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CYTED III Encontro Nacional de Tuberculose Uso de RFLP versus VNTR-MIRU ou Deligotyping para estudos de epidemiologia molecular.

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Presentation on theme: "CYTED III Encontro Nacional de Tuberculose Uso de RFLP versus VNTR-MIRU ou Deligotyping para estudos de epidemiologia molecular."— Presentation transcript:

1 CYTED III Encontro Nacional de Tuberculose Uso de RFLP versus VNTR-MIRU ou Deligotyping para estudos de epidemiologia molecular

2 molecular epidemiology molecular epidemiology phylogeny phylogeny virulence virulence –Spoligotyping –MIRU-VNTR –Deligotyping

3 I. Molecular epidemiology accuracy for strain definition accuracy for strain definition –Stability –Discriminative power robust/ease robust/ease represents recent transmission? represents recent transmission? –Spoligotyping –MIRU-VNTR –Deligotyping

4 M. tuberculosis strain typing: spoligotyping

5 M. tuberculosis strain typing: MIRU-VNTR

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12 Typing techniques IS6110-RFLP (+ 1,000 patterns) Spoligotyping (+ 5,000 patterns) MIRU-VNTR (+ 1,000 patterns) –ABI 3100/ABI 3730 Spoligo+MIRU-VNTR resolution vs RFLP?? –Depends on locallity –RJ (South East)/PA (North)/RS (South) Gelcompar/Bionumerics

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

14 Current projects for comparison of typing procedures Título do Projeto: GENOTIPAGEM DE ISOLADOS DE MYCOBACTERIUM TUBERCULOSIS COM INTUITO DE AVALIAR NOVOS METODOS DE TIPAGEM, VARIABILIDADE BIOGEOGRAFICA E PRESENCA DE DELECOES GENOMICAS NO BRASIL Coordenador do Projeto: Philip Suffys Instituição Executora: Instituto Oswaldo Cruz Edital: Edital MCT- CNPq / MS-SCTIE-DECIT – Nº 25/2006

15 Current projects for comparison of typing procedures RETROESPECTIVO –Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS). –Instituto Evandro Chagas (IEC) - UFPA –Hospital Universitário Clementino Fraga Filho (HUCFF) da UFRJ, PROSPECTIVO –Universidade Federal da Rondonia - –Faculdade de Medicina de Campinas –UFRJ –UFC –OUTROS ESTADOS…

16 Deligotyping: our study Collaborative study with Cristina Gutierrez (IP Paris) and Philip Supply (IP Lille) Compared use of spoligotyping, 15/24-MIRU-VNTR on ABI 3730 and deligotyping 369 isolates from BR (com health centers) –177 from Rio de Janeiro (UFRJ) –120 from Rio Grande do Sul (FEPPS) –72 from Para (IEC – UFPA) RESULTS:

17 Spol-24MIRU NJ tree

18 Spol-24MIRU municipio

19 24 MIRU MST

20 Deligotyping: our study 369 isolates with 24-MIRU-VNTR –307 patterns, 41 clusters and 266 unique + spoligotyping –321 patterns, 32 clusters and 289 unique Spoligotyping + 15-MIRUs –315 genotypes ~ 24-MIRUs Spol + 24-MIRU –1 cluster of 6, rest mostly n=2 –27 clusters isolates of the same state!!

21 II. Molecular phylogeny accuracy for strain definition accuracy for strain definition –Stability –Discriminative power robust/ease robust/ease represents recent transmission represents recent transmission –Spoligotyping –MIRU-VNTR –Deligotyping

22 Projects using spoligotyping Phylogeny Spol DB3 (Institut Pasteur, Guadeloupe; Filliol et al. JCM 2003) (SpolDB4) – strains (+/- 150 Brazilian) (39.000) –> 90 countries (140) –population genetics, biogeographic distribution and phylogeny of Mtb Complex –Rio: 400 patients >50% LAM 7% Haarlem 10% new LAM Only 2 Beijing Finetuning with MIRU-VNTR

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30 Spoligotypes of isolates with SNP G619A. StrainsFamilyTypePlace 15nnnnnnnnnnnnonoonnnnnnnnnnnnnnnnoooonnnnnnnNewWTPA 219nnnnoooonnnnonoonnnnnnnnnnnnnnnnoooonnnnnnnNewWTRJ 81nonnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnnNewWTSP 68nnnnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnnS34RJ 216nnnnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnnS34RJ 107nnnnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnnS34RJ 159nnnnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnnS34RJ 319nnnnnnnnnnnnnnnnnnnnnnnnnnnnononoooonnnnnnnHaarlen746RJ 47nnnnnnnnnnnnnnonnnnnoooonnnnnnnnoooonnnnnnnLAM150RS 38nnnnnnnnoonnnooooooooooooooooooooooooonnnnnLAM2571RS 68oooooooooooooooooooooooonnnnnnnnoooonnnnnnnLAM4RJ 294oooooooooooooooooooooooonnnnnnnnoooonnnnnnnLAM4RJ

31 Isolates Nat SNPs Nat SNPs FamilyType 5C312TnnnnnnnnnnnnnnnnnnnnnnnnnnnooooooonnnnnnnnnNew- 13C312TnnnoooooonnonnnnonnnnnoonnnnoooononnnnnnnnnEAI- 20C312TnnnoooooonnnnnnnonnnnnoonnnnoooononnnnnnnnnEAI C312TnnnoooooonnnnnnnonnnnnoonnnnoooononnnnnnnnnEAI5702 Table 7: Spoligotyping analysis of isolates with SNPs 312T

32 MTB Complex

33 Sympatric - allopatric

34 III. Virulence accuracy for strain definition accuracy for strain definition –Spoligotyping –MIRU-VNTR –Deligotyping

35 New typing technologies: microarrays Small et al. InDel detection ( bp-range)

36 New typing technologies: microarrays Association nr of deletions and decreased capacity of cavitation

37 MTB Complex

38 PE-PGRS50Rv3346cPPE 55Rv3348IS1561PPE 56Rv3351cRv3352cRv3353cRv3354Rv3355cfolD PE-PGRS50folDRv3346c/3355c Description of the deleted area in RD Rio Mtb strains. The recombination between 2 homologues 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 recombination (comprising a length of >26 kb).

39 Deletion studies Genome composition –RDrio, 26 kB, 30% strains –Mtb, Mbovis? –Deletions - influence on transmissibility/virulence - influence on disease evolution dynamics of deletions? dynamics of deletions? behaviour in transmission chains? behaviour in transmission chains? simultaneous/mechanism? simultaneous/mechanism? –Deligotyping...

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42 Deligotyping: original Probe design for 43 LSPs Compared to Genechips/Affimetrics 100 Mtb strains from SF Almost complete identical results No info on regions or strains

43 Deligotyping: our study Collaborative study with Cristina Gutierrez (IP Paris) and Philip Supply (IP Lille) Compared use of spoligotyping, 15/24-MIRU-VNTR on ABI 3730 and deligotyping 369 isolates from BR –177 from Rio de Janeiro –120 from Rio Grande do Sul –72 from Para ~ 150 selected for deligotyping

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45 Deligotyping reactions Reaction conditions –PCR Multiplex Primers from bp Melting temp degrees bp fragments –Hybridization 28 bp aminolink-probes on biodyne Most within ORFs of Rvs

46 Deligotyping deletions –43 LSPs We have the list! Can change LSP composition –hybridization Membranes available

47 Blotter lane Oligonucleotide Genomic Regions/Genes 1HT0921_1 Rv0921 2HT2812_1 Rv2812 3DMT0676_1 Rv3446c 4DMT1360_2b Rv2739c 5DS110_3 Rv0195 6DS117_2 Rv0405 (pks6) 7DS121_5 Rv0571c 8RD16_1 Rv3901c 9RD18BCG_6 Rv1189 (sigI) 10DS145_1b Rv1355c (moeY) 11RD6BCG_1b Rv1515 (epiA) 12RD5_1 Rv1582c 13DS150_1 Rv1672c 14RD6l_1 Rv1755c (plcD) 15RD6a_1 Rv1761c 16DS163_1 Rv1878 (glnA3) 17DS165_1 Rv1907c 18DS167_1b Rv RD15BCG_1b Rv1976c 20RD2BCG_1 RV1985c 21DS7a_1 intergenic 22DS174C_2 Rv1994c (cmtR) 23DS174A_2b Rv2002 (fabG3) 24RD20_1 Rv2075c (metH) 25DS182_1 Rv DS183_1 RV2314c 27RD9_2 Rv2349c (plcC) 28ORD9_1b RV2351c (plcA/mtp40 antigen) 29RD33_1 Rv2406c 30DS198a_1 valU (tRNA-Val) 31RD19_1 Rv3084(lip R ) 32RD5BCG_1 Rv RD16BCG_1b Rv3402c 34RD13_1 Rv3425 (PPE57) 35RD11BCG_1 Rv3429 (PPE59) 36L27_1 Rv RD14BCG_1 RV DS236_1 intergenic 39RD21_1 Rv3617 (ephA) 40DS239_1 Rv RD25BCG_1 intergenic 42RD18_1 RV RD1BCG_1 Rv3875 (esat-6/esxA) Deligotyping oligonucleotides with the corresponding genomic regions and genes.

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

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

50 M. tuberculosis genes plcC ( ), plcB ( ) and plcA ( ). The genes codify for: phospholipase C 3 (PLCC) phospholipase C 2 (PLCB) and phospholipase C 1 (MPT40 antigen), respectively.

51 NJ spol-24MIRU-DELIGO

52 Deligotyping results Cluster definition and phylogeny –Coherence of phylogenetic tree building with spol/MIRU –Definition of supremacy of LAM and definition of LSPs for this family and others Virulence/transmission?

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54 Deligotyping results Transmission and virulence –Absolute association of cmtR (Rv1994c - RD174) and RDrio –Phospholipase deletions in most EAI 7 isolates from Para lack PLA, PLB, PLC and PLD

55 LARGE SCALE DELETION STUDY Perform a large scale study of deligotyping of Mtb and Mbovis strains Other markers: spol/MIRU/SNPs different L-A countries Epi and mol characterization –RFLP etc –Clin/epi data Growth and transmission rate Virulence studies in cell and animal models


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