Transcript presentation

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
Proteins
- genomic
- proteomic
RNA
- transcriptomic
- metabolomic
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
Bacterial proteogenomic profiling as a
modern tool of the medical microbiology
Practical Resources
Microbiology
• morphology
• growth features
• drug susceptibility
Practical
Genomics
• identification
• genotyping
• drug resistance
determination
• protein profiles
• identification
• typing
Practical
Proteomics
Research Power
Genomics
NEW
information
about object
?
Transcriptomics
Proteomics
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 sequencing
DNA chip
technique
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
Microbiology
Practical Genomics
Practical Proteomics
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
a. i .
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Development and introduction of leading-edge
technologies of diagnostics and prediction
PCR
+
MALDI-TOF
MS
Reflex IV (Bruker
Daltonics)
DNA array
Transfer of technologies
Research Institute
Scientific potential, new technologies
for Physical–Chemical
Medicine
• 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
Murmansk
St. Petersburg
Arkhangelsk
Tver
Moscow
Tula
Ryazan
Yekaterinburg
N. Novgorod
Stavropol
Khabarovsk
Samara
Irkutsk
Regions of the Russian Federation – participants of
N.gonorrhoeae antibiotic-resistance monitoring program:
year 2005
•Murmansk
•St. Petersburg
•Kaliningrad
•Arkhangelsk
•Pskov •Izhevsk
•Moscow•Kazan
•Kostroma•Yekaterinburg
•Kaluga
•N. Novgorod
•Ryazan
•Cheboksary••Chelyabinsk
•Penza
Perm
•Kirov
•
Samara
•Saratov
•Voronezh
•Syktyvkar•Ufa
•Rostov-on-Don •Orenburg •Tumen
•Astrakhan
•Omsk
•Krasnodar
•Krasnoyarsk
•Stavropol
•Novosibirsk
•Irkutsk
•Chita
•Khabarovsk
•Vladivostok
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
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People infected
New TB cases
New ss+ TB cases
Change incidence rate
Prevalence HIV in new adult cases
Prevalence MDR in new cases
2 billion
8.8 million
3.9 million
1% per year
12 %
3.2 %
New problem: emergency of the Extensively Drug-Resistant
Tuberculosis (XDR-TB) strains
Deaths from TB (inc HIV infected)
~ 3 million peoples per year
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 Extensively Drug-Resistant
Tuberculosis (XDR-TB) strains
Prevalence XDR among MDR in Russia
~14%
TB problems for Russian Federation
•High morbidity level
•Enormous level of MDR spreading
•Threat of XDR-strains appearance and spreading
Integration scheme
Ministry of Public Health of
Russian Federation
Research
Institutes
Research Institute for
Physical-Chemical
Medicine
Central Tuberculosis
Research Institute, Moscow
The Institute of Cytology and
Genetics
Data flow
Ural Research Institute for
Phthisiopulmonology,
Ekaterinburg
Novosibirsk Tuberculosis
Research Institute
Development the monitoring and control systems of
the TB (including drug-resistance) spreading
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
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
Systems for detect genetic markers
of drug resistance
Score Oriented Dendrogram for Streptococcus _db
Streptococcus pneumoniae_63073P
Streptococcus pneumoniae_523SP
Streptococcus pneumoniae_522SP
Streptococcus pneumoniae_3103Irk
Streptococcus pneumoniae_265VL
Streptococcus viridans_2733SP
Streptococcus viridans_5045SP
Streptococcus viridans_2693SP
Streptococcus viridans_2462SP
Streptococcus pneumoniae_64004P
Streptococcus pneumoniae_4972Irk
Streptococcus pneumoniae_49619
Streptococcus pneumoniae_350T
Streptococcus pneumoniae_414Irk
Streptococcus pneumoniae_653Irk
Streptococcus pneumoniae_661Irk
Streptococcus pneumoniae_540_n7
Streptococcus pneumoniae_36P
Streptococcus pneumoniae_608P
Streptococcus pneumoniae_1874Irk
Streptococcus pneumoniae_635iar
Streptococcus pneumoniae_531SP
Streptococcus pneumoniae_1373_n3
Streptococcus pneumoniae_2992_n9
Streptococcus pneumoniae_1010_n3
Streptococcus pneumoniae_97VL
Streptococcus pneumoniae_95VL
Streptococcus pneumoniae_1349_n3
Streptococcus pneumoniae_1688_n3
Streptococcus pneumoniae_328VL
Streptococcus pneumoniae_135VL
Streptococcus pneumoniae_64336_2P
Streptococcus pneumoniae_1243Zil
Streptococcus pneumoniae_613_n3
Streptococcus pneumoniae_340_n7
Streptococcus pneumoniae_1538_n23
Streptococcus pneumoniae_42697P
Streptococcus pneumoniae_1779_n23
Streptococcus pneumoniae_965_n3
Streptococcus pneumoniae_1765_n23
Monitoring of M. tuberculosis
resistance spread
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Distance Level
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100
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Inter- and intra species
differentiation for
epidemiology, evolution
studies, studies of
microbial populations
Method: primer extension reaction
followed by MALDI ToF MS
DNA extraction
1. PCR
Dephosphorylation
Biological
samples
2. Primer extension reaction
Purification
TETRAD DNA ENGINE
(MJ Research, Inc.)
5440
Intensity, a.u.
3. MALDI-ToF MS measuring
5768
4814
6675
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5768
Intensity, a.u.
Analysis of mass spectra.
Conclusion about the presence of
known nucleotide mutations
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m/z
5440
Intensity, a.u.
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4814
6675
Reflex IV (Bruker Daltonics)
Judgment about resistance
Genotyping
4500
Microflex
(Bruker
Daltonics)
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6000
m/z
6500
7000
Tuberculosis clinical isolates genetic markers of drug
resistance monitoring (2006 – 2008)
DNA of M. tuberculosis strains were
collected from:
Central Region of Russia (383),
Three regions from which 976 M. tuberculosis strains were collected.
Ural Region (310),
West Siberian Region (283).
Central region
(n=383)
Reflex IV (Bruker Daltonics)
Ural region
(n=310)
West Siberian
region (n=283)
microflex (Bruker
Daltonics)
Total – 976 strains were studied
Distribution amino acid
substitutions in RRDR of rpoB gene
among M.tubercolosis strains in
different Russian regions
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40
30
20
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Distribution amino acid and
nucleotide substitutions in KatG and
fabG promoter among M.tubercolosis
strains in different Russian regions
% от числа штаммов, имеющих мутации в исследованных
позициях
% от числа штаммов, имеющих мутации в исследованных позициях
60
80
70
60
50
40
30
20
10
0
Molecular studies of drug-resistance
MALDI-ToF mass-spectrometry based technology
Molecular studies of drug-resistance
MALDI-ToF mass-spectrometry based technology
Genotyping of M. tuberculosis
MDR/XDR
THE MAIN GOAL: TO PREVENT SECONDARY RESISTANT CASES OF TB
EPIDEMIOLOGICAL MONITORING BASED ON MODERN MOLECULAR GENETIC METHODS
Research Institute for Physical-Chemical Medicine
Epidemiological typing by VNTR and spoligotyping
Molecular genetic typing method based
on 24 Variable Number of Tandem
Repeats (VNTR) loci.
High throughput system
Size fragment analysis by
ABI prism™ 3100
VNTR/MIRU-typing
24 MIRU (micobacterial interspersed repetitive units)
were selected for VNTR analysis
Central Region
100 M. tuberculosis strains
collected from Central Region
of Russia.
HGDI=0.97
Genotyping of M. tuberculosis
Spoligotyping
VNTR/MIRU typing
•18th European Congress of Clin.
Microb. Infect. Dis. -2008.
•39th Union World Conference on
Lung Health. -2008.
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
GIS technologies
Application of geographical informational systems in health service
The Institute of Cytology
and Genetics
60
50
40
30
20
10
0
Ново сиби
рск
Уральски
й регио
н
Московск
ий регио
н
Intens. [a.u.]
A
Leu
Trp 5
3
P 53 1
ro
A 5 1
rg 33
Leu 5
26
Tyr 5
26
sn 526
A 526
sp
Th 5
26
V r 52
al
G 516 6
ly
P 51
ro 6
Leu
+ Pro 513
Leu 53 Ph 5
1+
11
Leu 53 P e 5
1+ ro 14
Trp 53 T 53
1+ yr 3
5
Tyr 31 A 52
rg
52 + L 52 6
6+ eu 6
Va 526
l5
16
% от числа штаммов, имеющих
мутации в исследованных позициях
Research Institute for
Physical-Chemical
Medicine
5455.804
800
National microbiological and molecular genetic
monitoring and control systems of the TB
spread. (Geographic Informational Systems –
GIS)
600
5784.808
400
6693.290
200
4829.065
6394.616
0
4500
5000
5500
6000
6500
7000
m /z
gyrAwt, parCwt
100%
26,3
80%
36,2
40%
S
73,7
63,8
20%
46,3
0%
PEN
TET
31%
500
90,0%
53,7
60%
CIPR
R
450
88%
80,0%
70,0%
60,0%
82%
57%
50,0%
40,0%
30,0%
20,0%
10,0%
0,0%
54%
35%
46%
gyrAmut, parCmut
350
58%
42%
PIA
300
PIB
250
200
12%
РФ
18%
Германия
150
Греция
Англия
Швеция
2%
100
21%
50
0
1
Universal High-throughput
technological platforms for
typing and monitoring
2
3
4
5
6
7
8
Central Tuberculosis
Research Institute, Moscow
New technologies adoption
gyrAmut, parCwt
gyrAwt, parCmut
400
65%
Ural Research Institute for
Phthisiopulmonology,
Ekaterinburg
Novosibirsk Tuberculosis
Research Institute
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.
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
Med. statistic
Pathogen
Patient
Ecology
Topography
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 project
strains under investigation
Phenotype
R-№
R-894
R-849
R-898
R-975
RFLP
genotype
AI
KY
KY
KY
RIF
INH
EMB
STR
Pz
ETH
AMI
CAP
OFL
S
R
S
R
S
R
R
R
S
R
R
R
S
R
R
R
S
R
S
R
S
R
R
S
S
R
R
R
S
R
S
R
S
R
S
S
AI and KY genotypes are endemic for Russian Federation
Among KY-strains MDR and XDR are prevalent
Susceptible
XDR
Polyresistent
MDR
Genomic project - methodology
• 454 Life Sciences
technology
• ABI PRISM 3700
Genetic Analyzers
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)
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.
The Institute of cytology and genetics, RAS
Novosibirsk Tuberculosis Research Institute of Ministry of Public Health
of Russian Federation, Novosibirsk, Russia
Vladimir N. Kinsht, Andrey G. Cherednichenko.
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).
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.
[email protected]
tel. (495) 246-77-21