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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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

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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

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46%

gyrAwt, parCwt

gyrAmut, parCwt

gyrAwt, parCmut

gyrAmut, parCmut

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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.

govorun@hotmail.ru

tel. (495) 246-77-21

Thank you for attention,Any questions?