265VL_gki (0.0000)328VL_gki (0.0000)613_n3_gki (0.0000)965_n3_gki (0.0000)97VL_gki (0.0000)42697P_gki (0.0000)
Mass-spectrometry based minisequencing method Mass-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
Method: primer extension reaction followed by MALDI ToF MS
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1. PCR
2. Primer extension reaction
3. MALDI-ToF MS measuring
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, a.
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TETRAD DNA ENGINE TETRAD DNA ENGINE (MJ Research, Inc.)(MJ Research, Inc.)
Judgment about resistanceGenotyping
Reflex IV (Bruker Daltonics)
Microflex (Bruker
Daltonics)
BiologicBiological al samplessamples
DNA extraction
PurificationPurification
DephosphorylationDephosphorylation
Analysis of mass spectra. Conclusion about the presence of known nucleotide mutations
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
0
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% о
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а ш
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мею
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утац
ии
в и
ссл
едо
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ны
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ози
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шта
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утац
ии в
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пози
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яхDistribution 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
Molecular studies of drug-resistanceMALDI-ToF mass-spectrometry based technology
Molecular studies of drug-resistanceMALDI-ToF mass-spectrometry based technology
MDR/XDR
THE MAIN GOAL: TO PREVENT SECONDARY RESISTANT CASES OF TB
EPIDEMIOLOGICAL MONITORING BASED ON MODERN MOLECULAR GENETIC METHODS
Genotyping of M. tuberculosis
Molecular genetic typing method based on 24 Variable Number of Tandem
Repeats (VNTR) loci.
High throughput systemSize fragment analysis by
ABI prism™ 3100
Epidemiological typing by VNTR and spoligotyping
Research Institute for Physical-Chemical Medicine
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
Genotyping of M. tuberculosis
SpoligotypingVNTR/MIRU typing
•18th European Congress of Clin. Microb. Infect. Dis. -2008.
•39th Union World Conference on Lung Health. -2008.
GIS technologiesApplication 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%
21%2%
46%
gyrAwt, parCwt
gyrAmut, parCwt
gyrAwt, parCmut
gyrAmut, parCmut
73,7
26,3
63,8
36,2
46,3
53,7
0%
20%
40%
60%
80%
100%
PEN TET CIPR
S
R
5455.804
5784.808
6693.290
4829.0656394.616
0
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800
Inte
ns. [a
.u.]
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m/z
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мутац
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в и
ссл
ед
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ан
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ци
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0
50
100
150
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300
350
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1 2 3 4 5 6 7 8
12%
88%
18%
82%
35%
65%
54%
57% 58%
42%
0,0%
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90,0%
РФ Германия Греция Англия Швеция
PIA
PIB
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
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
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
Database “Epidemiology”:accumulation, storage, and analysis of information
Patient
Ecology
Topography
Statistic
Med. statistic
Operative data
Results of modeling
Modeling of gonorrhea distribution (unfavorable scenario)
Изменение числа заболеваний (в процентах)
Scabies in children (temporal dynamics)
Genomic Project –resequencing of clinical strains of M. TUBERCULOSIS
14% from MDR
Genomic projectstrains under investigation
R-№
RFLPgenotype
Phenotype
RIF INH EMB STR Pz ETH AMI CAP OFL
R-894 AI S S S S S S S S S SusceptibleR-849 KY R R R R R R R R R XDRR-898 KY S R R R S R R S S PolyresistentR-975 KY R R R R R S R R S MDR
AI and KY genotypes are endemic for Russian Federation
Among KY-strains MDR and XDR are prevalent
Genomic project - methodology• 454 Life Sciences
technology
• ABI PRISM 3700 Genetic Analyzers
PerspectivesGlobal 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)
Collaboration
Central Tuberculosis Research Institute, Russian Academy of Medical Sciences, Moscow, RussiaAleksey V. Kuz’min, Sofia N. Andreevskaya, Elena E. Larionova, Tat’yana G. Smirnova, Larisa N. Chernousova
Ural Research Institute for Phthisiopulmonology, Ekaterinburg, RussiaEugeny Yu. Kamaev, Sergey N. Skorniakov.
Novosibirsk Tuberculosis Research Institute of Ministry of Public Health of Russian Federation, Novosibirsk, RussiaVladimir N. Kinsht, Andrey G. Cherednichenko.
The Institute of cytology and genetics, RAS
Research works were partially supported by Bruker Daltonics, Germany (Development Contract No. BDALIPCM 270505).
Grants and funding
Research works were partially supported by Ministry of Public Health of Russian Federation
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.
tel. (495) 246-77-21
Thank you for attention,Any questions?