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

2. molecular epidemiology phylogeny virulence
Uso de RFLP versus VNTR-MIRU ou Deligotyping para estudos de epidemiologia molecular
molecular epidemiology
phylogeny
virulence
Spoligotyping
MIRU-VNTR
Deligotyping

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

4. M. tuberculosis strain typing: spoligotyping

5. M. tuberculosis strain typing: MIRU-VNTR

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. Dendrograma, gerado pelo programa GelCompar®, de isolados de Mtb.
mar20is2 16
mar25is1 11
mar10is1 18
mar17is2 13
mar19is3 7
mar20is2 3
mar21is5 12
mara9is1 19
100 90 80 70 60 50 40 30
0.50
1.00
1.50
2.00
15.00
10.00
5.00
3.00
2.50 kb
mar17is2 18
mar19is3 2
mar21is5 4
mar21is5 5
mar11is1 15
mar25is1 19
mar21is5 13
mar27is1 18
mar21is5 17
mar15is2 2
mar17is2 16
mar22is3 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
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
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
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
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
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
Stability
Discriminative power
robust/ease
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

30. Spoligotypes of isolates with SNP G619A.
Strains
Family
Type
Place 15
nnnnnnnnnnnnonoonnnnnnnnnnnnnnnnoooonnnnnnn
New WT PA
219
nnnnoooonnnnonoonnnnnnnnnnnnnnnnoooonnnnnnn RJ 81
nonnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn SP 68
nnnnnnnnoonnnnnnnnnnnnnnnnnnnnnnoooonnnnnnn S 34
216
107
159
319
nnnnnnnnnnnnnnnnnnnnnnnnnnnnononoooonnnnnnn
Haarlen
746 47
nnnnnnnnnnnnnnonnnnnoooonnnnnnnnoooonnnnnnn
LAM
150 RS 38
nnnnnnnnoonnnooooooooooooooooooooooooonnnnn
2571
oooooooooooooooooooooooonnnnnnnnoooonnnnnnn 4
294

31. Table 7: Spoligotyping analysis of isolates with SNPs 312T
Nat SNPs
Family
Type 5
C312T
nnnnnnnnnnnnnnnnnnnnnnnnnnnooooooonnnnnnnnn
New - 13
nnnoooooonnonnnnonnnnnoonnnnoooononnnnnnnnn
EAI 20
nnnoooooonnnnnnnonnnnnoonnnnoooononnnnnnnnn
EAI5
702 23

32. MTB Complex

33. Sympatric - allopatric

34. accuracy for strain definition
III. Virulence
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-PGRS50
Rv3346c
PPE 55
Rv3348
IS1561
PPE 56
Rv3351c
Rv3352c
Rv3353c
Rv3354
Rv3355c
folD
Rv3346c/3355c
Description of the deleted area in RDRio 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?
behaviour in transmission chains?
simultaneous/mechanism?
Deligotyping...

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

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. Genomic Regions/Genes
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 39
RD21_1
Rv3617 (ephA) 40
DS239_1
Rv3651 41
RD25BCG_1 42
RD18_1
RV3766 43
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?

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