Molecular

 epidemiology  of  
tuberculosis.  
Present  and  future  of  tuberculosis  
genotyping  methods
Dr  Vlad  Nikolayevskyy
UK  PHE  National  Mycobacterium  Reference  Service
Imperial  College
London,  United  Kingdom
 Purpose  of  TB  genotyping
Case  finding                                                                          (patient  care  /  public  health)
Ruling  in    transmission                                      (patient  care  /  public  health)
Outbreak  investigation                                      (patient  care  /  public  health)
Reactivation/reinfection                                  (patient  care  /  public  health)
Increasing
discrimination
Surveillance                                                                             (public  health)
requirements QC/identification  of  laboratory  cross-­‐contamination
Control  programme  assessment       (public  health  planning)
M.tb population  studies                                    (research)
M.tb phylogeny  studies                                      (research)

Courtesy  of  Dr  Tim  Brown

 Genotyping:  general  principles

• Differentiation  of  strains  of  Mycobacterium  

tuberculosis is  based  on  strain  specific  
differences  and  frequencies  and  order  of  certain  
DNA  sequences  in  M.  tuberculosis genome
• “Conventional”  typing  methods:
• Interrogate  tiny  portion(s)   of  the  genome
• Next  generation/Whole  Genome  Sequencing
• Interrogate  entire  genome
 Mycobacterium  tuberculosis genome
• FULL  sequences  are  now  available  for 1,000s  strains
• http://www.ncbi.nlm.nih.gov/genbank/
• De  novo  and  re-­‐sequencing  (NGS)
• M.tuberculosis complex  and  NTMs
• Quality  may  be  an  issue

• Consists  of  4.4  million  base  pairs

• Estimated  to  code  about  4000  proteins
• Very  rich  in  guanine  and  cytosine  

12/11/2016
 MTBC  genome  polymorphisms
M.tuberculosis polymorphisms Other  p olymorphisms
(associated  with  virulence  like  
pks15/1)
Repetitive  
sequences

SNPs LSP  (Large  sequence  polymorphisms) IS

VNTRs
sSNPs nsSNPs iSNPs UEP  (Unique   Non-­unique   Spacers  in  
Synonymous Non-­synonimous Intergenic events) (occurred   DR  region
Molecular  clock  s low Under  selection   Can  alter  t he  level   Big  d eletions  (RDs,   independently  
Phylogenetic  markers pressure of  gene  expression
TbD1  e tc) several  times)
(No  selection  pressure) Associated  with   Potentially  under  
drug  resistance selection  pressure
Generally  d o  n ot   Potentially  p lay  role  
involve  important   in  pathogenesis
genes (Associated  with   Generally  molecular  clock  is  
Potential   virulence?)  – Like   faster
phylogenetic   deletions  in  p lcA-­D
markers etc More  applicable  for  
3  Principal  genetic  groups  (PGG)     (Sreevatsan et  al,  1997)  2  SNPs Epidemiological  studies  rather  
4  lineages  +  M.bovis (Baker  et  al.,  2004)     37sSNPs
8  clusters  +  M.bovis (Gutacker et  al.,  2002)  230  s SNPs
than  for  p hylogenetical studies
9  clusters  +  M.bovis (Gutacker et  al.,  2006)  36sSNPs,  227nsSNPs,   Detection:
Concordant  to  some  d egree  
121  iSNPs By  individual  or  multiplex  PCRs  (Brosch et  al;;  Warren  et  al.,  2006) with  results  o f  S NP  a nalysis  
6  SCG  (SNP  Cluster  Groups)  +  M.bovis (Filliol et  al.,  2006)   Affymetrix chips  (Tsolaki et  al.,  2004) (particularly  for  M.bovis and  a  
159sSNPs,  35nsSNPs,  18iSNPs High  throughput  methods  using  DNA  probes  and  multiplex  PCRs   few  o ther  M.tuberculosis clades  
MORE  RECENT:  7  lineages  within  h uman-­adapted   (“Deligotyping”)  – de  la  Salmoniere et  al.,  2004;;  Alland et  al.,  2006   like  EAI  family
M.tuberculosis
Gagneux 2007;;  Coscolla 2014
Two   minimal  (although  representative)  s ets  of  SNPs  identified  f or  
May  be  identified  by:
future  s tudies
sequencing
12/11/2016 real-­time  PCR  with  TaqMan and/or  other  probes
multiplex  PCRs  with  specific  pairs  of  primers
Evolution  of  M.tuberculosis...  And  evolution  of  typing    tools!  (1)

12/11/2016
Evolution  of  M.tuberculosis...  And  evolution  of  typing  tools!  (2)

2 5 00 0

2 0 00 0 339.98
23","5
M IRU

591.61
10","7
M IRU
420.23
16","1
1 5 00 0 M IRU
374.02
24","1
M IRU

263.89
4","2
M IRU
1 0 00 0
286.76
2","2
M IRU

5 0 00

0
Dye  Signal

0 100 200 300 400 500 600 700

S ize  ( nt)

12/11/2016
 Spoligotyping
• Robust,  simple  and  
internationally  standardized  
method
• Data  in  digital  or  binary  
format,  easy  construction  of  
databases  and  data  eschange
• Particularly  useful  for  lineages  
Beijing identification
• May  be  used  for  
differentiation  between  
Mycobacteria  species

12/11/2016
 Spoligo family  and  country  of  birth

Brown  et  al.,  EID  2009

12/11/2016
 VNTR  Multilocus  typing
• Based  on  detection  of  variable  numbers   of  repeats  in  certain  loci  in  Mycobacteria   genome
• More  than  70  VNTR  loci  identified    
• Discriminative  power  varies  significantly   depending   on  loci  used,  number   of   loci  and  population   studied
• Standard  (24  VNTR)  sets  and  hypervariable  loci  (Supply   et  al.,  2006;   Allix-­‐Beguec et  al.,  2013)
• High   throughput   and  reproducibility;   extensively  evaluated  (Brown  et  al.,  2009;  Nikolayevskyy  et  al.,  2006  etc)
• Current  standard  in  many  EU  countries  and  worldwide

2 5 00 0

2 0 00 0 339.98
23","5
MIRU

591.61
10","7
MIRU
420.23
16","1
1 5 00 0 MIRU
374.02
24","1
MIRU

263.89
4","2
MIRU
1 0 00 0
286.76
2","2
MIRU

5 0 00

12/11/2016 0
Dye  Signal

0 100 200 300 400 500 600 700

S ize  ( nt)
 Spoligo/VNTR  data  analysis  and  portability
• International  databases
• Bionumerics tools  (Applied  Maths,  
Ghent,  Belgium)
• STs  (Shared  Types)  assignation
• Lineages  assignation  and  analysis
 Spoligotypes,  other  markers  and  lineages

Gagneux et  al.,  2007

Coscolla,   Gagneux 2014

12/11/2016
 Genomic  diversity  of  M.tuberculosis complex

Brosch et  al.,  PNAS,  2002

Arnold,  CMI,  2007 Coscolla,   Gagneux 2014

12/11/2016
Routine  use  of  TB  VNTR  typing  in  the  EU
 Current  activities

• 2009  – 2013  and  2014  – present

ERLN-­‐TB  and  ERLTB-­‐Net  projects  (funded  by  the  ECDC)

• 2014  -­‐ present

Annual  collection  of  MDR-­‐TB  VNTR  genotyping  data  through  TESSY-­‐MSS  
system
Production  of  annual  reports  and  newsletters
http://ecdc.europa.eu/en/publications/Publications/multidrug-­‐
resistant-­‐tuberculosis-­‐molecular-­‐typing-­‐surveillance.pdf
 Current  situation
Key  facts  2014
• No  of  MDR-­‐TB  strains  reported  to  TESSY-­‐MSS:  360  
• 24.6%  overall  coverage;  78.4%  coverage  for  countries  reporting
• Overall  coverage  remains  low
• No  of  EU  member  states  reporting:  17
No  of  countries  reporting
Coverage
18
30% 16
25% 14
12
20%
10
15% 8
10% 6
4
5%
2
0% 0
2012 2013 2014 2008 2009 2010 2011 2012 2013 2014

ECDC  molecular  surveillance  report,   2015

Current  situation  Key  facts  2014

ECDC  molecular  surveillance  report,   2015

 Cross  border  clusters  2014
• 155/360  MDR-­‐TB  strains  belonged  to  one  of  35  cross  border  clusters  
identified  in  2011-­‐2014  (assigned  MtbC15-­‐9  types)
• Cluster  size  varied  from  2  to  182  strains
• No  of  countries  involved  per  cluster  varied  from  2  to  14
• Two  most  common  clusters
• 100-­‐32  (Beijing)  N=51  (33.6%)
• 94-­‐32  (Beijing)  N=36  (23.7%)
• Known  limitations  of  24VNTR  genotyping  especially  for  Beijing  strains

ECDC  molecular  surveillance  report,   2015

 ERLTB-­‐Net  activities
TB  Genotyping  EQA  system  development
• Years  2013  – 2015
• No  of  countries  participating  (=  having  capacity/expertise  for  
genotyping)  remains  stable  (=low)
• Some  countries  (including  medium  TB/high  MDRTB)  do  not  
participate No  of  countries  participating
20

15

10

0
2013 2014 2015
RIVM,  Netherlands
ERLTB-­‐Net  report  unpublished
Whole  Genome/Next  generation  sequencing
 Classic  vs  Modern
• Sanger  Sequencing • Next  generation  sequencing

• M.tuberculosis H37Rv  genome,  S.Cole,  Nature  

1998

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 Analytical  pipelines    
• Heavily  dependant  on  the  purpose
• Detection  of  mutations  associated  with  drug  resistance
• Relatedness  of  strains/phylogeny 6.0E-5

• Good  news:  now  NOT  only  for  Linux  experts  J

• Windows-­‐based  tools  available
• Galaxy
• Visualising   software
• Data  analysis  (IGV,  Tablet,  Artemix etc etc)
• Online  databases  (PhyResSE,  TBDream,  MUBII-­‐TB-­‐DB)  and  tools  (TB  Profiler)  
available Sandgren et  al.,  PLoS Medicine  2009
Flandrois et  al.,  BMC  Bioinformatics  2014
Feuerriegel et  al.,  JAC  2014

12/11/2016 22
 Challenges
• Relatedness:
• No/little  agreement  on  SNP  distances  to  confirm  direct  transmission
• DST:
• Online  databases  (PhyResSE,  TBDream,  MUBII-­‐TB-­‐DB)  and  tools  (TB  Profiler)  
available  but  for  research  use  only  at  the  moment
• No/little  international  agreement  on  pipelines/databases
• General  problems
• Within  host  variation/evolution
• Mixed  populations
• Lack  of  reliable  evidence
• No  agreement  on  technical  parameters:  data  from  different  labs  may  not  be  
fully  compatible
Feuerriegel et  al.,  JAC  2014
Nikolayevskyy et  al.,  Tuberculosis  (Edinb)  2016
Coll et  al.,  Genome  Med  2015
Papaventsis et  al.,  CMI,  2016
12/11/2016 23
 Is  VNTR  genotyping  good  enough?
• Interrogate  only  small  fraction  of  genome
• Only  surrogate  markers
• Problems:
• Lack  of  discriminatory  power  (especially  in  highly  conserved  genotypes  eg
Beijing)
• Within-­‐host  evolution
• Can  rule  OUT  transmission
• Cannot  rule  IN  transmission
Allix-­‐Beguec et  al..,  JCM  2009
Niemann  et  al.,  PLoS ONE  2014
Walker  et  al.,  Nature  Genetics  2014

12/11/2016 24
Potential  role  of  WGS  in  TB  epidemiology  (2)
• First  WGS-­‐based  TB  epidemiology  study:  Gardy et  al.,   NEJM  2011   (Canada)
• 41  case  (previously  undistinguishable  using  24VNTR)  have  been  sequenced

12/11/2016 25
Potential  role  of  WGS  in  TB  epidemiology  (3)

• Walker  et  al.,  Lancet  ID  2013;  

12/11/2016 • Walker  et  al.,  Nature  Genetics  2014 26
Potential  role  of  WGS  in  TB  epidemiology  (4)
• Systematic  reviews  published  in  2016
• Nikolayevskyy et  al.,  Tuberculosis  (Edinb)  March  2016
• Hatherell  et  al.,  BMC  Medicine  April  2016
• Major  conclusions:
• 12  and  25  studies  reviewed,  respectively
• WGS  has  a  higher  discriminatory  power  and  most  likely  is  able  to  subdivide  
clusters  defined  by  classical   RFLP/VNTR  genotyping
• Proposed  cut-­‐off  value  of  <6  SNPs  between  isolates  predicts  links  established   by  
conventional  epidemiology  for  recent  transmission  events.  This  cut-­‐off  value  
could  be  used  for  identification  of  isolates  involved  in  direct  human-­‐to-­‐human  
TB  transmission  i.e.  TRANSMISSION  PROBABLY  COULD  BE  RULED  IN
• WGS  allowed  distinguishing   between  isolates  sharing  identical  genotyping  
patterns  potentially  separating  transmission  chains  within  RFLP/VNTR  clusters.  It  
identified  false  clustering  ruling  out  false  transmission  events

12/11/2016 27
 Potential  role  of  WGS  in  TB  epidemiology  (5)
• Problems  and  challenges  identified  
through  reviews:
• Study  design:  no  prospective  population-­‐
based  studies
• Lack  of  standardization  in  technology  
and  reporting
• Set  of  parameters  and  essential  
characteristics  proposed
• In  practical  terms:
• Is  there  any  added  value?
• Cost  effectiveness?
• Value  for  high  TB/DRTB  settings?
• The  impact  of  WGS  in  resolving  TB  
outbreaks,  real-­‐time  TB  epidemiology,  
and  molecular  surveillance   is  yet  to  be  
established
12/11/2016 28
 Acknowledgements
• Research  teams: • Selected  slides:  pictures  are  
• PHE/ECDC courtesy  of  Stefan  Niemann,  
• Yen  Holicka,  Dimitrios Papaventsis,   Germany;  Tim  Brown,  United  
Csaba Kodmon,  Marieke  van  der  Werf Kingdom
• Borstel,  Germany
• Stefan  Niemann,  Katharina  Kranzer
• QMUL/Imperial  College  London
• Francis  Drobniewski,  Nicola  Casali,  
Irina  Kontsevaya,  Agnieszka  Broda,  
Yanina   Balabanova • Funding:
• Samara,  Russia • ECDC
• Olga  Ignatyeva,  Alexander  Kovalyov,   • EU  FP7  Pannet
Katya  Koshkarova,  Anna  Isaeva • EU  FP7  Eurogen

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