cyted iii encontro nacional de tuberculose uso de rflp versus vntr-miru ou deligotyping para estudos...
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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
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