CYTEDCYTEDIII Encontro Nacional de III Encontro Nacional de

TuberculoseTuberculose

Uso de RFLP versus VNTR-MIRU Uso de RFLP versus VNTR-MIRU ou Deligotyping para estudos de ou Deligotyping para estudos de

epidemiologia molecular epidemiologia molecular

Uso de RFLP versus VNTR-MIRU ou Uso de RFLP versus VNTR-MIRU ou Deligotyping para estudos de epidemiologia Deligotyping para estudos de epidemiologia

molecular molecular

molecular epidemiologymolecular epidemiology

phylogenyphylogeny

virulencevirulence

– SpoligotypingSpoligotyping– MIRU-VNTRMIRU-VNTR– DeligotypingDeligotyping

I. Molecular epidemiologyI. Molecular epidemiology

accuracy for strain definitionaccuracy for strain definition– StabilityStability– Discriminative powerDiscriminative power

robust/easerobust/ease

represents recent transmission?represents recent transmission?

– SpoligotypingSpoligotyping– MIRU-VNTRMIRU-VNTR– DeligotypingDeligotyping

M. tuberculosisM. tuberculosis strain typing: spoligotyping strain typing: spoligotyping

M. tuberculosisM. tuberculosis strain typing: MIRU-VNTR strain typing: MIRU-VNTR

Typing techniquesTyping techniques

IS6110-RFLP IS6110-RFLP (+ 1,000 patterns)(+ 1,000 patterns)

Spoligotyping Spoligotyping (+ 5,000 patterns)(+ 5,000 patterns)

MIRU-VNTR MIRU-VNTR (+ 1,000 patterns)(+ 1,000 patterns)– ABI 3100/ABI 3730ABI 3100/ABI 3730

Spoligo+MIRU-VNTR resolution vs RFLP??Spoligo+MIRU-VNTR resolution vs RFLP??– Depends on locallityDepends on locallity– RJ (South East)/PA (North)/RS (South)RJ (South East)/PA (North)/RS (South)

Gelcompar/BionumericsGelcompar/Bionumerics

mar17is2 18

mar19is3 2

mar21is5 4

mar21is5 5

mar11is1 15

mar25is1 19

mar21is5 13

mar27is1 18

mar21is5 17

mar15is2 2

mar17is2 16

mar22is3 7

mar15is2 5

mar25is1 16

mara9is1 10

mar11is1 12

mar17is2 2

mar11is1 5

mar20is2 4

mara9is1 16

mar25is1 14

mar20is2 11

mar20is2 12

mar11is1 10

mar17is2 5

mar17is2 14

mar19is3 4

mar21is5 18

mar20is2 2

mar11is1 8

mar19is3 1

mar15is2 19

mar27is1 4

mar19is3 13

mar15is2 7

mar17is2 20

mar27is1 21

mar25is1 8

mar27is1 14

mar27is1 17

mar18is4 6

mar19is3 17

mar23is3 15

mar19is3 9

mar22is3 11

mar11is1 6

mar11is1 7

mar11is1 9

mar17is1 7

mar25is1 1

mar15is2 6

mar27is1 12

mar23is3 5

mara9is1 18

mar10is1 15

mar20is2 5

mar20is2 6

mar17is2 12

mar20is2 7

mar21is5 11

mar27is1 16

mar27is1 15

mar10is1 14

mar18is4 15

mar19is3 3

mar19is3 5

mar25is1 10

mar11is1 14

mara9is1 15

mar10is1 10

mar23is3 3

mar15is2 20

mar19is3 11

mar17is2 4

mar17is2 17

mar10is1 11

mar10is1 12

mar10is1 13

mar17is1 8

mar20is2 9

mar10is1 17

mar11is1 4

mara9is1 13

mar20is2 1

mar19is3 16

mar22is3 15

mar27is1 19

mar22is3 13

mar22is3 14

mar25is1 7

mar27is1 20

mar18is4 11

mara9is1 7

mar23is3 13

mar11is1 13

mar25is1 4

mar21is5 15

mar23is3 16

mar19is3 10

mar21is5 6

mar20is2 8

mar15is2 3

mar27is1 6

mar19is3 6

mar15is2 4

mar19is3 12

mar22is3 5

mara9is1 6

mar21is5 3

mar25is1 21

mar17is2 15

mar19is3 14

mara9is1 11

mara9is1 2

mara9is1 3

mara9is1 5

mar18is4 2

mara9is1 4

mara9is1 9

mar18is4 5

mara9is1 14

mara9is1 1

mar17is2 3

mar22is3 4

mar23is3 11

mar10is1 16

mar18is4 4

mar25is1 12

mar17is2 19

mar17is1 1

mar19is3 8

mara9is1 8

mar20is2 16

mar25is1 11

mar10is1 18

mar17is2 13

mar19is3 7

mar20is2 3

mar21is5 12

mara9is1 19

10090807060504030 0.50

1.00

1.50

2.00

2.50

3.00

5.00

10.0

0

15.0

0

kb

Dendrograma, gerado pelo programa GelCompar®, de isolados de Mtb.

Current projects for comparison of Current projects for comparison of typing procedurestyping procedures

Título do Projeto: Título do Projeto: GENOTIPAGEM DE ISOLADOS DE GENOTIPAGEM DE ISOLADOS DE MYCOBACTERIUM TUBERCULOSIS COM INTUITO MYCOBACTERIUM TUBERCULOSIS COM INTUITO DE AVALIAR NOVOS METODOS DE TIPAGEM, DE AVALIAR NOVOS METODOS DE TIPAGEM, VARIABILIDADE BIOGEOGRAFICA E PRESENCA DE VARIABILIDADE BIOGEOGRAFICA E PRESENCA DE DELECOES GENOMICAS NO BRASILDELECOES GENOMICAS NO BRASIL

Coordenador do Projeto: Coordenador do Projeto: Philip SuffysPhilip Suffys

Instituição Executora: Instituição Executora: Instituto Oswaldo CruzInstituto Oswaldo Cruz

Edital:Edital:

Edital MCT- CNPq / MS-SCTIE-DECIT – Nº 25/2006Edital MCT- CNPq / MS-SCTIE-DECIT – Nº 25/2006

Current projects for comparison of Current projects for comparison of typing procedurestyping procedures

RETROESPECTIVORETROESPECTIVO

– Fundação Estadual de Produção e Pesquisa em Saúde Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS(FEPPS).).

– Instituto Evandro Chagas (IEC) - UFPAInstituto Evandro Chagas (IEC) - UFPA

– Hospital Universitário Clementino Fraga Filho (HUCFF) da Hospital Universitário Clementino Fraga Filho (HUCFF) da UFRJUFRJ,,

PROSPECTIVOPROSPECTIVO

– Universidade Federal da Rondonia - Universidade Federal da Rondonia -

– Faculdade de Medicina de CampinasFaculdade de Medicina de Campinas

– UFRJUFRJ

– UFCUFC

– OUTROS ESTADOS…OUTROS ESTADOS…

Deligotyping: our studyDeligotyping: our study

Collaborative study with Cristina Gutierrez (IP Paris) and Collaborative study with Cristina Gutierrez (IP Paris) and Philip Supply (IP Lille)Philip Supply (IP Lille)

Compared use of spoligotyping, 15/24-MIRU-VNTR on Compared use of spoligotyping, 15/24-MIRU-VNTR on ABI 3730 and deligotypingABI 3730 and deligotyping

369 isolates from BR (com health centers)369 isolates from BR (com health centers)– 177 from Rio de Janeiro (UFRJ)177 from Rio de Janeiro (UFRJ)

– 120 from Rio Grande do Sul (FEPPS)120 from Rio Grande do Sul (FEPPS)

– 72 from Para (IEC – UFPA)72 from Para (IEC – UFPA)

RESULTS: RESULTS:

Spol-24MIRU NJ treeSpol-24MIRU NJ tree

Spol-24MIRU municipioSpol-24MIRU municipio

24 MIRU MST24 MIRU MST

Deligotyping: our studyDeligotyping: our study369 isolates with 24-MIRU-VNTR369 isolates with 24-MIRU-VNTR– 307 patterns, 41 clusters and 266 unique307 patterns, 41 clusters and 266 unique

+ spoligotyping+ spoligotyping– 321 patterns, 32 clusters and 289 unique321 patterns, 32 clusters and 289 unique

Spoligotyping + 15-MIRUsSpoligotyping + 15-MIRUs– 315 genotypes ~ 24-MIRUs315 genotypes ~ 24-MIRUs

Spol + 24-MIRUSpol + 24-MIRU– 1 cluster of 6, rest mostly n=21 cluster of 6, rest mostly n=2

– 27 clusters isolates of the same state!!27 clusters isolates of the same state!!

II. Molecular phylogenyII. Molecular phylogeny

accuracy for strain definitionaccuracy for strain definition– StabilityStability– Discriminative powerDiscriminative power

robust/easerobust/ease

represents recent transmissionrepresents recent transmission

– SpoligotypingSpoligotyping– MIRU-VNTRMIRU-VNTR– DeligotypingDeligotyping

Projects using spoligotypingProjects using spoligotyping

PhylogenyPhylogenySpol DB3 (Institut Pasteur, Guadeloupe; Filliol et al. JCM 2003) Spol DB3 (Institut Pasteur, Guadeloupe; Filliol et al. JCM 2003)

(SpolDB4)(SpolDB4)– 12.000 strains (+/- 150 Brazilian) 12.000 strains (+/- 150 Brazilian) (39.000)(39.000)– > 90 countries > 90 countries (140)(140)– population genetics, biogeographic distribution and population genetics, biogeographic distribution and

phylogeny of Mtb Complexphylogeny of Mtb Complex

– Rio: 400 patientsRio: 400 patients>50% LAM>50% LAM

7% Haarlem7% Haarlem

10% new LAM10% new LAM

Only 2 BeijingOnly 2 Beijing

Finetuning with MIRU-VNTRFinetuning with MIRU-VNTR

Spoligotypes of isolates with SNP G619A.

StrainsStrains FamilyFamily TypTypee

PlacePlace

1515 NewNew WTWT PAPA

219219 NewNew WTWT RJRJ

8181 NewNew WTWT SPSP

6868 SS 3434 RJRJ

216216 SS 3434 RJRJ

107107 SS 3434 RJRJ

159159 SS 3434 RJRJ

319319 HaarlenHaarlen 746746 RJRJ

4747 LAMLAM 150150 RSRS

3838 LAMLAM 25712571 RSRS

6868 LAMLAM 44 RJRJ

294294 LAMLAM 44 RJRJ

IsolatesIsolates Nat Nat SNPsSNPs

   Family Family TypeType

55 C312TC312T NewNew --

1313 C312TC312T EAIEAI --

2020 C312TC312T EAI5EAI5 702702

2323 C312TC312T EAI5EAI5 702702

Table 7: Spoligotyping analysis of isolates with SNPs 312T

MTB ComplexMTB Complex

Sympatric - allopatricSympatric - allopatric

III. VirulenceIII. Virulence

accuracy for strain definitionaccuracy for strain definition

– SpoligotypingSpoligotyping– MIRU-VNTRMIRU-VNTR– DeligotypingDeligotyping

New typing technologies: microarraysNew typing technologies: microarrays

Small et al. InDel detection (3000-300 bp-range)Small et al. InDel detection (3000-300 bp-range)

New typing technologies: microarraysNew typing technologies: microarrays

Association nr of deletions and decreased capacity of Association nr of deletions and decreased capacity of cavitationcavitation

MTB ComplexMTB Complex

PE-PGRS50 Rv3346c PPE 55 Rv3348 IS1561 PPE 56 Rv3351c Rv3352c Rv3353c Rv3354 Rv3355c folD

PE-PGRS50 folDRv3346c/3355c

Description of the deleted area in Description of the deleted area in RDRDRio Rio Mtb strains. The recombination between 2 homologues Mtb strains. The recombination between 2 homologues genes Rv3346c and Rv3355c formed a new in-frame fusion sequence (Rv3346c/Rv3355c). All genes Rv3346c and Rv3355c formed a new in-frame fusion sequence (Rv3346c/Rv3355c). All elements between these 2 genes were excised and lost during this process of reciprocal elements between these 2 genes were excised and lost during this process of reciprocal recombination (comprising a length of >26 kb). recombination (comprising a length of >26 kb).

Deletion studiesDeletion studies

Genome compositionGenome composition– RDrio, 26 kB, 30% strainsRDrio, 26 kB, 30% strains– Mtb, Mbovis?Mtb, Mbovis?– DeletionsDeletions

- influence on transmissibility/virulence- influence on transmissibility/virulence

- influence on disease evolution- influence on disease evolution

dynamics of deletions?dynamics of deletions?

behaviour in transmission chains?behaviour in transmission chains?

simultaneous/mechanism?simultaneous/mechanism?– Deligotyping...Deligotyping...

Deligotyping: originalDeligotyping: original

Probe design for 43 LSPsProbe design for 43 LSPs

Compared to Genechips/AffimetricsCompared to Genechips/Affimetrics

100 Mtb strains from SF100 Mtb strains from SF

Almost complete identical results Almost complete identical results

No info on regions or strainsNo info on regions or strains

Deligotyping: our studyDeligotyping: our study

Collaborative study with Cristina Gutierrez (IP Paris) and Collaborative study with Cristina Gutierrez (IP Paris) and Philip Supply (IP Lille)Philip Supply (IP Lille)

Compared use of spoligotyping, 15/24-MIRU-VNTR on Compared use of spoligotyping, 15/24-MIRU-VNTR on ABI 3730 and deligotypingABI 3730 and deligotyping

369 isolates from BR 369 isolates from BR – 177 from Rio de Janeiro177 from Rio de Janeiro

– 120 from Rio Grande do Sul120 from Rio Grande do Sul

– 72 from Para72 from Para

~ 150 selected for deligotyping~ 150 selected for deligotyping

Deligotyping reactionsDeligotyping reactions

Reaction conditionsReaction conditions– PCRPCR

MultiplexMultiplex

Primers from 20-24 bpPrimers from 20-24 bp

Melting temp 68-72 degreesMelting temp 68-72 degrees

113-282 bp fragments113-282 bp fragments

– HybridizationHybridization28 bp aminolink-probes on biodyne28 bp aminolink-probes on biodyne

Most within ORFs of RvsMost within ORFs of Rvs

Deligotyping deletionsDeligotyping deletions

– 43 LSPs43 LSPs

We have the list!We have the list!

Can change LSP compositionCan change LSP composition

– hybridizationhybridizationMembranes available Membranes available

Blotter lane Oligonucleotide Genomic Regions/Genes

1 HT0921_1 Rv0921

2 HT2812_1 Rv2812

3 DMT0676_1 Rv3446c

4 DMT1360_2b Rv2739c

5 DS110_3 Rv0195

6 DS117_2 Rv0405 (pks6)

7 DS121_5 Rv0571c

8 RD16_1 Rv3901c

9 RD18BCG_6 Rv1189 (sigI)

10 DS145_1b Rv1355c (moeY)

11 RD6BCG_1b Rv1515 (epiA)

12 RD5_1 Rv1582c

13 DS150_1 Rv1672c

14 RD6l_1 Rv1755c (plcD)

15 RD6a_1 Rv1761c

16 DS163_1 Rv1878 (glnA3)

17 DS165_1 Rv1907c

18 DS167_1b Rv1927

19 RD15BCG_1b Rv1976c

20 RD2BCG_1 RV1985c

21 DS7a_1 intergenic

22 DS174C_2 Rv1994c (cmtR)

23 DS174A_2b Rv2002 (fabG3)

24 RD20_1 Rv2075c (metH)

25 DS182_1 Rv2275

26 DS183_1 RV2314c

27 RD9_2 Rv2349c (plcC)

28 ORD9_1b RV2351c (plcA/mtp40 antigen)

29 RD33_1 Rv2406c

30 DS198a_1 valU (tRNA-Val)

31 RD19_1 Rv3084(lip R )

32 RD5BCG_1 Rv3121

33 RD16BCG_1b Rv3402c

34 RD13_1 Rv3425 (PPE57)

35 RD11BCG_1 Rv3429 (PPE59)

36 L27_1 Rv3519

37 RD14BCG_1 RV1769

38 DS236_1 intergenic

39 RD21_1 Rv3617 (ephA)

40 DS239_1 Rv3651

41 RD25BCG_1 intergenic

42 RD18_1 RV3766

43 RD1BCG_1 Rv3875 (esat-6/esxA)

Deligotyping oligonucleotides with the corresponding genomic regions and genes.

M. tuberculosis cmtR gene Rv1994c. Coordinates: 2.237.628-2.237.984. Metal sensor transcriptional regulator (ArsR-SmtB family). Represses transcription from the cmt operator-promoter.

M. tuberculosis plcD gene Rv1755c. Coordinates: 1.986.854-1.987.696. PROBABLE PHOSPHOLIPASE C 4 (fragment). PROBABLE VIRULENCE FACTOR IMPLICATED IN THE PATHOGENESIS OF M. tuberculosis.

M. tuberculosis genes plcC (2627172-2628698), plcB (2628781-2630319) and plcA (2630537-2632075). The genes codify for: phospholipase C 3 (PLCC) phospholipase C 2 (PLCB) and phospholipase C 1 (MPT40 antigen), respectively.

NJ spol-24MIRU-DELIGONJ spol-24MIRU-DELIGO

Deligotyping resultsDeligotyping results

Cluster definition and phylogenyCluster definition and phylogeny

– Coherence of phylogenetic tree building with Coherence of phylogenetic tree building with spol/MIRUspol/MIRU

– Definition of supremacy of LAM and definition Definition of supremacy of LAM and definition of LSPs for this family and othersof LSPs for this family and others

Virulence/transmission?Virulence/transmission?

Deligotyping resultsDeligotyping results

Transmission and virulenceTransmission and virulence

– Absolute association of Absolute association of cmtR (Rv1994c - cmtR (Rv1994c - RD174) and RDrio RD174) and RDrio

– Phospholipase deletions in most EAIPhospholipase deletions in most EAI7 isolates from Para lack PLA, PLB, PLC and PLD7 isolates from Para lack PLA, PLB, PLC and PLD

LARGE SCALE DELETION LARGE SCALE DELETION STUDYSTUDY

Perform a large scale study of Perform a large scale study of deligotypingdeligotyping of of MtbMtb and and MbovisMbovis strains strains

Other markers: spol/MIRU/Other markers: spol/MIRU/SNPsSNPs

different L-A countriesdifferent L-A countries

Epi and mol characterizationEpi and mol characterization– RFLP etcRFLP etc– Clin/epi dataClin/epi data

Growth and transmission rateGrowth and transmission rate

Virulence studies in cell and animal modelsVirulence studies in cell and animal models